menaquinone-6 and menatetrenone

Vitamin K acts as a cofactor and is required for post-translational γ-carboxylation of vitamin K-dependent proteins (VKDP). The current recommended daily intake (RDI) of vitamin K in most countries has been established based on normal coagulation requirements. Vitamin K1 and menaquinone (MK)-4 has been shown to decrease osteocalcin (OC) γ-carboxylation at RDI levels. Among the several vitamin K homologs, only MK-7 (vitamin K2) can promote γ-carboxylation of extrahepatic VKDPs, OC, and the matrix Gla protein at a nutritional dose around RDI. MK-7 has higher efficacy due to its higher bioavailability and longer half-life than other vitamin K homologs. As vitamin K1, MK-4, and MK-7 have distinct bioactivities, their RDIs should be established based on their relative activities. MK-7 increases bone mineral density and promotes bone quality and strength. Collagen production, and thus, bone quality may be affected by MK-7 or MK-4 converted from MK-7. In this review, we comprehensively discuss the various properties of MK-7.

Topics: Biological Availability; Bone and Bones; Bone Density; Collagen; Dietary Supplements; Humans; Osteocalcin; Recommended Dietary Allowances; Vitamin K 1; Vitamin K 2

Vitamin K is a cofactor for γ-glutamyl carboxylase, which catalyzes the posttranslational conversion of specific glutamyl residues to γ-carboxyglutamyl residues in a variety of vitamin K-dependent proteins (VKDPs) involved in blood coagulation, bone and cartilage metabolism, signal transduction, and cell proliferation. Despite the great advances in the genetic, structural, and functional studies of VKDPs as well as the enzymes identified as part of the vitamin K cycle which enable it to be repeatedly recycled within the cells, little is known of the identity and roles of key regulators of vitamin K metabolism in mammals and humans. This review focuses on new insights into the molecular mechanisms underlying the intestinal absorption and in vivo tissue conversion of vitamin K1 to menaquinone-4 (MK-4) with special emphasis on two major advances in the studies of intestinal vitamin K transporters in enterocytes and a tissue MK-4 biosynthetic enzyme UbiA prenyltransferase domain-containing protein 1 (UBIAD1), which participates in the in vivo conversion of a fraction of dietary vitamin K1 to MK-4 in mammals and humans, although it remains uncertain whether UBIAD1 functions as a key regulator of intracellular cholesterol metabolism, bladder and prostate tumor cell progression, vascular integrity, and protection from oxidative stress.

Topics: Animals; Cell Transformation, Neoplastic; Cholesterol; Dimethylallyltranstransferase; Enterocytes; Humans; Intestinal Absorption; Mice; Neoplasms; Oxidative Stress; Vitamin K; Vitamin K 2

Possible benefits of vitamin K on bone health, fracture risk, markers of bone formation and resorption, cardiovascular health, and cancer risk in postmenopausal women have been investigated for over three decades; yet there is no clear evidence-based universal recommendation for its use. Interventional studies showed that vitamin K1 provided significant improvement in undercarboxylated osteocalcin (ucOC) levels in postmenopausal women with normal bone mineral density (BMD); however, there are inconsistent results in women with low BMD. There is no study showing any improvement in bone-alkaline-phosphatase (BAP), n-telopeptide of type-1 collagen (NTX), 25-hydroxy-vitamin D, and urinary markers. Improvement in BMD could not be shown in the majority of the studies; there is no interventional study evaluating the fracture risk. Studies evaluating the isolated effects of menatetrenone (MK-4) showed significant improvement in osteocalcin (OC); however, there are inconsistent results on BAP, NTX, and urinary markers. BMD was found to be significantly increased in the majority of studies. The fracture risk was assessed in three studies, which showed decreased fracture risk to some extent. Although there are proven beneficial effects on some of the bone formation markers, there is not enough evidence-based data to support a role for vitamin K supplementation in osteoporosis prevention among healthy, postmenopausal women receiving vitamin D and calcium supplementation. Interventional studies investigating the isolated role of vitamin K on cardiovascular health are required. Longterm clinical trials are required to evaluate the effect of vitamin K on gynecological cancers. MK-4 seems safe even at doses as high as 45 mg/day.

Topics: Bone and Bones; Bone Density; Female; Fractures, Bone; Humans; Osteocalcin; Osteoporosis, Postmenopausal; Postmenopause; Vitamin K; Vitamin K 2; Vitamins

In contrast to other fat-soluble vitamins, dietary vitamin K is rapidly lost to the body resulting in comparatively low tissue stores. Deficiency is kept at bay by the ubiquity of vitamin K in the diet, synthesis by gut microflora in some species, and relatively low vitamin K cofactor requirements for γ-glutamyl carboxylation. However, as shown by fatal neonatal bleeding in mice that lack vitamin K epoxide reductase (VKOR), the low requirements are dependent on the ability of animals to regenerate vitamin K from its epoxide metabolite via the vitamin K cycle. The identification of the genes encoding VKOR and its paralog VKOR-like 1 (VKORL1) has accelerated understanding of the enzymology of this salvage pathway. In parallel, a novel human enzyme that participates in the cellular conversion of phylloquinone to menaquinone (MK)-4 was identified as UbiA prenyltransferase-containing domain 1 (UBIAD1). Recent studies suggest that side-chain cleavage of oral phylloquinone occurs in the intestine, and that menadione is a circulating precursor of tissue MK-4. The mechanisms and functions of vitamin K recycling and MK-4 synthesis have dominated advances made in vitamin K biochemistry over the last five years and, after a brief overview of general metabolism, are the main focuses of this review.

Topics: Animals; Biosynthetic Pathways; Diet; Dimethylallyltranstransferase; Humans; Intestinal Mucosa; Molecular Structure; Vitamin K 1; Vitamin K 2; Vitamin K Epoxide Reductases

Vitamin K may play an important role in the prevention of fractures in postmenopausal women with osteoporosis. Menatetrenone is the brand name of a synthetic vitamin K2 that is chemically identical to menaquinone-4. The present review study aimed to clarify the effect of menatetrenone on the skeleton in postmenopausal women with osteoporosis, by reviewing the results of randomized controlled trials (RCTs) in the literature. RCTs that investigated the effect of menatetrenone on bone mineral density (BMD), measured by dual-energy X-ray absorptiometry and fracture incidence in postmenopausal women with osteoporosis, were identified by a PubMed search for literature published in English. Eight studies met the criteria for RCTs. Small RCTs showed that menatetrenone monotherapy decreased serum undercarboxylated osteocalcin (ucOC) concentrations, modestly increased lumbar spine BMD, and reduced the incidence of fractures (mainly vertebral fracture), and that combined alendronate and menatetrenone therapy enhanced the decrease in serum ucOC concentrations and further increased femoral neck BMD. This review of the literature revealed positive evidence for the effects of menatetrenone monotherapy on fracture incidence in postmenopausal women with osteoporosis. Further studies are required to clarify the efficacy of menatetrenone in combination with bisphosphonates against fractures in postmenopausal women with osteoporosis.

Topics: Absorptiometry, Photon; Bone Density; Female; Fractures, Bone; Humans; Incidence; Osteocalcin; Osteoporosis, Postmenopausal; Postmenopause; Randomized Controlled Trials as Topic; Vitamin K 2

In this decade, the field of pharmacogenomics (PGx), which is related to pharmacokinetics (PK) or pharmacodynamics (PD), has attracted much attention because it may provide a possible explanation for individual differences in the clinical efficacy of drugs. For the development of personalized drug therapy, it is important to accumulate evidence from PK/PD/PGx analysis in clinical trials. Warfarin (WF) is one of the most widely prescribed anticoagulants for the prevention and treatment of venous and arterial thromboembolism. However, large interindividual and interethnic differences have been observed in the WF dose required to elicit the anticoagulant effect. We investigated the factors influencing the WF maintenance dose in Japanese patients. Our study confirmed a large interindividual variability in the WF maintenance dose that was due to a VKORC1 1639 G>A polymorphism and differences in body weight, age, and serum albumin. In addition, we found that the CYP4F2 genotype affects the plasma concentration of menaquinone-4, and that this finding was correlated with the WF sensitivity index in Japanese pediatric patients. Methotrexate (MTX) is an antifolate that is widely used to treat rheumatoid arthritis (RA) and cancer. The response to low-dose MTX demonstrated wide interpatient variability; however, the contributing factors remain unclear. We found that the frequency of the RFC1 80A allele was higher in RA patients treated with MTX alone compared with patients who received biological disease-modifying antirheumatic drugs (bDMARDs). This finding may support the combined use of bDMARDs and MTX. Further large-scale prospective clinical trials are required to confirm these findings.

Topics: Anticoagulants; Antirheumatic Agents; Arthritis, Rheumatoid; Asian People; Clinical Trials as Topic; Cytochrome P-450 Enzyme System; Cytochrome P450 Family 4; Drug Therapy, Combination; Gene Frequency; Humans; Maintenance Chemotherapy; Methotrexate; Pharmacokinetics; Polymorphism, Genetic; Precision Medicine; Replication Protein C; Thromboembolism; Vitamin K 2; Vitamin K Epoxide Reductases; Warfarin

One of the fat-soluble vitamins, vitamin K was initially discovered for its role in blood coagulation. Although several vitamin K-dependent hemostatic proteins are particularly important for the brain, other vitamin K-dependent proteins (VKDPs), not associated with blood coagulation, also contribute to the brain function. In addition to the VKDPs, vitamin K participates in the nervous system through its involvement in sphingolipid metabolism, a class of lipids widely present in brain cell membranes. Classically known for their structural role, sphingolipids are biologically potent molecules involved in a wide range of cellular actions. Also, there is growing evidence that the K vitamer, menaquinone-4, has anti-inflammatory activity and offers protection against oxidative stress. Finally, although limited in numbers, reports point to a modulatory role of vitamin K in cognition. This short review presents an overview of the known role of vitamin K in brain function to date.

Topics: Animals; Brain; Cognition; Humans; Intercellular Signaling Peptides and Proteins; Protein C; Protein S; Sphingolipids; Vitamin K; Vitamin K 2

The effect of the anti-osteoporosis medicine, menatetrenone (vitamin K(2); menaquinone-4) on the skeleton remains a matter of controversy. The objective of the present review study was to evaluate the effect of menatetrenone on the skeleton of postmenopausal women, men and glucocorticoid-treated patients.. PubMed was used to search the literature for randomized controlled trials (RCTs), meta-analyses and systematic reviews. Thirteen RCTs, one meta-analysis and one systematic review were available for analysis.. Except for one large Japanese RCT (Phase IV trial: Osteoporotic Fracture (OF) study, n = 4378), RCTs with small sample size showed non-significant or modest effect on bone mineral density (BMD) in postmenopausal women and patients treated with glucocorticoid, positive effect on hip geometry in postmenopausal women and efficacy against fractures (mainly vertebral fractures) in postmenopausal women with osteoporosis. A post hoc analysis of the OF study showed that the incidence of vertebral fractures decreased in postmenopausal women with at least five vertebral fractures. A meta-analysis study, but not a systematic review study, showed efficacy against vertebral and non-vertebral fractures mainly in postmenopausal women with osteoporosis. There was no available evidence for men with osteoporosis.. The present review of the literature revealed some evidence of a positive effect of menatetrenone on the skeleton of postmenopausal women and in patients treated with glucocorticoid.. Menatetrenone is considered to be a second-line medicine for postmenopausal osteoporotic women with an increased risk for vertebral fractures.

Topics: Bone Density; Clinical Trials as Topic; Humans; Osteocalcin; Osteoporosis; Spinal Fractures; Treatment Outcome; Vitamin K 2

Bisphosphonate-related osteonecrosis of the jaw (BRONJ) is an iatrogenic disease and a significant concern to patients and dentists. Bisphosphonates are used for millions of men and women with osteoporosis, with the rationale being that these medications increase bone mineral density and decrease fracture risk. Clinicians will undoubtedly encounter some patients who are taking bisphosphonate medications. Recognizing the risk factors for BRONJ, understanding the pathophysiology of BRONJ, and enacting a reasonable approach to prevent BRONJ in patients is crucial to providing safe and effective treatments. This article proposes a rational approach for moving BRONJ away from a risk management and quality assurance model that is currently being used by dentists, to a preventive model. The role of collagen as postulated in this article cannot be ignored in the pathophysiology and potential prevention and treatment of osteoporosis and BRONJ. Studies from the medical literature support the safety and efficacy of MK4 as a potential therapeutic agent in preventing and treating osteoporosis and BRONJ. While the approach outlined herein requires additional study, the conceptual framework based on a broad review of the osteonecrosis of the jaw (ONJ) literature provides a means to begin to address this situation in a proactive, rather than reactive, way.

Topics: Bisphosphonate-Associated Osteonecrosis of the Jaw; Collagen; Diphosphonates; Humans; Osteoporosis; Vitamin K 2

A high incidence of fractures, particularly of the hip, represents an important problem in patients with AD, who are prone to falls and may have osteoporosis. The odds ratio reported for fracture prevalence between elderly persons with and without AD is 6.9. We previously demonstrated that deficiency of 25-hydroxyvitamin D due to sunlight deprivation and vitamin K deficiency due to malnutrition contributed to reduced bone mineral density in patients with AD and were associated with a high risk of hip fracture. Treatment with menatetrenone, risedronate or regular sunlight exposure are safe and effective in increasing bone mass and reducing the risk of hip fracture in patients with AD.

Topics: Accidental Falls; Bone Density; Dementia; Etidronic Acid; Hip Fractures; Humans; Hyperparathyroidism, Secondary; Osteoporosis; Risedronic Acid; Risk; Risk Factors; Sunlight; Vitamin D Deficiency; Vitamin K 2; Vitamin K Deficiency

Patients with neurological diseases such as Alzheimer's disease, stroke and Parkinson's disease have been reported to have vitamin K deficiency secondary to malnutrition, which increases the risk of non-vertebral and hip fractures. The purpose of the present study was to clarify the efficacy of menatetrenone (vitamin K(2)) against non-vertebral and hip fractures in patients with neurological diseases.. A literature search was conducted on PubMed from January 1995 to July 2008 to identify randomized controlled trials (RCTs) of use of menatetrenone against non-vertebral and hip fractures in patients with neurological diseases. A meta-analysis of all RCTs meeting these criteria was then performed.. Three RCTs of patients with Alzheimer's disease (n = 178, mean age 78 years), stroke (n = 99, mean age 66 years) and Parkinson's disease (n = 110, mean age 72 years) met the criteria for meta-analysis. These RCTs did not include placebo controls but did have non-treatment controls. According to the meta-analysis, the overall relative risks (95% confidence intervals) for non-vertebral and hip fractures with menatetrenone treatment compared with non-treatment were 0.13 (0.05, 0.35) and 0.14 (0.05, 0.43), respectively, in patients with neurological diseases. No severe adverse events were reported with menatetrenone treatment.. The present meta-analysis of three RCTs suggests that there is efficacy for menatetrenone treatment against non-vertebral and hip fractures among patients with neurological diseases. Further larger placebo-controlled trials are needed to confirm the results of the present study.

Topics: Aged; Bone Density Conservation Agents; Brain Diseases; Female; Fractures, Bone; Hip Fractures; Humans; Male; Randomized Controlled Trials as Topic; Vitamin K 2

To determine the clinical and cost-effectiveness of vitamin K in preventing osteoporotic fractures in postmenopausal women.. Searches were conducted in May 2007 in MEDLINE, MEDLINE In-Process, EMBASE, Cochrane Database of Systematic Reviews, Cochrane Controlled Trials Register, BIOSIS, CINAHL, DARE, NHS EED and HTA databases, AMED, NRR, Science Citation Index and Current Controlled Trials. The MEDLINE search was updated in March 2009.. Selected studies were assessed and subjected to data extraction and quality assessment using standard methods. Where appropriate, meta-analysis was carried out. A mathematical model was constructed to estimate the cost-effectiveness of vitamin K1.. The electronic literature searches identified 1078 potentially relevant articles. Of these, 14 articles relating to five trials that compared vitamin K with a relevant comparator in postmenopausal women with osteoporosis or osteopenia met the review inclusion criteria. The double-blind ECKO trial compared 5 mg of phylloquinone (vitamin K1) with placebo in Canadian women with osteopenia but without osteoporosis. Four open-label trials used 45 mg of menatetrenone (vitamin K2) in Japanese women with osteoporosis; the comparators were no treatment, etidronate or calcium. The methodological quality of the ECKO trial was good; however, all four menatetrenone trials were poorly reported and three were very small (n < 100 in each group). Phylloquinone was associated with a statistically significant reduction in the risk of clinical fractures relative to placebo [relative risk 0.46, 95% confidence interval (CI) 0.22 to 0.99]; morphometric vertebral fractures were not reported. The smaller menatetrenone trials found that menatetrenone was associated with a reduced risk of morphometric vertebral fractures relative to no treatment or calcium; however, the larger Osteoporosis Fracture (OF) study found no evidence of a reduction in vertebral fracture risk. The three smaller trials found no significant difference between treatment groups in non-vertebral fracture incidence. In the ECKO trial, phylloquinone was not associated with an increase in adverse events. In the menatetrenone trials, adverse event reporting was generally poor; however, in the OF study, menatetrenone was associated with a significantly higher incidence of skin and skin appendage lesions. No published economic evaluations of vitamin K were found and a mathematical model was thus constructed to estimate the cost-effectiveness of vitamin K1. Comparators were alendronate, risedronate and strontium ranelate. Vitamin K1 and alendronate were markedly more cost-effective than either risedronate or strontium ranelate. The base-case results favoured vitamin K1, but this relied on many assumptions, particularly on the efficacy of preventing hip and vertebral fractures. Calculation of the expected value of sampled information was conducted assuming a randomised controlled trial of 5 years' duration comparing alendronate with vitamin K1. The costs incurred in obtaining updated efficacy data from a trial with 2000 women per arm were estimated to be a cost-effective use of resources.. There is currently large uncertainty over whether vitamin K1 is more cost-effective than alendronate; further research is required. It is unlikely that the present prescribing policy (i.e. alendronate as first-line treatment) would be altered.

Topics: Aged; Aged, 80 and over; Bone Density Conservation Agents; Cost-Benefit Analysis; Female; Fractures, Bone; Humans; Models, Econometric; Osteoporosis, Postmenopausal; Quality-Adjusted Life Years; Vitamin K; Vitamin K 1; Vitamin K 2; Vitamins

Phylloquinone is a major form (>90%) of dietary vitamin K, but the form of vitamin K that exists at the highest concentrations in tissues of animals and humans is menaquinone-4 (MK-4) . Despite this great difference, the origin of tissue MK-4 had not been clarified until recently. We demonstrated that deuterium-labeled phylloquinone was converted into deuterium-labeled MK-4 in mice and this conversion occurred following an oral or enteral administration, but not parenteral administration. By the oral route, the phylloquinone with the deuterium-labeled side chain (phytyl side-chain) was clearly converted into menaquinone-4 with a non-deuterium-labeled side chain (geranylgeranyl side-chain), implying that phylloquinone was converted into menaquinone-4 via integral side-chain removal. Our results suggest that cerebral menaquinone-4 originates from phylloquinone intake and the release of menadione from phylloquinone in the intestine followed by the prenylation of menadione into menaquinone-4 in the intestine or tissues.

Topics: Administration, Oral; Animals; Brain; Humans; Intestinal Absorption; Intestinal Mucosa; Mice; Neurons; Tissue Distribution; Vitamin K 1; Vitamin K 2

The mechanisms by which vitamin E interferes with vitamin K activity, especially blood clotting, are not known, but hypothetically this interference may involve metabolic pathways. Phylloquinone (K(1)) must be converted to menaquinone (MK-4, the most potent extrahepatic tissue vitamin K) by truncation of the K(1) side chain and replacement with geranylgeranyl. Possible mechanisms for the vitamin E and K interaction include: 1) vitamin E competes for the yet undiscovered enzyme that truncates the K(1) side chain; 2) vitamin E competes with K(1) for the hypothetical cytochrome P450 enzyme that omega-hydroxylates the K(1) side chain, thereby preventing its beta-oxidation and its removal for MK-4 formation; or 3) vitamin E increases xenobiotic pathways that increase hepatic metabolism and excretion of all vitamin K forms. Currently, the pathway for K(1) conversion to MK-4 is unknown, the process for regulating vitamin K metabolism to urinary excretion products is unknown, and why vitamin E supplements have such a dramatic effect, causing bleeding in some individuals and not in others, remains a mystery.

Topics: Blood Coagulation; Drug Interactions; Hemostatics; Humans; Hydroxylation; Oxidation-Reduction; Vitamin E; Vitamin K; Vitamin K 2; Xenobiotics

Bone loss and impaired bone quality have been proposed as major causes of increased bone fragility in osteoporosis. The proposed determinants of the material properties of bone are the degree of secondary mineralization, microdamage accumulation, and collagen cross-link formation that is affected by bone turnover rate. Recently, we demonstrated that (1) three years bisphosphonate increases the degree of mineralization, collagen maturity, and non-enzymatic cross-link, pentosidine without changing the amount of enzymatic cross-link and (2) the changes in collagen cross-link formation after treatment of bisphosphonate were associated with microdamage accumulation, which were independent of the mineral content (Saito M, Osteoporos Int, in press) and (3) urinary excretion of pentosidine predicts vertebral fracture (Shiraki M, Saito M, et al, J Bone Miner Metab, 2008) . In this review, I described effects of drugs for osteoporosis on bone material properties.

Topics: Animals; Bone and Bones; Bone Density Conservation Agents; Bone Remodeling; Calcification, Physiologic; Collagen; Diphosphonates; Glycation End Products, Advanced; Humans; Hydroxycholecalciferols; Osteoporosis; Raloxifene Hydrochloride; Vitamin K 2

Phylloquinone (vitamin K(1) = VK(1)) and the menaquinones (MK-n, or vitamin K(2) = VK(2)) are naturally occurring forms of VK. Most of the menaquinone series are synthesized by microorganisms, but we have reported that MK-4 is usual in being synthesized by the conversion of orally ingested VK(1) or MK-n in the major tissues of germfree rats and mice which lack their intestinal microflora. This result led us to deny 1960's Martius' hypothesis that described the participation of bacterial enzyme of the intestinal flora to this conversion. VK acts as a cofactor in the posttranslational synthesis of gamma-carboxyglutamic acid (Gla) from glutamic acid (Glu) residues in the nascent Gla-protein molecule. Therefore, VK is essential for blood coagulation (various clotting factors) and bone structure (as osteocalcin [OC = BGP] and matrix Gla-protein [MGP] in mammals. In addition to the liver, VK is found in the bone, brain, heart, testis, kidney, pancreas and salivary glands mainly as MK-4, and it has been reported that MK-4 itself has specific biological activities in these tissues beside Gla-protein formation. However, the physiological role of MK-4 in these organs has not been fully understood yet. Recently MK-4 has been attracted the attention of researchers due to its activities such as apoptotic activity on the osteoclast cells and leukemia cells, SXR/PXR ligand, and so on. We further review the potent important physiological role of MK-4 in the bone as well as other major tissues.

Topics: Animals; Apoptosis; Blood Coagulation; Bone and Bones; Humans; Intestinal Absorption; Ligands; Mice; Osteocalcin; Osteoclasts; Pregnane X Receptor; Protein Processing, Post-Translational; Rats; Receptors, Steroid; Vitamin K; Vitamin K 2

Menatetrenone (MK-4) is a form of vitamin K(2) (VK(2)) which is utilized for the treatment of osteoporosis. MK4 has a grade B as a total recommendation rate in the Japanese guideline for the prevention and treatment of osteoporosis in 2006 based on the current clinical evidences. The effects of fracture prevention by MK-4 are assumed to be based on the increase of bone mineral density as well as the improvement of bone quality. Recent researches suggest that VK affects bone metabolism via nuclear receptor in addition to the carboxylation of VK dependent proteins.

Topics: Bone and Bones; Bone Density; Evidence-Based Medicine; Fractures, Bone; Humans; Meta-Analysis as Topic; Osteoporosis; Practice Guidelines as Topic; Vitamin K 2

Topics: Bone and Bones; Bone Density; Bone Density Conservation Agents; Calcitonin; Diphosphonates; Femoral Neck Fractures; Humans; Hypercalcemia; Osteoporosis; Stroke; Vitamin D; Vitamin D Deficiency; Vitamin K 2

Vitamin K2 (menatetrenone) treatment was reported to significantly prevent new clinical fracture (chi2 = 10.935;p = 0.0273) in a 2-year group comparison study of patients with osteoporosis, although it only maintained the baseline bone mineral density. This result strongly suggested that another factor was involved in promoting bone strength apart from an increase in bone mineral density. With respect to the therapeutic effect of menatetrenone treatment on corticosteroid-induced osteoporosis over 2 years, the incidence of a new vertebral fracture was 13.3% in the menatetrenone treatment group versus 41% in the control group, indicating that this treatment could prevent fractures. Multivariate logistic regression analysis was performed to investigate independent risk factors for new vertebral fractures, and treatment with menatetrenone showed a preventive effect on fracture with an odds ratio of 0.03 and a risk rate of 0.003.

Topics: Animals; Bone and Bones; Bone Density; Fractures, Bone; Humans; Osteoporosis; Vitamin K 2

The kidney deeply takes part in phosphorus and vitamin D metabolism. The chronic kidney disease patients usually suffer from serious bone disease. They are prescribed the glucocorticoid medication for primary kidney disease, as nephrotic syndrome and collagen renal disease, with aging. The end of therapeutic target is normal bone structure with normal bone turnover in CKD patients. The control of serum Ca and phosphorus level is important step and also another target. This issue describes the problem and the advancement of treatment and the measurement of the bone mass from this viewpoint.

Topics: Absorptiometry, Photon; Bone Density; Calcitriol; Calcium; Chronic Kidney Disease-Mineral and Bone Disorder; Deferoxamine; Humans; Hyperparathyroidism, Secondary; Parathyroid Hormone; Parathyroidectomy; Phosphorus; Vitamin K 2

Osteoporosis with increased risk of bone fracture is a disabling syndrome that naturally occurs as long as one ages and moves on two legs. Recent progress in bone cell biology has shed light on the mechanisms underlying the anti-osteoporotic properties of drugs that have been in use for a long time, providing a fresh stage for novel pharmacotherapies. In addition, large scale clinical trials developed in the past decade appear not only to rationalize the clinical utilities of these drugs but also to provide new concepts for the development of new therapeutic modalities. Progress in the fields of basic and clinical research field is briefly reviewed herein.

Topics: Alendronate; Androgens; Animals; Binding Sites; Bone Remodeling; Clinical Trials as Topic; Estrogen Replacement Therapy; Estrogens; Estrogens, Non-Steroidal; Evidence-Based Medicine; Humans; Isoflavones; Osteoblasts; Osteoclasts; Osteoporosis; Phytoestrogens; Plant Preparations; Selective Estrogen Receptor Modulators; Vitamin D; Vitamin K 2

Topics: Alendronate; Calcitonin; Clinical Trials as Topic; Drug Approval; Estriol; Etidronic Acid; Europe; Humans; Isoflavones; Japan; Osteoporosis; Practice Guidelines as Topic; Raloxifene Hydrochloride; United States; Vitamin K 2

The effects of vitamin K (VK) on immune cells in ruminants are yet to be fully investigated. The objective of this study was to examine the effects of VK on peripheral blood mononuclear cells (PBMC) in Holstein dairy cows. A cell proliferation assay was performed to evaluate the effect of menaquinone-4 (MK-4, the biologically active form of VK) on immune response of PBMC. The proliferation of PBMC stimulated by MK-4 was significantly higher than that of nonstimulated controls. The expression of T cell-related genes in PBMC, stimulated with MK-4, was assessed by quantitative PCR. No significant changes were observed in the mRNA expression levels of both CD4 and CD8 as helper T cell and cytotoxic T cell markers, respectively. The present study demonstrated that MK-4 positively influenced cow PBMC proliferation and suggested the possibility of bovine-specific immune cell activation. The present study lays a foundation for understanding the physiological role of VK in cattle.

Topics: Animals; Cattle; Cell Proliferation; Female; Leukocytes, Mononuclear; Vitamin K; Vitamin K 2

Objective Vitamin K2 (menatetrenone) is an effective treatment for patients with postmenopausal osteoporosis. We herein performed a subanalysis of patients with systemic autoimmune diseases undergoing glucocorticoid therapy in our previous prospective study. Methods Sixty patients were categorized into a group with vitamin K2 treatment (n=20, Group A) and a group without vitamin K2 treatment (n=40, Group B). All patients were treated with bisphosphonates. Results Serum levels of osteocalcin and undercarboxylated osteocalcin decreased significantly after the start of glucocorticoid therapy in both groups, while the serum osteocalcin level was significantly higher in Group A than Group B during the third (p=0.0250) and fourth weeks (p=0.0155). The serum level of the N-terminal peptide of type I procollagen, a bone formation marker, decreased during glucocorticoid therapy, but was significantly higher in Group A than Group B during the fourth week (p=0.0400). The bone mineral density and fracture rate showed no significant differences between the two groups. Conclusion Although vitamin K2 improves bone turnover markers in patients with osteoporosis on glucocorticoid therapy, it has no significant effect on the bone mineral density and fracture rate after 1.5 years of treatment.

Topics: Autoimmune Diseases; Biomarkers; Bone Density; Collagen Type I; Diphosphonates; Female; Glucocorticoids; Humans; Male; Middle Aged; Osteoporosis; Prospective Studies; Vitamin K 2

Osteoporosis is one of the major complications of glucocorticoid therapy. Osteoporosis is usually defined by the levels of bone mineral density (BMD) assessed by dual energy X-ray absorptiometry (DEXA); however, glucocorticoids often induce fractures in patients with normal BMD. Thus, novel diagnostic approaches are required. In this study, we examined whether multidetector-row computed tomography (MDCT) is useful to assess the bone status in glucocorticoid-induced osteoporosis (GIO). Because bisphosphonates have been proven to prevent bone fracture in GIO, we tried to detect the therapeutic effects of bisphosphonates in GIO by MDCT. Fifteen Japanese patients with immunoglobulin A nephropathy who had normal renal function were enrolled in this open-label randomized trial. Patients were randomly divided into three groups-calcitriol (VD), menatetrenone (VK), or bisphosphonate (Bis). Bone conditions were analyzed twice by three different methods-bone turnover markers, DEXA, and MDCT-at the start and 6 months after the start of therapy. Both bone markers and DEXA could not detect significant differences among the therapeutic groups; however, MDCT-based analyses detected the preventive effects of bisphosphonates in GIO. Compared to VD, Bis improved structural indices, such as bone volume fraction, trabecular separation, marrow star volume, and structure model index whereas the difference between VD and VK was not significant. Finite element analysis revealed that simulated fracture load in the Bis group was significantly improved. These findings suggested that MDCT-based assessment is superior to bone markers and/or DEXA in assessing the therapeutic effect of bisphosphonates on GIO.

Topics: Adolescent; Adult; Biomarkers; Bone and Bones; Bone Density; Bone Density Conservation Agents; Calcitriol; Diphosphonates; Female; Glucocorticoids; Humans; Male; Middle Aged; Multidetector Computed Tomography; Osteoporosis; Prospective Studies; Vitamin K 2; Young Adult

To evaluate whether the efficacy and safety of menatetrenone for the treatment of osteoporosis is noninferior to alfacalcidol in Chinese postmenopausal women.. This multicenter, randomized, double-blinded, double-dummy, noninferiority, positive drug-controlled clinical trial was conducted in five Chinese sites. Eligible Chinese women with postmenopausal osteoporosis (N=236) were randomized to Group M or Group A and received menatetrenone 45 mg/day or alfacalcidol 0.5 μg/day, respectively, for 1 year. Additionally, all patients received calcium 500 mg/day. Posttreatment bone mineral density (BMD), new fracture onsets, and serum osteocalcin (OC) and undercarboxylated OC (ucOC) levels were compared with the baseline value in patients of both groups.. A total of 213 patients (90.3%) completed the study. After 1 year of treatment, BMD among patients in Group M significantly increased from baseline by 1.2% and 2.7% at the lumbar spine and trochanter, respectively (P<0.001); and the percentage increase of BMD in Group A was 2.2% and 1.8%, respectively (P<0.001). No difference was observed between groups. There were no changes in femoral neck BMD in both groups. Two patients (1.9%, 2/108) in Group M and four patients (3.8%, 4/105) in Group A had new fracture onsets (P>0.05). In Group M, OC and ucOC decreased from baseline by 38.7% and 82.3%, respectively (P<0.001). In Group A, OC and ucOC decreased by 25.8% and 34.8%, respectively (P<0.001). Decreases in serum OC and ucOC were more obvious in Group M than in Group A (P<0.001). The safety profile of menatetrenone was similar to alfacalcidol.. Menatetrenone is an effective and safe choice in the treatment of postmenopausal osteoporosis in Chinese women.

Topics: Aged; Alkaline Phosphatase; Bone Density; Bone Density Conservation Agents; Calcium; Double-Blind Method; Female; Fractures, Bone; Humans; Hydroxycholecalciferols; Middle Aged; Osteocalcin; Osteoporosis, Postmenopausal; Phosphorus; Treatment Outcome; Vitamin K 2

Long-term treatment with glucocorticoids can induce bone loss and increase fracture risks.. To compare the efficacy of a 12-month treatment between alfacalcidol and menatetrenone in preventing bone loss in children treated with long-term glucocorticoids.. Twenty children on a stable dosage of glucocorticoids were randomly divided into two groups (alfacalcidol or menatetrenone). Each group received the assigned treatment along with 400 mg of elemental calcium daily for 12 months. Patients receiving medications affecting bone metabolism or patients with impaired kidney function were excluded. Bone density parameters, including lumbar spine bone mineral content (BMC), bone mineral density (BMD), and BMD Z-score were assessed by dual-energy X-ray absorptiometry at baseline and at 12-month follow-up. Bone mineral apparent density (BMAD) was calculated as a size-adjusted measurement of BMD in growing children. Baseline characteristics and bone density parameters were similar between both groups.. After 12 months, BMC and BMD were significantly increased from baseline in both groups, but did not differ between the groups. The BMD Z-score at 12-month follow-up was significantly decreased from baseline in the menatetrenone group. BMAD was significantly increased from baseline in the alfacalcidol group.. Administration of long-term glucocorticoids in children justifies an intervention to preserve bone mass. Calcium supplementation along with alfacalcidol can prevent further bone loss to a greater extent than menatetrenone in this group of patients.

Topics: Absorptiometry, Photon; Adolescent; Bone Density; Bone Density Conservation Agents; Bone Diseases, Metabolic; Calcium; Female; Glucocorticoids; Hemostatics; Humans; Hydroxycholecalciferols; Lumbar Vertebrae; Male; Vitamin K 2

Vitamin K₂ contributes to bone and cardiovascular health. Therefore, two vitamin K₂ homologues, menaquinone-4 (MK-4) and menaquinone-7 (MK-7), have been used as nutrients by the food industry and as nutritional supplements to support bone and cardiovascular health. However, little is known about the bioavailability of nutritional MK-4. To investigate MK-4 and MK-7 bioavailability, nutritional doses were administered to healthy Japanese women.. Single dose administration of MK-4 (420 μg; 945 nmol) or MK-7 (420 μg; 647 nmol) was given in the morning together with standardized breakfast. MK-7 was well absorbed and reached maximal serum level at 6 h after intake and was detected up to 48 h after intake. MK-4 was not detectable in the serum of all subjects at any time point. Consecutive administration of MK-4 (60 μg; 135 nmol) or MK-7 (60 μg; 92 nmol) for 7 days demonstrated that MK-4 supplementation did not increase serum MK-4 levels. However, consecutive administration of MK-7 increased serum MK-7 levels significantly in all subjects.. We conclude that MK-4 present in food does not contribute to the vitamin K status as measured by serum vitamin K levels. MK-7, however significantly increases serum MK-7 levels and therefore may be of particular importance for extrahepatic tissues.

Topics: Adult; Bone Density Conservation Agents; Breakfast; Cardiovascular Agents; Chromatography, High Pressure Liquid; Dietary Supplements; Female; Food, Fortified; Humans; Intestinal Absorption; Kinetics; Limit of Detection; Nutritive Value; Spectrometry, Fluorescence; Vitamin K 2; Young Adult

The aim of this study was to investigate whether menatetrenone (MNT) suppresses hepatocellular carcinoma (HCC) recurrence in patients undergoing hepatectomy. Between January 2005 and September 2009, 101 patients who underwent curative hepatectomy for primary HCC were enrolled in the study. Patients were divided into two groups: a non-MNT group (n=51), and an MNT group (n=50) that was administered 45 mg of MNT daily. During the observation period, recurrence was observed in 33 patients in the non-MNT group and in 28 patients of the MNT group (p=0.545). In patients with a preoperative Des-γ-carboxy-prothrombin (DCP) level lower than 40 AU/l (n=38), the cumulative disease-free survival rates at 12, 36, and 60 months in the non-MNT group, were 81.3%, 0.0%, and 0.0%, respectively, while those in the MNT group were 78.3%, 58.1%, and 31.0%, respectively (p=0.060). MNT has a moderately suppressive effect on HCC recurrence after hepatectomy, especially in patients with a normal preoperative DCP level.

Topics: Aged; Carcinoma, Hepatocellular; Combined Modality Therapy; Female; Hepatectomy; Humans; Liver Neoplasms; Male; Neoplasm Recurrence, Local; Prospective Studies; Vitamin K 2

A low level of intact parathyroid hormone (PTH) is an indicator of adynamic bone disease in hemodialysis patients, and is associated with a significant increase of all-cause mortality. Thus, effective treatment for adynamic bone disease is required. We previously investigated the effect of vitamin K₂ on adynamic bone disease. In this study, we assessed the efficacy of oral vitamin K₂ in a controlled trial.. Forty hemodialysis patients with low intact PTH levels (<100 pg/ml) were randomly divided into two groups, which were a vitamin K₂ group receiving oral menatetrenone (45 mg/day) for 1 year and a control group without vitamin K₂. Venous blood samples were collected at baseline and during the study for measurement of bone metabolism parameters.. Thirty-three patients completed follow-up. There was a significant increase of the serum intact osteocalcin level after 1 month of vitamin K₂ administration. Serum levels of intact PTH, bone alkaline phosphatase, and cross-linked N-terminal telopeptide of type I collagen increased significantly after 12 months in the vitamin K₂ group. The serum osteoprotegerin level was decreased after 12 months in the vitamin K₂ group, but the change was not significant.. Vitamin K₂ therapy improves bone remodeling in hemodialysis patients with a low intact PTH level.

Topics: Aged; Alkaline Phosphatase; Biomarkers; Bone Diseases; Bone Remodeling; Collagen Type I; Female; Humans; Male; Middle Aged; Osteocalcin; Parathyroid Hormone; Peptides; Renal Dialysis; Statistics, Nonparametric; Vitamin K 2

The purpose of this study was to evaluate the influence of gamma-glutamyl transferase (GGCX) gene polymorphisms on the response of serum undercarboxylated osteocalcin (ucOC) and bone turnover markers 3 months after treatment with menatetrenone. One hundred and forty postmenopausal Thai women were enrolled and assigned to receive 45 mg/day treatment of menatetrenone (MK-4) concurrently with calcium 1.2 g and vitamin D 400 IU for 3 months. Demographic characteristics, GGCX genotyping, serum bone turnover markers and ucOC levels were obtained from all participants at baseline. We evaluated the reduction of ucOC at 3 months and the reduction of beta-CTx and P1NP at 1 and 3 months. The responses were compared between the different genotypes of GG and GA + AA groups. There was a significant reduction of serum ucOC, beta-CTx and P1NP from the baseline at 3 months (p < 0.001) though there was no significant difference between genotypes (GG vs. GA + AA; p > 0.05). Nonetheless, a subgroup analysis of postmenopausal women who 65 years of age or over (N = 37) revealed a significant difference between the two groups in the reduction of ucOC. Menatetrenone significantly reduced serum ucOC as well as beta-CTX and P1NP from the baseline. GGCX polymorphism appeared to have an influence over the reduction of ucOC especially in older women (age ≥65). Furthermore, the groups which have "A" allele trend to being more efficient in reducing the serum ucOC level than the group which does not have it.

Topics: Adult; Aged; Bone and Bones; Female; gamma-Glutamyltransferase; Genotype; Humans; Middle Aged; Osteocalcin; Polymorphism, Genetic; Postmenopause; Thailand; Vitamin K 2

The US Surgeon General's Report on Bone Health suggests America's bone-health is in jeopardy and issued a "call to action" to develop bone-health plans incorporating components of (1) improved nutrition, (2) increased health literacy, and (3) increased physical activity.. To conduct a Comparative Effectiveness Research (CER) study comparing changes in bone mineral density in healthy women over-40 with above-average compliance when following one of three bone health Plans incorporating the SG's three components.. Using an open-label sequential design, 414 females over 40 years of age were tested, 176 of whom agreed to participate and follow one of three different bone-health programs. One Plan contained a bone-health supplement with 1,000 IUs of vitamin D(3 )and 750 mg of a plant-sourced form of calcium for one year. The other two Plans contained the same plant form of calcium, but with differing amounts of vitamin D(3) and other added bone health ingredients along with components designed to increase physical activity and health literacy. Each group completed the same baseline and ending DXA bone density scans, 43-chemistry blood test panels, and 84-item Quality of Life Inventory (QOL). Changes for all subjects were annualized as percent change in BMD from baseline. Using self-reports of adherence, subjects were rank-ordered and dichotomized as "compliant" or "partially compliant" based on the median rating. Comparisons were also made between the treatment groups and two theoretical age-adjusted expected groups: a non-intervention group and a group derived from a review of previously published studies on non-plant sources of calcium.. There were no significant differences in baseline BMD between those who volunteered versus those who did not and between those who completed per protocol (PP) and those who were lost to attrition. Among subjects completing per protocol, there were no significant differences between the three groups on baseline measurements of BMD, weight, age, body fat and fat-free mass suggesting that the treatment groups were statistically similar at baseline. In all three treatment groups subjects with above average compliance had significantly greater increases in BMD as compared to the two expected-change reference groups. The group following the most nutritionally comprehensive Plan outperformed the other two groups. For all three groups, there were no statistically significant differences between baseline and ending blood chemistry tests or the QOL self-reports.. The increases in BMD found in all three treatment groups in this CER stand in marked contrast to previous studies reporting that interventions with calcium and vitamin D(3) reduce age-related losses of BMD, but do not increase BMD. Increased compliance resulted in increased BMD levels. No adverse effects were found in the blood chemistry tests, self-reported quality of life and daily tracking reports. The Plans tested suggest a significant improvement over the traditional calcium and vitamin D(3) standard of care.

Topics: Adipose Tissue; Adult; Aged; Aged, 80 and over; Ascorbic Acid; Blood Glucose; Body Weight; Bone Density; Boron; C-Reactive Protein; Calcium; Cholecalciferol; Comparative Effectiveness Research; Dietary Supplements; Female; Humans; Lipids; Magnesium; Middle Aged; Minerals; Motor Activity; Patient Education as Topic; Plant Extracts; Quality of Life; Strontium; Treatment Outcome; Vitamin K 2

This study aimed to model the atypical absorption of menatetrenone and its epoxide metabolite and to examine the influence of the single nucleotide polymorphism (SNP) of vitamin K2 epoxide reductase complex subunit 1 (VKORC1) on the pharmacokinetics.. After the administration of 30 mg of menatetrenone to 26 healthy subjects, the plasma concentrations of menatetrenone and its epoxide were measured using LC-MS/MS. For the haplotype analysis, the SNP of the VKORC1 gene was investigated in the 26 volunteers. The model parameters were estimated using the ADAPT II program.. A two-compartment model with Weibull-type absorption and saturable elimination described the pharmacokinetics of menatetrenone and its epoxide. The plasma concentrations of both tended to be lower in the H1/H7 genotype group than in the wild-type H1/H1 group.. We present the first detailed pharmacokinetic modelling of menatetrenone in relation to VKORC1 genotype. This study suggests that VKORC1 genotype is unlikely to be helpful in dose-selection because of the very high inter-individual variation in systemic exposure within each genotype group, and the small inter-group difference observed.

Topics: Adult; Biotransformation; Epoxy Compounds; Gene Frequency; Genetic Association Studies; Half-Life; Humans; Intestinal Absorption; Male; Mixed Function Oxygenases; Models, Biological; Polymorphism, Single Nucleotide; Reproducibility of Results; Republic of Korea; Software; Vitamin K 2; Vitamin K Epoxide Reductases; Young Adult

A high incidence of fractures, particularly of the hip, represents an important problem in patients with Alzheimer disease (AD), who are prone to falls and have osteoporosis. We previously found that vitamin K deficiency and low 25-hydroxyvitamin D (25-OHD) with compensatory hyperparathyroidism cause reduced bone mineral density (BMD) in female patients with AD. This may modifiable by intervention with menatetrenone (vitamin K2) and risedronate sodium; we address the possibility that treatment with menatetrenone, risedronate and calcium may reduce the incidence of nonvertebral fractures in elderly patients with AD. A total of 231 elderly patients with AD were randomly assigned to daily treatment with 45 mg of menatetrenone or a placebo combined with once weekly risedronate sodium, and followed up for 12 months. At baseline, patients of both groups showed high undercarboxylated osteocalcin (ucOC) and low 25-OHD insufficiency with compensatory hyperparathyroidism. During the study period, BMD in the treatment group increased by 5.7% and increased by 2.1% in the control group. Nonvertebral fractures occurred in 15 patients (10 hip fractures) in the control group and 5 patients (2 hip fractures) in the treatment group. The relative risk in the treatment group compared with the control group was 0.31 (95% confidence interval, 0.12-0.81). Elderly AD patients with hypovitaminosis K and D are at increased risk for hip fracture. The study medications were well tolerated with relatively few adverse events and effective in reducing the risk of a fracture in elderly patients with AD.

Topics: Aged; Aged, 80 and over; Alzheimer Disease; Bone Density; Bone Density Conservation Agents; Etidronic Acid; Female; Hemostatics; Hip Fractures; Humans; Hyperparathyroidism; Male; Osteoporosis; Risedronic Acid; Vitamin D; Vitamin K; Vitamin K 2

An open-label study with blinded evaluation was performed to compare the preventive effect of a calcium supplement alone (monotherapy) or calcium supplement plus menatetrenone (combined therapy) on fracture in osteoporotic postmenopausal women aged 50 years or older. Patients were randomized to receive monotherapy (n = 2,193) or combined therapy (n = 2,185). Before randomization, the subjects were stratified into a subgroup without vertebral fractures (n = 2,986; no-fracture subgroup) and a subgroup with at least one vertebral fracture (n = 1,392; fracture subgroup). The incidence rate of new vertebral fractures during 36 months of treatment (primary endpoint) did not differ significantly between either subgroup of the two treatment groups. Although the cumulative 48-month incidence rate of new clinical fractures (secondary endpoint) was lower in the combined therapy group, the difference was not significant. There was a lower risk of new vertebral fractures in patients with at least five baseline fractures who received combined therapy. Also, the loss of height was less with combined therapy than with monotherapy among patients 75 years of age or older at enrollment, those whose last menstrual period occurred 30 years or more before enrollment, and those with at least five vertebral fractures at enrollment. Adverse events and adverse reactions were more frequent in the combined therapy group. In conclusion, menatetrenone therapy was not effective for preventing vertebral fractures in the full analysis set of this study, but the results suggested that it may prevent vertebral fractures in patients with more advanced osteoporosis.

Topics: Aged; Aged, 80 and over; Calcium, Dietary; Dietary Supplements; Drug Therapy, Combination; Female; Hemostatics; Humans; Middle Aged; Osteoporosis, Postmenopausal; Spinal Fractures; Vitamin K 2

Low vitamin K status is associated with low BMD and increased fracture risk. Additionally, a specific menaquinone, menatetrenone (MK4), may reduce fracture risk. However, whether vitamin K plays a role in the skeletal health of North American women remains unclear. Moreover, various K vitamers (e.g., phylloquinone and MK4) may have differing skeletal effects. The objective of this study was to evaluate the impact of phylloquinone or MK4 treatment on markers of skeletal turnover and BMD in nonosteoporotic, postmenopausal, North American women. In this double-blind, placebo-controlled study, 381 postmenopausal women received phylloquinone (1 mg daily), MK4 (45 mg daily), or placebo for 12 mo. All participants received daily calcium and vitamin D(3) supplementation. Serum bone-specific alkaline phosphatase (BSALP) and n-telopeptide of type 1 collagen (NTX) were measured at baseline and 1, 3, 6, and 12 mo. Lumbar spine and proximal femur BMD and proximal femur geometry were measured by DXA at baseline and 6 and 12 mo. At baseline, the three treatment groups did not differ in demographics or study endpoints. Compliance with calcium, phylloquinone, and MK4 treatment was 93%, 93%, and 87%, respectively. Phylloquinone and MK4 treatment reduced serum undercarboxylated osteocalcin but did not alter BSALP or NTX. No effect of phylloquinone or MK4 on lumbar spine or proximal femur BMD or proximal femur geometric parameters was observed. This study does not support a role for vitamin K supplementation in osteoporosis prevention among healthy, postmenopausal, North American women receiving calcium and vitamin D supplementation.

Topics: Absorptiometry, Photon; Alkaline Phosphatase; Bone Density; Bone Remodeling; Double-Blind Method; Female; Humans; Osteocalcin; Placebos; Postmenopause; Vitamin K; Vitamin K 1; Vitamin K 2

The effect of vitamin K(2) (menatetrenone) on bone turnover was investigated in postmenopausal patients with osteoporosis. A 6-month open-label, randomized prospective study was conducted in 109 patients. The control group (n = 53) received calcium aspartate (133.8 mg of elemental calcium daily), while the menatetrenone group (n = 56) received 45 mg of menatetrenone daily for 6 months. Serum and urinary levels of bone turnover markers were monitored. The serum level of undercarboxylated osteocalcin (uc-OC) was significantly lower (P < 0.001) in the menatetrenone group than in the control group (at 1 month), while there was a higher level of osteocalcin containing gamma-carboxylated glutamic acid (Gla-OC) in the menatetrenone group than the control group (P = 0.018). Significant differences of uc-OC and Gla-OC between the two groups were observed from 1 month onward. In addition, a higher level of intact osteocalcin was found in the menatetrenone group compared with the control group after 6 months (P = 0.006). Assessment of bone resorption markers showed that menatetrenone therapy was associated with significantly higher urinary N-telopeptide of type I collagen (NTX) excretion compared with the control group after 6 months, while there was no significant difference of urinary deoxypyridinoline excretion between the two groups. In conclusion, one month of menatetrenone therapy enhanced the secretion and gamma-carboxylation of osteocalcin, while urinary NTX excretion was increased after 6 months of treatment. Further investigations are required to determine whether the effects of menatetrenone on bone turnover are associated with fracture prevention.

Topics: Aged; Biomarkers; Bone Density; Bone Remodeling; Calcium; Female; Hemostatics; Hormones; Humans; Lumbar Vertebrae; Osteocalcin; Osteoporosis, Postmenopausal; Protein Processing, Post-Translational; Time Factors; Treatment Outcome; Vitamin K 2

No chemopreventive agent has been approved against hepatocellular carcinoma (HCC) yet. Since neovascularization plays a pivotal role in HCC, an angiostatic agent is considered as one of the promising approaches. The aim of this study was to elucidate the combined effect of the clinically used vitamin K(2) (VK) and angiotensin-converting enzyme inhibitor (ACE-I) on cumulative recurrence after curative treatment on a total of 87 patients, especially in consideration of neovascularization.. VK (menatetrenone; 45 mg/day) and/or ACE-I (perindopril; 4 mg/day) were administered for 36-48 months after curative therapy for HCC. The cumulative recurrence and several indices were analyzed.. A 48-month follow-up revealed that the combination treatment with VK and ACE-I markedly inhibited the cumulative recurrence of HCC in association with suppression of the serum level of the vascular endothelial growth factor (VEGF); a central angiogenic factor. The serum level of lectin-reactive alpha-fetoprotein was also suppressed almost in parallel with VEGF. These beneficial effects were not observed with single treatment using VK or ACE-I.. The combination treatment of VK and ACE-I may suppress the cumulative recurrence of HCC after the curative therapy, at least partly through suppression of the VEGF-mediated neovascularization.

Topics: Aged; Angiotensin-Converting Enzyme Inhibitors; Carcinoma, Hepatocellular; Catheter Ablation; Drug Synergism; Drug Therapy, Combination; Female; Humans; Liver Neoplasms; Male; Middle Aged; Neoplasm Recurrence, Local; Neovascularization, Pathologic; Perindopril; Survival Rate; Vascular Endothelial Growth Factor A; Vitamin K 2

The recommended dose of Menatretenone is 45 mg three times a day; however the compliant in daily practice is not convenient. This study shows the twice dose per day is inferior to the recommended dose. This study used the level of Gla protein in osteocalcin as a parameter for the comparison. The mean of three-time dose a day is 11.27 nanogram per milliliter while the mean of the other group is 6.07 nanogram per milliliter after the three-month treatment.

Topics: Aged; Calcium; Female; Humans; Middle Aged; Osteoporosis; Treatment Outcome; Vitamin D; Vitamin K 2

Despite the progression of therapeutic approaches, a high frequency of recurrence is what determines the long-term prognosis of patients with hepatocellular carcinoma (HCC). In this study, the chemopreventive effects of vitamin K2 on the recurrence and survival of patients with HCC after curative therapy were evaluated.. Sixty patients who were diagnosed to be free of HCC after radiofrequency ablation therapy or surgery were randomly assigned to either the vitamin K2 group (n = 30 patients) or the control group (n = 30 patients). All patients were positive for the hepatitis C virus (HCV) antibody and hepatitis B surface antigen positive patients were excluded from this study. Patients in the vitamin K2 group received an oral dose of menatetrenone at 45 mg per day. Disease recurrence and the survival rates were analyzed in patients with HCC.. The cumulative recurrence-free rates in the vitamin K2 group were 92.3% at 12 months, 48.6% at 24 months and 38.8% at 36 months; and those in the control group were 71.7%, 35.9% and 9.9%, respectively (P = 0.045). The cumulative survival rates in the vitamin K2 group were 100% at 12 months, 95.0% at 24 months and 77.5% at 36 months; and those in the control group were 95.8%, 90.2% and 66.4%, respectively (P = 0.70).. Vitamin K2 may have a suppressive effect on the recurrence of HCC and a beneficial effect on tumor recurrence. However, there was no significant difference in the survival rates. The chemopreventive effects of vitamin K2 are not sufficient. The development of a further regimen such as combination therapy is required.

Topics: Aged; Carcinoma, Hepatocellular; Catheter Ablation; Female; Hepatitis C Antibodies; Humans; Liver Neoplasms; Male; Middle Aged; Proportional Hazards Models; Secondary Prevention; Survival Rate; Vitamin K 2

The high recurrence rate of hepatocellular carcinoma (HCC) determines the long-term prognosis for patients with HCC. In the current study, the authors tested the effects of menatetrenone, a vitamin K2 analog, on recurrent HCC and survival after curative treatment.. Sixty-one patients who were diagnosed as free of HCC after surgical resection or percutaneous local ablation were assigned randomly assigned to either a menatetrenone group (n = 32 patients) or a control group (n = 29 patients). Patients in the menatetrenone group received a daily oral dose of 45 mg of menatetrenone. Disease recurrence and survival rates were analyzed in patients with HCC.. The cumulative recurrence rates in the menatetrenone group were 12.5% at 12 months, 39.0% at 24 months, and 64.3% at 36 months; and the corresponding recurrence rates in the control group were 55.2%, 83.2%, and 91.6%, respectively (P = 0.0002). Similar results were obtained even for patients who had low baseline levels of serum des-gamma-carboxy-prothrombin. Univariate and multivariate Cox proportional hazard analyses showed that the administration of menatetrenone was the only factor related to the recurrence rate of HCC. The cumulative survival rates for the patients who received menatetrenone were 100% at 12 months, 96.6% at 24 months, and 87.0% at 36 months; and the corresponding survival rates for patients in the control group were 96.4%, 80.9%, and 64.0%, respectively (P = 0.051).. The current study findings suggested that menatetrenone may have a suppressive effect on recurrence of HCC and a beneficial effect on survival, although a larger, placebo-controlled trial will be required to prove these effects.

Topics: Administration, Oral; Aged; Carcinoma, Hepatocellular; Female; Hemostatics; Humans; Liver Neoplasms; Male; Middle Aged; Neoplasm Recurrence, Local; Survival Analysis; Vitamin K 2

This article has been retracted: please see Elsevier Policy on Article Withdrawal (https://www.elsevier.com/about/our-business/policies/article-withdrawal).\ \ This article has been retracted at the request of the Corresponding Author, Yoshihiro Sato, and the co-authors have been informed.\ \ Dr. Sato wishes to retract this article on the grounds that it contains fabricated clinical trial data, which he was responsible for producing. In addition, Dr. Sato claims he listed all of the named co-authors without their consent. The co-authors were therefore unaware of the presence of fabricated data in this publication and their participation in the publication. This retraction was initiated by Dr. Sato, and the Editor-in-Chief of Bone was informed by the author directly.

Topics: Aged; Aged, 80 and over; Alzheimer Disease; Bone Density; Calcium; Ergocalciferols; Female; Fractures, Bone; Humans; Vitamin K 2

Osteoporosis is a common complication of anorexia nervosa (AN). Although weight recovery and resumption of menses are important goals in AN treatment, they are often achieved only after a prolonged period of recovery. Therefore, it becomes important to find therapies with the potential to prevent further decreases in bone mineral density (BMD). We conducted a non-randomized study of the effects of menatetrenone (vitamin K2) on bone loss in patients with AN. Lumbar BMD was longitudinally measured by Dual Energy X-ray Absorptiometry (DXA) in 10 patients with AN who chose to receive menatetrenone treatment (MED+ group) and 11 patients who did not (MED- group). During the mean 0.9-year follow-up period, the BMD of the lumbar vertebrae of the MED+ group decreased significantly less than that of the MED- group (-2.8% and -6.9%, respectively). Among bone metabolism markers, gamma-carboxyglutamic acid osteocalcin significantly increased (128.6% and 28.3%, respectively) and urine deoxypyridinoline significantly decreased (-44.5% and -13.7%, respectively) more in the MED+ group than in the MED- group. These differences in BMD and bone metabolism markers may be attributable to menatetrenone treatment. The results suggest that menatetrenone may be beneficial in the prevention of bone loss in patients with AN. Randomized placebo-controlled studies are needed to confirm these findings.

Topics: Adult; Anorexia Nervosa; Blood Chemical Analysis; Bone Density; Female; Follow-Up Studies; Hemostatics; Humans; Male; Osteoporosis; Urinalysis; Vitamin K 2; Weight Gain

Significant reduction in bone mineral density (BMD) occurs in patients with Parkinson's disease (PD), correlating with immobilization and with vitamin D deficiency, and increasing the risk of hip fracture, especially in elderly women. As a biological indicator of compromised vitamin K status, an increased serum concentration of undercarboxylated osteocalcin (Oc) has been associated with reduced BMD in the hip and an increased risk of fracture in otherwise healthy elderly women. We evaluated treatment with vitamin K(2) (menatetrenone; MK-4) in maintaining BMD and reducing the incidence of nonvertebral fractures in elderly female patients with PD. In a random and prospective study of PD patients, 60 received 45 mg of MK-4 daily for 12 months, and the remaining 60 (untreated group) did not. At baseline, patients of both groups showed vitamin D and K(1) deficiencies, high serum levels of ionized calcium, and glutaminic residue (Glu) Oc, and low levels of parathyroid hormone (PTH) and 1,25-dihydroxyvitamin D [1,25-(OH)(2)D], indicating that immobilization-induced hypercalcemia inhibits renal synthesis of 1,25-(OH)(2)D and compensatory PTH secretion. BMD in the second metacarpals increased by 0.9% in the treated group and decreased by 4.3% in the untreated group (p < 0.0001). Vitamin K(2) level increased by 259.8% in the treated group. Correspondingly, significant decreases in Glu Oc and calcium were observed in the treated group, in association with an increase in both PTH and 1,25-(OH)(2)D. Ten patients sustained fractures (eight at the hip and two at other sites) in the untreated group, and one hip fracture occurred among treated patients (p = 0.0082; odds ratio = 11.5). The treatment with MK-4 can increase the BMD of vitamin D- and K-deficient bone by increasing vitamin K concentration, and it can also decrease calcium levels through inhibition of bone resorption, resulting in an increase in 1,25-(OH)(2)D concentration.

Topics: Aged; Aged, 80 and over; Analysis of Variance; Bone Density; Chi-Square Distribution; Female; Humans; Osteoporosis; Parkinson Disease; Patients; Prospective Studies; Statistics, Nonparametric; Vitamin D Deficiency; Vitamin K 2

Vitamin K2 induces differentiation of leukemic cell lines and apoptosis of immature blasts in myelodysplastic syndrome (MDS). We recently reported a case of MDS-refractory anemia (MDS-RA) with trilineage hematologic response to oral administration of menatetrenone, a vitamin K2 analog. To determine a possible role of this agent in treatment of MDS-RA, we conducted a prospective randomized trial assessing the safety and efficacy of menatetrenone. A total of 18 consecutive patients newly diagnosed with MDS-RA were randomized to receive either 45 mg of oral menatetrenone (group 1) or no menatetrenone (group 2). Administration of menatetrenone was well tolerated. Of the nine patients in group 1 (56%), five improved with menatetrenone treatment while only one (11%) of the group 2 patients improved. Three patients (33%) showed a major response in absolute neutrophil count (ANC), two (22%) showed a major response in hemoglobin concentration, and two of the nine (22%) showed a major response in platelet count. The ANC of group 1 patients rose after treatment, while that of group 2 patients decreased slightly at follow-up after 16 weeks ( p=0.03). Significant improvement was also seen in final platelet count ( p=0.01), but not in hemoglobin concentration. Given the absence of toxicity, menatetrenone can be recommended for all patients with MDS-RA.

Topics: Administration, Oral; Aged; Aged, 80 and over; Anemia, Refractory; Cell Count; Female; Hematopoiesis; Hemostatics; Humans; Male; Middle Aged; Myelodysplastic Syndromes; Vitamin K 2

Bone loss frequently appears in the natural history of liver disease. The effects of therapy for osteoporosis associated with cirrhosis of the liver are still controversial. We evaluated the effects of vitamin K2 on osteopenia in women with cirrhosis.. The subjects were 50 women with cirrhosis who had underlying hepatitis viral infections. Half of the patients were randomly assigned to receive vitamin K2 (menatetrenone). The bone mineral density (BMD) of the lumbar vertebrae was measured by dual-energy X-ray absorptiometry at entry and at 1-yr intervals for 2 yr.. The percentages of change from the initial BMD at 1 and 2 yr after initiation of the study were, respectively, +0.1 +/- 2.6% and -0.5 +/- 3.5% for the vitamin K2-treated group and -2.2 +/- 2.4% and -4.6 +/- 3.9% for the control group. The changes in BMD at each timepoint differed significantly between the control and treated groups (p = 0.008 for 1 yr and p = 0.002 for 2 yr). In the vitamin K2-treated group, the ratio of osteocalcin to undercarboxylated osteocalcin in those patients with increases in BMD after 1 yr of treatment was significantly lower than that in patients showing decreases in BMD (p = 0.017). No adverse effects of vitamin K2 were noted.. Vitamin K2 can prevent bone loss and may therefore be useful in the management of bone disease in women with cirrhosis of the liver.

Topics: Absorptiometry, Photon; Adult; Aged; Bone Density; Female; Hemostatics; Hepatitis B; Hepatitis C; Humans; Liver Cirrhosis; Lumbar Vertebrae; Middle Aged; Osteoporosis; Time Factors; Vitamin K 2

The effect of maternal phylloquinone supplementation on vitamin K in breast milk was studied to establish: (1) if phylloquinone is the source of menaquinone-4 in breast milk; (2) the dose-effect relationship between intake and obtainable levels. Four groups of lactating mothers with a full-term healthy infant participated and took oral phylloquinone supplements of 0.0 (n 8), 0.8 (n 8), 2.0 (n 8), and 4.0 (n 7) mg/d for 12d, starting at day 4 post-partum. Milk samples were collected on days 4, 8, 16, and 19. Blood samples were collected on days 4 and 16. Vitamin K and vitamin E concentrations, the latter for reason of comparison, were assayed. Phylloquinone and menaquinone-4 were present in all milk samples: 5.84 (SD 2.31) and 2.98 (SD 1.51) nmol/l (n 31) respectively, in colostrum (day 4 sample). A strong correlation between the vitamers was found (r 0.78, P<0.001). Breast-milk phylloquinone levels were raised in a dose-dependent manner: 4-, 12-, and 30-fold on day 16 for the 08, 2.0, and 4.0 mg group respectively. In addition, menaquinone-4 levels were higher: 2.5- (P<0.05) and 7-fold (P<0.001) in the 2.0 and 4.0 mg groups respectively. Plasma of supplemented subjects contained 3-, 5-, and 10-fold higher phylloquinone levels on day 16. Detectable menaquinone-4 was found in ten of thirty-one day 4 plasma samples. All day 16 plasma samples of the 4mg supplemented group contained the vitamin. There was no correlation between the K-vitamers in plasma. Vitamin E and phylloquinone appear to differ in their distribution in breast milk, milk:plasma concentration ratios were < or =1 and 3-5 for vitamin E and phylloquinone respectively. The milk:plasma concentration ratio of menaquinone-4 was >10. In conclusion, dietary phylloquinone is a source of menaquinone-4 in breast milk. Phylloquinone supplementation to lactating mothers may be of benefit to the newborn infant, since both phylloquinone and menaquinone-4 are raised by supplementation.

Topics: Antifibrinolytic Agents; Dietary Supplements; Dose-Response Relationship, Drug; Female; Humans; Milk, Human; Vitamin E; Vitamin K 1; Vitamin K 2

We have reported that alfacalcidol plus menatetrenone, a vitamin K2 with four isoprene units (menaquinone-4), treatment is useful for improving bone problems in children with skeletal unloading. The aim of this study was to evaluate the effect of menatetrenone on bone metabolism in long-term glucocorticoid-treated children with alfacalcidol treatment. Twenty children who had been treated with fixed dosages of prednisolone and alfacalcidol (0.03 microg/kg/day) for 24 weeks were enrolled in a prospective pilot study, and assigned to receive alfacalcidol (0.03 microg/kg/day) or alfacalcidol (0.03 microg/kg/day) plus menatetrenone (approximately 2 mg/kg/day). Bone biochemical markers and bone mineral density (BMD) were measured at baseline and after the 12-week treatment. In the group receiving alfacalcidol plus menatetrenone, serum carboxylated osteocalcin (OC) (p =0.0022) and lumbar BMD (p=0.0029) increased and serum undercarboxylated OC (p=0.0004) decreased significantly in comparison to the group receiving alfacalcidol; further, the change of lumbar BMD showed an inverse correlation to the change of serum undercarboxylated OC (r=-0.744, p=0.0134) and positive correlations to the baseline values of bone turnover markers such as serum levels of intact OC, bone-specific alkaline phosphatase and type I procollagen carboxyl extension peptide and urinary levels of deoxypyridinoline and N-telopeptide of type I collagen. No adverse effect was observed. This is a small short-term study, but its results suggest that menatetrenone effectively and safely increases lumbar BMD probably through carboxylation of OC in long-term prednisolone-treated children with alfacalcidol treatment who have a high bone turnover. Randomized double-blind controlled trials are needed to confirm our findings.

Topics: Adolescent; Bone Density; Child; Child, Preschool; Female; Glucocorticoids; Humans; Hydroxycholecalciferols; Lumbar Vertebrae; Male; Osteocalcin; Osteoporosis; Prednisolone; Prospective Studies; Vitamin K; Vitamin K 2

The purpose of the present study was to compare the effects of etidronate and menatetrenone on bone mineral density (BMD) and the incidence of vertebral fractures in postmenopausal women with osteoporosis. Seventy-two osteoporotic women, more than 5 years after menopause, 53-78 years of age, were randomly divided into three administration groups: E group; intermittent cyclical etidronate (200 mg/day, 14 days per 3 months; n = 25); M group; menatetrenone (45 mg/day, daily; n = 23); and C group (control); calcium lactate (2 g/day, daily; n = 24). Forearm BMD was measured by dual-energy X-ray absorptiometry at 0, 6, 12, 18, and 24 months after the treatment started. There were no significant differences in age, body mass index, years since menopause, and initial BMD among the three groups. One-way analysis of variance (ANOVA) with repeated measurements showed a significant decrease in BMD in the C group (P < 0.0001). Two-way ANOVA with repeated measurements showed a significant increase in BMD in the M group compared with that in the C group (P < 0.0001), and a significant increase in BMD in the E group compared with that in the C and M groups (P < 0.0001 and P < 0.01, respectively). The indices of new vertebral fractures/1000 patient-years in the E and M groups were significantly higher than that in the C group (chi(2) = 47.7; P < 0.0001 and chi(2) = 42.4; P < 0.0001, respectively), and did not differ significantly between the E and M groups. The present preliminary study provides evidence to suggest that, despite the lower increase in BMD produced by menatetrenone, this agent, as well as etidronate, may have the potential to reduce osteoporotic vertebral fractures in postmenopausal women with osteoporosis.

Topics: Absorptiometry, Photon; Aged; Bone Density; Etidronic Acid; Female; Humans; Middle Aged; Osteoporosis, Postmenopausal; Prospective Studies; Spinal Fractures; Vitamin K 2

The administration of menaquinone-4 (MK-4), one of subclasses of vitamin K2, significantly reduces bone loss in postmenopausal osteoporotic women. However, concerns have been raised about whether vitamin K administration alters the hemostatic balance by inducing a thrombotic tendency. We investigated were whether the administration of vitamin K in the form of MK-4 induced a thrombotic tendency in 29 elderly patients with osteoporosis (5 men, 24 women; age range 78.7+/-5.1 years). Patients were administered 45 mg/day (three times a day, 30 min after each meal) of MK-4 for 12 weeks. Blood samples were obtained from the patients at 0, 4 and 12 weeks after the start of MK-4 administration. A number of hemostatic parameters remained stable under the markedly increased plasma levels of MK-4. However, in patients with suspected vitamin K deficiency, whose plasma levels of vitamin K or factor VII were low, vitamin-K-dependent clotting factors such as factor VII and prothrombin were gradually increased after administration of MK-4. No changes in the sensitive molecular markers such as TAT and F1+2, which reflect the amount of thrombin generated in the blood stream, were observed, even in those patients with suspected vitamin K deficiency. These results indicate that MK-4 can be administered safely, with regard to maintaining the hemostatic balance, to osteoporotic patients receiving no anticoagulant therapy.

Topics: Aged; Aged, 80 and over; Blood Coagulation Factors; Cyanoacrylates; Female; Hemostasis; Hemostatics; Humans; Male; Osteoporosis; Osteoporosis, Postmenopausal; Vitamin K 1; Vitamin K 2; Vitamin K Deficiency

Clinical and comparative study of the efficacy and adverse events of Menatetrenone-4. The control group (n=40) received elemental calcium carbonate 800 mg/day and the Menatetrenone-4 treated group received elemental calcium carbonate 800 mg/day plus vitamin K2 45 mg/day (n=43). The vitamin K2 treated group showed a marked decrease of undercarboxylated osteocalcin at 2 weeks, six months (51.52% p=0.0001) and twelve months (87.26% p=0.0001) compared to the calcium treated group. At the end of the sixth and twelve months both groups did not increase bone mass of the hip but the vitamin K2 treated group increased 0.6 per cent of bone mass of the lumbar spine and decreased bone resorption 65.42 per cent (p=0.0001) compared to the calcium treated group. The calcium treated group was switched to the vitamin K2 treated group at the end of six months and showed a decrease of the level of undercarboxylated osteocalcin the same as the former vitamin K2 treated group. The adverse events were 2 cases of mild skin rash which subsided after cessation of medication.

Topics: Bone Density; Bone Resorption; Calcium Carbonate; Female; Humans; Osteocalcin; Postmenopause; Vitamin K 2

Glucocorticoid-induced osteoporosis has been reported to be caused by enhanced bone resorption and suppressed bone formation. To clarify whether administration of vitamin K, which enhances bone formation, prevents prednisolone-induced loss of bone mineral density (BMD), a randomized, prospective, controlled study was conducted on 20 patients with chronic glomerulonephritis scheduled for treatment with prednisolone. All patients were initially treated with 0.8 mg/kg body weight/day of prednisolone (maximum of 40 mg) for 4 weeks, tapering to 20 mg/day over approximately 6 weeks. Ten patients received prednisolone alone (Group 1), and the other 10 patients received prednisolone plus 15 mg of menatetrenone, vitamin K, three times per day (Group 2). BMD of the lumbar spine measured by dual-energy X-ray absorptiometry (DXA) and biochemical markers of bone metabolism in blood and urine were evaluated before and 10 weeks after administration of prednisolone alone or with menatetrenone. In Group 1, treatment with prednisolone significantly reduced BMD of the lumbar spine from 1.14 +/- 0.12 to 1.10 +/- 0.11 g/cm2 (P = 0.0029). Serum intact osteocalcin and procollagen type I C-peptide (PICP) concentrations, biochemical markers of bone formation, were markedly reduced. A biochemical marker of bone resorption, urinary excretion of deoxypyridinoline, was significantly reduced. In Group 2, prednisolone-induced reduction of BMD was prevented by menatetrenone administration (1.09 +/- 0.09 to 1.07 +/- 0.07 g/cm2, P = 0.153). Menatetrenone prevented reduction of PICP concentration by prednisolone but not in serum intact osteocalcin concentration and urinary excretion of deoxypyridinoline. Thus, treatment with prednisolone resulted in loss of BMD of the lumbar spine associated with suppression of both bone formation and bone resorption. Menatetrenone is a useful agent in preventing prednisolone-induced loss of BMD.

Topics: Absorptiometry, Photon; Adolescent; Adult; Anti-Inflammatory Agents; Bone Density; Calcium; Chronic Disease; Drug Therapy, Combination; Female; Glomerulonephritis; Hemostatics; Humans; Male; Middle Aged; Osteoporosis; Phosphates; Prednisolone; Vitamin K; Vitamin K 2

We attempted to investigate whether vitamin K2 (menatetrenone) treatment effectively prevents the incidence of new fractures in osteoporosis. A total of 241 osteoporotic patients were enrolled in a 24-month randomized open label study. The control group (without treatment; n = 121) and the vitamin K2-treated group (n = 120), which received 45 mg/day orally vitamin K2, were followed for lumbar bone mineral density (LBMD; measured by dual-energy X-ray absorptiometry [DXA]) and occurrence of new clinical fractures. Serum level of Glu-osteocalcin (Glu-OC) and menaquinone-4 levels were measured at the end of the follow-up period. Serum level of OC and urinary excretion of deoxypyridinoline (DPD) were measured before and after the treatment. The background data of these two groups were identical. The incidence of clinical fractures during the 2 years of treatment in the control was higher than the vitamin K2-treated group (chi2 = 10.935; p = 0.0273). The percentages of change from the initial value of LBMD at 6, 12, and 24 months after the initiation of the study were -1.8 +/- 0.6%, -2.4 +/- 0.7%, and -3.3 +/- 0.8% for the control group, and 1.4 +/- 0.7%, -0.1 +/- 0.6%, and -0.5 +/- 1.0% for the vitamin K2-treated group, respectively. The changes in LBMD at each time point were significantly different between the control and the treated group (p = 0.0010 for 6 months, p = 0.0153 for 12 months, and p = 0.0339 for 24 months). The serum levels of Glu-OC at the end of the observation period in the control and the treated group were 3.0 +/- 0.3 ng/ml and 1.6 +/- 0.1 ng/ml, respectively (p < 0.0001), while the serum level of OC measured by the conventional radioimmunoassay (RIA) showed a significant rise (42.4 +/-6.9% from the basal value) in the treated group at 24 months (18.2 +/- 6.1% for the controls;p = 0.0081). There was no significant change in urinary DPD excretion in the treated group. These findings suggest that vitamin K2 treatment effectively prevents the occurrence of new fractures, although the vitamin K2-treated group failed to increase in LBMD. Furthermore, vitamin K2 treatment enhances gamma-carboxylation of the OC molecule.

Topics: Absorptiometry, Photon; Amino Acids; Biomarkers; Bone Density; Bone Remodeling; Female; Fractures, Spontaneous; Humans; Incidence; Lumbar Vertebrae; Osteocalcin; Osteoporosis; Radionuclide Imaging; Treatment Outcome; Vitamin K; Vitamin K 2

To investigate the effect of vitamin K2 treatment for a year on spinal bone mineral density (BMD) in postmenopausal women, comparing with vitamin D3 hormone replacement therapy and to determine the factors which affect the efficacy of vitamin K2 therapy.. Seventy-two postmenopausal women were randomized into four groups and treated with respective agents. Before the therapy, 6 and 12 months after the treatment, their lumbar spine BMD were measured by dual energy X-ray absorptiometry. The rates of change in BMD (delta BMD) were calculated. Correlations of BMD with age, year since menopause and the initial BMD were determined.. Vitamin K2 suppressed the decrease in spinal BMD as compared with no treatment group. BMD in women treated with vitamin K2 was inversely correlated with their age (r = -0.54; P < 0.05).. Vitamin K2 therapy may be a useful method for preventing postmenopausal spinal bone mineral loss. In addition, the therapy should be started early in postmenopausal period.

Topics: Bone Density; Cholecalciferol; Estrogen Replacement Therapy; Female; Humans; Longitudinal Studies; Lumbar Vertebrae; Middle Aged; Osteoporosis, Postmenopausal; Vitamin K; Vitamin K 2

The purpose of this study is to evaluate the efficacy of vitamin K2 and 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3] in preventing bone loss induced by estrogen deficiency during therapy with the GnRH agonist (GnRH-a) leuprolide. One hundred ten women (mean age, 46.2+/-0.5 yr), receiving leuprolide therapy for estrogen-dependent diseases (such as endometriosis and uterine leiomyomas), were randomly allocated into four groups (group A, leuprolide only; group B, leuprolide with vitamin K2; group C, leuprolide with 1,25-(OH)2D3; and group D, leuprolide with vitamin K2 and 1,25-(OH)2D3). Bone mineral density of the lumbar spine was measured by dual-energy x-ray absorptiometry before and after 6 months of treatment. Bone formation and resorption markers were also measured before and after 6 months of treatment. There were no significant differences in the background parameters among the four groups. Bone mineral density was reduced in all four groups, but the percent changes varied slightly, at - 5.25% (group A), -3.72% (P < 0.05 vs. group A) (group B), -4.13% (group C), and -3.59% (P < 0.01 vs. group A) (group D), respectively. Bone formation markers were significantly increased in all four groups, and the percent changes of bone formation markers were highest in group B. Bone resorption markers also increased significantly in all four groups after treatment of 6 months. Group B tended to have the highest percent changes of bone resorption markers among the four groups, but these increases were not significantly different between any of the groups. Vitamin K2, especially when combined with 1,25-(OH)2D3, can partially prevent bone loss caused by estrogen deficiency. However, because this effect is attributable mainly to the activation of bone formation, it is not sufficient to eliminate bone loss induced by GnRH-a therapy.

Topics: Adult; Bone Density; Bone Resorption; Calcitriol; Female; Humans; Leuprolide; Middle Aged; Vitamin K; Vitamin K 2

Significant reduction in bone mineral density (BMD) occurs in stroke patients on the hemiplegic and contralateral sides, correlating with the degree of paralysis and vitamin D and K deficiency due to malnutrition, and increasing the risk of hip fracture. We evaluated the efficacy of vitamin K2 (menatetrenone: menaquinone-4; MK-4) in maintaining BMD by comparing serum biochemical indices of bone metabolism between treated and untreated patients. In a random and prospective study, of 108 hemiplegic patients following stroke, 54 received 45 mg menatetrenone daily (MK-4 group, n = 54) for 12 months, and the remaining 54 (untreatment group) did not. Nine patients excluded from the study. The BMD in the second metacarpals and serum indices of bone metabolism were determined. BMD on the hemiplegic side increased by 4.3% in the MK-4 group and decreased by 4.7% in the untreated group (p < 0.0001), while BMD on the intact side decreased by 0.9% in the MK-4 group and by 2.7% in the untreated group (p < 0.0001). At baseline, patients of both groups showed vitamin D and K1 deficiencies, high serum levels of ionized calcium, pyridinoline cross-linked carboxyterminal telopeptide of type I collagen (ICTP), and low levels of parathyroid hormones (PTH) and bone Gla proteins (BGP), indicating that immobilization-induced hypercalcemia inhibits renal synthesis of 1, 25-dihydroxyvitamin D (1, 25-[OH]2D) and compensatory PTH secretion. Both vitamins K1 and K2 increased by 97.6% and 666.9%, respectively, in the MK-4 group. Correspondingly, a significant increase in BGP and decreases in both ICTP and calcium were observed in the MK-4 group, in association with a simultaneous increase in both PTH and 1, 25-[OH]2D. One patient in the untreated group suffered from a hip fracture, compared with none in the MK-4 group. The treatment with MK-4 can increase the BMD of disused and vitamin D- and K-deficient hemiplegic bone by increasing the vitamin K concentration, and it also can decrease calcium levels through inhibition of bone resorption, resulting in an increase in 1, 25-[OH]2D concentration.

Topics: Aged; Biomarkers; Bone Density; Bone Diseases, Metabolic; Cerebrovascular Disorders; Female; Hemiplegia; Hemostatics; Humans; Male; Metacarpus; Middle Aged; Prospective Studies; Vitamin D Deficiency; Vitamin K; Vitamin K 1; Vitamin K 2; Vitamin K Deficiency

The efficacy and side effects of oral vitamin K2 therapy were examined in 20 children and adults with probable secondary osteoporosis who had been hospitalized for a long period, due to severe mental and/or physical handicaps. Vitamin K2 was given for 12 months. Bone mineral density significantly increased 4 months after starting oral vitamin K2 therapy (p = 0.0038) and it retained the elevated level after 12 months of treatment. This therapy was particularly effective in patients with severe locomotor dysfunction. Serum total protein levels significantly decreased following the start of this therapy (p = 0.0012). The reasons of this decrease and its causal relationship to the administration of vitamin K2 are unknown, and should be investigated further.

Topics: Adolescent; Adult; Bone Density; Disabled Persons; Female; Humans; Male; Motor Activity; Osteoporosis; Vitamin K; Vitamin K 2

The purpose of this investigation was to determine the effects of fat content and frequency of meals on the oral bioavailability of menatetrenone (2-methyl-3-all-trans-tetraprenyl-1,4-naphthoquinone), a vitamin K2 with four isoprene units. In the first series of studies, menatetrenone (15 mg) was administered at breakfast time to 18 healthy male volunteers after meals with three different fat contents (meals A, B, and C) on three occasions in a crossover design. The three types of meals had almost the same calorie content (721-746 kcal) with varied fat contents (A, 8.8 g; B, 20.0 g; C, 34.9 g). The area under the plasma menatetrenone concentration-time curve within the first 24 h (AUC0-24) increased with increase of fat content: 371 +/- 194, 485 +/- 150, and 1024 +/- 341 ng.h/mL (mean +/- SD, n = 18) after meals A, B, and C, respectively. On the fourth occasion, the same dose of menatetrenone was administered to all volunteers after taking meal B, but in this case the lunch 5 h after drug administration was omitted from the protocol. The time profile of plasma menatetrenone showed a single peak when lunch was not taken, whereas it showed two peaks with lunch. On the fifth occasion, 12 out of 18 volunteers took the same dose of menatetrenone after a meal with the highest fat content (53.8 g of fat and 789 kcal; meal D), showing that AUC0-24 was almost the same as that for meal C, 1027 +/- 389 and 991 +/- 392 ng.h/mL (n = 12) for meals C and D, respectively. The oral bioavailability of lipid-soluble vitamin K was influenced by the fat content of a meal, although the increase in bioavailability seemed to reach a peak when the lipid content of the meal was > 35 g.

Topics: Administration, Oral; Adult; Biological Availability; Dietary Fats; Humans; Male; Middle Aged; Vitamin K; Vitamin K 2

Patients (40 cases) were treated with daily dosage of warfarin of 2-7 mg after being undergone artificial valve replacements. Twenty one days after administration of warfarin, we examined the patients for kinetics of K vitamins and vitamin K-dependent coagulation factors in blood, and intestinal flora in feces, as well as the relationship between K vitamins and coagulation activity. The following results were obtained. (1) In warfarin-administered patients (Group B), blood levels of vitamin K1 and menaquinone-7, a vitamin K2 homologue, were similar to those in non-warfarin-administered patients. Therefore, administration of warfarin did not significantly decreased the levels. (2) In patients selected randomly from Group B (Group C), the vitamin K1 level in feces was higher than that in non-warfarin-administered patients. The menaquinone-7 level in feces was similar to that in non-warfarin-administered patients. For the total counts of bacteria and the detection rate of vitamin K2-producing bacteria, there was no significant difference between Group C and non-warfarin-administered patients. (3) The above mentioned results of (1) and (2) suggest that it is important for development of anticoagulant effects by warfarin to inhibit conversion from vitamin K1 to reduced vitamin K1, as well as to inhibit the reducing process from vitamin K1-epoxide to vitamin K1. (4) Vitamin K1-epoxide, a metabolite of vitamin K1, appeared in blood after administration of warfarin; there was a lower correlation between the blood level of vitamin K1-epoxide and the warfarin dosage. Further, PIVKA-II appeared in blood after administration of warfarin; there was a inverse lower correlation between the level of PIVKA-II and HPT, and between PIVIKA-II and TT. In conclusion, it has been clarified that vitamin K1-epoxide and PIVKA-II are useful parameters to evaluate anticoagulant effect of warfarin.

Topics: Adult; Aged; Biomarkers; Blood Coagulation; Blood Coagulation Factors; Feces; Female; Humans; Male; Middle Aged; Protein Precursors; Prothrombin; Vitamin K; Vitamin K 1; Vitamin K 2; Warfarin

The effect of vitamin K on bovine endometrial epithelial cells has not been thoroughly investigated. The objective of this study was to examine the effect of the biologically active form of vitamin K, menaquinone-4, on gene expression in bovine endometrial epithelial cells. First, we examined the mRNA and protein expression levels of UBIAD1, a menaquinone-4 biosynthetic enzyme. Second, we screened for potential target genes of menaquinone-4 in bovine endometrial epithelial cells using RNA-sequencing. We found 50 differentially expressed genes; 42 were upregulated, and 8 were downregulated. Among them, a dose-dependent response to menaquinone-4 was observed for the top three upregulated (TRIB3, IL6, and TNFAIP3) and downregulated (CDC6, ORC1, and RRM2) genes. It has been suggested that these genes play important roles in reproductive events. In addition, GDF15 and VEGFA, which are important for cellular functions as they are commonly involved in pathways, such as positive regulation of cell communication, cell differentiation, and positive regulation of MAPK cascade, were upregulated in endometrial epithelial cells by menaquinone-4 treatment. To the best of our knowledge, this is the first study showing the expression of UBIAD1 in the bovine uterus. Moreover, the study determined menaquinone-4 target genes in bovine endometrial epithelial cells, which may positively affect pregnancy with alteration of gene expression in cattle uterus.

Topics: Animals; Cattle; Endometrium; Epithelial Cells; Female; Vitamin K; Vitamin K 2

Myocardial infarction is among the leading causes of mortality. Menaquinone-4 (MK-4), a vitamin K2 analog, might play a role in rescuing cardiac ischemia/reperfusion (I/R) injury. This work aimed to discover the potential cardioprotective role of MK-4 against myocardial I/R injury in rats. Thirty-two rats were categorized into 3 groups: (I/R) control group: subjected to I/R protocol (received vehicle), MK-4 preconditioning group: MK-4 infusion for 20 minutes before the I/R protocol, and MK-4 postconditioning group: MK-4 infusion for 20 minutes at the start of the reperfusion phase. The hearts were placed in the Langendorff apparatus, and the left ventricular developed pressure (LVDP), heart rate (HR), + (LV dP/dt) max, - (LV dP/dt) max, and Tau were calculated. The necrotic mass was determined by staining it with nitro blue tetrazolium. Creatine kinase-MB (CK-MB), lactate dehydrogenase (LDH), tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), and C- reactive protein (CRP), as well as cardiac superoxide dismutase (SOD), nitric oxide (NOx), malondialdehyde (MDA), and glutathione (GSH) levels were all evaluated. MK-4 postconditioning significantly reduced myocardial infarct size; increased LVDP, + (LV dp/dt) max, - (LV dp/dt) max, and HR; reduced Tau, CK-MB, LDH, CRP, IL-6, TNF-α, MDA, and NOx levels; and increased SOD activity, whereas no significant difference in the GSH level was detected. In conclusion, these data imply that MK-4 may protect the heart from the consequences of I/R.

Topics: Animals; Glutathione; Interleukin-6; Myocardial Reperfusion Injury; Myocardium; Oxidative Stress; Rats; Rats, Sprague-Dawley; Superoxide Dismutase; Tumor Necrosis Factor-alpha; Vitamin K 2

Vitamin K is a term that comprises a family of structurally related quinones, phylloquinone (PK) and the menaquinones (MKn), that share a common naphthoquinone ring but vary in sidechain length (n) and saturation. Dietary PK is a biosynthetic precursor to tissue menaquinone-4 (MK4), but little is known about the absorption and metabolism of dietary MKn.. To characterize the absorption and metabolism of dietary MKn relative to PK.. In the 4-week diet study, 10-week-old male and female C57BL/6 mice were pair-fed a vitamin K deficient diet (control) or a diet supplemented with 5.0 μmol/kg total PK, MK4, and/or MK9 (separately and in combination). In the 1-week stable isotope study, 12-week-old mice were pair-fed diets containing 2.2 μmol/kg PK (unlabeled control), 2H7PK, 13C11MK4, 2H7MK7, or 2H7MK9. Vitamin K tissue content was quantified by HPLC and/or LC-MS, and concentrations were compared by sex and diet group using 2-factor ANOVA.. Regardless of the form(s) of vitamin K provided in the diet, tissue MK4 concentrations did not differ across equimolar supplemented groups in the kidney, adipose, reproductive organ, bone, or pancreas in either males or females in the diet study (all P values > 0.05). Isotopic labeling confirmed the naphthoquinone ring of MK4 in tissues originated from the administered dietary PK or MKn. Despite equimolar supplementation, accumulation of the administered dietary form differed across diet groups in small intestinal segments (all P values < 0.002) and the liver (P < 0.001). Female mice had greater total vitamin K than males in every tissue examined (P < 0.05).. Dietary PK, MK4, MK7, and MK9 all served as precursors to tissue MK4 in mice. This study expands our understanding of vitamin K metabolism and supports a common conversion mechanism of all dietary vitamin K forms to MK4. Further investigation of the metabolism and physiological roles of MK4 that may be independent of classical vitamin K function is warranted.

Topics: Animals; Diet; Female; Male; Mice; Mice, Inbred C57BL; Vitamin K; Vitamin K 1; Vitamin K 2

Topics: Nutritional Status; Vitamin K; Vitamin K 2

Vitamin K and metabolites have a beneficial role in blood coagulation, bone metabolism and growth. However, the determination of vitamin K concentrations in the blood in patients consuming a diet with naturally occurring vitamin K is currently challenging. We aim to develop a cost-effective and rapid method to measure vitamin K metabolites with potential application for clinics and research.. We developed a simple liquid chromatography-tandem mass spectrometric (LC-MS/MS) method for the determination of vitamin K1, menaquinone-4 (MK-4), menaquinone-7 (MK-7) and vitamin K1-2,3 epoxide in human serum and validated the method in a study cohort of 162 patients tested for carbohydrate malabsorption and in 20 patients with oral phenprocoumon intake.. The overall precision (CVs) ranged between 4.8 and 17.7% in the specified working range (0.06-9.0 nmol/L for all analytes except for MK-7 with 0.04-6.16 nmol/L). In the malabsorption cohort samples, measured values were obtained for all different vitamin K metabolites except for vitamin K1-2,3 epoxide. This metabolite could be detected only in patients with phenprocoumon intake. The good performance of the method is especially achieved by the interaction of three factors: the use of lipase in the sample preparation, the use of an atypical fluorinated reversed phase column, and a logarithmic methanol gradient.. The described method is able to determine the concentration of four vitamin K metabolites in a time-efficient, simple and cost-effective manner. It can be suitable for both routine clinics and research.

Topics: Chromatography, High Pressure Liquid; Chromatography, Liquid; Epoxy Compounds; Humans; Phenprocoumon; Tandem Mass Spectrometry; Vitamin K; Vitamin K 1; Vitamin K 2

Topics: Nutritional Status; Vitamin K; Vitamin K 1; Vitamin K 2

The content and composition of dietary supplements is of great interest due to their increasing consumption and variety of available brand offered in the market. Accurate determination of vitamins is important for the improvement of dietary supplement quality and nutrition assessments. In this regard, the simultaneous determination of vitamin D3 (calcitriol-CT and cholecalciferol-CHL) and K2 (menaquinone-4-MK-4 and menaquinone-7-MK-7) in dietary supplements was developed by using ultra-high-pressure liquid chromatography (UHPLC). The overall runtime per sample was above 35 min, with the retention times of 2.40, 6.59, 7.06, and 32.6 min for vitamin D3 (CT and CHL) and vitamin K2 (MK-4 and MK-7), respectively. The limits of detection and limits of quantification for the target nutritional compounds ranged between 0.04-0.05 µg/mL, respectively. The validation results indicated that the method had reasonable linearity (

Topics: Calcitriol; Cholecalciferol; Chromatography, High Pressure Liquid; Dietary Supplements; Vitamin K 2

New chemotherapeutics are needed to treat hepatocellular carcinoma (HCC), and menaquinones, homologs of vitamin K consisting of a 1,4-naphthoquinone core and a (poly)isoprene chain, are potential candidates. In this study, we designed and synthesized a series of phthalazine-1,4-dione-based menaquinone analogs. Among them, compounds bearing the intact isoprene chain exhibited selective antiproliferative activity towards HCC cell line JHH7, as compared with normal hepatocytes. The geranyl derivative 10 showed submicromolar potency, and might be a promising lead compound for anticancer agents.

Topics: Antineoplastic Agents; Cell Line, Tumor; Cell Proliferation; Dose-Response Relationship, Drug; Drug Design; Drug Screening Assays, Antitumor; Humans; Molecular Structure; Phthalazines; Structure-Activity Relationship; Vitamin K 2

Topics: Animals; Biopharmaceutics; Cadmium; Cation Transport Proteins; Disease; Ecotoxicology; Fatty Acids, Omega-3; Food; Food Analysis; Glutathione; Humans; Lysophospholipids; Manganese; Mice; Nutrients; Nutritional Sciences; Organoselenium Compounds; Preventive Medicine; Sphingosine; Trans Fatty Acids; Vitamin K; Vitamin K 2

Topics: Bacillus subtilis; Gene Expression Regulation, Bacterial; Metabolic Engineering; Quorum Sensing; Vitamin K 2

The pregnane X receptor (PXR) is the key regulator of our defense mechanism against foreign substances such as drugs, dietary nutrients, or environmental pollutants. Because of increased health consciousness, the use of dietary supplements has gradually increased, and most of them can activate PXR. Therefore, an analysis of the interaction between drugs and nutrients is important because altered levels of drug-metabolizing enzymes or transporters can remarkably affect the efficiency of a co-administered drug. In the present study, we analyzed the effect of vitamin K-mediated PXR activation on drug metabolism-related gene expression in intestine-derived LS180 cells via gene expression studies and western blotting analyses. We demonstrated that menaquinone 4 (MK-4), along with other vitamin Ks, including vitamin K

Topics: ATP Binding Cassette Transporter, Subfamily B; Carcinoma; Cell Line, Tumor; Cytochrome P-450 CYP3A; Gene Expression; Humans; Intestinal Neoplasms; Nutritional Physiological Phenomena; Pregnane X Receptor; Rifampin; Vitamin K; Vitamin K 1; Vitamin K 2

Vitamin K (VK) exists in the form of phylloquinone (PK) and menaquinones (MKs). Roles of VK on cognitive health in the elderly are emerging, but there is limited evidence on VK uptake and metabolism in human brain.. The primary objective of this study was to characterize VK distribution in brains of an elderly population with varied cognitive function. In addition, associations among circulating (a biomarker of VK intake) and cerebral VK concentrations and cognition were investigated.. Serum or plasma (n = 27) and brain samples from the frontal cortex (FC; n = 46) and the temporal cortex (TC; n = 33) were acquired from 48 decedents (aged 98-107 y; 25 demented and 23 nondemented) enrolled in the Georgia Centenarian Study. Both circulating and brain VK concentrations were measured using HPLC with fluorescence detection. Cognitive assessment was performed within 1 y prior to mortality. Partial correlations between serum/plasma or cerebral VK concentrations and cognitive function were performed, adjusting for covariates and separating by dementia and antithrombotic use.. MK-4 was the predominant vitamer in both FC (mean ± SD = 4.92 ± 2.31 pmol/g, ≥89.15% ± 5.09% of total VK) and TC (4.60 ± 2.11 pmol/g, ≥89.71% ± 4.43% of total VK) regardless of cognitive status. Antithrombotic users had 34.0% and 53.9% lower MK-4 concentrations in FC (P < 0.05) and TC (P < 0.001), respectively. Circulating PK was not correlated with cerebral MK-4 or total VK concentrations. Circulating PK concentrations were significantly associated with a wide range of cognitive measures in nondemented centenarians (P < 0.05). In contrast, cerebral MK-4 concentrations were not associated with cognitive performance, either before or after exclusion of antithrombotic users.. Circulating VK concentrations are not related to cerebral MK-4 concentrations in centenarians. Cerebral MK-4 concentrations are tightly regulated over a range of VK intakes and cognitive function. Circulating PK may reflect intake of VK-rich foods containing other dietary components beneficial to cognitive health. Further investigation of VK uptake and metabolism in the brain is warranted.

Topics: Aged, 80 and over; Cerebral Cortex; Cognition; Female; Humans; Male; Vitamin K 1; Vitamin K 2

Cerebral ischemia/reperfusion (I/R) injury causes cognitive deficits, excitotoxicity, neuroinflammation, oxidative stress and brain edema. Vitamin K2 (Menaquinone 4, MK-4) as a potent antioxidant can be a good candidate to ameliorate I/R consequences. This study focused on the neuroprotective effects of MK-4 for cerebral I/R insult in rat's hippocampus. The rat model of cerebral I/R was generated by transient bilateral common carotid artery occlusion for 20 min. Rats were divided into control, I/R, I/R+DMSO (solvent (1% v/v)) and I/R+MK-4 treated (400 mg/kg, i.p.) groups. Twenty-four hours after I/R injury induction, total brain water content, superoxide dismutase (SOD) activity, nitrate/nitrite concentration and neuronal density were evaluated. In addition to quantify the apoptosis processes, TUNEL staining, as well as expression level of Bax and Bcl2, were assessed. To evaluate astrogliosis and induced neurotoxicity by I/R GFAP and GLT-1 mRNA expression level were quantified. Furthermore, pro-inflammatory cytokines including IL-1β, IL-6 and TNF-α were measured. Seven days post I/R, behavioral analysis to quantify cognitive function, as well as Nissl staining for surviving neuronal evaluation, were conducted. The findings indicated that administration of MK-4 following I/R injury improved anxiety-like behavior, short term and spatial learning and memory impairment induced by I/R. Also, MK-4 was able to diminish the increased total brain water content, apoptotic cell density, Bax/ Bcl2 ratio and GFAP mRNA expression following I/R. In addition, the high level of nitrate/nitrite, IL-6, IL-1β and TNF-α induced by I/R was reduced after MK-4 administration. However, MK-4 promotes the level of SOD activity and GLT-1 mRNA expression in I/R rat model. The findings demonstrated that MK-4 can rescue transient global cerebral I/R consequences via its anti-inflammatory and anti-oxidative stress features. MK-4 administration ameliorates neuroinflammation, neurotoxicity and neuronal cell death processes and leads to neuroprotection.

Topics: Animals; Apoptosis; Brain; Brain Ischemia; Excitatory Amino Acid Transporter 2; Glial Fibrillary Acidic Protein; Interleukin-1beta; Interleukin-6; Male; Neuroprotective Agents; Rats; Rats, Wistar; Reperfusion Injury; Spatial Learning; Superoxide Dismutase; Tumor Necrosis Factor-alpha; Vitamin K 2

The autosomal dominant disorder Schnyder corneal dystrophy (SCD) is caused by mutations in UbiA prenyltransferase domain-containing protein-1 (UBIAD1), which uses geranylgeranyl pyrophosphate (GGpp) to synthesize the vitamin K

Topics: Autophagy; Cell Line; Corneal Dystrophies, Hereditary; Dimethylallyltranstransferase; Genetic Variation; Humans; Intracellular Space; Protein Transport; Proteolysis; Vitamin K 2

The acid-base and redox properties of the menaquinones MK-4, MK-7, and MK-9 (vitamin K

Topics: Dimyristoylphosphatidylcholine; Electrodes; Hydrogen-Ion Concentration; Mercury; Oxidation-Reduction; Vitamin K 2

Tacrolimus, a calcineurin inhibitor, affects bone metabolism and increases the risk of fracture due to marked bone loss. Bisphosphonates increase the bone mineral density (BMD) in osteoporosis patients. Menatetrenone has less positive effects on BMD but reduces the risk of fracture by improving bone quality. In this study, we investigated the effectiveness of the combined administration of risedronate and menatetrenone against bone loss induced by tacrolimus. Wistar rats were divided into four groups: [1] control, [2] tacrolimus at 1.5 mg/kg, [3] tacrolimus + risedronate at 1.0 mg/kg, and [4] tacrolimus + risedronate + menatetrenone at 20 mg/kg. After the drugs were administered for 4 weeks, bone histomorphometric analysis was performed and bone strength was evaluated using a three point bending method. BMD was measured using quantitative computed tomography. Tacrolimus significantly reduced the BMD and strength properties of the lower limb bones. These tacrolimusinduced decreases were suppressed by risedronate treatment. The combined administration of risedronate and menatetrenone more significantly improved bone strength properties than risedronate alone. Bone histomorphometric analysis revealed a significant increase in bone resorption with tacrolimus. Risedronate alone significantly suppressed the tacrolimus-induced increase in bone resorption but simultaneously reduced bone formation. On the other hand, the combined administration of risedronate and menatetrenone suppressed the tacrolimus-induced increase in bone resorption, in addition to the significant risedronate-induced decrease in bone formation. This study suggests that the combined administration of risedronate and menatetrenone improves bone strength in tacrolimus-treated rats by preventing and ameliorating the risedronate-induced suppression of bone formation.

Topics: Animals; Bone Density; Bone Diseases, Metabolic; Drug Therapy, Combination; Femur; Immunosuppressive Agents; Male; Osteogenesis; Rats, Wistar; Risedronic Acid; Tacrolimus; Tibia; Vitamin K 2

The absence of vitamin E from the diet can lead to cardiovascular disease, cancer, cataracts, and premature aging. Vitamin K deficiency can lead to bleeding disorders. These fat-soluble vitamins are important nutritional factors that can be determined in different methods in vegetables. In this work, the simultaneous determination of α-tocopherol, α-tocopheryl acetate, phylloquinone, and menaquinone-4 by gas chromatography-mass spectrometry (GC-MS) has been optimized using both direct injection and solid phase microextraction (SPME). Three different sample pre-treatment approaches based on: (A) solid-liquid-liquid-liquid extraction (SLE-LLE), (B) SLE, and (C) SPME were then applied to extract the target analytes from vegetables samples using menaquinone as internal standard. All the procedures allowed the determination of the target analytes in onion, carrot, celery, and curly kale samples. Similar results were obtained with the three different approaches, even if the one based on SPME offers the best performance, together with a reduced use of solvent, time consumption, and experimental complexity, which makes it the preferable option for industrial applications.

Topics: alpha-Tocopherol; Gas Chromatography-Mass Spectrometry; Temperature; Vegetables; Vitamin E; Vitamin K; Vitamin K 1; Vitamin K 2

Vitamin K2 in the form of menatetrenone has clinical benefits for osteoporosis and cytopenia. Given the dominant role of mesenchymal-osteolineage cells in the regulation of hematopoiesis, we investigated whether menatetrenone alters the hematopoiesis-supportive capability of human bone marrow mesenchymal stromal/stem cells (BM-MSCs). Menatetrenone up-regulated fibronectin protein expression in BM-MSCs without affecting their proliferation and differentiation capabilities. In addition, menatetrenone treatment of BM-MSCs enhanced generation of the CD34

Topics: Antigens, CD34; Bone Marrow Cells; Cell Communication; Cell Differentiation; Cell Proliferation; Cells, Cultured; Coculture Techniques; Fibronectins; Gene Expression; Hematopoiesis; Humans; Mesenchymal Stem Cells; Stem Cell Niche; Vitamin K 2

Topics: Female; Fractures, Compression; Humans; Osteocalcin; Osteoporosis; Prospective Studies; Spinal Fractures; Vitamin K 2; Vitamins

Menaquinone-4 (MK4), a vitamin K metabolite, is converted from phylloquinone through a process that requires intermediates of endogenous cholesterol production. Recent evidence suggests that MK4 is involved in kidney function.. The purpose of this study was to determine the effect of atorvastatin treatment on MK4 formation in young and old male mice.. C57BL/6 male mice (4-mo-old and 20-mo-old) were randomly assigned to either a diet containing 300 mg atorvastatin/kg with 3 mg phylloquinone/kg or a control diet containing 3 mg phylloquinone/kg for 8 wk. During week 8, all mice received deuterium-labeled phylloquinone in the diet. Labeled and unlabeled phylloquinone and MK4 in liver, kidney, brain, and intestine were measured by atmospheric pressure chemical ionization LC/MS. 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase gene expression was quantified by reverse transcriptase-PCR. Tissue MK4 and phylloquinone concentrations were compared between atorvastatin treatment groups with use of general linear models.. There was no age-treatment interaction on MK4 tissue concentrations. In atorvastatin-treated mice, total MK4 and percentage of deuterium-labeled MK4 in kidney were both approximately 45% lower compared to values in mice not given atorvastatin (all P < 0.05). MK4 concentrations did not differ between groups in any other tissue measured.. In male mice, atorvastatin reduced endogenous MK4 formation in the kidney, but not other organs. These observations are consistent with our hypothesis that cholesterol metabolism is involved in the generation of MK4. Further research is needed to understand potential regulatory mechanisms and the unique functions of MK4 in the kidney.

Topics: Animals; Atorvastatin; Cholesterol; Gene Expression Regulation; Male; Mice; Mice, Inbred C57BL; Triglycerides; Vitamin K 2

Vascular calcification (VC) is an active and cell-mediated process that shares many common features with osteogenesis. Knowledge demonstrates that in the presence of risk factors, such as hypertension, vascular smooth muscle cells (vSMCs) lose their contractile phenotype and transdifferentiate into osteoblastic-like cells, contributing to VC development. Recently, menaquinones (MKs), also known as Vitamin K2 family, has been revealed to play an important role in cardiovascular health by decreasing VC. However, the MKs' effects and mechanisms potentially involved in vSMCs osteoblastic transdifferentiation are still unknown. The aim of this study was to investigate the possible role of menaquinone-4 (MK-4), an isoform of MKs family, in the modulation of the vSMCs phenotype. To achieve this, vascular cells from spontaneously hypertensive rats (SHR) were used as an in vitro model of cell vascular dysfunction. vSMCs from Wistar Kyoto normotensive rats were used as control condition. The results showed that MK-4 preserves the contractile phenotype both in control and SHR-vSMCs through a γ-glutamyl carboxylase-dependent pathway, highlighting its capability to inhibit one of the mechanisms underlying VC process. Therefore, MK-4 may have an important role in the prevention of vascular dysfunction and atherosclerosis, encouraging further in-depth studies to confirm its use as a natural food supplement.

Topics: Animals; Atherosclerosis; Blood Pressure; Carbon-Carbon Ligases; Cell Proliferation; Cell Transdifferentiation; Disease Models, Animal; Humans; Hypertension; Muscle, Smooth, Vascular; Myocytes, Smooth Muscle; Osteogenesis; Rats; Rats, Inbred SHR; Signal Transduction; Vitamin K 2

Vitamin K2 is indispensable for blood coagulation and bone metabolism. Menaquinone-4 (MK-4) is the predominant homolog of vitamin K2, which is present in large amounts in the pancreas, although its function is unclear. Meanwhile, β-cell dysfunction following insulin secretion has been found to decrease in patients with type 2 diabetes mellitus. To elucidate the physiological function of MK-4 in pancreatic β-cells, we studied the effects of MK-4 treatment on isolated mouse pancreatic islets and rat INS-1 cells. Glucose-stimulated insulin secretion significantly increased in isolated islets and INS-1 cells treated with MK-4. It was further clarified that MK-4 enhanced cAMP levels, accompanied by the regulation of the exchange protein directly activated by the cAMP 2 (Epac2)-dependent pathway but not the protein kinase A (PKA)-dependent pathway. A novel function of MK-4 on glucose-stimulated insulin secretion was found, suggesting that MK-4 might act as a potent amplifier of the incretin effect. This study therefore presents a novel potential therapeutic approach for impaired insulinotropic effects.

Topics: Animals; Cell Line, Tumor; Cyclic AMP; Glucose; Insulin Secretion; Insulinoma; Islets of Langerhans; Mice; Pancreatic Neoplasms; Rats; Signal Transduction; Vitamin K 2

Topics: Animals; Cell Line; Cell Nucleus; Humans; Inflammation; Inflammation Mediators; Lipopolysaccharides; Mice; Microglia; NF-kappa B; Phosphorylation; Protein Transport; Signal Transduction; Time Factors; Transcription Factor RelA; Tumor Necrosis Factor-alpha; Vitamin K; Vitamin K 2

Menaquinones, also known as Vitamin K2 family, regulate calcium homeostasis in a 'bone-vascular cross-talk' and recently received particular attention for their positive effect on bone formation. Given that the correlation between menaquinones and bone metabolism to date is still unclear, the objective of our study was to investigate the possible role of menaquinone-4 (MK-4), an isoform of the menaquinones family, in the modulation of osteogenesis. For this reason, we used a model of human amniotic fluid mesenchymal stem cells (hAFMSCs) cultured both in two-dimensional (2D) and three-dimensional (3D; RCCS™bioreactor) in vitro culture systems. Furthermore, to mimic the 'bone remodelling unit' in vitro, hAFMSCs were co-cultured in the 3D system with human monocyte cells (hMCs) as osteoclast precursors. The results showed that in a conventional 2D culture system, hAFMSCs were responsive to the MK-4, which significantly improved the osteogenic process through γ-glutamyl carboxylase-dependent pathway. The same results were obtained in the 3D dynamic system where MK-4 treatment supported the osteoblast-like formation promoting the extracellular bone matrix deposition and the expression of the osteogenic-related proteins (alkaline phosphatase, osteopontin, collagen type-1 and osteocalcin). Notably, when the hAFMSCs were co-cultured in a 3D dynamic system with the hMCs, the presence of MK-4 supported the cellular aggregate formation as well as the osteogenic function of hAFMSCs, but negatively affected the osteoclastogenic process. Taken together, our results demonstrate that MK-4 supported the aggregate formation of hAFMSCs and increased the osteogenic functions. Specifically, our data could help to optimize bone regenerative medicine combining cell-based approaches with MK-4 treatment.

Topics: Amniotic Fluid; Carbon-Carbon Ligases; Cell Culture Techniques; Cells, Cultured; Female; Humans; Mesenchymal Stem Cells; Osteoclasts; Osteogenesis; Pregnancy; Vitamin K 2

The relationship between dietary vitamin K and plasma PIVKA-II concentration, a biomarker of hepatic vitamin K status, in a Yup'ik study population in southwestern Alaska is investigated.. A total of 659 male and female, self-reported Yup'ik people, ≥14 years of age, were enrolled. Blood is collected for genotyping and plasma PIVKA-II biomarker analysis. A Yup'ik-specific dietary food frequency questionnaire is used to assess vitamin K intake. Among the participants, 22% report not consuming foods rich in vitamin K during the past year and 36% have a PIVKA-II concentration ≥ 2 ng mL. A substantial proportion of the Yup'ik population exhibits vitamin K insufficiency, which is associated with low consumption of vitamin K rich foods and which might affect an individual's response to anticoagulant drugs such as warfarin that target the vitamin K cycle.

Topics: Adolescent; Adult; Aged; Aged, 80 and over; Alaska; Alaskan Natives; Cytochrome P450 Family 4; Diet; Female; Humans; Liver; Male; Middle Aged; Nutritional Status; Prothrombin; Vegetables; Vitamin K; Vitamin K 1; Vitamin K 2

Vitamin (V) K deficiency may cause severe bleeding tendencies, which necessitates extreme caution. We report a case of a 30-year-old man diagnosed with VK deficiency of unknown etiology. He was treated with intravenous menatetrenone three times a week in an outpatient setting for about 1 year and 9 months. Eventually, he developed an allergic reaction to intravenous menatetrenone and was under steroid therapy. In order to reduce his hospital visits and discontinue steroid use, the pharmacist proposed to change the method of menatetrenone administration from intravenous to oral (high dose). The change in treatment method has greatly improved the patient's quality of life.

Topics: Administration, Intravenous; Administration, Oral; Adult; Drug Hypersensitivity; Hemostatics; Humans; Male; Quality of Life; Steroids; Vitamin K 2; Vitamin K Deficiency

Pregnane X receptor (PXR) is a nuclear receptor activated by various compounds, including prescribed drugs and dietary ingredients. Ligand-specific activation of PXR alters drug metabolism and affects many other physiological conditions. Species-specific ligand preference is a considerable challenge for studies of PXR function. To increase translational value of the results of mouse studies, humanized mouse model expressing human PXR (hPXR) has been developed. Menaquinone-4 (MK-4), one of vitamin K₂ analogs prescribed in osteoporosis, is a PXR ligand. We hypothesized that MK-4 could modulate the physiological conditions endogenously influenced by PXR, including those that have not been yet properly elucidated. In the present study, we investigated the effects of a single oral treatment with MK-4 on hepatic gene expression in wild-type and hPXR mice by using quantitative RT-PCR and DNA microarray. MK-4 administration altered mRNA levels of genes involved in drug metabolism (

Topics: Animals; Animals, Genetically Modified; ATP-Binding Cassette Transporters; Bile Acids and Salts; Cytochrome P-450 Enzyme System; Energy Metabolism; Female; Gene Expression; Glucose; Glucose Transport Proteins, Facilitative; Glucose Transporter Type 5; Hep G2 Cells; Homeostasis; Humans; Lipid Metabolism; Liver; Membrane Transport Proteins; Mice; Oligonucleotide Array Sequence Analysis; Polymerase Chain Reaction; Pregnane X Receptor; Receptors, Steroid; Sulfotransferases; Vitamin K 2; Vitamins

Previous studies have shown that α-tocopherol intake lowers phylloquinone (PK) concentration in some extrahepatic tissues in rats. The study's aim was to clarify the effect of α-tocopherol intake on vitamin K concentration in bone, as well as the physiological action of vitamin K. Male Wistar rats were divided into 4 groups. Over a 3-mo period, the K-free group was fed a vitamin K-free diet with 50 mg RRR-α-tocopherol/kg, the E-free group was fed a diet containing 0.75 mg PK/kg without vitamin E, the control group was fed a diet containing 0.75 mg PK/kg with 50 mg RRR-α-tocopherol/kg, and the E-excess group was fed a diet containing 0.75 mg PK/kg with 500 mg RRR-α-tocopherol/kg. PK concentration in the liver was higher in E-excess rats than in E-free rats, was lower in the tibias of control rats than in those of E-free rats, and was lower in E-excess rats than in control rats. Menaquinone-4 (MK-4) concentration in the liver was higher in E-excess rats than in E-free and control rats. However, MK-4 concentrations in the tibias of E-free, control, and E-excess rats were almost the same. Blood coagulation activity was lower in K-free rats than in the other rats but was not affected by the level of α-tocopherol intake. Additionally, dietary intake of PK and α-tocopherol did not affect uncarboxylated-osteocalcin concentration in the serum, femur density, or expression of the genes related to bone resorption and formation in the femur. These results suggest that α-tocopherol intake decreases PK concentration in bone but does not affect bone metabolism in rats.

Topics: alpha-Tocopherol; Animals; Biomarkers; Bone and Bones; Bone Density; Bone Development; Diet; Dietary Supplements; Energy Metabolism; Gene Expression Regulation, Developmental; Liver; Male; Organ Specificity; Osteocalcin; Rats, Wistar; Specific Pathogen-Free Organisms; Tibia; Vitamin K 1; Vitamin K 2; Vitamin K Deficiency; Vitamin K Deficiency Bleeding; Weight Gain

Topics: Blood Coagulation Tests; Colectomy; Female; Hemostatics; Hirschsprung Disease; Humans; Infant, Newborn; Male; Mothers; Short Bowel Syndrome; Vitamin K 2; Vitamin K Deficiency Bleeding; Young Adult

We have reported that vitamin E intake lowers phylloquinone (PK) concentration in extrahepatic tissues of rats. In this study, we aimed to clarify the characteristic of the distribution of menaquinone-7 (MK-7), a vitamin K contained in fermented foods, by comparison with other vitamin K distributions and to clarify the effect of vitamin E intake on MK-7 concentration in rats. Rats were fed a vitamin K-free diet (Free group), a diet containing 0.75 mg PK/kg (PK group), a 0.74 mg menaquinone-4 (MK-4)/kg diet (MK-4 group), a 1.08 mg MK-7/kg diet (MK-7 group), or a 0.29 mg menadione (MD)/kg diet (MD group) for 16 wk. MK-7 mainly accumulated in the liver, spleen, and adrenal gland of the MK-7 group, although PK accumulated in the serum and all tissues of the PK group. Conversely, MK-4 was present in all tissues of the PK, MK-4, MK-7, and MD groups. MK-4 concentration in the serum, liver, adipose tissue, and spleen was higher in the MK-4 group than in the other groups; however, MK-4 concentration in the kidney, testis, tibia, and brain was lower in the MK-4 group than in the PK, MK-7, and MD groups. Next, vitamin E- and K-deficient rats were orally administered MK-7 with or without α-tocopherol. α-Tocopherol did not affect MK-7 or MK-4 concentration in the serum and various tissues. These results suggested that MK-7 is particularly liable to accumulate in the liver, and MK-7 concentration is not affected by vitamin E intake.

Topics: alpha-Tocopherol; Animals; Diet; Fermented Foods; Liver; Male; Nutritional Status; Rats, Wistar; Tissue Distribution; Vitamin K 1; Vitamin K 2; Vitamin K 3; Vitamin K Deficiency

The aim of the present study was to determine the molecular basis of the beneficial effects of genistein and/or menaquinone‑4 (MK‑4) on bone quality. Initially, 1 µM genistein was applied to MC3T3‑E1 cells for 24 h and the upregulated mRNAs that were detected by microarray were selected for further examination by reverse transcription‑quantitative‑polymerase chain reaction. Among them, alterations were observed in the level of GATA‑binding protein 6 (GATA6), Notch gene homolog 2 (NOTCH2), Wnt family member 5A (WNT5A), bone γ‑carboxyglutamate protein (BGLAP), chondroadherin (CHAD), dipeptidyl peptidase 4 (DPP4), ectonucleotide pyrophosphatase/phosphodiesterase 2 (ENPP2), alkaline phosphatase (ALP) 3 and ATPase phospholipid‑transporting 11A (ATP11A) in response to treatment with 0.1 µM 17‑β‑estradiol, 1 µM genistein, and/or 1 µM MK‑4. GATA6, NOTCH2 and WNT5A are considered to be associated with osteoclast, but not osteoblast, function; however, increases in osteoblastic mRNAs, including BGLAP and CHAD, were observed in each of the treatment groups at 48 h. Immunocytochemical analysis confirmed an increase in CHAD and DPP4 proteins following the administration of genistein + MK‑4. Furthermore, genistein + MK‑4 led to alterations in cell morphology to spindle or oval shapes, and increased the intensity of ALP staining. Although the level of ALP mRNA was not consistently altered in response to the treatments, a marked increase in ALP activity was observed following 96 h treatment with genistein + MK‑4. Therefore, the simultaneous intake of genistein and MK‑4 appears to be beneficial for the maintenance of bone quality.

Topics: Animals; Biomarkers; Cell Line; Estradiol; Gene Expression Regulation; Genistein; Immunohistochemistry; Mice; Osteoblasts; RNA, Messenger; Vitamin K 2

Patients with chronic kidney disease (CKD) have very high levels of uncarboxylated, inactive, extra-hepatic vitamin K-dependent proteins measured in circulation, putting them at risk for complications of vitamin K deficiency. The major form of vitamin K found in the liver is phylloquinone (K1). Menaquinone-4 (MK-4) is the form of vitamin K that is preferentially found in extra-hepatic tissues.. In the present study, we assessed tissue concentrations of K1 and MK-4 and the expression of vitamin K-related genes in a rat model of adenine-induced CKD.. It was found that rats with both mild and severe CKD had significantly lower amounts of K1 measured in liver, spleen and heart and higher levels of MK-4 measured in kidney cortex and medulla. All animals treated with high dietary K1 had an increase in tissue levels of both K1 and MK-4; however, the relative increase in K1 differed suggesting that the conversion of K1 to MK-4 may be a regulated/limiting process in some tissues. There was a decrease in the thoracic aorta expression of vitamin K recycling (Vkor) and utilization (Ggcx) enzymes, and a decrease in the kidney level of vitamin K1 to MK-4 bioconversion enzyme Ubiad1 in CKD.. Taken together, these findings suggest that CKD impacts vitamin K metabolism, and this occurs early in the disease course. Our findings that vitamin K metabolism is altered in the presence of CKD provides further support that sub-clinical vitamin K deficiency may represent a modifiable risk factor for vascular and bone health in this population.

Topics: Adenine; Animals; Aorta, Thoracic; Carbon-Carbon Ligases; Dimethylallyltranstransferase; Disease Models, Animal; Gene Expression; Kidney; Male; Rats; Real-Time Polymerase Chain Reaction; Renal Insufficiency, Chronic; Vitamin K; Vitamin K 1; Vitamin K 2; Vitamin K Epoxide Reductases

Vitamin K is the collective term for compounds that share a 2-methyl-1,4-naphthoquinone ring, but differ in the side-chain at the 3-position. We synthesized novel 2-methyl-1,4-naphthoquinone derivatives with different side chain length at the 3-position. Derivatives with C-14 and C-16 tails showed the highest in vitro bioactivity resulting in 2.5 and 2-fold higher carboxylated osteocalcin synthesis in MG63 cells than menaquinone-4 (MK-4, form of vitamin K2). Longer side chain lengths resulted in lower bioactivity. The in vivo vitamin K activity of the C-14 tail derivative was further tested in WKY rats receiving a vitamin K-deficient diet that resulted in a 40% decrease of prothrombin activity. The C-14 tail derivative was able to counteract the effects on vitamin K deficiency induced by the diet and resulted in the complete restoration of prothrombin activity. Compared to naturally occurring forms of vitamin K, synthetic vitamin K derivatives may have higher bioactivity and different pharmacological characteristics that are more favorable for use as supplements or in clinical settings.

Topics: Animals; Carbon-Carbon Ligases; Cell Line, Tumor; Enzyme Activators; Humans; Molecular Structure; Osteocalcin; Prothrombin; Rats, Inbred WKY; Vitamin K; Vitamin K 2; Vitamin K Deficiency

We synthesized novel vitamin K

Topics: Aniline Compounds; Animals; Cells, Cultured; Female; Mice; Mice, Inbred C57BL; Neural Stem Cells; Neurogenesis; Vitamin K; Vitamin K 2; Vitamins

Vascular calcification is an important risk factor associated with mortality among patients with chronic kidney disease. Intracellular cholesterol metabolism is involved in the process of vascular cell calcification. In this study, we investigated the role of UbiA prenyltransferase domain containing 1 (UBIAD1) in intracellular cholesterol metabolism and vascular cell calcification, and identified its subcellular location. Primary human umbilical vein smooth muscle cells (HUVSMCs) were incubated with either growth medium (1.4 mmol/L Pi) or calcification medium (CM) (3.0 mmol/L Pi). Under treatment with CM, HUVSMCs were further incubated with exogenous cholesterol, or menaquinone-4, a product of UBIAD1. The plasmid and small interfering RNA were transfected in HUVSMCs to alter the expression of UBIAD1. Matrix calcium quantitation, alkaline phosphatase activity, intracellular cholesterol level and menaquinone-4 level were measured. The expression of several genes involved in cholesterol metabolism were analyzed. Using an anti-UBIAD1 antibody, an endoplasmic reticulum marker and a Golgi marker, the subcellular location of UBIAD1 in HUVSMCs was analyzed. CM increased matrix calcium, alkaline phosphatase activity and intracellular cholesterol level, and reduced UBIAD1 expression and menaquinone-4 level. Addition of cholesterol contributed to increased matrix calcification and alkaline phosphatase activity in a dose-dependent manner. Elevated expression of UBIAD1 or menaquinone-4 in HUVSMCs treated with CM significantly reduced intracellular cholesterol level, matrix calcification and alkaline phosphatase activity, but increased menaquinone-4 level. Elevated expression of UBIAD1 or menaquinone-4 reduced the gene expression of sterol regulatory element-binding protein-2, and increased gene expression of ATP binding cassette transporters A1, which are in charge of cholesterol synthesis and efflux. UBIAD1 co-localized with the endoplasmic reticulum marker and the Golgi marker in HUVSMCs. In conclusion, high intracellular cholesterol content contributes to phosphate-induced vascular cell differentiation and calcification. UBIAD1 or menaquinone-4 could decrease vascular cell differentiation and calcification, probably via its potent role of inversely modulating cellular cholesterol.

Topics: Alkaline Phosphatase; Cell Differentiation; Cell Membrane Permeability; Cholesterol; Culture Media; Dimethylallyltranstransferase; Endoplasmic Reticulum; Extracellular Matrix; Gene Expression Regulation; Golgi Apparatus; Humans; Intracellular Space; Myocytes, Smooth Muscle; Osteoblasts; Phosphorus; Umbilical Veins; Vascular Calcification; Vitamin K 2

UbiA prenyltransferase domain-containing protein-1 (UBIAD1) utilizes geranylgeranyl pyrophosphate (GGpp) to synthesize the vitamin K2 subtype menaquinone-4. Previously, we found that sterols trigger binding of UBIAD1 to endoplasmic reticulum (ER)-localized HMG-CoA reductase, the rate-limiting enzyme in synthesis of cholesterol and nonsterol isoprenoids, including GGpp. This binding inhibits sterol-accelerated degradation of reductase, which contributes to feedback regulation of the enzyme. The addition to cells of geranylgeraniol (GGOH), which can become converted to GGpp, triggers release of UBIAD1 from reductase, allowing for its maximal degradation and permitting ER-to-Golgi transport of UBIAD1. Here, we further characterize geranylgeranyl-regulated transport of UBIAD1. Results of this characterization support a model in which UBIAD1 continuously cycles between the ER and medial-trans Golgi of isoprenoid-replete cells. Upon sensing a decline of GGpp in ER membranes, UBIAD1 becomes trapped in the organelle where it inhibits reductase degradation. Mutant forms of UBIAD1 associated with Schnyder corneal dystrophy (SCD), a human eye disease characterized by corneal accumulation of cholesterol, are sequestered in the ER and block reductase degradation. Collectively, these findings disclose a novel sensing mechanism that allows for stringent metabolic control of intracellular trafficking of UBIAD1, which directly modulates reductase degradation and becomes disrupted in SCD.

Topics: Cell Membrane; Corneal Dystrophies, Hereditary; Dimethylallyltranstransferase; Endoplasmic Reticulum; Golgi Apparatus; Humans; Hydroxymethylglutaryl CoA Reductases; Lipid Metabolism; Polyisoprenyl Phosphates; Protein Transport; Proteolysis; Terpenes; Vitamin K; Vitamin K 2

Dietary intake of vitamin K has been reported to reduce coronary artery calcification (CAC) and cardiovascular events. However, it is unknown whether supplemental menaquinone (MK)-4 can reduce CAC or arterial stiffness. To study the effect of MK-4 supplementation on CAC and brachial ankle pulse wave velocity (baPWV).. This study is a single arm design to take 45 mg/day MK-4 daily as a therapeutic drug for 1 year. Primary endpoint was CAC score determined using 64-slice multislice CT (Siemens), and the secondary endpoint was baPWV measured before and 1 year after MK-4 therapy.. A total of 26 patients were enrolled. The average age was 69 ± 8 years and 65 % were female. Plasma levels of phylloquinone (PK), MK-7, and MK4 were 1.94 ± 1.38 ng/ml, 14.2 ± 11.9 ng/ml and 0.4 ± 2.0 ng/ml, respectively, suggesting that MK-7 was the dominant vitamin K in the studied population. Baseline CAC and baPWV were 513 ± 773 and 1834 ± 289 cm/s, respectively. At 1 year following MK-4 supplementation, the values were 588 ± 872 (+14 %) and 1821 ± 378 cm/s (-0.7 %), respectively. In patients with high PIVKA-2, -18 % annual reduction of baPWV was observed.. Despite high dose MK-4 supplementation, CAC increased +14 % annually, but baPWV did not change (-0.7 %). The benefits of MK-4 supplementation were only observed in patients with vitamin K insufficiencies correlated with high PIVKA-2 baseline levels, reducing baPWV but not CAC.. This study was registered as UMIN 000002760.

Topics: Aged; Ankle Brachial Index; Body Mass Index; Cardiomyopathies; Coronary Vessels; Dietary Supplements; Endpoint Determination; Female; Hemostatics; Humans; Male; Middle Aged; Pilot Projects; Prospective Studies; Pulse Wave Analysis; Risk Factors; Vascular Stiffness; Vitamin K 1; Vitamin K 2

Increasing evidence has shown that environmental factors such as rotenone and paraquat induce neuroinflammation, which contributes to the pathogenesis of Parkinson's disease (PD). In this study, we investigated the molecular mechanisms underlying the repression by menaquinone-4 (MK-4), a subtype of vitamin K2, of rotenone-induced microglial activation in vitro.. A microglial cell line (BV2) was exposed to rotenone (1 μmol/L) with or without MK-4 treatment. The levels of TNF-α or IL-1β in 100 μL of cultured media of BV2 cells were measured using ELISA kits. BV2 cells treated with rotenone with or without MK4 were subjected to mitochondrial membrane potential, ROS production, immunofluorescence or immunoblot assays. The neuroblastoma SH-SY5Y cells were treated with conditioned media (CM) of BV2 cells that were exposed to rotenone with or without MK-4 treatment, and the cell viability was assessed using MTT assay.. In rotenone-treated BV2 cells, MK-4 (0.5-20 μmol/L) dose-dependently suppressed the upregulation in the expression of iNOS and COX-2 in the cells, as well as the production of TNF-α and IL-1β in the cultured media. MK-4 (5-20 μmol/L) significantly inhibited rotenone-induced nuclear translocation of NF-κB in BV2 cells. MK-4 (5-20 μmol/L) significantly inhibited rotenone-induced p38 activation, ROS production, and caspase-1 activation in BV2 cells. MK-4 (5-20 μmol/L) also restored the mitochondrial membrane potential that had been damaged by rotenone. Exposure to CM from rotenone-treated BV2 cells markedly decreased the viability of SH-SY5Y cells. However, this rotenone-activated microglia-mediated death of SH-SY5Y cells was significantly attenuated when the BV2 cells were co-treated with MK-4 (5-20 μmol/L).. Vitamin K2 can directly suppress rotenone-induced activation of microglial BV2 cells in vitro by repressing ROS production and p38 activation.

Topics: Animals; Cell Culture Techniques; Cell Line; Cell Survival; Dose-Response Relationship, Drug; Environmental Pollutants; Interleukin-1beta; Membrane Potential, Mitochondrial; Mice; Microglia; Neuroimmunomodulation; NF-kappa B; Reactive Oxygen Species; Rotenone; Tumor Necrosis Factor-alpha; Vitamin K 2

Warfarin is characterized by a large inter-individual variability in dosage requirement. This study aimed to analyze the contribution of the CYP4F2 genetic polymorphism and plasma vitamin K concentration on the warfarin pharmacodynamics in patients and to clarify the plasma vitamin K concentration affecting warfarin sensitivity index in rats.. Genetic analyses of selected genes were performed and plasma concentrations of warfarin, vitamin K1 (VK1) and menaquinone-4 (MK-4) were measured in 217 Japanese patients. We also assessed the association of plasma VK1 and MK-4 concentrations with the warfarin sensitivity index (INR/Cp) in rats.. Patients with the CYP4F2 (rs2108622) TT genotype had significantly higher plasma VK1 and MK-4 concentrations than those with CC and CT genotypes. The multiple linear regression model including VKORC1, CYP4F2, and CYP2C9 genetic variants, age, and weight could explain 42% of the variability in warfarin dosage. The contribution of CYP4F2 polymorphism was estimated to be 2.2%. In contrast, plasma VK1 and MK-4 concentrations were not significantly associated with warfarin dosage in patients. Nevertheless, we were able to demonstrate that the warfarin sensitivity index was attenuated and negatively correlated with plasma VK1 concentration by the oral administration of VK1 in rats, as it resulted in a higher VK1 concentration than that in patients.. The plasma VK1 and MK-4 concentrations are significantly influenced by CYP4F2 genetic polymorphism but not associated with warfarin therapy at the observed concentration in Japanese patients. The CYP4F2 polymorphism is poorly associated with inter-individual variability of warfarin dosage requirement.

Topics: Adult; Aged; Aged, 80 and over; Alleles; Animals; Anticoagulants; Asian People; Biotransformation; Cytochrome P-450 CYP2C9; Cytochrome P-450 Enzyme System; Cytochrome P450 Family 4; Drug Resistance; Female; Genetic Variation; Genotype; Humans; International Normalized Ratio; Male; Middle Aged; Polymorphism, Single Nucleotide; Rats; Rats, Sprague-Dawley; Thrombophilia; Vitamin K 1; Vitamin K 2; Vitamin K Epoxide Reductases; Warfarin; Young Adult

The liver is the main organ responsible for the modification, clearance, and transformational toxicity of most xenobiotics owing to its abundance in cytochrome P450 (CYP450) enzymes. However, the scarcity and variability of primary hepatocytes currently limits their utility. Human pluripotent stem cells (hPSCs) represent an excellent source of differentiated hepatocytes; however, current protocols still produce fetal-like hepatocytes with limited mature function. Interestingly, fetal hepatocytes acquire mature CYP450 expression only postpartum, suggesting that nutritional cues may drive hepatic maturation. We show that vitamin K2 and lithocholic acid, a by-product of intestinal flora, activate pregnane X receptor (PXR) and subsequent CYP3A4 and CYP2C9 expression in hPSC-derived and isolated fetal hepatocytes. Differentiated cells produce albumin and apolipoprotein B100 at levels equivalent to primary human hepatocytes, while demonstrating an 8-fold induction of CYP450 activity in response to aryl hydrocarbon receptor (AhR) agonist omeprazole and a 10-fold induction in response to PXR agonist rifampicin. Flow cytometry showed that over 83% of cells were albumin and hepatocyte nuclear factor 4 alpha (HNF4α) positive, permitting high-content screening in a 96-well plate format. Analysis of 12 compounds showed an R(2) correlation of 0.94 between TC50 values obtained in stem cell-derived hepatocytes and primary cells, compared to 0.62 for HepG2 cells. Finally, stem cell-derived hepatocytes demonstrate all toxicological endpoints examined, including steatosis, apoptosis, and cholestasis, when exposed to nine known hepatotoxins.. Our work provides fresh insights into liver development, suggesting that microbial-derived cues may drive the maturation of CYP450 enzymes postpartum. Addition of these cues results in the first functional, inducible, hPSC-derived hepatocyte for predictive toxicology.

Topics: Cell Culture Techniques; Cell Differentiation; Cells, Cultured; Cytochrome P-450 CYP2C9; Cytochrome P-450 CYP3A; Embryonic Stem Cells; Hepatocytes; Humans; Lithocholic Acid; Pluripotent Stem Cells; Pregnane X Receptor; Receptors, Steroid; Sequence Analysis, RNA; Toxicity Tests, Acute; Vitamin K 2

Breast cancer is the most prevalent cancer type worldwide. Continued efforts to improve treatment strategies for patients with breast cancer will be instrumental in reducing the death rates associated with this disease. In particular, the triple-negative breast cancer subtype of breast cancer has no targeted therapy available so it is essential to continue to work on any potential therapies. Vitamin K (VK) is known for its essential role in the clotting cascade. The antitumor properties of VK derivatives have been reported in both hepatocellular carcinoma and glioblastoma. Our hypothesis was that menaquinone-4, the most common form of vitamin K2 (VK2), is an effective anticancer agent against breast cancer cell types. In this study, we used a novel impedance-based live cell monitoring platform (xCELLigence) to determine the effects of VK derivatives on the triple-negative breast cancer cell line, MDA-MB-231, and the HER2+ breast cancer cell line, MDA-MB-453. Cells were treated with varying concentrations of menaquinone-4 (VK2) previously reported to have an antiproliferative effect on human glioblastoma cells. After initial testing, these concentrations were adjusted to 100, 125, and 150 μmol/L. A significant dose-dependent, growth inhibitory effect was found when cells were treated at these concentrations. These effects were seen in both adhesion and proliferation phases and show a dramatic reduction in cell growth. Additional analysis of MDA-MB-231 cells treated with VK2 (100 μmol/L) in combination with a low-glucose nutrient media showed a further decrease in adhesion and viability. This is the first study of its kind showing the real-time effects of VK derivatives on breast cancer cells and suggests that dietary factors may be an important consideration for patients.

Topics: Antineoplastic Agents; Breast Neoplasms; Cell Adhesion; Cell Line, Tumor; Cell Proliferation; Cell Survival; Female; Humans; Vitamin K 2

To further understand the correlation between vitamin K and bone metabolism, the effects of vitamins K1, menaquinone-4 (MK-4), and menaquinone-7 (MK-7) on RANKL-induced osteoclast differentiation and bone resorption were comparatively investigated. Vitamin K2 groups (MK-4 and MK-7) were found to significantly inhibit RANKL-medicated osteoclast cell formation of bone marrow macrophages (BMMs) in a dose-dependent manner, without any evidence of cytotoxicity. The mRNA expression of specific osteoclast differentiation markers, such as c-Fos, NFATc1, OSCAR, and TRAP, as well as NFATc1 protein expression and TRAP activity in RANKL-treated BMMs were inhibited by vitamin K2, although MK-4 exhibited a significantly greater efficiency compared to MK-7. In contrast, the same dose of vitamin K1 had no inhibitory effect on RANKL-induced osteoclast cell formation, but increased the expression of major osteoclastogenic genes. Interestingly, vitamins K1, MK-4 and MK-7 all strongly inhibited osteoclastic bone resorption (p < 0.01) in a dose dependent manner. These results suggest that vitamins K1, MK-4 and MK-7 have anti-osteoporotic properties, while their regulation effects on osteoclastogenesis are somewhat different.

Topics: Acid Phosphatase; Animals; Bone Marrow Cells; Bone Resorption; Cell Differentiation; Isoenzymes; Macrophages; Male; Mice; Mice, Inbred ICR; NFATC Transcription Factors; Osteoclasts; Proto-Oncogene Proteins c-fos; RANK Ligand; Receptors, Cell Surface; RNA, Messenger; Signal Transduction; Tartrate-Resistant Acid Phosphatase; Vitamin K; Vitamin K 1; Vitamin K 2

Decreased concentration of menaquinone-4 (MK-4) seems to be an important risk factor of vascular calcification in haemodialysis (HD) patients. Optimal dietary intake, as well as serum MK-4 reference range, in HD has not been determined, yet. The aim of the present study was to assess daily vitamin K1 and MK-4 intakes and their relation to serum MK-4 concentration in HD patients.. Daily vitamin K1 and MK-4, micro- and macronutrients and energy intakes were assessed using 3-day food diary completed by patients and serum MK-4 concentration was measured by HPLC [limit of quantification (LOQ): 0.055 ng/mL] in 85 HD patients (51 males) and 22 apparently healthy subjects.. Daily MK-4 intake was significantly lower (by 29%) among HD, while K1 consumption was similar in both groups. Daily MK-4 intake was associated with fat and protein consumption in HD (r=0.43, p<0.001 and r=0.33, p=0.004, respectively). In HD serum MK-4 concentration was more frequently below LOQ (in 41% HD and 5% controls, p<0.001) and in those HD with quantifiable values was lower than in the controls (by 42%). The correlations between MK-4 concentrations and both MK-4 and K1 daily intakes were weaker in HD (r=0.38 and r=0.30 respectively) than in the control group (r=0.47 and r=0.45, respectively). In multiple regression analysis the variability of serum MK-4 concentrations in HD patients was explained by its daily intake.. Decreased serum MK-4 concentration in HD patients is caused by lower dietary MK-4 intake, mainly due to diminished meat consumption, and in addition, probably reduced K1 conversion.

Topics: Case-Control Studies; Chromatography, High Pressure Liquid; Diet Records; Dietary Fats; Dietary Proteins; Energy Intake; Female; Hemostatics; Humans; Limit of Detection; Male; Middle Aged; Recommended Dietary Allowances; Reference Values; Renal Dialysis; Renal Insufficiency, Chronic; Vascular Calcification; Vitamin K 1; Vitamin K 2

Unlike the other fat-soluble vitamins, vitamin K circulates in the human bloodstream at very low levels because of a low intake in the diet. Mammals have developed an efficient recycling system, known as vitamin K-epoxide cycle, which involve quinone, hydroquinone and epoxide forms of the vitamin. Phylloquinone (K(1)) is the main homologue, while menaquinone-4 (MK-4) is both a member of the vitamin K(2) family and metabolite of K(1) in extra-hepatic tissues. Notwithstanding the recent advances, many aspects of the complex vitamin K physiology still remain to be investigated. Therefore, there is a critical need to develop more reliable analytical methods for determining the vitamin K and its metabolites in biological fluids and tissues. Nevertheless, relatively low concentrations, unavailability of some authentic standards and occurrence of interfering lipids make this a challenging task. The method proposed in the present paper can directly and accurately estimate K(1), K(1) 2,3-epoxide (K(1)O), and MK-4 in human serum and plasma at concentrations in the ng/L-μg/L range, using labelled internal standards and a quadrupole linear ion trap instrument operated in multiple reaction monitoring (MRM) mode. High sensitivity was achieved by removing signal "endogenous suppressors" and making the composition of the non-aqueous mobile phase suitable to support the positive atmospheric pressure chemical ionization of the analytes. An excellent selectivity resulted from the combination of some factors: the MRM acquisition, the adoption of an identification point system, an extraction optimized to remove most of the lipids and a tandem-C18 column-system necessary to separate isobaric interferences from analytes. The method was validated according to the Food and Drug Administration (FDA) guidelines and its accuracy was assessed by analysing 9 samples from the Vitamin K External Quality Assessment Scheme (KEQAS). Its feasibility in evaluating vitamin K status in human serum was also tested by monitoring a group of six healthy subjects and a group of six patients under oral anticoagulant therapy (OAT). Warfarinised patients did not show deficiency of K1 but levels comparable with those of healthy people and an accumulation of K1O up to 3.760μg/L. MK-4 was not detected in either of the two groups.

Topics: Chromatography, High Pressure Liquid; Epoxy Compounds; Humans; Tandem Mass Spectrometry; Time Factors; Vitamin K; Vitamin K 1; Vitamin K 2; Vitamins

The effects of vitamin E on vitamin K metabolism were elucidated by comparing the effect of tocopherol intake on vitamin K concentrations in rats fed phylloquinone (PK) or menaquinone (MK)-4.. Initially, the dietary effect of RRR-α-tocopherol, but not RRR-γ-tocopherol, in decreasing extrahepatic PK concentrations was confirmed. Subsequently, rats were fed a PK or MK-4-containing diet (0.75 mg/kg) with RRR-α-tocopherol (0, 10, 50, or 500 mg/kg) for 6 weeks. In rats fed PK, α-tocopherol consumption decreased PK in kidney, lung, heart, muscle, testis, and brain but not in serum and liver. However, in rats fed MK-4, α-tocopherol consumption did not decrease MK-4 in serum and tissues. Finally, vitamin K- and E-depleted rats were administered PK or MK-4 (0.2 mg) with RRR-α-tocopherol (0, 1, or 10 mg) by gavage. After PK administration, α-tocopherol was observed to decrease PK in kidney, adrenal gland, lung, testis, and brain but not in serum and liver, whereas, after MK-4 administration, α-tocopherol did not affect MK-4 in serum and tissues.. Excess α-tocopherol decreased extrahepatic PK in rats fed PK but not MK-4 in rats fed MK-4.

Topics: alpha-Tocopherol; Animals; Dietary Supplements; Down-Regulation; gamma-Tocopherol; Male; Organ Specificity; Rats, Wistar; Specific Pathogen-Free Organisms; Vitamin E Deficiency; Vitamin K 1; Vitamin K 2; Vitamin K Deficiency

The influence of excess α-tocopherol (α-T) on tissue depletion of phylloquinone (PK) and menaquinone-4 (MK-4) was evaluated.. Rats (n = 5 per group) were fed deuterium-labeled PK (2 μmol/kg diet) for 17 days, thereby labeling the conversion from deuterium-labeled PK to d₄-MK-4. Then they were injected subcutaneously daily for the last 7 days with saline, vehicle, or α-T (100 mg/kg body weight). α-T injections (i) increased α-T concentrations by tenfold in liver, doubled them in plasma and most tissues, but they were unchanged in brain; (ii) increased the α-T metabolite, carboxyethyl hydroxychromanol (α-CEHC) concentrations: >25-fold in liver and kidney, tenfold in plasma and lung, and 50-fold in heart; brain contained detectable α-CEHC (0.26 ± 0.03 nmol/g) only in α-T-injected animals; and (iii) depleted most tissues' vitamin K. Compared with vehicle-injected rats, brains from α-T rats contained half the total vitamin K (10.3 ± 0.5 versus 21 ± 2 pmol/g, p = 0.0002) and one-third the d₄-MK-4 (5.8 ± 0.5 versus 14.6 ± 1.7 pmol/g, p = 0.0002). Tissues with high PK concentrations (liver, 21-30 pmol/g and heart, 28-50 pmol/g) were resistant to K depletion.. We propose that α-T-dependent vitamin K depletion is likely mediated at an intermediate step in MK-4 production; thus, tissues with high PK are unaffected.

Topics: alpha-Tocopherol; Animals; Biotransformation; Brain; Deuterium; Injections, Subcutaneous; Kidney; Liver; Male; Neurons; Organ Specificity; Rats, Sprague-Dawley; Vitamin K 1; Vitamin K 2; Vitamin K Deficiency; Vitamins

UbiA prenyltransferase domain containing 1 (UBIAD1) is a novel vitamin K2 biosynthetic enzyme screened and identified from the human genome database. UBIAD1 has recently been shown to catalyse the biosynthesis of Coenzyme Q10 (CoQ10) in zebrafish and human cells. To investigate the function of UBIAD1 in vivo, we attempted to generate mice lacking Ubiad1, a homolog of human UBIAD1, by gene targeting. Ubiad1-deficient (Ubiad1(-/-)) mouse embryos failed to survive beyond embryonic day 7.5, exhibiting small-sized body and gastrulation arrest. Ubiad1(-/-) embryonic stem (ES) cells failed to synthesize vitamin K2 but were able to synthesize CoQ9, similar to wild-type ES cells. Ubiad1(+/-) mice developed normally, exhibiting normal growth and fertility. Vitamin K2 tissue levels and synthesis activity were approximately half of those in the wild-type, whereas CoQ9 tissue levels and synthesis activity were similar to those in the wild-type. Similarly, UBIAD1 expression and vitamin K2 synthesis activity of mouse embryonic fibroblasts prepared from Ubiad1(+/-) E15.5 embryos were approximately half of those in the wild-type, whereas CoQ9 levels and synthesis activity were similar to those in the wild-type. Ubiad1(-/-) mouse embryos failed to be rescued, but their embryonic lifespans were extended to term by oral administration of MK-4 or CoQ10 to pregnant Ubiad1(+/-) mice. These results suggest that UBIAD1 is responsible for vitamin K2 synthesis but may not be responsible for CoQ9 synthesis in mice. We propose that UBIAD1 plays a pivotal role in embryonic development by synthesizing vitamin K2, but may have additional functions beyond the biosynthesis of vitamin K2.

Topics: Animals; Dimethylallyltranstransferase; Embryo Culture Techniques; Embryonic Development; Female; Gene Deletion; Male; Mice, Inbred C57BL; Ubiquinone; Vitamin K 2

Menaquinone-4 is a type of vitamin K that has a physiological function in maintaining bone quality via γ-carboxylation of osteocalcin. However, little is known about the beneficial effect of intake of dosages below1500 μg/day.. Fifteen healthy males aged 25.0 years (median) participated in a non-placebo-controlled dose-examination study. They received menaquinone-4 daily for 5 weeks at 0, 300, 600, 900, and 1500 μg/day in weeks 1, 2, 3, 4, and 5, respectively. Compared with baseline, serum γ-carboxylated osteocalcin levels were significantly greater at an intake of 900 μg/day or more; serum undercarboxylated osteocalcin levels and the ratio of serum undercarboxylated osteocalcin to γ-carboxylated osteocalcin were significantly lower than baseline at doses of 600 μg/day or more.. This preliminary graded-dose study suggested that menaquinone-4 supplementation at 600 μg/day or more is likely to be important in terms of vitamin K requirements for bone health.

Topics: Adult; Biomarkers; Body Mass Index; Body Weight; Bone and Bones; Dietary Supplements; Dose-Response Relationship, Drug; Healthy Volunteers; Humans; Male; Osteocalcin; Vitamin K 1; Vitamin K 2; Young Adult

Topics: Dimethylallyltranstransferase; Humans; Neurodegenerative Diseases; Neuroprotective Agents; Vitamin K; Vitamin K 2

We have shown that intake of sesame seed and its lignan increases vitamin E concentrations and decreases urinary excretion levels of vitamin E metabolites in male Wistar rats, suggesting inhibition of vitamin E catabolism by sesame lignan. The aim of this study was to examine whether dietary sesame seed also increased vitamin K concentrations, because its metabolic pathway is similar to that of vitamin E. To test the effect of sesame lignan on vitamin K concentrations, male Wistar rats were fed a control diet or a diet with 0.2% sesamin (a sesame lignan) for 7 d in experiment 1. Liver phylloquinone (PK), menaquinone-4 (MK-4), and γ-tocopherol were greater in rats fed sesamin than in control rats. To test the effect of sesame seed on vitamin K concentrations, male Wistar rats were fed a control diet or a diet with 1, 5, or 10% sesame seed for 3 d in experiment 2. Liver and kidney PK and γ-tocopherol but not MK-4 were greater in rats fed sesame seed than in control rats, although differences in dietary amounts of sesame seed did not affect the PK concentrations. For further confirmation of the effect of sesame seed, male Wistar rats were fed a control diet or a diet with 20% sesame seed for 40 d in experiment 3. Kidney, heart, lung, testis, and brain PK and brain MK-4 were greater in rats fed sesame seed than in control rats. The present study revealed for the first time, to our knowledge, that dietary sesame seed and sesame lignan increase not only vitamin E but also vitamin K concentrations in rat tissues.

Topics: Animals; Brain; Diet; Dioxoles; gamma-Tocopherol; Heart; Kidney; Lignans; Liver; Lung; Male; Rats; Rats, Wistar; Seeds; Sesamum; Testis; Vitamin K 1; Vitamin K 2

Mice have the ability to convert dietary phylloquinone (vitamin K1) into menaquinone-4 (vitamin K2) and store the latter in tissues. A prenyltransferase enzyme, UbiA prenyltransferase domain-containing 1 (UBIAD1), is involved in this conversion. There is evidence that UBIAD1 has a weak side chain cleavage activity for phylloquinone but a strong prenylation activity for menadione (vitamin K3), which has long been postulated as an intermediate in this conversion. Further evidence indicates that when intravenously administered in mice phylloquinone can enter into tissues but is not converted further to menaquinone-4. These findings raise the question whether phylloquinone is absorbed and delivered to tissues in its original form and converted to menaquinone-4 or whether it is converted to menadione in the intestine followed by delivery of menadione to tissues and subsequent conversion to menaquinone-4. To answer this question, we conducted cannulation experiments using stable isotope tracer technology in rats. We confirmed that the second pathway is correct on the basis of structural assignments and measurements of phylloquinone-derived menadione using high resolution MS analysis and a bioassay using recombinant UBIAD1 protein. Furthermore, high resolution MS and (1)H NMR analyses of the product generated from the incubation of menadione with recombinant UBIAD1 revealed that the hydroquinone, but not the quinone form of menadione, was an intermediate of the conversion. Taken together, these results provide unequivocal evidence that menadione is a catabolic product of oral phylloquinone and a major source of tissue menaquinone-4.

Topics: Animals; Dimethylallyltranstransferase; Female; Intestinal Mucosa; Male; Mice; Rats; Rats, Sprague-Dawley; Recombinant Proteins; Species Specificity; Vitamin K 1; Vitamin K 2; Vitamin K 3; Vitamins

Vitamin K plays an essential role in many biological processes including blood clotting, maintenance of bone health, and inhibition of arterial calcification. A menaquinone form of vitamin K, MK4, is increasingly recognized for its key roles in mitochondrial electron transport, as a ligand for the nuclear receptor SXR, which controls the expression of genes involved in transport and metabolism of endo- and xenobiotics, and as a pharmacotherapeutic in the treatment of osteoporosis. Although cytochrome P450 (CYP) 4F2 activity is recognized as an important determinant of phylloquinone (K1) metabolism, the enzymes involved in menaquinone catabolism have not been studied previously. CYP4F2 and CYP4F11 were expressed and purified and found to be equally efficient as in vitro catalysts of MK4 ω-hydroxylation. CYP4F2, but not CYP4F11, catalyzed sequential metabolism of MK4 to the ω-acid without apparent release of the intermediate aldehyde. The ω-alcohol could also be metabolized to the acid by microsomal NAD(+)-dependent alcohol and aldehyde dehydrogenases. LC-MS/MS analysis of trypsinized human liver microsomes (using a surrogate peptide approach) revealed the mean concentrations of CYP4F2 and CYP4F11 to be 14.3 and 8.4 pmol/mg protein, respectively. Microsomal MK4 ω-hydroxylation activities correlated with the CYP4F2 V433M genotype but not the CYP4F11 D446N genotype. Collectively, these data expand the lexicon of vitamin K ω-hydroxylases to include the 'orphan' P450 CYP4F11 and identify a common variant, CYP4F2 (rs2108622), as a major pharmacogenetic variable influencing MK4 catabolism.

Topics: Cytochrome P-450 CYP4A; Cytochrome P-450 Enzyme System; Cytochrome P450 Family 4; Humans; Hydroxylation; Kinetics; Microsomes, Liver; Vitamin K 2

We aimed to evaluate the clinical efficacy of monotherapy with alendronate and combined therapy with alendronate and menatetrenone (vitamin K2 [VitK2]) in postmenopausal rheumatoid arthritis (RA) patients with osteoporosis or osteopenia.. Sixty-two postmenopausal RA patients with untreated osteoporosis or osteopenia (lumbar spine bone density ≤80 % of young adult mean [YAM]) were enrolled: 39 had abnormal serum undercarboxylated osteocalcin (ucOC) levels (>4.5 ng/mL) and received combined therapy with alendronate (35 mg/week) and VitK2 (45 mg/day) (ALN + K group); 23 had normal ucOC levels (≤4.5 ng/mL) and received alendronate monotherapy (35 mg/week) (ALN group). The clinical results for the 57 patients in both groups were evaluated after 1-year treatment.. The mean baseline/follow-up (FU) lumbar spine bone density (%YAM) values were 73.0/76.8 % (P < 0.01) in the ALN + K group and 77.0/80.3 % (P < 0.01) in the ALN group; a significant increase was shown in both groups. Mean proximal femoral bone density values at baseline/FU were 71.4/73.8 (P < 0.01) in the ALN + K group and 71.4/71.6 % (not significant; NS) in the ALN group; a significant increase was shown in the ALN + K group only. Serum ucOC levels were normalized in the ALN + K group at FU. At FU, bone metabolism markers [bone-specific alkaline phosphatase (BAP) and N-terminal cross-linked telopeptides of type I collagen] were decreased in both groups. One patient in the ALN + K group and three in the ALN group suffered new fractures.. Combined therapy with alendronate and VitK2 decreases bone metabolism marker levels and serum ucOC levels, and increases lumbar spine and femoral neck bone density in postmenopausal RA patients with abnormal ucOC levels and osteoporosis or osteopenia.

Topics: Aged; Alendronate; Arthritis, Rheumatoid; Bone Density; Bone Density Conservation Agents; Drug Therapy, Combination; Female; Humans; Middle Aged; Osteoporosis, Postmenopausal; Postmenopause; Prospective Studies; Treatment Outcome; Vitamin K 2

Schnyder corneal dystrophy (SCD) is an autosomal dominant disease characterized by germline variants in UBIAD1 introducing missense alterations leading to deposition of cholesterol in the cornea, progressive opacification, and loss of visual acuity. UBIAD1 was recently shown to synthesize menaquinone-4 (MK-4, vitamin K(2) ), but causal mechanisms of SCD are unknown. We report a novel c.864G>A UBIAD1 mutation altering glycine 177 to glutamic acid (p.G177E) in six SCD families, including four families from Finland who share a likely founder mutation. We observed reduced MK-4 synthesis by UBIAD1 altered by SCD mutations p.N102S, p.G177R/E, and p.D112N, and molecular models showed p.G177-mutant UBIAD1 disrupted transmembrane helices and active site residues. We show UBIAD1 interacts with HMGCR and SOAT1, enzymes catalyzing cholesterol synthesis and storage, respectively, using yeast two-hybrid screening and immunoprecipitation. Docking simulations indicate cholesterol binds to UBIAD1 in the substrate-binding cleft and substrate-binding overlaps with GGPP binding, an MK-4 substrate, suggesting potential competition between these metabolites. Impaired MK-4 synthesis is a biochemical defect identified in SCD suggesting UBIAD1 links vitamin K and cholesterol metabolism through physical contact between enzymes and metabolites. Our data suggest a role for endogenous MK-4 in maintaining cornea health and visual acuity.

Topics: Aged; Aged, 80 and over; Amino Acid Sequence; Cholesterol; Cornea; Corneal Dystrophies, Hereditary; Dimethylallyltranstransferase; Female; Finland; Genetic Variation; Glutamic Acid; Glycine; Humans; Hydroxymethylglutaryl CoA Reductases; Immunoprecipitation; Japan; Lipid Metabolism; Male; Middle Aged; Models, Molecular; Molecular Sequence Data; Mutation, Missense; Pedigree; Protein Conformation; Sequence Analysis, DNA; Sterol O-Acyltransferase; Turkey; Vitamin K 2

The leading cause of death in patients with chronic kidney disease (CKD) is cardiovascular disease, with vascular calcification being a key modifier of disease progression. A local regulator of vascular calcification is vitamin K. This γ-glutamyl carboxylase substrate is an essential cofactor in the activation of several extracellular matrix proteins that inhibit calcification. Warfarin, a common therapy in dialysis patients, inhibits the recycling of vitamin K and thereby decreases the inhibitory activity of these proteins. In this study, we sought to determine whether modifying vitamin K status, either by increasing dietary vitamin K intake or by antagonism with therapeutic doses of warfarin, could alter the development of vascular calcification in male Sprague-Dawley rats with adenine-induced CKD. Treatment of CKD rats with warfarin markedly increased pulse pressure and pulse wave velocity, as well as significantly increased calcium concentrations in the thoracic aorta (3-fold), abdominal aorta (8-fold), renal artery (4-fold), and carotid artery (20-fold). In contrast, treatment with high dietary vitamin K1 increased vitamin K tissue concentrations (10-300-fold) and blunted the development of vascular calcification. Thus, vitamin K has an important role in modifying mechanisms linked to the susceptibility of arteries to calcify in an experimental model of CKD.

Topics: Adenine; Animals; Anticoagulants; Arteries; Biomarkers; Blood Pressure; Dietary Supplements; Disease Models, Animal; Disease Progression; Male; Osteocalcin; Pulse Wave Analysis; Rats; Rats, Sprague-Dawley; Renal Insufficiency, Chronic; Time Factors; Vascular Calcification; Vitamin K 1; Vitamin K 2; Warfarin

We synthesized new vitamin K analogues with demethylation or reduction of the double bonds of the side chain of menaquinone-4 (MK-4) and evaluated their SXR-mediated transcriptional activity as well as the extent of their conversion to MK-4. The results indicated that the analogue with the methyl group deleted at the 7' site of the side chain part affected conversion activity to MK-4. In contrast, a decrease in the number of the double bonds in the side chain moiety appeared to decrease the SXR-mediated transcriptional activity.

Topics: Cell Line, Tumor; Deuterium; Genes, Reporter; Humans; Luciferases; Pregnane X Receptor; Protein Binding; Protein Multimerization; Receptors, Steroid; Retinoid X Receptors; Structure-Activity Relationship; Transcription, Genetic; Vitamin K 2

Phylloquinone (PK) is converted into menaquinone-4 (MK-4) via side chain removal-addition. Stable isotope use is an effective approach to identify the tissue location of this conversion, which is currently unknown. Following a 14-d PK-deficient diet, male Fischer 344 rats (8 mo; n = 15) were fed 1.6 mg deuterium-labeled PK (L-PK) per kg diet for 0 (control), 1 d (PK-1d), and 7 d (PK-7d). Both L-PK and deuterium-labeled MK-4 (L-MK-4) were detected in tissues in PK-1d and PK-7d, although the results varied. Whereas some tissues had an overall increase in MK-4 in response to L-PK, total brain, testes, and fat MK-4 concentrations did not. In contrast, L-MK-4 concentrations increased in all 3 tissues. The deuterium label was found only on the L-MK-4 naphthoquinone ring, confirming the need for side chain removal for the formation of MK-4. Labeled menadione (MD) was detected in urine and serum in PK-1d and PK-7d, confirming its role as an intermediate. A Caco-2 cell monolayer model was used to study the role of the enterocytes in the conversion process. Neither MK-4 nor MD was detected in Caco-2 cells treated with PK. However, when Caco-2 cells were treated with MD, MK-4 was formed. Similarly, MK-4 was formed in response to MD-treated 293T kidney cells, but not HuH7 liver cells. These data demonstrate that MK-4 is the predominant form of vitamin K in multiple tissues, but there appears to be a tissue-specific regulation for the conversion of PK to MK-4.

Topics: Animal Feed; Animals; Brassica; Caco-2 Cells; Creatinine; Deuterium; Enterocytes; HEK293 Cells; Humans; Liver; Male; Proteinuria; Rats; Rats, Inbred F344; Tissue Distribution; Vitamin K 1; Vitamin K 2; Vitamins

This study analyzed associations between plasma vitamin D(3) (25OHD(3)) and bone mineral density (BMD) and whether the effects of conjugated equine estrogens (CEE) on BMD are modulated by 25OHD(3).. Fifty cynomolgus monkeys were fed a diet containing 25OHD(3) (providing a woman's equivalent of 1000 IU/day of 25OHD3). The monkeys underwent bilateral oophorectomy and were randomized to either CEE (equivalent of 0.45 mg/day) (n=25) or placebo (n=25) and continued receiving the same diet. 25OHD(3) and BMD were measured at randomization and after 6 months. BMD also was measured after 20 months (equivalent to 6 human years). Associations between 25OHD(3) and BMD were subsequently analyzed.. Baseline 25OHD(3) plasma concentrations varied from 26 to 95 ng/mL (mean±standard deviation [SD] 54 ± 15 ng/mL). Higher plasma concentrations of 25OHD(3) were associated with a significantly increased BMD. Monkeys on both CEE and placebo had increased BMD over 20 months; however, the increase was not significantly different (0.034 g/cm(2) vs. 0.020 g/cm(2), respectively; p=0.064). The 20-month BMD increased significantly with CEE treatment in those with higher vs. lower 25OHD(3) concentrations (p=0.027). The percent change in BMD over 20 months also increased significantly with CEE treatment in those with higher vs. lower 25OHD(3) concentrations (p=0.018). A higher 25OHD(3) concentration had no significant effect on BMD in those receiving placebo.. Monkeys fed a diet containing 1000 IU/day equivalent of 25OHD(3) have a wide range of plasma 25OHD(3) concentrations. Those receiving CEE with higher 25OHD(3) concentrations had higher BMDs, suggesting 25OHD(3) and CEE have synergistic effects on BMD.

Topics: Animals; Antifibrinolytic Agents; Body Mass Index; Bone Density; Cholecalciferol; Cohort Studies; Dose-Response Relationship, Drug; Estradiol; Estrogen Replacement Therapy; Estrogens; Estrogens, Conjugated (USP); Hemostatics; Longitudinal Studies; Macaca fascicularis; Models, Animal; Postmenopause; Vitamin K 2; Vitamins

The mechanism for increased bleeding and decreased vitamin K status accompanying vitamin E supplementation is unknown. We hypothesized that elevated hepatic α-tocopherol (α-T) concentrations may stimulate vitamin K metabolism and excretion. Furthermore, α-T may interfere with the side chain removal of phylloquinone (PK) to form menadione (MN) as an intermediate for synthesis of tissue-specific menaquinone-4 (MK-4).. In order to investigate these hypotheses, rats were fed phylloquinone (PK) or menadione (MN) containing diets (2 μmol/kg) for 2.5 weeks. From day 10, rats were given daily subcutaneous injections of either α-T (100 mg/kg) or vehicle and were sacrificed 24 h after the seventh injection. Irrespective of diet, α-T injections decreased MK-4 concentrations in brain, lung, kidney, and heart; and PK in lung. These decreases were not accompanied by increased excretion of urinary 5C- or 7C-aglycone vitamin K metabolites, however, the urinary α-T metabolite (α-CEHC) increased ≥ 100-fold. Moreover, α-T increases were accompanied by downregulation of hepatic cytochrome P450 expression and modified expression of tissue ATP-binding cassette transporters.. Thus, in rats, high tissue α-T depleted tissue MK-4 without significantly increasing urinary vitamin K metabolite excretion. Changes in tissue MK-4 and PK levels may be a result of altered regulation of transporters.

Topics: Administration, Oral; alpha-Tocopherol; Animals; ATP-Binding Cassette Transporters; Biotransformation; Chromans; Cytochrome P-450 Enzyme System; Dietary Supplements; Gene Expression Regulation; Injections, Subcutaneous; Liver; Male; Propionates; Random Allocation; Rats; Rats, Sprague-Dawley; RNA, Messenger; Tissue Distribution; Vitamin E; Vitamin K 1; Vitamin K 2; Vitamin K 3

A novel gram-staining-negative, rod-shaped, non-motile bacterium, strain AMV16(T), was isolated from a soil sample collected from a mud volcano located in the Andaman Islands, India. The cell suspension was pale orange. Cells of strain AMV16(T) were positive for catalase, oxidase, lipase, ornithine decarboxylase and lysine decarboxylase and negative for gelatinase and urease. The fatty acids present were anteiso-C(11 : 0) (5.4 %), anteiso-C(12 : 0) (4.1 %), C(12 : 0) (7.0 %), iso-C(15 : 0) (14.4 %), anteiso-C(15 : 0) (3.4 %), anteiso-C(16 : 0) (3.0 %), C(16 : 0) (2.6 %), anteiso-C(17 : 0) (3.7 %), iso-C(19 : 0) (9.7 %), C(13 : 1) (13.8 %), iso-C(15 : 1) G (15.9 %), iso-C(16 : 1) G (11.1 %) and summed feature 5 (anteiso-C(18 : 0) and/or C(18 : 2)ω6,9c; 5.9 %). Strain AMV16(T) contained MK-4 is [corrected] the major respiratory quinone and diphosphatidylglycerol and phosphatidylethanolamine made up the phospholipids. The G+C content of DNA of strain AMV16(T) was 50.9 mol%. blast sequence similarity searches based on the 16S rRNA gene sequence indicated that species of the genus Marivirga were the nearest phylogenetic neighbours, with pairwise sequence similarity ranging from 89.9 to 90.0 %. Phylogenetic analyses indicated that strain AMV16(T) clustered with the type strains of Marivirga tractuosa and Marivirga sericea at a phylogenetic distance of 14.6 % (85.4 % similarity), distinct from clades representing other genera of the family 'Flammeovirgaceae'. Based on the above-mentioned phenotypic and phylogenetic characteristics, strain AMV16(T) is proposed as a representative of a new genus and novel species, Cesiribacter andamanensis gen. nov., sp. nov. The type strain of Cesiribacter andamanensis is AMV16(T) ( = DSM 22818(T)  = CCUG 58431(T)).

Topics: Bacterial Typing Techniques; Bacteroidetes; Base Composition; DNA, Bacterial; Fatty Acids; India; Molecular Sequence Data; Phospholipids; Phylogeny; Pigmentation; RNA, Ribosomal, 16S; Sequence Analysis, DNA; Soil Microbiology; Vitamin K 2

The effect of several vitamin K homologs on plasma vitamin K concentration was determined to assess their potential as a vitamin K supplement for adult horses. Sixteen Thoroughbred horses consisting of 8 mares and 8 geldings, aged 8.4 ± 3.6 yr and weighing 520.8 ± 36.1 kg, were allocated to 4 groups (n = 4). Each group was given phylloquinone, menaquinone-4, or menadione at 58 µmol/d, or no vitamin K supplement for 7 d. Plasma samples were collected before feeding, and 2, 4, and 8 h after feeding on d 7, and plasma concentrations of phylloquinone and menaquinone-4 were determined. Plasma phylloquinone concentration was greater in the phylloquinone group than in the other groups (P < 0.001). The phylloquinone concentration quadratically increased (P < 0.001) after feeding in the phylloquinone group but no changes in the plasma phylloquinone concentration were observed after feeding in the other groups. Plasma menaquinone-4 concentration was greater (P < 0.001) in the menadione group than the other groups, including the menaquinone-4 group. Menaquinone-4 concentration did not change (P = 0.192) after feeding in each group. Menaquinone-4 has been considered the most potent vitamin K homolog for bone metabolism; therefore, the present experiment indicates that menadione is a good source of vitamin K for bone health in horses because it is the only vitamin K homolog that increased the plasma concentrations of menaquinone-4.

Topics: Animals; Bone and Bones; Diet; Dietary Supplements; Female; Horses; Male; Vitamin K; Vitamin K 1; Vitamin K 2; Vitamin K 3

Vitamin K is a cofactor for γ-glutamyl carboxylase, which is an essential enzyme for the γ-carboxylation of vitamin K-dependent proteins such as osteocalcin and matrix Gla protein. Although it has been suggested that vitamin K plays an important role in the improvement of bone metabolism, the relationship between dietary vitamin K intake and bone metabolism has not been thoroughly investigated. Moreover, vitamin K is thought to have other actions beyond influencing the γ-carboxylation status. In the present study, we examined the effects of the long-term addition of phylloquinone (PK) or menaquinone-4 (MK-4) to a control diet on bone mineral density, bone strength, body composition, and serum parameters in rats. A total of 23 female Sprague-Dawley strain rats (6 weeks old) were divided into three groups: basic control diet group, PK diet (PK: 600mg/kg diet) group, and MK diet (MK-4: 600mg/kg diet) group. Three months after starting the experimental diet, the addition of PK to the basic control diet significantly increased the bone mineral density (BMD) of the femur (p<0.05). In the MK group, there was no significant difference in the BMD of the femur. However, two types of bone strength parameter: the minimum cross-sectional moment of inertia and the polar moment of inertia, were significantly higher in the MK group than in the control (p<0.05, respectively). Furthermore, the femoral bone parameters (the width, dry weight and ash weight, and cortical, cancellous, trabecular, and total bone mineral contents) in the MK group were increased significantly compared with the control. Interestingly, the addition of PK or MK-4 significantly decreased the total fat accumulation (p<0.01 and p<0.05, respectively), and serum triglycerides were reduced by 48% in the PK group and 29% in the MK group compared with the control. There were no significant differences in the levels of serum calcium, phosphorus, alkaline phosphatase, growth hormone, insulin-like growth hormone-1, insulin-like growth hormone binding protein-3, and cross-linked N-teleopeptide of type I collagen among the three groups. This is the first study to demonstrate the effect of the long-term addition of PK or MK-4 to the control diet on body composition and serum parameters in an in vivo system using rats. Further studies on the mechanism of vitamin K supplementation in the regulation of bone metabolism would provide valuable data on the prevention of lifestyle-related disorders, including osteoporosi

Topics: Adipose Tissue; Animals; Blood Glucose; Body Composition; Bone Density; Cholesterol; Cytokines; Diet; Dietary Supplements; Female; Femur; Hormones; Organ Size; Rats; Rats, Sprague-Dawley; Serum; Time Factors; Tomography, X-Ray Computed; Triglycerides; Vitamin K 1; Vitamin K 2

A biodegradable drug delivery system with perforated macro pores was established using an apatite/collagen composite cement containing menatetrenone (VK2). The drug-release capabilities of the device were investigated in vitro under osteoblast and osteoclast-like conditions (SOB and SOC). A bulk powder of apatite cement containing 2.5% VK2 and 20% bovine collagen was obtained by grinding, kneaded with phosphoric acid, and poured into molds, producing fixed blocks with 0-60 perforated macro pores. The characteristics of these samples were measured by X-ray powder diffraction analysis and Fourier-transformed and infrared spectroscopy, and found to be very similar to those of natural bone. Drug release tests were performed under SOB in simulated body fluid (pH 7.8), and then under SOC in acetate buffer (pH 4.5) at 37.0±0.1 °C, and the process repeated twice. The device released almost no drug in SOB, but a significant amount in SOC. The drug release in SOC was not proportional to the number of macro pores in the first test, but was in the second. The device showed dissolution medium-responsive drug release.

Topics: Animals; Apatites; Biocompatible Materials; Bone and Bones; Bone Cements; Collagen; Drug Delivery Systems; Nanocomposites; Osteoblasts; Osteoclasts; Porosity; Rats; Spectroscopy, Fourier Transform Infrared; Vitamin K 2; X-Ray Diffraction

Vitamin K is essential for the posttranslational modification of various Gla proteins. Although it is widespread in several organs, including the testis, the function of vitamin K in these organs is not well characterized. In this study, we investigated the function of vitamin K in the testis and analyzed its role in steroidogenesis.. Eight-week-old male Wistar rats were fed a diet supplemented with menaquinone-4 (MK-4, 75 mg/kg diet), one of the predominant K₂ vitamins present in the testis, for 5 weeks. In vivo testosterone levels of the rats' plasma and testes were measured by enzyme-linked immunosorbent assay, and in vitro testosterone levels of testis-derived tumor cells (I-10 cells) maintained in Ham's F-10 medium with 10% fetal bovine serum were measured following treatment with MK-4 (0 to 100 μM) at several time points. Testosterone and cellular protein levels were analyzed with respect to their effects on steroidogenesis.. Testosterone levels in the plasma and testes of MK-4-fed rats were significantly increased compared to those of control rats, with no obvious differences in plasma luteinizing hormone levels. Secreted testosterone levels from I-10 cells were elevated by MK-4, but not by vitamin K₁, in a dose-dependent manner independent of cAMP treatment. Western blot analysis revealed that expression of CYP11A, the rate-limiting enzyme in steroidogenesis, and phosphorylation levels of protein kinase A (PKA) and the cAMP response element-binding protein were all stimulated by the presence of MK-4. Enhancement of testosterone production was inhibited by H89, a specific inhibitor of PKA, but not by warfarin, an inhibitor of γ-glutamylcarboxylation.. MK-4 stimulates testosterone production in rats and testis-derived tumor cells via activation of PKA. MK-4 may be involved in steroidogenesis in the testis, and its supplementation could reverse the downregulation of testosterone production in elders.

Topics: Animals; Carbon-Carbon Ligases; Cell Line, Tumor; Cholesterol Side-Chain Cleavage Enzyme; Cyclic AMP Response Element-Binding Protein; Cyclic AMP-Dependent Protein Kinases; Leydig Cells; Liver; Male; Mice; Phosphorylation; Protein Kinase Inhibitors; Protein Processing, Post-Translational; Rats; Rats, Wistar; Specific Pathogen-Free Organisms; Testis; Testosterone; Tissue Distribution; Up-Regulation; Vitamin K 1; Vitamin K 2

Dysregulation of myelin sulfatides is a risk factor for cognitive decline with age. Vitamin K is present in high concentrations in the brain and has been implicated in the regulation of sulfatide metabolism. Our objective was to investigate the age-related interrelation between dietary vitamin K and sulfatides in myelin fractions isolated from the brain regions of Fischer 344 male rats fed one of two dietary forms of vitamin K: phylloquinone or its hydrogenated form, 2',3'-dihydrophylloquinone (dK), for 28 days. Both dietary forms of vitamin K were converted to menaquinone-4 (MK-4) in the brain. The efficiency of dietary dK conversion to MK-4 compared to dietary phylloquinone was lower in the striatum and cortex, and was similar to that in the hippocampus. There were significant positive correlations between sulfatides and MK-4 in the hippocampus (phylloquinone-supplemented diet, 12 and 24 months; dK-supplemented diet, 12 months) and cortex (phylloquinone-supplemented diet, 12 and 24 months). No significant correlations were observed in the striatum. Furthermore, sulfatides in the hippocampus were significantly positively correlated with MK-4 in serum. This is the first attempt to establish and characterize a novel animal model that exploits the inability of dietary dK to convert to brain MK-4 to study the dietary effects of vitamin K on brain sulfatide in brain regions controlling motor and cognitive functions. Our findings suggest that this animal model may be useful for investigation of the effect of the dietary vitamin K on sulfatide metabolism, myelin structure and behavior functions.

Topics: Age Factors; Animals; Brain; Diet; Dietary Supplements; Male; Models, Animal; Myelin Sheath; Rats; Rats, Inbred F344; Sulfoglycosphingolipids; Vitamin K; Vitamin K 1; Vitamin K 2

To reveal an essential biological role of menaquinone-4, we have clarified that dietary PK was converted to menaquinone-4 (MK-4) in animal tissues using deuterated vitamin K analogues. However, the kinds of analogue converted into MK-4 have not been elucidated. In this study, we examined structure-activity relationships in the conversion of several vitamin K analogues, with a substituted side chain, into MK-4 using cultured human cell lines. The results differed with the side chain of the analogues, that is, (1) the length of the isoprene unit and (2) the number of double bonds in the side chain. These findings would be useful for clarifying the mechanism of conversion of other vitamin K homologs into MK-4 as well as related enzymes.

Topics: Cell Line; Cell Line, Tumor; Chromatography, High Pressure Liquid; Hemostatics; Humans; Mass Spectrometry; Molecular Structure; Structure-Activity Relationship; Substrate Specificity; Vitamin K; Vitamin K 2

The purpose of the present study was to examine the effects of vitamin K2 on cortical and cancellous bone mass, cortical osteocyte and lacunar system, and porosity in sciatic neurectomized rats. Thirty-four female Sprague-Dawley retired breeder rats were randomized into three groups: age-matched control, sciatic neurectomy (NX), and NX + vitamin K2 administration (menatetrenone, 30 mg/kg/day p.o., three times a week). At the end of the 8-week experiment, bone histomorphometric analysis was performed on cortical and cancellous bone of the tibial diaphysis and proximal metaphysis, respectively, and osteocyte lacunar system and porosity were evaluated on cortical bone of the tibial diaphysis. NX decreased cortical and cancellous bone mass compared with age-matched controls as a result of increased endocortical and trabecular bone erosion and decreased trabecular mineral apposition rate (MAR). Vitamin K2 ameliorated the NX-induced increase in bone erosion, prevented the NX-induced decrease in MAR, and increased bone formation rate (BFR/bone surface) in cancellous bone, resulting in an attenuation of NX-induced cancellous bone loss. However, vitamin K2 did not significantly influence cortical bone mass. NX also decreased osteocyte density and lacunar occupancy and increased porosity in cortical bone compared with age-matched controls. Vitamin K2 ameliorated the NX-induced decrease in lacunar occupancy by viable osteocytes and the NX-induced increase in porosity. The present study showed the efficacy of vitamin K2 for cancellous bone mass and cortical lacunar occupancy by viable osteocytes and porosity in sciatic NX rats.

Topics: Animals; Bone and Bones; Bone Density; Cell Count; Denervation; Female; Femur; Osteocytes; Osteoporosis; Rats; Rats, Sprague-Dawley; Sciatic Nerve; Tibia; Vitamin K 2

Vitamin K occurs in the natural world in several forms, including a plant form, phylloquinone (PK), and a bacterial form, menaquinones (MKs). In many species, including humans, PK is a minor constituent of hepatic vitamin K content, with most hepatic vitamin K content comprising long-chain MKs. Menaquinone-4 (MK-4) is ubiquitously present in extrahepatic tissues, with particularly high concentrations in the brain, kidney and pancreas of humans and rats. It has consistently been shown that PK is endogenously converted to MK-4 (refs 4-8). This occurs either directly within certain tissues or by interconversion to menadione (K(3)), followed by prenylation to MK-4 (refs 9-12). No previous study has sought to identify the human enzyme responsible for MK-4 biosynthesis. Previously we provided evidence for the conversion of PK and K(3) into MK-4 in mouse cerebra. However, the molecular mechanisms for these conversion reactions are unclear. Here we identify a human MK-4 biosynthetic enzyme. We screened the human genome database for prenylation enzymes and found UbiA prenyltransferase containing 1 (UBIAD1), a human homologue of Escherichia coli prenyltransferase menA. We found that short interfering RNA against the UBIAD1 gene inhibited the conversion of deuterium-labelled vitamin K derivatives into deuterium-labelled-MK-4 (MK-4-d(7)) in human cells. We confirmed that the UBIAD1 gene encodes an MK-4 biosynthetic enzyme through its expression and conversion of deuterium-labelled vitamin K derivatives into MK-4-d(7) in insect cells infected with UBIAD1 baculovirus. Converted MK-4-d(7) was chemically identified by (2)H-NMR analysis. MK-4 biosynthesis by UBIAD1 was not affected by the vitamin K antagonist warfarin. UBIAD1 was localized in endoplasmic reticulum and ubiquitously expressed in several tissues of mice. Our results show that UBIAD1 is a human MK-4 biosynthetic enzyme; this identification will permit more effective decisions to be made about vitamin K intake and bone health.

Topics: Animals; Baculoviridae; Bone and Bones; Cell Line; Dimethylallyltranstransferase; Humans; Magnetic Resonance Imaging; Mice; Osteoblasts; Proteins; RNA, Small Interfering; Spodoptera; Vitamin K; Vitamin K 1; Vitamin K 2; Warfarin

Herein, we show that intraerythrocytic stages of Plasmodium falciparum have an active pathway for biosynthesis of menaquinone. Kinetic assays confirmed that plasmodial menaquinone acts at least in the electron transport. Similarly to Escherichia coli, we observed increased levels of menaquinone in parasites kept under anaerobic conditions. Additionally, the mycobacterial inhibitor of menaquinone synthesis Ro 48-8071 also suppressed menaquinone biosynthesis and growth of parasites, although off-targets may play a role in this growth-inhibitory effect. Due to its absence in humans, the menaquinone biosynthesis can be considered an important drug target for malaria.

Topics: Anaerobiosis; Animals; Benzophenones; Electrons; Erythrocytes; Life Cycle Stages; Malaria; Molecular Targeted Therapy; Plasmodium falciparum; Vitamin K 2

Vitamin K is an essential nutrient and a cofactor for the carboxylation of specific glutamyl residues of proteins to gamma-glutamyl residues, which activates osteocalcin related to bone formation. Among vitamin K homologues, menaquinone-4 (MK-4) is the most active biologically, up-regulating the gene expression of bone markers, and thus has been clinically used in the treatment of osteoporosis in Japan. Recently, we confirmed that MK-4 was converted from dietary phylloquinone (PK), and then accumulated in various tissues at high concentrations. This system should play an important role in biological functions including bone formation, however, the pathway by which MK-4 is converted remains unclear. In this study, we studied the mechanism of MK-4's conversion with chemical techniques using deuterated analogues.

Topics: Humans; Hydroquinones; Japan; Molecular Structure; Osteoblasts; Osteogenesis; Vitamin K 1; Vitamin K 2

Oxidative mechanisms of injury are important in many neurological disorders. Developing oligodendrocytes (pre-OLs) are particularly sensitive to oxidative stress-mediated injury. We previously demonstrated a novel function of phylloquinone (vitamin K(1)) and menaquinone 4 (MK-4; a major form of vitamin K2) in protecting pre-OLs and immature neurons against glutathione depletion-induced oxidative damage (Li et al. [ 2003] J. Neurosci. 23:5816-5826). Here we report that vitamin K at nanomolar concentrations prevents arachidonic acid-induced oxidative injury to pre-OLs through blocking the activation of 12-lipoxygenase (12-LOX). Arachidonic acid metabolism is a potential source for reactive oxygen species (ROS) generation during ischemia and reperfusion. Exposure of pre-OLs to arachidonic acid resulted in oxidative cell death in a concentration-dependent manner. Administration of vitamin K (K(1) and MK-4) completely prevented the toxicity. Consistent with our previous findings, inhibitors of 12-LOX abolished ROS production and cell death, indicating that activation of 12-LOX is a key event in arachidonic acid-induced pre-OL death. Vitamin K(1) and MK-4 significantly blocked 12-LOX activation and prevented ROS accumulation in pre-OLs challenged with arachidonic acid. However, vitamin K itself did not directly inhibit 12-LOX enzymatic activity when assayed with purified 12-LOX in vitro. These results suggest that vitamin K, or likely its metabolites, acts upstream of activation of 12-LOX in pre-OLs. In summary, our data indicate that vitamin K prevents oxidative cell death by blocking activation of 12-LOX and ROS generation.

Topics: Animals; Arachidonate 12-Lipoxygenase; Arachidonic Acid; Cell Death; Cell Survival; Cells, Cultured; Cytoprotection; Enzyme Activation; Oligodendroglia; Oxidative Stress; Rats; Rats, Sprague-Dawley; Reactive Oxygen Species; Stem Cells; Vitamin K; Vitamin K 2

Prior 8-week treatment with menatetrenone, MK-4, followed by 8-week risedronate prevented the shortcomings of individual drugs and significantly increased the strength of ovariectomized ICR mouse femur compared to the ovariectomized (OVX) controls. Neither MK-4 following risedronate nor the concomitant administration may be recommended because they brought the least beneficial effect.. The objective of this study was to determine the best combinatory administration of risedronate at 0.25 mg/kg/day (R) with vitamin K(2) at approximately 100 microg MK-4/kg/day (K) to improve strength of osteoporotic mouse bone.. Thirteen-week-old ICR mice, ovariectomized at 9-week, were treated for 8 weeks with R, K, or R plus K (R/K), and then, either the treatment was withdrawn (WO) or switched to K or R in the case of R and K. After another 8 weeks, the mice were killed, and mechanical tests and analyses of femur properties by peripheral quantitative computed tomography, microfocus X-ray tube computed tomography, and confocal laser Raman microspectroscopy were carried out.. The K to R femur turned out superior in parameters tested such as material properties, bone mineral density, BMC, trabecular structure, and geometry of the cortex. The increased cross-sectional moment of inertia, which occurred after K withdrawal, was prevented by risedronate in K to R. In addition to K to R, some properties of R to WO diaphysis and K to WO epiphysis were significantly better than OVX controls.. Prior treatment with MK-4 followed by risedronate significantly increased femur strength in comparison to the OVX controls.

Topics: Animals; Body Weight; Bone Density Conservation Agents; Drug Administration Schedule; Drug Evaluation, Preclinical; Drug Therapy, Combination; Etidronic Acid; Female; Femur; Mice; Mice, Inbred ICR; Osteoporosis; Ovariectomy; Risedronic Acid; Vitamin K 2

In this study, we focused on collagen metabolism as a factor involved in menatetrenone (MK-4)-related improvement in bone quality. Using rats with a congenital ascorbic acid (AA) deficiency, osteogenic disorder Shionogi (ODS) rats, we established a model in which abnormal collagen metabolism reduced bone mechanical properties, and investigated the effects of MK-4. We divided 13-week-old ODS rats into four groups: Pre, AA sufficiency (AA(+)), AA deficiency-control (AA(-)control), and AA deficiency+ MK-4-treated (AA(-)MK-4). MK-4 was given as a dietary supplement (30 mg/kg). At the beginning (pre) and after two, three, and four weeks, seven rats in each group were killed to measure plasma bone metabolism and femoral bone mass data and bone mechanical properties. In the rats killed after four weeks, histomorphometric data of the tibiae, the total amino acid level in bone collagen, and rates of proline and lysine hydroxylation were determined. In the AA(+)group, both the cortical bone mass data and bone mechanical properties were serially increased. However, in the AA(-)control group, the cortical bone mass data were similar for four weeks and the bone mechanical properties decreased after three to four weeks. After four weeks, the total level of amino acids in bone collagen and rates of proline and lysine hydroxylation were significantly lower in the AA(-)control group than in the AA(+)group. MK-4 increased bone mechanical properties after four weeks without influencing cortical bone mass. Simultaneously, it inhibited decreases in the total level of amino acids in collagen (P = 0.017). The rates of proline and lysine hydroxylation were higher in the AA(-)MK-4 group than in the AA(-)control group, but not significantly. These results suggest the level of collagen and abnormalities of hydroxylation are involved in the AA deficiency-related reduction in bone mechanical properties, and that MK-4 improves bone mechanical properties by restoring collagen metabolism.

Topics: Amino Acids; Animals; Ascorbic Acid Deficiency; Biomechanical Phenomena; Body Weight; Bone and Bones; Chromatography, High Pressure Liquid; Collagen; Collagen Type I; Femur; Organ Size; Osteocalcin; Osteogenesis; Peptides; Rats; Regression Analysis; Vitamin K 2

Vitamin K2, known as a cofactor for gamma-carboxylase, also serves as a ligand of a nuclear receptor, Steroid and Xenobiotic Receptor (SXR). Several clinical trials revealed that vitamin K2 reduced de novo formation and recurrence of hepatocellular carcinoma (HCC). To examine the role of SXR in HCC as a receptor activated by vitamin K2, the cells stably overexpressing SXR were established using a HCC cell line, HuH7. Overexpression of SXR resulted in reduced proliferation and motility of the cells. Further suppression of proliferation and motility was observed when SXR overexpressing clones were treated with vitamin K2. These results suggest that the activation of SXR could contribute to tumor suppressive effects of vitamin K2 on HCC cells.

Topics: Carcinoma, Hepatocellular; Cell Line, Tumor; Cell Movement; Cell Proliferation; Hep G2 Cells; Humans; Liver Neoplasms; Pregnane X Receptor; Receptors, Steroid; Rifampin; Vitamin K 2

Vitamin K2 (VK2) has been shown to have a potent anti-tumor effect against several cancer types including hepatocellular carcinoma (HCC), but the mechanisms remain to be elucidated. Matrix metalloproteinase (MMP) plays an important role in the invasion and metastasis of cancer cells, but it is not known whether VK2 regulates the expression of MMPs. Human HCC cell lines were treated with VK2 combined with 12-O-tetradecanoyl phorbol-13 acetate (TPA) and the expression of MMPs was examined by reporter gene assay, RT-PCR and Western blotting. VK2 inhibited the basal and TPA-induced expression of MMP-1, -3 and -7 at the transcriptional, mRNA and protein levels in a dose-dependent manner. VK2 also inhibited the TPA-induced activation of NF-kappaB and AP-1 activity. The inhibitors against NF-kappaB and mitogen-activated protein kinases (MAP kinase) including ERK and JNK pathways suppressed TPA-induced luciferase activity of MMP-1, -3 and -7 promoters. These data suggest that VK2 inhibits MMP expression by suppressing NF-kappaB and MAP kinase activity and might be potentially useful in the treatment of HCC.

Topics: Carcinoma, Hepatocellular; Humans; Liver Neoplasms; MAP Kinase Signaling System; Matrix Metalloproteinase Inhibitors; Matrix Metalloproteinases; NF-kappa B; Protease Inhibitors; Protein Kinase C; Tetradecanoylphorbol Acetate; Transcription Factor AP-1; Vitamin K 2

The surprising lack of the cytochrome bc1 complex in the filamentous anoxygenic phototrophic bacterium Chloroflexus aurantiacus suggests that a functional replacement exists to link the cyclic electron transfer chain. Earlier work identified the alternative complex III (ACIII) as a substitute of cytochrome bc1 complex. Herein, the enzymatic activity of ACIII is studied. The results strongly support the view that the ACIII functions as menaquinol:auracyanin oxidoreductase in the C. aurantiacus electron transfer chain. Among all the substrates tested, auracyanin is the most efficient electron acceptor of ACIII, suggesting that ACIII directly transfers the electron to auracyanin instead of cytochrome c-554. The lack of sensitivity to common inhibitors of the cytochrome bc1 complex indicates a different catalytic mechanism for the ACIII complex.

Topics: Bacterial Proteins; Chloroflexus; Electron Transport; Electron Transport Complex III; Metalloproteins; Quinone Reductases; Vitamin K 2

The growth of human breast cancer-derived MCF-7 cells was affected by oil-in-water lipid emulsions prepared with fish oil (FO) rich in n-3 fatty acids (FAs) and egg-yolk phosphatides (EYP) (FO-emulsions), but not by lipid emulsions prepared with soybean oil (SO) and EYP (SO-emulsions). On the other hand, the growth of human hepatocarcinoma HepG2 cells was affected by neither SO-emulsions nor FO-emulsions. The growth inhibition of MCF-7 cells in the presence of FO-emulsions was not affected by trolox, but was inhibited by alpha-lipoic acid, and was even potentiated by ebselen, which works as an antioxidant as well as a lipoxygenase inhibitor. Since prostaglandin E(3), generated from n-3 FAs by cyclooxygenases, has a suppressive effect on tumour cell growth, and increases when lipoxygenases are inhibited, these findings suggest that lipid emulsions incorporating triglycerides of n-3 FAs might be effective in suppressing the growth of MCF-7 cells, possibly via oxidative stress and through eicosanoid production with anti-proliferating activity against cancer cells.

Topics: Antioxidants; Breast Neoplasms; Cell Line, Tumor; Cell Proliferation; Dose-Response Relationship, Drug; Eicosanoids; Emulsions; Epoxy Compounds; Female; Fish Oils; Humans; Liver Neoplasms; Oxidative Stress; Particle Size; Soybean Oil; Time Factors; Vitamin K 2; Water

It has been reported that the Mg-insufficient bone is fragile upon mechanical loading, despite its high bone mineral density, while vitamin K2 (MK-4: menatetrenone) improved the mechanical strength of Mg-insufficient bone. Therefore, we aimed to elucidate the ultrastructural properties of bone in rats with dietary Mg insufficiency with and without MK-4 supplementation. Morphological examinations including histochemistry, transmission electron microscopy, electron probe microanalysis (EPMA) and X-ray diffraction were conducted on the femora and tibiae of 4-week-old Wistar male rats fed with 1) a normal diet (control group, 0.09% Mg), 2) a Mg-insufficient diet (low Mg group, 0.006% Mg), or 3) a Mg-insufficient diet supplemented with MK-4 (MK-4 group, 0.006% Mg, 0.03% MK-4). MK-4 appeared to inhibit the osteoclastic bone resorption that is stimulated by Mg insufficiency. EPMA analysis, however, revealed an increased concentration of Ca paralleling Mg reduction in the low Mg group. Assessment by X-ray diffraction revealed an abundance of a particular synthetic form of hydroxyapatite in the low Mg group, while control bones featured a variety of mineralized crystals. In addition, Mg-deficient bones featured larger mineral crystals, i.e., crystal overgrowth. This crystalline aberration in Mg-insufficient bones induced collagen fibrils to mineralize easily, even in the absence of mineralized nodules, which therefore led to an early collapse of the fibrils. MK-4 prevented premature collagen mineralization by normalizing the association of collagen fibrils with mineralized nodules. Thus, MK-4 appears to rescue the impaired collagen mineralization caused by Mg insufficiency by promoting a re-association of the process of collagen mineralization with mineralized nodules.

Topics: Animals; Biomechanical Phenomena; Bone Resorption; Calcification, Physiologic; Calcium; Collagen; Disease Models, Animal; Electron Probe Microanalysis; Femur; Immunohistochemistry; Magnesium Deficiency; Male; Osteocalcin; Osteoclasts; Phosphorus; Rats; Rats, Wistar; Tibia; Vitamin K 2; X-Ray Diffraction

We have prepared lipid emulsions of approximately 200 nm in diameter with soybean oil (SO) and a series of Pluronics with various numbers of oxyethylene units and about 60 oxypropylene units (SO/Pluronics), and studied the pharmacokinetics of menatetrenone incorporated into SO/Pluronics in rats. Emulsions of approximately 200 nm in diameter were obtained when SO contents were 2.5% and 20% (w/w) for 2.4% (w/w) PL101 and Pluronics that more than 30% was made up by oxyethylene units, respectively. The half-life of menatetrenone in plasma when oxyethylene units made up less than 30% of the Pluronic (SO/PL101 and SO/PP103) was similar to that for SO/egg yolk phosphatides (SO/EYP), but longer than that when oxyethylene units composed more than 40% of the Pluronic (SO/PP104 and SO/PF108, by 3- and 10-fold, respectively). Pretreatment with dextran sulfate 500000, an inhibitor of emulsion uptake by the reticuloendothelial system (RES), resulted in a higher plasma concentration and a lower liver uptake of menatetrenone as SO/PL101 at 10 min and SO/PP103 at 60 min, indicating that both SO/PL101 and SO/PP103 were taken up by the RES, although SO/PP103 required some time to be recognized by the RES. These findings suggested that larger numbers of oxyethylene units of Pluronics with 60 oxypropylene units were required for the longer plasma circulation of SO/Pluronics due to evasion of the RES.

Topics: Animals; Dextran Sulfate; Emulsions; Excipients; Hemostatics; Injections, Intravenous; Liver; Male; Particle Size; Poloxalene; Poloxamer; Rats; Rats, Wistar; Soybean Oil; Structure-Activity Relationship; Tissue Distribution; Triglycerides; Vitamin K 2

There are two forms of naturally occurring vitamin K, phylloquinone and the menaquinones. Phylloquinone (vitamin K(1)) is a major type (>90%) of dietary vitamin K, but its concentrations in animal tissues are remarkably low compared with those of the menaquinones, especially menaquinone-4 (vitamin K(2)), the major form (>90%) of vitamin K in tissues. Despite this great difference, the origin of tissue menaquinone-4 has yet to be exclusively defined. It is postulated that phylloquinone is converted into menaquinone-4 and accumulates in extrahepatic tissues. To clarify this, phylloquinone with a deuterium-labeled 2-methyl-1,4-naphthoquinone ring was given orally to mice, and cerebra were collected for D NMR and liquid chromatography-tandem mass spectrometry analyses. We identified the labeled menaquinone-4 that was converted from the given phylloquinone, and this conversion occurred following an oral or enteral administration, but not parenteral or intracerebroventricular administration. By the oral route, the phylloquinone with the deuterium-labeled side chain in addition to the labeled 2-methyl-1,4-naphthoquinone was clearly converted into a labeled menaquinone-4 with a non-deuterium-labeled side chain, implying that phylloquinone was converted into menaquinone-4 via integral side-chain removal. The conversion also occurred in cerebral slice cultures and primary cultures. Deuterium-labeled menadione was consistently converted into the labeled menaquinone-4 with all of the administration routes and the culture conditions tested. Our results suggest that cerebral menaquinone-4 originates from phylloquinone intake and that there are two routes of accumulation, one is the release of menadione from phylloquinone in the intestine followed by the prenylation of menadione into menaquinone-4 in tissues, and another is cleavage and prenylation within the cerebrum.

Topics: Animals; Cerebrum; Chromatography, Liquid; Epoxy Compounds; Female; Gene Expression Regulation; Magnetic Resonance Spectroscopy; Male; Mice; Mice, Inbred C57BL; Models, Biological; Tissue Distribution; Vitamin K 1; Vitamin K 2

It has been reported that vitamin K supplementation effectively prevents fractures and sustains bone mineral density in osteoporosis. However, there are only limited reported data concerning the association between vitamin K nutritional status and bone mineral density (BMD) or fractures in Japan. The objectives were to evaluate the association between plasma phylloquinone (K1) or menaquinone (MK-4 and MK-7) concentration and BMD or fracture in Japanese women prospectively. A total of 379 healthy women aged 30-88 years (mean age, 63.0 years) were consecutively enrolled. Plasma K1, MK-4, MK-7, and serum undercarboxylated osteocalcin (ucOC) concentrations, BMD, and incidence of vertebral fractures were evaluated. In stepwise multiple linear regression analyses, L2-4 BMD and a bone turnover marker, log K1, concentrations were independently correlated with vertebral fracture incidence. When subjects were divided into low and high K1 groups by plasma K1 concentration, the incidence of vertebral fracture in the low K1 group (14.4%) was significantly higher than that in the high K1 group (4.2%), and its age-adjusted RR was 3.58 (95% CI, 3.26-3.93). L2-4 BMD was not different between the two groups. These results suggest that subjects with vitamin K1 insufficiency in bone have increased susceptibility for vertebral fracture independently from BMD.

Topics: Adult; Age Factors; Aged; Aged, 80 and over; Asian People; Female; Fractures, Bone; Humans; Incidence; Japan; Middle Aged; Spinal Injuries; Vitamin K 1; Vitamin K 2

The transformation of smooth muscle cells (VSMCs) in the vessel wall to osteoblast like cells is known to precede arterial calcification which may cause bleeding complications. The vitamin K-dependent protein MGP has been identified as an inhibitor of this process by binding BMP-2, a growth factor known to trigger the transformation. In this study, we determined if the vitamin K-dependent Gla region in MGP by itself can inhibit the growth factor activity of BMP-2 and if menaquinone-4 (MK4) regulates gene expression in VSMCs.. A synthetic gamma-carboxyglutamic acid (Gla) containing peptide covering the Gla region in human MGP was used to test its ability to inhibit BMP-2 induced transformation of mouse pro-myoblast C2C12 cells into osteoblasts. MK4 was tested by microarray analysis as a gene regulatory molecule in VSMCs.. The results show that the Gla - but not the Glu-peptide inhibited the transformation which provide evidence that the Gla region in MGP is directly involved in the BMP-2/MGP interaction and emphasizes the importance of the vitamin K-dependent modification of MGP. From the data obtained from the microarray analysis, we focused on two quantitatively altered cDNAs representing proteins known to be associated with vessel wall calcification. DT-diaphorase of the vitamin K-cycle, showed increased gene expression with a 4.8-fold higher specific activity in MK4 treated cells. Osteoprotegrin gene expression was down regulated and osteoprotegrin protein secretion from the MK4 treated cells was lowered to 1.8-fold. These findings suggest that MK4 acts as an anti-calcification component in the vessel wall.

Topics: Animals; Antifibrinolytic Agents; Aorta, Thoracic; Bone Morphogenetic Protein 2; Bone Morphogenetic Proteins; Calcinosis; Calcium-Binding Proteins; Cell Differentiation; Cell Line; Extracellular Matrix Proteins; Humans; Matrix Gla Protein; Mice; Muscle, Smooth, Vascular; Myoblasts; Osteoblasts; Peptide Fragments; Rats; Transforming Growth Factor beta; Vitamin K; Vitamin K 2

Phylloquinone, the primary dietary form of vitamin K, is converted to menaquinone-4 (MK-4) in certain tissues. MK-4 may have tissue-specific roles independent of those traditionally identified with vitamin K. Fischer 344 male rats of different ages (2, 12, and 24 mo, n = 20 per age group) were used to compare the conversion of phylloquinone to MK-4 with an equivalent dose of another dietary form of vitamin K, 2',3'-dihydrophylloquinone. Rats were age- and diet-group pair-fed phylloquinone (198 +/- 9.0 microg/kg diet) or dihydrophylloquinone (172 +/- 13.0 microg/kg diet) for 28 d. MK-4 was the primary form of vitamin K in serum, spleen, kidney, testes, bone marrow, and brain myelin fractions, regardless of age group. MK-4 concentrations were significantly lower in kidney, heart, testes, cortex (myelin), and striatum (myelin) in the dihydrophylloquinone diet group compared with the phylloquinone diet group (P < 0.05). The MK-4 concentrations in 2-mo-old rats were lower in liver, spleen, kidney, heart, and cortex (myelin) but higher in testes compared with 24-mo-old rats (P < 0.05). However, there were no age-specific differences in MK-4 concentrations among the rats fed the 2 diets. These data suggest that dihydrophylloquinone, which differs from phylloquinone in its side phytyl chain, is absorbed but its intake results in less MK-4 in certain tissues. Dihydrophylloquinone may be used in models for the study of tissue-specific vitamin K deficiency.

Topics: Aging; Animals; Bone Marrow; Brain Chemistry; Diet; Dose-Response Relationship, Drug; Kidney; Liver; Male; Myocardium; Rats; Rats, Inbred F344; Spleen; Testis; Vitamin K; Vitamin K 2

We experienced a case of epidural hematoma caused by coagulopathy 3 days after surgery. A 72-year-old man, who had undergone a total gastrectomy, suffered from nausea and vomiting by ileus. He underwent repair of ileus under general anesthesia with thoracic epidural anesthesia. Three days after surgery, abnormal bleeding followed by disorder of prothrombin activity (PT) and activated partial thromboplastin time (aPTT) and paralysis due to thoracic epidural hematoma developed. It was suspected that these coagulopathies were the results of vitamin K deficiency. Vitamin K deficiency in this patient was considered to have been caused by cephem antibiotics containing N-methyl-thiotetrazole (NMTT) side chain and no oral intake of food for a few days preoperatively. The patient was treated with fresh frozen plasma and intravenous menatetrenon, which improved abnormal bleeding and disorder of PT and aPTT within 24hr. After a discussion with orthopedic consultants, we selected a conservative therapy rather than surgical removal of the hematoma. Thoracic epidural hematoma disappeared two months after surgery, but motor paralysis requiring rehabilitation remained. In conclusion, when patients have not eaten anything for a few days and antibiotics with an NMTT sidechain has been administered, care must be taken to prevent vitamin K deficiency and coagulopathy.

Topics: Aged; Anesthesia, Epidural; Anesthesia, General; Carbapenems; Gastrectomy; Hematoma, Epidural, Spinal; Humans; Ileus; Infusions, Intravenous; Male; Plasma; Postoperative Complications; Stomach Neoplasms; Tetrazoles; Vitamin K 2; Vitamin K Deficiency

We examined whether phosphatidylcholine inhibited growth of hepatic cancer, as previously shown for menaquinone-4 (vitamin K2).. Growth inhibitions by phosphatidylcholine and/or menaquinone-4 and apoptosis induction by phosphatidylcholine were evaluated in vitro using human hepatic cancer cell lines (Hep-3B, Hep-G2, HuH-7, and Alexander). Effects of these agents were then investigated in male Sprague-Dawley rats against hepatocarcinogenesis induced by diethylnitrosamine plus phenobarbital. All rats were killed to examine livers to evaluate inhibitory potential macroscopically and immunohistochemically using an antibody against the marker of carcinogenesis, glutathione S-transferase and apoptotic induction by phosphatidylcholine using TUNEL staining. Blood samples were obtained by cardiac puncture.. In vitro, phosphatidylcholine and menaquinone-4 each inhibited cancer cell growth and phosphatidylcholine induced apoptosis dose-dependently. Moreover, exposure to both synergistically inhibited growth in Hep-3B. In vivo, diets containing phosphatidylcholine with or without menaquinone-4 significantly reduced the number of macroscopic hepatic tumor nodules and the extent of abnormally immunoreactive foci conserving hepatic function on serum examinations compared with controls given only the carcinogens. Moreover, phosphatidylcholine supplementation induced apoptosis on TUNEL staining of liver sections.. Given together, phosphatidylcholine and menaquinone-4 may exhibit synergy against hepatocarcinogenesis conserving hepatic function that could benefit patients at high risk for hepatocellular carcinoma.

Topics: Animals; Apoptosis; Carcinoma, Hepatocellular; Cell Line, Tumor; Cell Proliferation; Cell Transformation, Neoplastic; Diet; Diethylnitrosamine; Drug Synergism; Humans; Liver Neoplasms; Liver Neoplasms, Experimental; Male; Phenobarbital; Phosphatidylcholines; Rats; Rats, Sprague-Dawley; Vitamin K 2

Menatetrenone, a vitamin K2 analogue, plays an important role in the production of blood coagulation factors. Menatetrenone has also bee shown to have antineoplastic effects against several cancer cell lines including hepatocellular carcinoma (HCC) cells. However, the mechanisms by which vitamin K2 inhibits HCC cell growth have not bee fully clarified, and we therefore investigated the molecular basis of vitamin K2-induced growth inhibition of HCC cells.. HCC cells were treated with vitamin K2 and the expression of several growth-related genes including cyclin-dependent kinase inhibitors and cyclin D1 was examined at the mRNA and protein levels. A reporter gene assay of the cyclin D1 promoter was done under vitamin K2 treatment. The regulation of nuclear factor kappaB (NF-kappaB) activation was investigated by a NF-kappaB reporter gene assay, an electrophoretic mobility shift assay, a Western blot for phosphorylated IkappaB, and an in vitro kinase assay for IkappaB kinase (IKK). We also examined the effect of vitamin K2 on the growth of HCC cells transfected with p65 or cyclin D1.. Vitamin K2 inhibited cyclin D1 mRNA and protein expression in a dose-dependent manner in the HCC cells. Vitamin K2 also suppressed the NF-kappaB binding site-dependent cyclin D1 promoter activity and suppressed the basal, 12-O-tetradecanoylphorbol-13-acetate (TPA)-, TNF-alpha-, and interleukin (IL)-1-induced activation of NF-kappaB binding and transactivation. Concomitant with the suppression of NF-kappaB activation, vitamin K2 also inhibited the phosphorylation and degradation of IkappaBalpha and suppressed IKK kinase activity. Moreover, HCC cells overexpressing cyclin D1 and p65 became resistant to vitamin K2 treatment.. Vitamin K2 inhibits the growth of HCC cells via suppression of cyclin D1 expression through the IKK/IkappaB/NF-kappaB pathway and might therefore be useful for treatment of HCC.

Topics: Antineoplastic Agents; Blotting, Western; Carcinoma, Hepatocellular; Cell Line, Tumor; Cell Proliferation; Cell Separation; Cyclin D1; Electrophoretic Mobility Shift Assay; Enzyme Activation; Flow Cytometry; Humans; I-kappa B Kinase; Liver Neoplasms; NF-kappa B; Reverse Transcriptase Polymerase Chain Reaction; Transcription, Genetic; Transfection; Vitamin K 2

A sensitive and highly selective high-performance liquid chromatography (HPLC) method was developed for the determination of vitamin K homologues including phylloquinone (PK), menaquinone-4 (MK-4) and menaquinone-7 (MK-7) in human plasma using post-column peroxyoxalate chemiluminescence (PO-CL) detection following on-line ultraviolet (UV) irradiation. The method was based on ultraviolet irradiation (254 nm, 15 W) of vitamin K to produce hydrogen peroxide and a fluorescent product at the same time, which can be determined with PO-CL detection. The separation of vitamin K by HPLC was accomplished isocratically on an ODS column within 35 min. The method involves the use of 2-methyl-3-pentadecyl-1,4-naphthoquinone as an internal standard. The detection limits (signal-to-noise ratio = 3) were 32, 38 and 85 fmol for PK, MK-4 and MK-7, respectively. The recoveries of PK, MK-4 and MK-7 were greater than 82% and the inter- and intra-assay R.S.D. values were 1.9-5.4%. The sensitivity and selectivity of this method were sufficient for clinical and nutritional applications.

Topics: Chromatography, High Pressure Liquid; Female; Humans; Luminescence; Male; Oxalates; Reproducibility of Results; Ultraviolet Rays; Vitamin K 1; Vitamin K 2; Vitamins

A simple and rapid quantification method was developed for determining menatetrenone and its epoxide metabolite in human plasma. After a simple protein precipitation with methanol, the analytes were chromatographed on a reversed-phase C(18) column and detected by LC/MS/MS with atmospheric pressure chemical ionization. The coefficient of variation of the assay precision was less than 10.1%, and the accuracy ranged from 98.0 to 106.5%. The limit of detection of menatetrenone and its epoxide was 0.5 and 0.2 ng/ml, respectively. This method was used to simultaneously measure the plasma concentration of menatetrenone and its epoxide metabolite from healthy subjects after a single 30 mg oral dose of menatetrenone.

Topics: Chromatography, Liquid; Epoxy Compounds; Hemostatics; Humans; Methanol; Reference Standards; Reproducibility of Results; Sensitivity and Specificity; Solvents; Tandem Mass Spectrometry; Time Factors; Vitamin K 2

Vitamin K is a fat-soluble vitamin that serves as a coenzyme for vitamin K-dependent carboxylase. Besides its canonical action, vitamin K binds to the steroid and xenobiotic receptor (SXR)/pregnane X receptor (PXR) and modulates gene transcription. To determine if the osteoprotective action of vitamin K is the result of the PXR/SXR pathway, we screened by two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis the PXR/SXR target genes in an osteoblastic cell line (MC3T3-E1) treated with a vitamin K2 (menaquinone 4 [MK4]). Osteoblastic differentiation of MC3T3-E1 cells was induced by MK4. Msx2, an osteoblastogenic transcription factor, was identified as an MK4-induced gene. Functional analysis of the Msx2 gene promoter mapped a vitamin K-responsive element (PXR-responsive element [PXRE]) that was directly bound by a PXR/retinoid X receptor alpha heterodimer. In a chromatin immunoprecipitation analysis, PXR was recruited together with a coactivator, p300, to the PXRE in the Msx2 promoter. MK4-bound PXR cooperated with estrogen-bound estrogen receptor alpha to control transcription at the Msx2 promoter. Knockdown of either PXR or Msx2 attenuated the effect of MK4 on osteoblastic differentiation. Thus, the present study suggests that Msx2 is a target gene for PXR activated by vitamin K and suggests that the osteoprotective action of MK4 in the human mediates, at least in part, a genomic pathway of vitamin K signaling.

Topics: Animals; Biomarkers; Cell Differentiation; Cells, Cultured; DNA-Binding Proteins; Estradiol; Estrogen Receptor alpha; Gene Expression Regulation; Hemostatics; Homeodomain Proteins; Humans; Mice; Mice, Knockout; Osteoblasts; Pregnane X Receptor; Promoter Regions, Genetic; Receptors, Steroid; Response Elements; Retinoid X Receptor alpha; Signal Transduction; Transcription, Genetic; Vitamin K; Vitamin K 2

Vitamin K is known as a critical nutrient required for bone homeostasis and blood coagulation, and it is clinically used as a therapeutic agent for osteoporosis in Japan. Besides its enzymatic action as a cofactor of vitamin K-dependent gamma-glutamyl carboxylase (GGCX), we have previously shown that vitamin K(2) is a transcriptional regulator of bone marker genes and extracellular matrix-related genes, by activating the steroid and xenobiotic receptor (SXR). To explore a novel action of vitamin K in osteoblastic cells, we identified genes up-regulated by a vitamin K(2) isoform menaquinone-4 (MK-4) using oligonucleotide microarray analysis. Among these up-regulated genes by MK-4, growth differentiation factor 15 (GDF15) and stanniocalcin 2 (STC2) were identified as novel MK-4 target genes independent of GGCX and SXR pathways in human and mouse osteoblastic cells. The induction of GDF15 and STC2 is likely specific to MK-4, as it was not exerted by another vitamin K(2) isoform MK-7, vitamin K(1), or the MK-4 side chain structure geranylgeraniol. Investigation of the involved signaling pathways revealed that MK-4 enhanced the phosphorylation of protein kinase A (PKA), and the MK-4-dependent induction of both GDF15 and STC2 genes was reduced by the treatment with a PKA inhibitor H89 or siRNA against PKA. These results suggest that vitamin K(2) modulates its target gene expression in osteoblastic cells through the PKA-dependent mechanism, which may be distinct from the previously known vitamin K signaling pathways.

Topics: Animals; Bone Morphogenetic Proteins; Carbon-Carbon Ligases; Cells, Cultured; Cyclic AMP-Dependent Protein Kinases; Gene Expression Profiling; Gene Expression Regulation; Glycoproteins; Growth Differentiation Factor 15; Humans; Intercellular Signaling Peptides and Proteins; Mice; Oligonucleotide Array Sequence Analysis; Osteoblasts; Phosphorylation; Pregnane X Receptor; Receptors, Steroid; Vitamin K 2

Several reports indicate an important role for vitamin K in bone health as well as blood coagulation. However, the current Adequate Intakes (AI) might not be sufficient for the maintenance of bone health. To obtain a closer estimate of dietary intake of phylloquinone (PK) and menaquinones (MKs), PK, MK-4 and MK-7 contents in food samples (58 food items) were determined by an improved high-performance liquid chromatography method. Next, we assessed dietary vitamin K intake in young women living in eastern Japan using vitamin K contents measured here and the Standard Tables of Food Composition in Japan. PK was widely distributed in green vegetables and algae, and high amounts were found in spinach and broccoli (raw, 498 and 307 microg/100 g wet weight, respectively). Although MK-4 was widely distributed in animal products, overall MK-4 content was lower than PK. MK-7 was observed characteristically in fermented soybean products such as natto (939 microg/100 g). The mean total vitamin K intake of all subjects (using data from this study and Japanese food composition tables) was about 230 microg/d and 94% of participants met the AI of vitamin K for women aged 18-29 y in Japan, 60 microg/d. The contributions of PK, MK-4 and MK-7 to total vitamin K intake were 67.7, 7.3 and 24.9%, respectively. PK from vegetables and algae and MK-7 from pulses (including fermented soybean foods) were the major contributors to the total vitamin K intake of young women living in eastern Japan.

Topics: Adolescent; Adult; Chromatography, High Pressure Liquid; Dairy Products; Edible Grain; Eukaryota; Fabaceae; Female; Food Analysis; Humans; Japan; Meat; Nutrition Assessment; Spices; Tea; Vegetables; Vitamin K 1; Vitamin K 2

The purpose of this study was to determine the contents of three forms of vitamin K [phylloquinone, dihydrophylloquinone, and menaquinone-4 (MK-4)] in representative samples (including different samples within the same food category) of meat (n = 128), dairy and eggs (n = 24), and fast foods (n = 169) common to the U.S. diet. The findings of our analysis indicate that no single food item in these categories is a rich dietary source of any one form of vitamin K. However, these foods are often consumed in large quantities; hence, they may be of importance in overall contribution to total vitamin K intake. The presence of MK-4 in meat, eggs, and dairy foods could be important as physiologic functions unique to MK-4 are identified.

Topics: Animals; Dairy Products; Diet; Food Analysis; Meat; Restaurants; United States; Vitamin K; Vitamin K 1; Vitamin K 2

Phylloquinone is converted into menaquinone-4 and accumulates in extrahepatic tissues. Neither the route nor the function of the conversion is known. One possible metabolic route might be the release of menadione from phylloquinone by catabolic activity. In the present study we explored the presence of menadione in urine and the effect of vitamin K intake on its excretion. Menadione in urine was analysed by HPLC assay with fluorescence detection. Urine from healthy male volunteers was collected before and after administration of a single dose of K vitamins. Basal menadione excretion in non-supplemented subjects (n 6) was 5.4 (sd 3.2) microg/d. Urinary menadione excretion increased greatly after oral intake of the K vitamins, phylloquinone and menaquinone-4 and -7. This effect was apparent within 1-2 h and peaked at about 3 h after intake. Amounts of menadione excreted in 24 h after vitamin K intake ranged, on a molar basis, from 1 to 5 % of the administered dose, indicating that about 5-25 % of the ingested K vitamins had been catabolized to menadione. Menadione excretion was not enhanced by phylloquinone administered subcutaneously or by 2',3'-dihydrophylloquinone administered orally. In archived samples from a depletion/repletion study (Booth et al. (2001) Am J Clin Nutr 74, 783-790), urinary menadione excretion mirrored dietary phylloquinone intake. The present study shows that menadione is a catabolic product of K vitamins formed after oral intake. The rapid appearance in urine after oral but not subcutaneous administration suggests that catabolism occurs during intestinal absorption. The observations make it likely that part of the menaquinone-4 in tissues results from uptake and prenylation of circulating menadione.

Topics: Administration, Cutaneous; Administration, Oral; Cell Line; Cells, Cultured; Dietary Supplements; Hemostatics; Humans; Male; Vitamin K; Vitamin K 1; Vitamin K 2; Vitamin K 3; Vitamins

Vitamin K deficiency is associated with low bone mineral density and increased risk of bone fracture. Phylloquinone (K1) and menaquinone 4 (MK-4) and 7 (MK-7) are generally observed in human plasma; however, data are limited on their circulating concentrations and their associations with bone metabolism or with gamma-carboxylation of the osteocalcin molecule.. The objectives were to measure the circulating concentrations of K1, MK-4, and MK-7 in women and to ascertain whether each form of vitamin K is significantly associated with bone metabolism.. Plasma concentrations of K1, MK-4, MK-7, undercarboxylated osteocalcin (ucOC; measured by using the new electrochemiluminescence immunoassay), intact osteocalcin (iOC), calcium, and phosphorus; bone-derived alkaline phosphatase activity; and concentrations of urinary creatinine, N-terminal telopeptide, and deoxypyridinoline were measured in healthy women (n = 396).. On average, MK-7 and MK-4 were the highest and lowest, respectively, of the 3 vitamers in all age groups. K1 and MK-7 correlated inversely with ucOC, but associations between nutritional basal concentration of MK-4 and ucOC were not observed. Multiple regression analysis indicated that not only K1 and MK-7 concentrations but also age were independently correlated with ucOC concentration and the ratio of ucOC to iOC. The plasma K1 or MK-7 concentration required to minimize the ucOC concentration was highest in the group aged > or =70 y, and it decreased progressively for each of the younger age groups.. The definite role of ucOC remains unclear. However, if submaximal gamma-carboxylation is related to the prevention of fracture or bone mineral loss, circulating vitamin K concentrations in elderly people should be kept higher than those in young people.

Topics: Adult; Age Factors; Aged; Aged, 80 and over; Aging; Biomarkers; Bone and Bones; Bone Density; Carboxylic Acids; Female; Fractures, Bone; Humans; Japan; Middle Aged; Nutritional Requirements; Nutritional Status; Osteocalcin; Risk Factors; Vitamin K; Vitamin K 1; Vitamin K 2; Vitamin K Deficiency; Vitamins

It is generally accepted that the availability of vitamin K in vivo depends on its homologues, the biological activities of which would differ among organs. To test this hypothesis, we examined the uptake, metabolism, and utilization of menaquinone-4 (MK-4) and phylloquinone (PK) using 18O-labeled compounds in two cultured human cell lines (HepG2 and MG-63). Lipid extracts were prepared from the cells and media after 1, 3, and 6h of incubation. The detection of the vitamin K analogues (18O-, 16O-quinone, and epoxide forms) was carried out with LC-APCI-MS/MS as previously reported. The 18O of vitamin K was replaced with atmospheric 16O2 during the formation of vitamin K epoxide with a carboxylative catalytic reaction. As a result, a significant difference was observed between MK-4 and PK in the amounts taken up into the cells. The 18O-labeled MK-4 was rapidly and remarkably well absorbed into the cells and metabolized to the epoxide form via a hydroquinone form as compared to the 18O-labeled PK. The difference in uptake of MK-4 and PK was not affected by treatment with warfarin although the metabolism of both compounds was markedly inhibited. This methodology should be utilized to clarify some of the actions of vitamin K in target cells and facilitate the development of new vitamin K drugs.

Topics: Calibration; Cell Line; Cell Line, Tumor; Chromatography, High Pressure Liquid; Humans; Indicators and Reagents; Mass Spectrometry; Reference Standards; Vitamin K; Vitamin K 1; Vitamin K 2

A HPLC fluorescence determination method for Vitamin K derivatives (Vitamin K(1), phylloquinone, PK and K(2), menaquinones, MK-4 and MK-7) using post-column reduction and internal standards was developed. Selectivity and reproducibility were increased by optimized chromatography conditions and satisfactory precision and accuracy were attained by using synthetic internal standards. After addition of internal standards to plasma samples, lipids were extracted with ethanol and hexane. Chromatography was performed by isocratic reverse phase separation on a C18 column. Vitamin K derivatives were detected at 430 nm with excitation at 320 nm for MK-4 and 240 nm for PK and MK-7. The detection limits for MK-4, PK and MK-7 were 4, 2 and 4 pg, respectively. The recoveries of MK-4, PK and MK-7 were greater than 92% and the inter- and intra-assay R.S.D. values were 5.7-9.2% for MK-4, 4.9-9.6% for PK and 6.3-19.3% for MK-7. The data showed good correlation between proposed method and LC-APCI/MS method for MK-4 (R(2)=0.988), PK (R(2)=0.979) and MK-7 (R(2)=0.986). The method allows the determination of Vitamin K for evaluating their clinical and nutritional status.

Topics: Chromatography, High Pressure Liquid; Fluorescence; Humans; Osteoporosis; Reference Standards; Spectrometry, Mass, Electrospray Ionization; Vitamin K; Vitamin K 2

We report here the development of a precise and sensitive method for the determination of vitamin K homologues including phylloquinone (PK), menaquinone-4 (MK-4), and menaquinone-7 (MK-7) in human plasma using HPLC-tandem mass-mass spectrometry with atmospheric pressure chemical ionization (LC-APCI-MS/MS). The method involves the use of stable isotope (18)O-labeled internal standard compounds, which were synthesized in our laboratory, and the selection of a precursor and product ion with a MS/MS multiple reaction monitoring method. The average intraassay and interassay variation values for PK, MK-4, and MK-7 were <10%. Average spiked recoveries from authentic compounds added to normal human plasma samples for PK, MK-4, and MK-7 were 98-102%. Mean plasma concentrations of PK, MK-4, and MK-7 from healthy subjects (n = 20) were 1.22 +/-0.57, 0.39 +/- 0.46, and 6.37 +/- 7.45 ng/mL, respectively. We conclude that this novel LC-APCI-MS/MS method should be useful for the evaluation of vitamin K status in postmenopausal women and elderly subjects and provides useful information for the treatment and prevention of osteoporosis with vitamin K.

Topics: Calibration; Chromatography, High Pressure Liquid; Humans; Mass Spectrometry; Osteoporosis; Oxygen Isotopes; Reference Standards; Reproducibility of Results; Sensitivity and Specificity; Spectrometry, Fluorescence; Vitamin K; Vitamin K 1; Vitamin K 2

Menatetrenone (MK-4) is a vitamin K2 homologue that has been used as a therapeutic agent for osteoporosis in Japan. However, there is no far any reported evidence that MK-4 ameliorates a pre-existing condition of reduced bone mineral density (BMD) in vivo. In this study, we evaluated the effect of MK-4 in a rat model of established bone loss through immobilization caused by sciatic neurectomy. Unilateral sciatic neurectomy (SNx) was performed in rats, and 10 or 30 mg/kg of MK-4 or vehicle was administered to the rats three weeks after operation. Seven weeks after operation, the rats were sacrificed and BMD and bone histomorphometric parameters were measured to assess the effects of MK-4. While BMD of the distal femoral metaphysis was significantly decreased after SNx, MK-4 administration increased BMD in the neurectomized rats. Bone formation was decreased continuously and bone resorption was initially increased in SNx rats. Four weeks treatment of MK-4 increased bone formation and suppressed bone resorption. In addition, increased carboxylated osteocalcin and decreased undercarboxylated osteocalcin in serum were observed in MK-4-administered rats. These results indicated that MK-4 rescued bone volume by improving osteoblast dysfunction and accelerating gamma carboxylation of osteocalcin. MK-4 may be useful for treating disuse osteopenia.

Topics: Animals; Bone Density; Bone Resorption; Disease Models, Animal; Male; Osteoblasts; Osteocalcin; Osteogenesis; Osteoporosis; Rats; Rats, Sprague-Dawley; Sciatic Nerve; Vitamin K 2

The secondary electron acceptor of photosystem (PS) I in the cyanobacterium Gloeobacter violaceus PCC 7421 was identified as menaquinone-4 (MQ-4) by comparing high performance liquid chromatograms and absorption spectra with an authentic compound. The MQ-4 content was estimated to be two molecules per one molecule of chlorophyll (Chl) a', a constituent of P700. Comparative genomic analyses showed that six of eight men genes, encoding phylloquinone/MQ biosynthetic enzymes, are missing from the G. violaceus genome. Since G. violaceus clearly synthesizes MQ-4, the combined results indicate that this cyanobacterium must have a novel pathway for the synthesis of 1,4-dihydroxy-2-naphthoic acid.

Topics: Chlorophyll; Cyanobacteria; Genome, Bacterial; Naphthols; Photosystem I Protein Complex; Vitamin K 1; Vitamin K 2

Vitamin K is used for protecting against osteoporosis. Recently, it has been reported that the inhibitory effect of vitamin K(2) (menatetrenone) on bone resorption may be related to its side chain. Geranylgeranylacetone (GGA), known as teprenone, an antiulcer drug, has almost the same chemical structure as that of the side chain of menatetrenone. We hypothesized that GGA also has an inhibitory effect on osteoclastogenesis both in vitro and in vivo. GGA in pharmacological concentrations directly inhibited osteoclastogenesis from human monocytes induced by soluble receptor activator of nuclear factor-kappaB ligand. In addition, GGA induced degradation of actin rings in mature osteoclasts, which was reversed by adding geranylgeranylpyrophosphatase. Moreover, GGA increased the bone mineral density of total femur, proximal metaphysis, and diaphysis of femur in ovariectomized rats. GGA also prevented bone loss induced by hindlimb unloading in tail-suspended rats. These results indicate that GGA prevents bone loss by maintaining a positive balance of bone turnover through suppression of both the formation and the activity of osteoclasts. Thus, GGA could be used to prevent and improve osteoporosis.

Topics: Animals; Anti-Ulcer Agents; Bone Density; Bone Diseases, Metabolic; Bone Resorption; Cells, Cultured; Disease Models, Animal; Diterpenes; Dose-Response Relationship, Drug; Female; Femur; Hindlimb Suspension; Humans; NF-kappa B; Osteoclasts; Ovariectomy; Rats; Rats, Inbred F344; Tibia; Vitamin K 2

Studies with animals support a role for vitamin K (VK) in the biosynthesis of sphingolipids, a class of complex lipids present in high concentrations in the brain. In mice and rats, VK deficiency decreases levels of brain sulfatides and causes behavioral alterations. In light of its heterogeneity and to better understand the role of VK in the brain, we characterized the distribution of the two main VK vitamers, phylloquinone (K1) and menaquinone-4 (MK-4), in nine distinct brain regions. Weaning female Sprague-Dawley rats (n=5/dietary group) were fed diets containing either low (L, 80 microg/kg diet), adequate (A, 500 microg/kg diet) or high (H, 2000 microg/kg diet) levels of K1 for 6 mo. The main form of VK in the brain was MK-4, and it was present in significantly higher concentrations in myelinated regions (the pons medulla and midbrain) than in nonmyelinated regions. Both regional K1 and MK-4 increased with K1 intake (P<0.05). Sphingolipid distribution varied across brain regions (P<0.001) but was not affected by K1 intake. In the L and A groups but not the H group, brain MK-4 concentration was positively correlated with the concentrations of sulfatides (L, r=0.518; A, r=0.479) and sphingomyelin (L, r=0.515; A, r=0.426), and negatively correlated with ganglioside concentration (L, r=-0.398); A, r=-0.353). Sphingolipids are involved in major cellular events such as cell proliferation, differentiation and survival. The strong associations reported here between brain MK-4 and sphingomyelin, sulfatides and gangliosides suggest that this vitamer may play an important role in the brain.

Topics: Animals; Brain Chemistry; Diet; Female; Gangliosides; Rats; Rats, Sprague-Dawley; Sphingolipids; Sphingomyelins; Sulfoglycosphingolipids; Tissue Distribution; Vitamin K; Vitamin K 1; Vitamin K 2; Weaning

Previously, we reported that plasma half-lives of a drug incorporated in lipid emulsions prepared with soybean oil (SO), a long-chain triglyceride, and hydrogenated castor oils (HCOs) (SO/HCOs) were markedly longer, while those as SO/polyoxyethylene sorbitan esters (SO/PSs) were similar, compared to that as SO/egg yolk phosphatides (SO/EYP) [J. Pharm. Pharmacol. 54 (2002) 1357; J. Drug Target. 11 (2003) 37]. In the present study, lipid emulsions were prepared with Miglyol 812 (MO), a medium-chain triglyceride, and HCOs, and the kinetics of the incorporated drug, menatetrenone, were examined. The plasma half-lives and the liver uptake of menatetrenone as MO/polyoxyethylene-(10)-hydrogenated castor oils (MO/HCO10s) were similar to and larger than those as MO/EYP, respectively. On the other hand, the plasma half-lives and liver uptake of menatetrenone as MO/polyoxyethylene-(20)-hydrogenated castor oils (MO/HCO20s) or MO/polyoxyethylene-(60)-hydrogenated castor oils (MO/HCO60s) were markedly longer and lower than those as MO/EYP, respectively. The pretreatment of dextran sulfate 500,000, a reticuloendothelial system suppressor, raised the plasma concentration and inhibited liver uptake of menatetrenone as MO/HCO10, but not for MO/HCO20. These findings suggest that the minimum number of oxyethylene units within HCOs for the prolonged plasma circulation of menatetrenone was 20 for MO/HCOs, similarly to SO/HCOs.

Topics: Animals; Area Under Curve; Castor Oil; Delayed-Action Preparations; Emulsions; Half-Life; Hemostatics; Hydrogenation; Injections, Intravenous; Liver; Male; Particle Size; Pharmaceutic Aids; Rats; Rats, Wistar; Tissue Distribution; Triglycerides; Vitamin K 2

We conducted a cross-sectional examination of the role of serum vitamin K levels as they relate to bone metabolism in elderly women with type II diabetes mellitus (DM). Eighty-five elderly women with type II DM were enrolled. Three fractions of vitamin K, phylloquinone (PK), menaquinone 4 (menatetrenone; MK 4), and menaquinone 7 (MK 7), along with undercarboxylated osteocalcin (UcOC), intact osteocalcin (IOC), urinary deoxypyridinoline (udpd), urinary type I collagen N-telopeptide (NTx), and intact parathyroid hormone (IPTH) were measured. Bone mineral density was measured in the lumbar spine (LSBMD) by dual-energy X-ray absorptiometry (DXA), and T scores or Z scores were calculated. The patients were divided into two groups by T score, under -2.5 (osteoporotic group) and over -2.5 (non-osteoporotic group). UcOC levels in osteoporotics patients were significantly higher than those in the non-osteoporotic group (3.09 +/- 3.94 vs 1.82 +/- 1.76 ng/ml, P = 0.02). The correlation between Z score and logarithmic UcOC/IOC levels in type II DM showed a negative trend ( P = 0.07) and a significantly and negatively association with logarithmic NTx ( r = -0.38; P = 0.001). In osteoporotic DM, the UcOC/IOC ratio was significantly correlated with the Z score ( r = -0.61; P << 0.05). Furthermore, logarithmic UcOC/IOC showed a negative correlation with logarithmic MK 7 ( r = -0.50; P = 0.001). In conclusion, the reduction in LSBMD in elderly women with type II DM may be associated, in part, with a defect in Gamma-glutamylcarboxylation by vitamin K.

Topics: Aged; Biomarkers; Bone Density; Diabetes Mellitus, Type 2; Female; Humans; Osteocalcin; Vitamin K; Vitamin K 2

The exocrine pancreas releases secretory products essential for nutrient assimilation. In addition to digestive enzymes, the release of lipoprotein-like particles containing the membrane trafficking protein caveolin-1 from isolated pancreatic explants has been reported. The present study examined: (1) if gastrointestinal hormones induce the apical secretion of phospholipid in vivo and (2) a potential association of caveolin-1 and the lipid-soluble vitamin K analog menaquinone-4 (MK-4) with these structures. Analysis of isolated acinar cells, purified zymogen granules, and pancreatic juice collected in vivo indicated the presence a caveolin-1 immunoreactive protein that was acutely released in response hormone stimulation. Chloroform-extracted fractions of pancreatic juice also contained high concentrations of MK-4 that was secreted in parallel to protein and phospholipid. The presence of caveolin-1 and MK-4 in the phospholipid fraction of pancreatic juice places these molecules in the secretory pathway of exocrine cells and suggests a physiological role in digestive enzyme synthesis and/or processing.

Topics: Animals; Caveolin 1; Caveolins; Cells, Cultured; Cholecystokinin; Hemostatics; Humans; Male; Pancreas; Peptide Fragments; Phospholipids; Rats; Rats, Sprague-Dawley; Secretin; Tissue Extracts; Vitamin K 2

Oil-in-water (O/W) lipid emulsions were prepared with phosphatidylcholines (PCs) of various acyl chains and soybean oil (SO) using a microfluidizer system, and the pharmacokinetics of menatetrenone incorporated in these oil particles were examined at the clinical injection volume (0.1 mL kg(-1)) in rats. The plasma half-life of menatetrenone incorporated in the oil particles prepared with SO and dipalmitoylphosphatidylcholine (DPPC) (SO/DPPC) was longer than that prepared with SO and eggyolk phosphatides (EYP) (SO/EYP) by 3 fold, while those of menatetrenone as oil particles prepared with SO and either dilauroyl phosphatidylcholine (DLPC), dimyristoyl phosphatidylcholine (DMPC), distearoyl phosphatidylcholine (DSPC), dioleoyl phosphatidylcholine (DOPC) or dilinoleoyl phosphatidylcholine (DLoPC) (SO/DLPC, SO/DMPC, SO/DSPC, SO/DOPC and SO/DLoPC, respectively) were similar to that of menatetrenone as SO/EYP. The menatetrenone uptake by the liver was not significantly different from that as SO/EYP in all SO/PCs examined, but the menatetrenone uptake by the spleen as SO/DPPC and SO/DSPC was higher than that as SO/EYP. The menatetrenone uptake by the lungs as SO/DPPC was also higher than that as SO/EYP. These findings suggest that SO/DPPC is a good candidate drug carrier for the prolonged plasma circulation of lipophilic drugs.

Topics: Animals; Chemistry, Pharmaceutical; Delayed-Action Preparations; Drug Carriers; Emulsions; Half-Life; Hemostatics; Injections, Intravenous; Male; Particle Size; Phosphatidylcholines; Rats; Rats, Wistar; Soybean Oil; Tissue Distribution; Vitamin K 2

To evaluate the biological effects of vitamin K2 (menatetrenone, MK-4) on ovariectomy (OVX)-induced bone loss, we have examined histological alterations of femoral metaphyses of sham-operated (sham group), ovariectomized (OVX group), and MK-4 dietary-supplemented OVX (MK-4 group; 50 mg/kg per day) female Fischer rats 1, 2, 5, and 8 weeks after OVX. In the first week, rats of the OVX and MK-4 groups showed discontinuous trabeculae compared with sham-operated rats. At 2 weeks after OVX, the OVX rats revealed many large tartrate resistant acid phosphatase (TRAP)-positive osteoclasts, while osteoclasts in the MK-4-treated rats were similar in size to those of the sham group. At 5 weeks, the OVX and MK-4 groups revealed fragmented trabeculae in femoral metaphyses. The cartilage matrix was partially exposed due to stimulated bone resorption in the OVX group, but not in the MK-4 group. After 8 weeks, the OVX rats had little metaphyseal trabeculae, whereas the MK-4-treated rats had maintained short trabeculae. Despite the presence of intense alkaline phosphatase-positive osteoblasts on trabeculae in the MK-4 group, TRAP-positive osteoclasts were flattened without developing ruffled borders. Therefore, MK-4 appeared to lessen the increase in osteoclastic bone resorption induced by OVX, as well as to maintain the accelerated osteoblastic activity. It is of importance to identify the target cells for MK-4 in bone. Autoradiography localized [3H]-labeled MK-4 mainly in osteoblasts and adjacent bone matrices, but not in osteoclasts, indicating that MK-4 targets osteoblasts. Thus, MK-4 appears to target osteoblasts, consequently inhibiting bone loss induced by ovariectomy.

Topics: Animals; Animals, Newborn; Autoradiography; Bone Density; Female; Femur; Mice; Microscopy, Electron; Osteocalcin; Osteoclasts; Ovariectomy; Rats; Rats, Inbred F344; Time Factors; Vitamin K 2

In this study, we examined changes in bone parameters and bone strength in rats fed low-Mg diets (experiment 1) and the effects of vitamin K2 (MK-4, experiment 3) and alendronate (ALN, experiment 2) in this model. In experiment 1, 5-week-old male Wistar rats were fed three low-Mg diets (Mg 9, 6, 3 mg/100 g diet) for 4 weeks. Although the cortical bone mineral content (CtBMC) and cortical thickness (CtTh) of the femoral diaphysis in all low-Mg-diet groups were the same as or greater than those in the intact group (Mg: 90 mg/100 g diet), the maximum load and elastic modulus were significantly reduced in the 3-mg-Mg group. In experiment 2, 4-week-old Wistar rats were fed a 6-mg-Mg diet for 8 weeks, and the effect of ALN (2, 20, and 200 microg/kg twice a week) was evaluated. The administration of ALN at 200 microg/kg increased the cortical bone mineral content (CtBMC), CtTh, and maximum load, but had no effect on the elastic modulus, as compared with the low-Mg-control group. In experiment 3, the effect of MK-4 was evaluated under the same conditions as in experiment 2. The administration of MK-4 had no effect on CtBMC, CtTh, or bone components of the femoral diaphysis. However, MK-4 inhibited the decreases in maximum load and elastic modulus due to the low-Mg diet. Since there is no other experimental model in which there is a decrease in bone mechanical properties without a decrease in bone mineral content, the low-Mg diet model is considered to be an excellent model for examining bone quality. Our results from this model suggest that MK-4 and ALN affect bone mechanical properties by different mechanisms.

Topics: Alendronate; Alkaline Phosphatase; Animals; Body Weight; Bone and Bones; Bone Density; Calcitriol; Calcium; Elasticity; Femur; Hydroxyproline; Magnesium; Magnesium Deficiency; Male; Osteocalcin; Parathyroid Hormone; Rats; Rats, Wistar; Stress, Mechanical; Urine; Vitamin K 2

The purpose of the present study was to compare the effect of vitamin K(2) on cortical and cancellous bones in orchidectomized young rats.. Forty male Sprague-Dawley rats, 6 weeks of age, were randomized by stratified weight method into four groups with 10 rats in each group: baseline controls (BLC), age-matched controls (AMC), orchidectomy (ORX), and ORX+vitamin K(2) administration (K). Vitamin K(2) (menatetrenone) was administered subcutaneously twice a week at dose of 30 mg/kg each. The experimental period was 8 weeks, and cortical and cancellous bone histomorphometry was performed on the tibial shaft and the proximal tibia, respectively.. Cortical area (Ct Ar) and cancellous bone volume (BV/TV) were significantly greater in the AMC group than in the BLC group. Ct Ar was significantly lower in the ORX group than in the AMC group, and cancellous BV/TV was also significantly lower in the ORX group than in the AMC group as a result of significantly increased eroded surface (ES/BS). Although Ct Ar in the ORX+K group did not differ significantly from that in the ORX group, cancellous BV/TV was significantly greater in the ORX+K group than in the ORX group, but still significantly lower than in the AMC group. This protective effect of vitamin K(2) on cancellous bone was attributable to normalizing increased ES/BS.. Vitamin K(2) appears to act more strongly on cancellous bone than on cortical bone in ORX young rats. High dose vitamin K(2) could partially prevent the reduction of cancellous bone gain by normalizing raised bone resorption in ORX young rats.

Topics: Animals; Bone and Bones; Bone Density; Bone Resorption; Male; Orchiectomy; Rats; Rats, Sprague-Dawley; Tibia; Vitamin K 2

Accumulating evidence indicates that menaquinone-4 (MK-4), a vitamin K(2) with four isoprene units, inhibits osteoclastogenesis in murine bone marrow culture, but the reason for this inhibition is not yet clear, especially in human bone marrow culture. To clarify the inhibitory mechanism, we investigated the differentiation of colony-forming-unit fibroblasts (CFU-Fs) and osteoclasts in human bone marrow culture, to learn whether the enhancement of the differentiation of CFU-Fs from progenitor cells might relate to inhibition of osteoclast formation. Human bone marrow cells were grown in alpha-minimal essential medium with horse serum in the presence of MK-4 until adherent cells formed colonies (CFU-Fs). Colonies that stained positive for alkaline phosphatase activity (CFU-F/ALP(+)) were considered to have osteogenic potential. MK-4 stimulated the number of CFU-F/ALP(+) colonies in the presence or absence of dexamethasone. The stimulation was also seen in vitamin K(1) treatment. These cells had the ability to mineralize in the presence of alpha-glycerophosphate. In contrast, both MK-4 and vitamin K(1) inhibited 1,25 dihydroxyvitamin D(3)-induced osteoclast formation and increased stromal cell formation in human bone marrow culture. These stromal cells expressed ALP and Cbfa1. Moreover, both types of vitamin K treatment decreased the expression of receptor activator of nuclear factor kappaB ligand/osteoclast differentiation factor (RANKL/ODF) and enhanced the expression of osteoprotegerin/osteoclast inhibitory factor (OPG/OCIF) in the stromal cells. The effective concentrations were 1.0 microM and 10 microM for the expression of RANKL/ODF and OPG/OCIF respectively. Vitamin K might stimulate osteoblastogenesis in bone marrow cells, regulating osteoclastogenesis through the expression of RANKL/ODF more than through that of OPG/OCIF.

Topics: Alkaline Phosphatase; Bone Marrow Cells; Carrier Proteins; Cell Division; Cells, Cultured; Depression, Chemical; Dexamethasone; Fibroblasts; Glucocorticoids; Glycoproteins; Humans; Lectins, C-Type; Membrane Glycoproteins; Membrane Proteins; Osteoblasts; Osteoclasts; Osteoprotegerin; RANK Ligand; Receptor Activator of Nuclear Factor-kappa B; Receptors, Cytoplasmic and Nuclear; Receptors, Tumor Necrosis Factor; Stimulation, Chemical; Vitamin K; Vitamin K 2

We investigated the effects of phenytoin, an antiepileptic drug, and vitamin K2 (menatetrenone) on bone mineral density and the changes in the levels of menaquinone derivatives (MK-1 approximately MK-14) in the sera and femurs of growing male rats.. Levels of menaquinone derivatives were measured with high-performance liquid chromatography with an electrochemical detector.. Bone mineral density values decreased significantly in all parts of the femoral bones measured (diaphysis and metaphysis) in the phenytoin-treated group. When the serum and bone levels of menatetrenone and MK-6 decreased due to phenytoin administration, we observed bone loss in rats. Conversely, when bone loss was prevented by the combined administration of phenytoin and menatetrenone, serum and bone levels of menatetrenone and MK-6 increased to the levels of vehicle-treated rats.. Long-term phenytoin exposure may inhibit bone formation concomitantly with insufficient vitamin K, which, at least in part, contributes to bone loss in rats.

Topics: Animals; Anticonvulsants; Bone Density; Bone Diseases, Metabolic; Femur; Male; Phenytoin; Random Allocation; Rats; Rats, Wistar; Vitamin K 2

Oxidative stress is believed to be the cause of cell death in multiple disorders of the brain, including perinatal hypoxia/ischemia. Glutamate, cystine deprivation, homocysteic acid, and the glutathione synthesis inhibitor buthionine sulfoximine all cause oxidative injury to immature neurons and oligodendrocytes by depleting intracellular glutathione. Although vitamin K is not a classical antioxidant, we report here the novel finding that vitamin K1 and K2 (menaquinone-4) potently inhibit glutathione depletion-mediated oxidative cell death in primary cultures of oligodendrocyte precursors and immature fetal cortical neurons with EC50 values of 30 nm and 2 nm, respectively. The mechanism by which vitamin K blocks oxidative injury is independent of its only known biological function as a cofactor for gamma-glutamylcarboxylase, an enzyme responsible for posttranslational modification of specific proteins. Neither oligodendrocytes nor neurons possess significant vitamin K-dependent carboxylase or epoxidase activity. Furthermore, the vitamin K antagonists warfarin and dicoumarol and the direct carboxylase inhibitor 2-chloro-vitamin K1 have no effect on the protective function of vitamin K against oxidative injury. Vitamin K does not prevent the depletion of intracellular glutathione caused by cystine deprivation but completely blocks free radical accumulation and cell death. The protective and potent efficacy of this naturally occurring vitamin, with no established clinical side effects, suggests a potential therapeutic application in preventing oxidative damage to undifferentiated oligodendrocytes in perinatal hypoxic/ischemic brain injury.

Topics: Animals; Antioxidants; Cell Death; Cells, Cultured; Cystine; Dose-Response Relationship, Drug; gamma-Glutamylcyclotransferase; Glutathione; Neurons; Neuroprotective Agents; Oligodendroglia; Oxidative Stress; Rats; Rats, Sprague-Dawley; Reactive Oxygen Species; Stem Cells; Structure-Activity Relationship; Vitamin K 1; Vitamin K 2

Lipid emulsions with particle sizes of 190-270 nm were prepared with soybean oil (SO) and a series of hydrogenated castor oils (HCOs) with various oxyethylene numbers, and the effect of oxyethylene numbers of HCOs on the pharmacokinetics of menatetrenone incorporated into the lipid emulsions was studied in rats. Plasma half-life of menatetrenone after administration as the lipid emulsions prepared by HCO with 10 oxyethylene units (SO/HCO10) was similar to that after the administration as SO/egg yolk phosphatides (SO/EYP), but was shorter than that as the lipid emulsions prepared by HCOs with > 20 oxyethylene units (SO/HCO20, SO/HCO30, SO/HCO60, SOHC and SO/HCO100). Menatetrenone incorporated in SO/HCO10, SO/HCO20 and SO/HCO60 was not taken up by the blood cells in vitro, and the plasma level of menatetrenone incorporated in SO/HCO10 was similar to that of triglycerides, suggesting that menatetrenone was not released from the oil particles even after entering the circulation. Menatetrenone uptake by the liver for SO/HCO10 was similar to that for SO/EYP, while those for SO/HCO20, SO/HCO30, SO/HCO60 and SO/HCO100 was less than that for SO/EYP. These findings clearly demonstrate that 20 oxyethylene units in HCOs is the minimum requirement for the prolongation of the plasma circulation time of menatetrenone incorporated in SO/HCOs.

Topics: Animals; Castor Oil; Chemistry, Pharmaceutical; Emulsions; Hydrogenation; Lipids; Male; Rats; Rats, Wistar; Soybean Oil; Vitamin K 2; Water

This work was performed to investigate the role of vitamin K (VK) in the pathogenesis of ossification of posterior longitudinal ligament (OPLL), by analyzing the biochemical markers of the blood samples of OPLL patients and responses of ligament cells derived from OPLL lesion to VK2.. The pathogenesis of OPLL, classified as a form of diffuse idiopathic skeletal hyperostosis, is still unclear. In this study, we investigated the role of menaquinone (VK2) in patients with OPLL (OPLL patients) and the effects of VK2 on ligament cells isolated from OPLL lesion.. Serum levels of intact osteocalcin, glu-osteocalcin, MK-4, -7 (VK2 variants) and other minerals in spot blood samples were measured in 24 OPLL patients and in 24 age-matched control patients (non-OPLL patients). The cultured cells isolated from an OPLL patient were treated with MK-4. Alkaline phosphatase (Al-p) activity and osteocalcin release were measured after 2 weeks of culture.. In the clinical study, the serum MK-4 in male OPLL patients was significantly higher than that in male non-OPLL patients. However, among female patients, the difference was not significant. Although the serum osteocalcin in females was significantly higher than that in males, there was no significant difference between the OPLL and non-OPLL groups. In in vitro study, MK-4 did not increase Al-p activity in the ligament cells isolated from nonossified region of OPLL patient. Osteoblastic activity of the cultured cells was not stimulated by MK-4.. From these results and previous reports, we propose the possibility of the impediment in VK2 metabolism in OPLL patients. The results also implicate the gender tendency in OPLL, because the difference of serum level of MK-4 in OPLL patients was significant only in male.

Topics: Aged; Alkaline Phosphatase; Dose-Response Relationship, Drug; Female; Humans; Longitudinal Ligaments; Male; Middle Aged; Ossification of Posterior Longitudinal Ligament; Osteocalcin; Sex Factors; Time Factors; Vitamin K 2

Previously, we prepared lipid emulsions with soybean oil (SO; 20%) as oil phase and hydrogenated castor oils (HCOs; 2.4%) as emulsifiers (SO(20)/HCOs(2.4)), and found that the lipid emulsions prepared with HCO of 10 oxyethylene units (SO(20)/HCO10(2.4)) were quickly cleared from the plasma, while those prepared with HCO of 20 oxyethylene units (SO(20)/HCO20(2.4)) showed prolonged plasma circulation of the incorporated drug (Ueda et al., 2003). In the present study, the pharmacokinetics of menatetrenone incorporated into SO/HCO10s and SO/HCO20s of different particle sizes (100-280 nm), obtained by altering the SO contents, were examined in rats. The plasma half-lives of menatetrenone as SO/HCO10s were similar to each other, irrespective of particle size, even though the liver uptake of menatetrenone as SO(2.5)/HCO10(2.4) was larger than that as SO(20)/HCO10(2.4). The menatetrenone half-lives were also similar to each other for SO/HCO20s. The pretreatment with dextran sulfate 500,000 (DS500), a suppressor of the reticuloendothelial system (RES), increased the plasma concentration and inhibited the liver uptake of menatetrenone as SO/HCO10s, but not for those as SO/HCO20s. These findings indicated that the particle sizes did not affect the minimum oxyethylene units within HCOs for the prolonged plasma circulation of menatetrenone as SO/HCOs, which was 20.

Topics: Animals; Castor Oil; Fat Emulsions, Intravenous; Hydrogenation; Liver; Male; Particle Size; Rats; Rats, Wistar; Soybean Oil; Vitamin K 2

Vitamin K2 (menaquinone) acts on the bone metabolism. Menatetrenon (MK-4) is a vitamin K2 homologue that has been used as a therapeutic agent for osteoporosis in Japan. Rat models of immobilization induced by sciatic neurectomy are characterized by transiently increased bone resorption and sustained reduction in bone formation. Using such a rat model, we investigated the efficacy of MK-4 on bone loss. Male Sprague-Dawley rats were subjected to unilateral sciatic neurectomy and administered MK-4 for 28 d beginning day 21 after operation. The effect of MK-4 on the immobilized bone was assessed by measuring the bone mineral density of the femur, breaking force of the femoral diaphysis, and bone histomorphometry in tibial diaphysis. The BMD on both the femoral distal metaphysis and diaphysis was reduced by sciatic neurectomy. The administration of MK-4 ameliorated this reduction in a dose-dependent manner. The administration of 30 mg/kg MK-4 ameliorated the reduction in bone strength. An improvement in bone formation was observed following the administration of MK-4. These results suggest that MK-4 has a therapeutic potential for immobilization-induced osteopenia.

Topics: Absorptiometry, Photon; Animals; Bone Density; Bone Resorption; Disease Models, Animal; Dose-Response Relationship, Drug; Immobilization; Male; Osteoporosis; Rats; Rats, Sprague-Dawley; Sciatic Nerve; Tensile Strength; Vitamin K 2

Emerging evidence suggests neuroprotective functions of vitamin K and/or vitamin K-dependent proteins. We investigated the effect of dietary vitamin K on retina aging (thinning). Female Sprague-Dawley rats were maintained from weaning on low (80 microg kg(-1) diet), adequate (500 microg kg(-1) diet) or high (2000 microg kg(-1) diet) levels of vitamin K1 (phylloquinone). Relative concentrations of brain vitamin K associated with these diets were 1: 3.3: 25 (K1) and 1: 2.7: 9.0 (menaquinone-4). Histomorphometry of old (21 month) rats revealed positive associations between vitamin K and thickness of retina layers, especially in the equatorial/peripheral retina. No association of diet and retina thickness was detected among young (6 month) animals. The sparing effect of vitamin K in the retina was most evident in the inner plexiform layer and in the photoreceptor inner and outer segments. Surprisingly, we observed no effect of vitamin K on the age-dependent loss of photoreceptor cells, interneurons or ganglion cells. These data suggest a role for vitamin K in maintaining the aging retina and suggest that the sparing effect of vitamin K does not reflect the survival-promoting (anti-apoptotic) activities of vitamin K-dependent proteins.

Topics: Aging; Animals; Brain Chemistry; Cell Count; Diet; Female; Neurons; Nutritional Status; Photoreceptor Cells; Rats; Rats, Sprague-Dawley; Retina; Vitamin K 1; Vitamin K 2

In view of the fact that a deficient calcium (Ca) intake results in osteoporosis in elderly males, we conducted an animal experiment on aged male Wistar rats given a Ca-deficient diet. The rats were divided into 2 groups according to diet: a Ca-deficient diet group (Ca content, 0.08% to 0.1%) and a regular diet group (Ca content, 0.8% to 1.2%). The Ca-deficient diet reduced bone mineral density (BMD) by approximately 12%. Administration of menatetrenone or elcatonin was able to reverse the reduction in BMD induced by Ca deficiency. The mean estradiol level in sera of rats fed the Ca-deficient diet was significantly increased to 4.3 times that in the regular diet group. However, the increased estradiol concentration was reduced after the administration of menatetrenone or elcatonin. The estrone concentrations in sera of menatetrenone- or elcatonin-treated rats fed the Ca-deficient diet decreased to a level lower than that of animals fed the regular diet. Testicular aromatase cytochrome P450 (P450(arom); estrogen synthetase) activity was significantly increased by 2.4-fold in the Ca-deficient diet group compared to that in the regular diet group, and the aromatase mRNA level was also significantly increased 1.45-fold. Testicular aromatase activity was strongly correlated with aromatase mRNA level and serum estradiol level. These data suggest that the change in testicular aromatase expression might be, in part, a compensatory mechanism for the bone mineral deficiency induced by the Ca-deficient diet in aged male rats.

Topics: Aging; Animals; Aromatase; Bone and Bones; Bone Density; Calcitonin; Calcium; Calcium, Dietary; Diet; Estrogens; Male; Rats; Rats, Wistar; RNA, Messenger; Testis; Vitamin K 2

Menatetrenone (MK-4) inhibits bone resorption and enhances osteoblast-induced mineralization. In this study, we examined whether MK-4 administration had beneficial effects on osteoblast dysfunction and trabecular microstructure as well as on bone volume loss in a rat model of osteopenia. Male Sprague-Dawley rats were neurectomized and administered MK-4 as a daily supplement. On Day 21 after neurectomy, significant bone loss was observed in the positive control rats. MK-4 prevented the decrease in bone mineral density of the distal metaphysis of the femur. The osteoclast surface per bone surface (Oc.S/BS) and the number of osteoclasts per bone perimeter (N.Oc/B.Pm) were reduced and the mineral apposition rate (MAR) decreased in the immobilized rats on Day 42, suggesting suppression of bone turnover. In contrast, administration with a low dose of menatetrenone led to an increase of MAR and bone formation rate (BFR), while Oc.S/BS and N.Oc/B.Pm remained at normal levels. These data suggested that MK-4 reduced the loss of trabecular bone, prevented osteoblast dysfunction to a certain extent, and contributed to preservation of the trabecular microstructure in this rat model of osteopenia induced by sciatic neurectomy.

Topics: Animals; Bone Density; Bone Diseases, Metabolic; Male; Osteoblasts; Rats; Rats, Sprague-Dawley; Sciatic Neuropathy; Vitamin K 2

Oil-in-water (O/W) lipid emulsions are suitable drug carriers for lipophilic drugs; however, the effects of numbers or chains of oxyethylene units within a surfactant molecule such as polyoxyethylene sorbitan esters (PSs) on the biological fate of these lipid emulsions have not yet been clarified. In this study, a series of PSs and soybean oil (SO) were utilized to prepare menatetrenone-incorporated lipid emulsions (SO/PSs), and the biological fate of menatetrenone administered as SO/PSs was studied at a clinical injection volume (0.1 mL kg(-1)) in rats. The plasma concentration and organ uptake of menatetrenone administered as SO/20OE-PSs (PSs with 20 oxyethylene units) was similar to that of SO/egg-yolk phosphatides (SO/EYP). The plasma concentration of menatetrenone was extensively lower for SO/6OE-PSs (PSs with 6 oxyethylene units) and SO/20OE-3FA-PSs (PSs with 20 oxyethylene units and 3 fatty acid chains) than that for SO/EYP, and menatetrenone uptake by the liver and spleen was higher for SO/6OE-PSs and SO/20OE-3FA-PSs, respectively, than those for SO/EYP. Furthermore, menatetrenone uptake by the lungs was also increased for SO/6OE-PS and SO/20OE-3FA-PS with double bonds in the fatty acid moieties of the PSs. These findings suggested that shortening the oxyethylene units or decreasing the oxyethylene chain numbers of emulsifiers resulted in a rapid clearance of the lipid emulsions from the circulation by extensive uptake via the liver, spleen or lungs.

Topics: Animals; Chemical Phenomena; Chemistry, Physical; Emulsions; Excipients; Half-Life; Hemostatics; Injections, Intravenous; Male; Molecular Weight; Oxygen Consumption; Particle Size; Polysorbates; Rats; Rats, Wistar; Soybean Oil; Vitamin K 2

Topics: Aged; Arachidonic Acids; Blood Transfusion; Bone Marrow; Cyclosporine; Hemostatics; Humans; Male; Myelodysplastic Syndromes; Platelet Count; Treatment Outcome; Vitamin K 2

Vitamin K2 (menatetrenone) has been used for the treatment of osteoporosis in Japan. We investigated the effects of ovariectomy (OVX) and vitamin K2 on the calcium (Ca) balance in 20-week-old female Fischer rats. Vitamin K2 (31 mg/kg per day) was given to animals as a dietary supplement. At weeks 4 and 8 after OVX, a Ca balance study was performed for 5 days. The intestinal Ca transport was determined using the everted gut-sac technique at week 9. The Ca balance was poorer in the OVX-control group than in the sham-control group at weeks 4 and 8 after OVX. The Ca balance improved significantly in the vitamin K2 groups as compared with the sham- and OVX-control groups. The intestinal Ca transport decreased due to OVX and was higher in the vitamin K2 administration groups than in the sham- and OVX-control groups, but not to a significant extent. The bone mineral density in the femoral metaphysis as well as the cortical area and cortical thickness in the femoral diaphysis in the OVX-control group were lower than in the sham-control group. The administration of vitamin K2 significantly inhibited an OVX-induced decrease in cortical area and cortical thickness in the femur. These findings suggest that the poor Ca balance observed in ovariectomized rats may be improved by vitamin K2; vitamin K2 may be involved in preventing bone loss in vivo.

Topics: Animals; Biological Transport; Body Weight; Bone Density; Calcium; Feces; Female; Femur; Homeostasis; Intestinal Mucosa; Intestines; Organ Size; Osteoporosis; Ovariectomy; Rats; Rats, Inbred F344; Uterus; Vitamin K 2

Vitamin K is a group name for K1 (phylloquinone) and K2 (menaquinones). Both forms contribute to the tissue vitamin K status. Following intestinal absorption, the serum transport of these lipophilic compounds to their target tissues takes place via lipoproteins. In previous studies we have found that K1 is preferentially accumulated in the liver, whereas menaquinones have a more widespread distribution pattern. Here we have tested whether these differences may be explained by the different liposolubility of the various K-vitamers, resulting in their association with different lipoprotein particles. Six healthy male volunteers received a mixture containing 2 micromol of each of three K vitamers (K1, MK-4, and MK-9) dissolved in corn oil. Blood was obtained at baseline and at different time intervals after intake for the measurement of vitamin K in serum and in the lipoprotein fractions. During the first 4 h after intake all K-vitamins were found to be associated predominantly with the triacylglycerol-rich lipoprotein (TGRLP) fraction. Since the TGRLP fraction is mainly cleared by the liver, this suggests that initially most of the K-vitamins are transported to the liver. In contrast to K1, however, both menaquinones investigated were also found in TGRLP and low-density lipoprotein, whereas MK-4 was even present in high-density lipoprotein. This explains why menaquinones may have a different distribution profile and suggests a relatively large impact of menaquinones on extra-hepatic vitamin K status than generally assumed. Moreover, the very long half-life time of MK-9 in the circulation indicates that it may form a more constant source of vitamin K than are either K1 or MK-4.

Topics: Humans; Lipoproteins; Lipoproteins, HDL; Lipoproteins, LDL; Liver; Postprandial Period; Protein Transport; Triglycerides; Vitamin K; Vitamin K 1; Vitamin K 2

Mice transgenic for granulocyte colony-stimulating factor (G-CSF) exhibit severe osteopenia with an increase of osteoclast number and acceleration of bone resorption in adult mice. To examine the effect of G-CSF overexpression on developing bone, bone mineral density levels were examined from 4 weeks through 36 weeks after birth. Peak bone mass was observed at around 24 weeks of age irrespective of G-CSF expression. Apparent osteopenia was observed as early as 4 weeks of age without detectable developmental retardation in bone length and skeletal structure. Morphological examination confirmed a reduction of cancellous bone and cortical bone at this early stage of life, indicating that overexpression of G-CSF results in apparent osteopenia in developing mice, similar to that in adult animals. The effect of vitamin K2 (menatetrenone) (MK4) on bone phenotypes during development was then examined. Mice were fed chow containing either 0.05 mg MK-4 per 100 g or 20.0 mg MK-4 per 100 g for 12 weeks as the control and experimental diets, respectively. This treatment did not change bone length, irrespective of the type of mouse or diet. Peripheral quantitative computed tomography (pQCT) revealed an increase of in CT value bone of MK4-treated mice. Taken together, these results indicate that overexpression of G-CSF induces an apparent reduction of bone mass and results in osteopenia in developing mice. The bone reduction was partially restored by feeding the mice MK4, suggesting a choice for treatment on the osteopenia induced by G-CSF.

Topics: Animals; Bone and Bones; Bone Diseases, Metabolic; Granulocyte Colony-Stimulating Factor; Mice; Mice, Transgenic; Vitamin K 2

Vitamin K may be important in bone metabolism. Notably, high-dose menaquinone-4 (menatetrenone, MK4) has been reported to reduce ovariectomy (ovx)-induced bone loss in rats and to decrease osteoporotic fracture in postmenopausal women. However, it is unclear whether these beneficial effects reflect a physiologic effect of vitamin K, or indicate direct pharmacologic activity of MK4. To further evaluate this, 60 6-month-old nulliparous Sprague-Dawley rats were randomized by distal femur bone mineral density (BMD) in a 3:1 ratio to ovx or sham groups. The sham and one ovx group's diet contained 1% calcium and 1300 microg/kg of vitamin K1, phylloquinone. Diets of the other two ovx groups were supplemented with 882 mg phylloquinone or MK4 per kilogram chow. Distal femur bone mineral density (DFBMD) in an 8 mm region of interest was measured at baseline, 1 and 3 months postoperatively, utilizing dual-energy X-ray absorptiometry (DXA). All animals were killed at 3 months, their right femurs excised, ex vivo BMD measured by DXA, and biomechanical testing performed. No effect of phylloquinone or MK4 supplementation on ovx-induced bone loss was observed. Specifically, DFBMD declined 10.5%, 9.2%, and 11.2% at 1 month and 14.4%, 10.6%, and 13.9% at 3 months in the ovx control, high phylloquinone, and high MK4 groups, respectively. In addition, serum osteocalcin was elevated by ovx; this was not altered by phylloquinone or MK4. Finally, femoral biomechanical properties were not affected by phylloquinone or MK4. To conclude, in this study, neither high-dose phylloquinone nor MK4 reduced the ovx-associated increase in bone turnover or decline in DFBMD.

Topics: Animals; Bone Density; Bone Remodeling; Female; Femur; Ovariectomy; Rats; Rats, Sprague-Dawley; Vitamin K; Vitamin K 1; Vitamin K 2

The difference between vitamin K metabolism in the liver and that in the bone of vitamin K-deficient rats was examined. After 17 d administration of vitamin K-deficient food, vitamin K in the liver was almost depleted, and prothrombin time (PT) was prolonged. Serum total osteocalcin level was slightly decreased by vitamin K deficiency, whereas serum undercarboxylated osteocalcin level did not change. The level of menaquinone (MK)-4 as well as that of phylloquinone was decreased, but approximately 40 % of the initial level still existed in the femur after the 17 d period. A single-dose administration of vitamin K (250 nmol/kg body weight) markedly increased vitamin K level in the liver but not in the femur. These results suggest that the turnover of vitamin K in the bone is slower than that in the liver, and bone metabolism may be little affected by the short period of intake of vitamin K-deficient food. However, intake of a larger amount of vitamin K is required for its accumulation in the bone than in the liver. Furthermore, the counteracting effect of MK-7 on prolonged PT in vitamin K-deficient rats was found to be higher than phylloquinone or MK-4.

Topics: Animals; Bone and Bones; Cyanoacrylates; Indoleacetic Acids; Liver; Male; Osteocalcin; Partial Thromboplastin Time; Prothrombin Time; Rats; Rats, Sprague-Dawley; Vitamin K; Vitamin K 1; Vitamin K 2; Vitamin K Deficiency

We conducted this study to evaluate the characteristic effects of alfacalcidol (ALF) and menatetrenone (VK) in preventing bone loss using an ovariectomized rat model of osteoporosis. Bilateral ovariectomy (OVX) or sham operation was performed on 10-month-old female Wistar rats. OVX caused a significant decrease in the bone mass and the mechanical strength of the lumbar vertebra as well as the femur 6 months after surgery. VK treatment (30 mg/kg, food intake) required a 6-month period to prevent the bone loss induced by estrogen deficiency, whereas ALF (0.1 or 0.2 mg/kg, p.o.) increased the bone mass and the mechanical strength of the lumbar vertebra as well as the femur in a 3-month treatment period, far above the level in the sham-operated rats. Neither ALF or VK caused hypercalcemia, despite administration for as long as 6 months. By doing a micro-CT analysis of the vertebral trabecular microstructure, it was revealed that ALF treatment increased the interconnections and the plate-like structures and that VK significantly increased the trabecular number. It was also indicated that the increase in spinal strength by ALF treatment was closely associated with improvement of the microstructure, but not VK. The results of histomorphometric analysis showed that ALF caused a significant suppression of bone resorption yet maintained formation in the endocortical perimeter, and also stimulated bone formation in the periosteal perimeter, thereby causing an increase in cortical area. No marked effect of VK on histomorphometric parameters was observed, whereas VK as well as ALF maintained the material strength at femoral midshaft of the normal level, suggesting that VK affected bone quality and thereby prevented the decrease in mechanical strength of femur caused by OVX. In conclusion, it was demonstrated that the two drugs, ALF and VK, differed markedly in their potency and mechanisms for improving bone strength. These results have important implications in understanding the characteristic actions of vitamin K and active vitamin D on bone metabolism.

Topics: Animals; Anticarcinogenic Agents; Biomechanical Phenomena; Bone Density; Bone Resorption; Disease Models, Animal; Female; Femur; Hydroxycholecalciferols; Lumbar Vertebrae; Osteoporosis; Ovariectomy; Rats; Rats, Wistar; Reference Values; Vitamin K 2

The modulation of tissue concentrations of vitamin K by a lutein supplement preserved with natural vitamin E was studied in Fischer 344 rats. Vitamin K is necessary for blood coagulation and may be essential for tissue and bone health. Weanling male rats were fed the AIN-93G diet (control) or modified AIN-93G diets containing 0.3, 0.6, 1.2, 2.4 and 4.8 g supplement/100 g diet for 8 weeks. The supplement contained 5% lutein, 0.22% zeaxanthin and 2.2% natural vitamin E as a preservative. Concentrations of trans-phylloquinone in the plasma (nmol/mmol triglycerides) and heart were significantly reduced (P < or = 0.05) in rats fed the supplement. The reductions in trans-phylloquinone in the heart ranged from approximately 20 to 60% of the control. Concentrations of phylloquinone in the liver were significantly lower in the rats fed the supplement at levels > or = 1.2 g/100 g diet than in the control rats. Ratios of cis/trans phylloquinone in liver and heart increased and concentrations of menaquinone-4 in heart decreased as the dietary level of the lutein supplement increased. The results suggest that the lutein supplement affected the absorption, tissue uptake and/or turnover rate of vitamin K. The presence of other components in the supplement confounded the interpretation of the biological effects of lutein alone on vitamin K metabolism.

Topics: Animal Feed; Animals; Antifibrinolytic Agents; Dietary Supplements; Hemostatics; Liver; Lutein; Male; Myocardium; Rats; Rats, Inbred F344; Tissue Distribution; Vitamin E; Vitamin K; Vitamin K 1; Vitamin K 2

The antinociceptive effect of vitamin K2 (menatetrenone) in diabetic mice was examined using a tail-pressure test. Intraperitoneal injection of menatetrenone (10-100 mg/kg) produced a dose-dependent increase in the nociceptive threshold in diabetic mice. There was no significant difference between non-diabetic and diabetic mice in the menatetrenone-induced changes in the nociceptive threshold. The results suggest the therapeutical usefulness of menatetrenone for treating painful diabetic neuropathy and osteoporosis.

Topics: Analgesics; Animals; Diabetic Neuropathies; Male; Mice; Mice, Inbred ICR; Pain; Pain Measurement; Tail; Vitamin K; Vitamin K 2

Recently, vitamin K has become increasingly of interest in the bone metabolism field because of its role as a cofactor in the carboxylation of osteocalcin. Although the role of osteocalcin is not clear, noncarboxylated osteocalcin is one risk factor in hip fractures. It has been reported that the circulating levels of vitamin K1 in osteoporotic patients were significantly lower than those of age-matched control subjects. In this study, we measured circulating levels of vitamin K1, menaquinone-4 (MK-4) and menaquinone-7 (MK-7) in 23 normal healthy women aged 52-93 years (mean +/- SD: 80.1 +/- 3.5), 13 female patients with vertebral fractures aged 66-93 years (80.3 +/- 7.8) and 38 female patients with hip fractures aged 76-87 years (79.8 +/- 9.2), (all Japanese), in order to make sure whether these vitamin K levels were different in these three groups. Serum circulating levels of MK-4 was undetectable in most subjects (only one out of 74). Appreciable numbers from these three groups had undetectable levels of MK-7 (52% of the control group, 23% of the vertebral fracture group and 24% of the hip fracture group). Eight subjects from the normal control group (35%) and five patients from the vertebral group (38%) had undetectable levels of vitamin K1. We did not find a significant difference in the measurable levels of vitamin K1, MK-4 and MK-7 in patients with vertebral fractures or patients with hip fractures compared to age-matched normal controls. Undetectable levels of measured vitamin K1, MK-4 and MK-7 in most of subjects may significantly affect the results.

Topics: Aged; Aged, 80 and over; Alkaline Phosphatase; Female; Hip Fractures; Humans; Japan; Middle Aged; Osteocalcin; Serum Albumin; Spinal Fractures; Vitamin K 1; Vitamin K 2

Oil-in-water lipid emulsions are promising drug carriers for lipophilic drugs, however, the pharmacokinetics after entering the circulation should be clarified at clinical injection volume in order to utilize them in a clinical situation. In the present study, the standard lipid emulsions, consisting of soybean oil, egg yolk phosphatides and menatetrenone with diameters of about 150 nm, were prepared using a microfluidizer system. The pharmacokinetics of menatetrenone and the oil particles after intravenous injection as standard lipid emulsions at various injection volumes, from the clinical injection volume (0.1 ml/kg) to the experimental injection volume (3.0 ml/kg), were examined in rats. The plasma concentrations of menatetrenone and the oil particles were similar after administration, showing that menatetrenone was not released even after entering the circulation. Menatetrenone was delivered to the liver and spleen at the clinical injection volume, and more menatetrenone was delivered to the liver at clinical injection volume compared with the experimental volume. Moreover, additional information on injection volume-dependency was also obtained from these findings. These results at various injection volumes suggested that the standard lipid emulsions can be utilized as a useful drug delivery system at the clinical injection volume, especially for liver and spleen targeting.

Topics: Animals; Drug Carriers; Fat Emulsions, Intravenous; Hemostatics; Injections, Intravenous; Male; Oils; Phospholipids; Rats; Rats, Wistar; Tissue Distribution; Triglycerides; Vitamin K 2; Water

Topics: Adolescent; Bone Remodeling; Child; Drug Therapy, Combination; Female; Humans; Hydroxycholecalciferols; Hypokinesia; Male; Vitamin K; Vitamin K 2; Weight-Bearing

The effect of the prolonged intake of dietary vitamin K2 (menaquinone-7, MK-7) on bone loss in ovariectomized (OVX) rats was investigated. OVX rats were freely given experimental diets containing the fermented soybean (natto; including 9.4 micrograms MK-7/100 g diet) without or with supplemental MK-7 (containing 14.1 or 18.8 micrograms of MK-7 as total per 100 g diet) for 150 days. Feeding produced a significant elevation of MK-7 concentration in the serum of OVX rats. In this case, the femoral MK-4 content was significantly increased, but MK-7 was not detected in the femoral tissues, indicating degradation of MK-7. Serum gamma-carboxylated osteocalcin concentration was significantly decreased by OVX. This decrease was significantly prevented by the feeding of the natto diets with supplemental MK-7 (18.8 micrograms/100 g diets). OVX caused a significant decrease in femoral dry weight, femoral calcium content, and mineral density. These decreases were significantly prevented by feeding with diets containing natto with MK-7 (total, 18.8 micrograms/100 g diets). This study demonstrates that the prolonged intake of natto dietary including MK-7 has a preventive effect on bone loss induced by OVX. Dietary MK-7 may be useful in the prevention of osteoporosis.

Topics: Animal Feed; Animals; Bone Density; Calcium; Diet; Female; Femur; Fermentation; Glycine max; Humans; Organ Size; Osteocalcin; Osteoporosis, Postmenopausal; Ovariectomy; Rats; Rats, Wistar; Vitamin K; Vitamin K 2

Topics: Acute Disease; Adult; Aged; Female; Humans; Leukemia, Myeloid; Male; Middle Aged; Multicenter Studies as Topic; Myelodysplastic Syndromes; Pilot Projects; Vitamin K; Vitamin K 2

Congeners of vitamin K have been found to inhibit growth in various rodent and human tumor cells, but the mechanisms of the inhibitory action are still not well understood. To investigate the modes of actions of vitamin K, we used several vitamin K analogs and examined their cytotoxic effect for human glioma cell lines RBR17T and U251. The analogs included vitamin K1 (VK1), vitamin K2 (VK2), vitamin K3 (VK3), and geranylgeraniol (GGO) which form an unsaturated side chain of VK2. Cell viability was estimated by MTT assay. DNA fragmentation was demonstrated by gel electrophoresis and flow cytometry. In order to study the mechanism of apoptosis, we measured the changes of intracellular reactive oxygen intermediates (ROI) and Fas/APO-1 expression by flow cytometry. The results showed: (1) VK2, VK3, and GGO inhibited cell growth; (2) VK3 had a more potent cytotoxic effect than VK2, and VK3 enhanced the cytotoxic effect of antitumor agents (ACNU and IFN-beta) in RBR17T cells; (3) VK2, VK3, and GGO induce apoptosis: (4) VK3 increased the expression of Fas/APO-1 although VK2 and GGO did not increase its expression in glioma cells; (5) VK3 increased the production of intracellular ROI. Catalase and reduced glutathione (GSH) inhibited production of intracellular ROI and antagonized inhibition of cell-growth induced by VK3, but failed to antagonize that of VK2 and GGO. We hypothesize that VK3 induces apoptosis by promoting the generation of intracellular ROI and Fas/APO-1 expression. On the other hand, VK2 and GGO induce apoptosis but most likely by some other unknown pathway.

Topics: Antifibrinolytic Agents; Apoptosis; Catalase; Cell Division; Cytotoxins; Diterpenes; DNA Fragmentation; fas Receptor; Glioma; Glutathione; Hemostatics; Humans; Peroxides; Reactive Oxygen Species; Tumor Cells, Cultured; Vitamin K; Vitamin K 1; Vitamin K 2

Menatetrenone, a vitamin K2 with four isoprene units, has been reported to improve osteoporotic bone loss. The purpose of this investigation was to clarify the effect of menatetrenone on the three-dimensional (3D) trabecular microarchitecture in ovariectomized (OVX) rats by using microcomputed tomography (MCT). Forty-two 13-week-old female rats were used and divided into four groups: the OVX (OVX + MK-4) group treated with menatetrenone, the (OVX untreated) group, the sham-operated (Sham + MK-4) group treated with menatetrenone, and the sham-operated group not treated with menatetrenone (Sham untreated) group. OVX rats were fed a calcium-deficient diet. Menatetrenone treatment was begun just after the ovariectomy, and the mean menatetrenone oral intake over the 8-week period was adjusted to 30 mg/kg BW per day. The proximal metaphyseal region of the right tibia was evaluated by dual X-ray absorptiometry (DXA) and MCT. A parametric analysis of the reconstructed trabecular volume was carried out using bone volume fractions, the fractal dimension calculated by the 3D box-counting method, and the connectivity density as determined by topological analysis. Menatetrenone significantly increased the trabecular bone volume, fractal dimension, and connectivity in the OVX + MK-4 group compared with the OVX-untreated group (p < 0.01). Our results suggest that an 8-week administration of menatetrenone protects against the loss of trabecular bone volume and its connectivity when treatment is begun just after the ovariectomy. Despite this apparent protection, it remains unknown whether it is possible to reestablish trabecular connectivity if therapeutic intervention occurs after the trabecular connectivity has been lost.

Topics: Alkaline Phosphatase; Animals; Anthropometry; Body Weight; Bone Density; Calcium; Computer Simulation; Disease Models, Animal; Female; Humans; Osteoporosis, Postmenopausal; Ovariectomy; Rats; Rats, Inbred F344; Reproducibility of Results; Tibia; Tomography, X-Ray Computed; Vitamin K; Vitamin K 2

Several lines of evidence suggest that vitamin K has nutritional and pharmacological effects against bone loss. To clarify effects of vitamin K on bone marrow cells, which contains progenitors of both osteoblasts and osteoclasts, we examined mouse bone marrow cell cultures in the presence of vitamin K(1) (K1) and menatetrenone (MK4), a vitamin K(2) with four isoprene units. Treatment with MK4 but not K1 inhibited the formation of adipocytes and stimulated alkaline phosphatase activity, an early differentiation marker of osteoblast. Although nuclear receptor PPARgamma2 plays a pivotal role in adipogenesis, MK4 had no effects on the expression of PPARgamma2 mRNA and PPARgamma2-dependent transcriptional activity. MK4 inhibited the expression of osteoclast differentiation factor (ODF)/RANK ligand and the formation of osteoclast-like cells induced by 1,25-dihydroxyvitamin D(3). These results suggest that MK4 specifically influences differentiation and functions of bone marrow cells to inhibit adipogenesis and osteoclastogenesis. At the expense of adipogenesis, MK4 might stimulate osteoblastogenesis in bone marrow cells. Therefore, MK4 may favor bone metabolism to spare bone mass as a compound that modulates cellular differentiation and functions in bone marrow in addition to as a nutrient factor.

Topics: Adipocytes; Animals; Antineoplastic Agents; Bone and Bones; Bone Marrow Cells; Calcitriol; Calcium Channel Agonists; Carrier Proteins; Cell Differentiation; Cells, Cultured; Chromans; Gene Expression; Hemostatics; Male; Membrane Glycoproteins; Mice; Osteoclasts; RANK Ligand; Receptor Activator of Nuclear Factor-kappa B; Receptors, Cytoplasmic and Nuclear; RNA, Messenger; Thiazoles; Thiazolidinediones; Transcription Factors; Troglitazone; Vitamin K; Vitamin K 2

The antinociceptive effect of vitamin K2 (menatetrenone) in mice was examined using tail-flick and formalin test. Menatetrenone at doses of 10, 50 and 100 mg/kg, i.p. produced a dose-dependent and significant inhibition of the tail-flick response in mice. Menatetrenone (50 and 100 mg/kg, i.p.) had no significant effect on the duration of the first phase of the formalin-induced flinching. However, menatetrenone (100 mg/kg, i.p.) significantly inhibited the second phase of the formalin-induced flinching. I.p. administration of menatetrenone (100 mg/kg) significantly reduced the duration of nociceptive responses induced by i.t. injection of bradykinin, but not of substance P, prostaglandin E2 or N-methyl-D-aspartate (NMDA). These present data suggest that i.p. pretreatment with menatetrenone produced dose-dependent antinociceptive effect in mice. This effect may be, at least in part, mediated by the inhibition of bradykinin dependent nociceptive transmission in the spinal cord.

Topics: Analgesics; Animals; Behavior, Animal; Bradykinin; Dinoprostone; Injections, Intraperitoneal; Injections, Spinal; Male; Mice; Mice, Inbred ICR; N-Methylaspartate; Substance P; Vitamin K; Vitamin K 2

We studied the effects of vitamin K2 (menatetrenone) on the bone metabolic markers and bone mineral density (BMD) in 17 with low level of BMD cases of severely motor and intellectual disabilities (SMID). Markers of bone formation and resorption as well as BMD were determined before, and 16, 32, and 48 weeks after administration of vitamin K2. After 48 weeks of treatment, serum bone alkaline phosphatase and oseteocalcin levels of significantly increased (p < 0.05, p < 0.01, respectively), but urinary excretion of neither pyridinoline nor deoxypyridinoline decreased. BMD determined by the computed X-ray densitometer method tended to increase in cases with severe motor dysfunction (p = 0.07). There were no side effects. These data demonstrate that vitamin K2 may improve bone formation in patients with SMID.

Topics: Absorptiometry, Photon; Adult; Alkaline Phosphatase; Biomarkers; Bone and Bones; Bone Density; Bone Resorption; Disabled Persons; Female; Humans; Male; Osteocalcin; Osteogenesis; Severity of Illness Index; Vitamin K; Vitamin K 2

Gender differences in relation to vitamin K were investigated in the rat. Hepatic phylloquinone and menaquinone (MK-1 to MK-10) concentrations, gamma-carboxyglutamic acid (Gla) excretion, plasma phylloquinone and percent prothrombin were measured in male and female rats on a chow diet (24.5 ng phylloquinone and 8.8 microgram menadione), and on phylloquinone-deficient and -supplemented purified diets (0.38 and 1400 ng phylloquinone/g, respectively). Mean hepatic phylloquinone concentrations varied with dietary intake and ranged from 6.8+/-9.0 pmol/g in the deficient male, to 171. 1+/-56.9 pmol/g in the supplemented female. Menaquinones accounted for a large proportion of total vitamin K in the liver of males and females with MK-4, MK-6, and MK-10 present in highest concentrations. On the chow and supplemented diets, females had significantly higher MK-4, MK-6, and MK-10 concentrations in their livers (P<0.05). On the phylloquinone-deficient diet (-K1), hepatic phylloquinone, MK-4, and to a lesser extent MK-6 (but not MK-10) were significantly reduced (P<0.05). In the phylloquinone-supplemented male and female groups, which did not receive menadione during the experimental period, MK-4 increased above that in the chow groups suggesting synthesis of MK-4 from phylloquinone which was statistically significant in the female (P<0.01). A significant gender difference (P<0.05) was also observed for urinary Gla excretion with less Gla excreted by the females indicating that females may require less dietary phylloquinone than males of the same body weight.

Topics: 1-Carboxyglutamic Acid; Animals; Chromatography, High Pressure Liquid; Diet; Humans; Liver; Male; Prothrombin; Rats; Rats, Sprague-Dawley; Sex Factors; Vitamin K; Vitamin K 1; Vitamin K 2; Vitamin K Deficiency

The effect of dietary vitamin K2 (menaquinone-7) on bone loss in ovariectomized (OVX) rats was investigated. OVX rats were freely given experimental diets containing menaquinone-4 (MK-4; 12mg/100g diet) or menaquinone-7 (MK-7; 18.1mg/100g diet) for 24 days; MK-4 and MK-7 were equal in molar concentrations. This feeding caused a remarkable increase of MK-4 and MK-7 concentrations in the serum and femur of OVX rats. OVX-induced decrease in the femoral dry weight and femoral calcium content was prevented by the feeding of dietary MK-4 or NK-7. In separate experiments, OVX rats were freely given experimental diets containing the fermented soybean (natto; including 9.4 microg MK-7/100g diet) without or with added MK-7 (37.6 microg/100g diet) for 77 days. Feeding produced a significant elevation of MK-4 and MK-7 concentrations in the serum of OVX rats. In this case, a significant increase in the femoral MK-4 content was observed but MK-7 was not detected in the femoral tissues. OVX-induced decreases in the femoral dry weight and femoral calcium content were significantly prevented by the feeding of diets containing natto with MK-7 added (37.6 microg/100g diets). This study demonstrates that the intake of dietary MK-7 has a preventive effect on bone loss caused by OVX. This effect may be partly caused by MK-4, which is formed by degradation of MK-7.

Topics: Animals; Bone and Bones; Bone Density; Calcium; Disease Models, Animal; Female; Fermentation; Glycine max; Osteocalcin; Osteoporosis; Ovariectomy; Rats; Rats, Wistar; Vitamin K; Vitamin K 2

To clarify how vitamin K2 prevents bone loss in vivo, it was given to ovariectomized 20-week-old rats for 2 weeks. Bone mineral density (BMD) in the whole femur and in 7 specific portions (F1 to F7 from the proximal to the distal end) was determined by dual-energy X-ray absorptiometry, and histomorphometry was also performed in proximal tibial metaphysis. Ovariectomy (OVX) resulted in significant decreases in the BMD in the whole femur and the F1, F2, F6 and F7 portions. Histomorphometrical analysis of the tibia showed that the bone volume/tissue volume (BV/TV), trabecular thickness (Tb.Th) and trabecular number (Tb.N) were decreased, while trabecular separation (Tb.Sp) and osteoclast number/bone surface (Oc.N/BS) were increased by OVX. The parameters for bone formation were not changed by OVX. These data indicate that the bone loss within 2 weeks is due to the enhancement of bone resorption. Vitamin K2 at 50 mg/kg inhibited the decrease in the BMD of the whole femur together with the F6 and F7 portions. Vitamin K2 also inhibited the decrease in Tb.N and the increases in Tb.Sp, Oc.N/BS and osteoclast surface/bone surface (Oc.S/BS) caused by OVX. These results suggest that vitamin K2 prevents bone loss through the inhibition of bone resorption and osteoclast formation in vivo.

Topics: Absorptiometry, Photon; Animals; Bone Density; Bone Resorption; Female; Femur; Hemostatics; Ovariectomy; Rats; Rats, Inbred F344; Tibia; Vitamin K; Vitamin K 2

The importance of vitamin K in bone metabolism has been suggested previously. The binding protein of vitamin K2 (menatetrenone, 2-methyl-3-all-trans-tetraphenyl-1,4-naphthoquinone, menaquinone-4), found in nuclear extract of human osteoblasts, binds to vitamin K1 and K2, but not K3. Since the binding protein does not bind to other steroids or vitamins, such as hydrocortisone, vitamin A, 1,25(OH)2vitamin D3, trolox (a derivative of vitamin E), and warfarin, a specific binding protein to vitamin K1 and vitamin K2 in osteoblasts was suggested. The size of the specific binding protein was revealed to be 6S by sucrose density gradient and about 40,000 daltons by SDS-PAGE. Twenty amino acid residues from the N-terminal were the same as human glyceraldehyde-3-phosphate dehydrogenase (GAPDH), but the 21st residue, alanine, was replaced with serine. The binding protein was precipitated with anti-human GAPDH antibody, and authentic human GAPDH could bind vitamin K2. We propose that the nuclear binding protein for vitamin K2 exists in nuclei similarly to other vitamin receptors and that the molecular structure is very close to human GAPDH.

Topics: Amino Acid Sequence; Animals; Calcium-Binding Proteins; Centrifugation, Density Gradient; Glyceraldehyde-3-Phosphate Dehydrogenases; Humans; Mice; Molecular Sequence Data; Nuclear Proteins; Osteoblasts; Protein Binding; Sequence Analysis; Sequence Homology, Amino Acid; Tritium; Vitamin K; Vitamin K 2

This paper presents a design for randomized clinical trials in which incomplete data are collected on the occurrence of potentially recurrent events through periodic monitoring. In particular, events are assumed to arise according to a point process, but information is available at the times of monitoring only if one or more events has occurred since the preceding monitoring point. The event process is modelled via a piecewise Poisson process, and a proportional rates model is introduced to represent the difference in event rates between treatment groups. The design was developed on the basis of a Wald-type test derived from the generalized estimating equations of Liang and Zeger (Biometrika 73, 13-22 (1986)). Robustification of the variance of the estimator of the treatment effect was considered under a random effects model with a semi-parametric mixture distribution. The design was adopted to address issues which arose in an osteoporosis trial conducted in Japan.

Topics: Calcium; Computer Simulation; Hemostatics; Humans; Osteoporosis; Poisson Distribution; Proportional Hazards Models; Radiography; Randomized Controlled Trials as Topic; Research Design; Spinal Injuries; Spine; Vitamin K; Vitamin K 2

Two experiments were carried out in 4-week-old rats. First, the effect of dietary calcium (Ca) content (0.05, 0.1, 0.2, 0.5 and 1.16%) on bone loss was assessed for 3 weeks. Dry weight of the femur showed a Ca-content-dependent decrease. Significant decrease in body weight and plasma Ca level was observed in the 0.05 and 0.1% Ca diet groups, but not in other groups. Second, the curative effect of V.K2 on bone strength was examined. After being fed a 0.2%-Ca diet for 3 weeks, rats were fed 0.2%- or 0.5%-Ca diets for the next 6 weeks with or without V.K2 treatment. At the beginning and after 3 and 6 weeks of treatment, femurs and lumbar vertebra (L3) were collected. In the 0.2%-Ca group, bone mineral density (BMD) and bone strength in the femur gradually increased, but were much lower than those in the intact group. In the 0.5%-Ca group, both parameters in the femur and L3 were rapidly increased. V.K2 treatment did not affect the BMD or bone strength in the femur at either point. However, the bone strength in L3 in the V.K2 group was higher than that in the 0.5%-Ca group at 3 weeks and in the 0.2%-Ca group at 6 weeks than that in the respective control group. These findings suggest that V.K2 has curative effect on bone strength in the vertebra.

Topics: Animals; Body Weight; Bone and Bones; Bone Density; Calcium; Femur; Lumbar Vertebrae; Male; Rats; Rats, Sprague-Dawley; Vitamin K; Vitamin K 2

The expression of thrombomodulin (TM) and tissue factor (TF) antigens by estradiol and vitamin K2 were studied in human leukemic cell lines including U937 (monoblastic leukemia), NB4 (acute promyelocytic leukemia), and HL60 (acute myeloblastic leukemia). Combined stimulation with estradiol-17beta and menaquinone 4 (MK4), homologue of vitamin K2, showed a remarkable increase of total TM antigen level only in U937 cells among these leukemic cell lines, whereas a single treatment of each agent showed a modest or a moderate increase. A synergistic effect of cotreatment with estradiol-17beta and MK4 was observed in an optimum concentration of 1.0 micromol of estradiol-17beta and 1.0 micromol of MK4. Estrogen receptors were detected only in U937 cells among these cell lines, and the competitive assay with an antiestrogenic agent showed a suppression on TM expression in a dose-dependent manner. In the mean time, concerning expression of TF antigens, if at all, only a very slight decrease was observed by costimulation with estradiol-17beta and MK4 in U937 and NB4 cells, whereas all-trans-retinoic acid (ATRA) showed a remarkable decrease in surface TF antigen levels in NB4 cells and also a modest decrease in U937 cells. These findings suggest that estradiol-17beta would up-regulate TM antigen expression via estrogen receptors and in cooperation with MK4, showing a different mechanism from ATRA.

Topics: Antigens; Antigens, Surface; Cell Division; Estradiol; Flow Cytometry; Hemostatics; Humans; Receptors, Estrogen; Solubility; Tamoxifen; Thrombomodulin; Thromboplastin; Tumor Cells, Cultured; U937 Cells; Vitamin K; Vitamin K 2

Although vitamin K2 is an inducer of the in vitro differentiation of myeloid leukemic cell lines, its clinical efficacy in the treatment of myelodysplastic syndrome (MDS) is unclear. We administered a vitamin K2 analog, menatetrenone, at 45 mg daily to an 80-year-old woman with MDS (refractory anemia) heavily dependent on red-cell transfusions. The patient's pancytopenia gradually improved, and she became transfusion-independent after 14 months. Pancytopenia recurred when menatetrenone was discontinued but recovered again with readministration. Administration of menatetrenone at a dose effective in improving osteoporosis may also be useful in restoring hematopoiesis in MDS patients, possibly by way of inducing differentiation.

Topics: Aged; Aged, 80 and over; Anemia, Refractory; Apoptosis; Calcifediol; Cell Differentiation; Drug Therapy, Combination; Female; Humans; Osteoporosis, Postmenopausal; Vitamin K; Vitamin K 2

It has been recently reported that vitamin K2 (menaquinone-4: menatetrenone, VK2) has an anti-atherogenic effect as well as the ability to produce clotting factors and improve osteoporosis. However, the mechanism by which VK2 acts on atherosclerosis is still unclear. In this paper, we investigated the effects of vitamin K and its side chain on NO production as an anti-atherogenic substance in a cultured vascular system. Treatment of bovine vascular smooth muscle cells (SMC) with VK2 (30 microM) caused a time-dependent (24-72 h) increase in the nitrite (NO2) level in the conditioned medium, but not in bovine vascular endothelial cells. Classical NOS inhibitor (L-nitro arginine) and iNOS-specific inhibitors completely blocked the increased nitrite level induced by VK2 treatment, but D-nitro arginine could not it. Immunostaining and Western blotting analysis showed that VK2 induced iNOS protein in the SMC. VK2 has a naphtoquinone nucleus, which is identical in menadione (VK3), and an unsaturated side chain, which is called geranylgeraniol (GGO). To determine whether the structure of VK2 was related to an increasing nitrite level, we investigated the nitrite level in conditioned medium treated with VK3 or GGO. Neither VK3 nor GGO treatment of SMC increased the nitrite level. In addition, warfarin, an inhibitor of VK2-dependent gamma-carboxylation, did not affect the increased nitrite level induced by VK2 in SMC. In conclusion, VK2 caused NO production through iNOS induction in bovine SMC, that was not related to the structure of VK2, naphtoquinone nucleus or its side chain, independently of gamma-carboxylation.

Topics: Animals; Carboxylic Acids; Cattle; Cells, Cultured; Dose-Response Relationship, Drug; Endothelium, Vascular; Enzyme Induction; Hemostatics; Muscle, Smooth, Vascular; NF-kappa B; Nitric Oxide; Nitric Oxide Synthase; Nitric Oxide Synthase Type II; Vitamin K; Vitamin K 2

Two forms of vitamin K [phylloquinone (K1) and menaquinone-4 (MK-4)] were added to vitamin K-deficient rat food in varying amounts. These diets were given as the sole source of nutrition to rats for one week. The minimal dietary requirements (MDR) to attain maximal prothrombin synthesis were determined to be 0.6 and 6-10 microg/g of food for K1 and MK-4, respectively. The difference between both vitamers could be explained by the limited hepatic accumulation of MK-4. Next, vitamin K was offered to rats at concentrations ranging between 0.6 and 3000 microg/g of food, and the tissue distribution of vitamin K was investigated after one week of administration. Accumulation of K1 and MK-4 was found in all tissues investigated, but both the absolute tissue concentration and the ratio between K1 and MK-4 were tissue-dependent. Highest values were found in liver and in heart, but since the heart contains no gamma-glutamylcarboxylase, the function of vitamin K in this tissue remains obscure. High tissue concentrations of MK-4 were also found in pancreas and testis after a diet containing K1 exclusively. The data indicate that this conversion is tissue-specific, but neither the reason nor its mechanism are known.

Topics: Animal Nutritional Physiological Phenomena; Animals; Carbon-Carbon Ligases; Diet; Dietary Supplements; Liver; Male; Myocardium; Prothrombin; Rats; Rats, Inbred Strains; Vitamin K; Vitamin K 1; Vitamin K 2; Vitamin K Deficiency

To elucidate the role of intestinal bacteria in the conversion of phylloquinone into menaquinone-4 (MK-4) we investigated the tissue distribution of vitamin K in germ-free rats. The rats were made vitamin K deficient by feeding a vitamin K-free diet for 13 days. In a subsequent period of 6 days, phylloquinone and menadione were supplied via the drinking water in concentrations of 10 and 50 micromol l(-1). Menadione supplementation led to high levels of tissue MK-4, particularly in extrahepatic tissues like pancreas, aorta, fat and brain. Liver and serum were low in MK-4. Phylloquinone supplementation resulted in higher phylloquinone levels in all tissues when compared with vitamin K-deficient values. The main target organs were liver, heart and fat. Remarkably, tissue MK-4 levels were also higher after the phylloquinone supplementation. The MK-4 tissue distribution pattern after phylloquinone intake was comparable with that found after menadione intake. Our results demonstrate that the conversion of phylloquinone into MK-4 in extrahepatic tissues may occur in the absence of an intestinal bacterial population and is tissue specific. A specific function for extrahepatic MK-4 or a reason for this biochemical conversion of phylloquinone into MK-4 remains unclear thus far.

Topics: Animals; Diet; Germ-Free Life; Intestines; Male; Rats; Rats, Wistar; Vitamin K; Vitamin K 1; Vitamin K 2; Vitamin K Deficiency

Vitamin K is a group name for a number of prenylated 2-methyl-1,4-naphtoquinones, which may differ in their ability to function as a cofactor for prothrombin biosynthesis. To quantify the bioactivity of different forms of vitamin K, two experimental animal systems are frequently used: vitamin K-deficient rats and anticoagulated rats. In this paper both models are compared, and it is shown that the results obtained depend on the model used. The main reason for this discrepancy is the difference in recycling of vitamin K-epoxide, which results in a 500 times higher vitamin K requirement in anticoagulated rats. Absorption and hepatic accumulation of long chain menaquinones seem to be restricted to a maximum, whereas also the lipophilic nature of long chain menaquinones may hamper the quinone-quinol reduction in anticoagulated animals. If these data may be extrapolated to patients, food items rich in K1 and MK-4 would be expected to influence the stability of oral anticoagulation to a much larger extent than food items primarily containing higher menaquinones.

Topics: 4-Hydroxycoumarins; Absorption; Animals; Anticoagulants; Blood Coagulation; Disease Models, Animal; Male; Prothrombin; Rats; Rats, Inbred Lew; Vitamin K; Vitamin K 1; Vitamin K 2; Vitamin K Deficiency

Topics: Aged; Antineoplastic Combined Chemotherapy Protocols; Female; Humans; Leukemia, Promyelocytic, Acute; Tretinoin; Vitamin K; Vitamin K 2

The potential usefulness of oil-in-water (O/W) lipid emulsions as injectable drug delivery systems was examined. Plasma concentrations of oil particles after intravenous injection of a standard lipid emulsion composed of soybean oil and egg yolk phosphatides were monitored based on the plasma concentrations of phospholipids and triglycerides, and the light scattering intensity of the plasma. Their time profiles were similar to each other, and the oil particle size decreased time-dependently. Pretreatment with dextran sulfate, a known reticuloendothelial system (RES) suppressor, resulted in marked reduction of the plasma clearance of the oil particles and of the time-dependent alteration of oil particle size, suggesting that oil particles were trapped by RES. The lipophilicity of the drug needed for its incorporation in the oil particles even after intravenous injection was found to be clog P > 8, where clog P is the calculated logarithm of the partition coefficient between n-octanol and water. In the case of sudan II (clog P = 5.4), the release from the oil particles was very quick after intravenous injection, resulting in slight alteration in biodistribution when compared with its micellar solution. In contrast, menatetrenone (clog P = 9.5) was selectively delivered to the liver, lungs and spleen, being consistent with the oil particles taken up by RES.

Topics: Anesthesia; Animals; Azo Compounds; Dextran Sulfate; Drug Delivery Systems; Egg Yolk; Fat Emulsions, Intravenous; Hemostatics; In Vitro Techniques; Male; Mononuclear Phagocyte System; Phospholipids; Rats; Rats, Wistar; Scattering, Radiation; Solubility; Soybean Oil; Time Factors; Triglycerides; Vitamin K; Vitamin K 2

The pharmacokinetics and pharmacodynamics of antihemorrhagic vitamin, menatetrenone after intravenous injection as the lipid emulsion, were compared to those as the micellar solutions. Menatetrenone was selectively delivered to the liver, lungs and spleen and retained in them. Hepatic and splenetic concentration at 6 h (C6h) increased 21.6- and 27.1-fold, respectively, and the area under the tissue concentration-time curve up to 6 h (AUC(0-6h)) were 2.3- and 11.4-fold, respectively, when compared with its micellar solution. Antihemorrhagic effect of menatetrenone was assessed using warfarin-induced hypoprothrombinemic rats. The lipid emulsion of menatetrenone decreased the prothrombin time at 6h after intravenous injection more effectively than micellar solution. The dose response curves indicated that the efficacy of the lipid emulsion was 2.4-2.9 times that of a micellar solution, and this was correlated with AUC(0-6h) rather than C6h. The plasma level of clotting factor VII and the hepatic level of descarboxyprothrombin were also recovered more effectively, while no significant differences were noted between the two formulations for the plasma level of factor II or descarboxyprothrombin at the dose levels examined. Although selective delivery of menatetrenone in the liver by the lipid emulsion was due to phagocytosis by non-parenchymal cells, menatetrenone in the whole liver appeared to contribute to recovery from hypoprothrombinemia.

Topics: Analysis of Variance; Animals; Area Under Curve; Biomarkers; Dose-Response Relationship, Drug; Drug Delivery Systems; Emulsions; Factor VII; Hemostatics; Hypoprothrombinemias; Male; Protein Precursors; Prothrombin; Prothrombin Time; Rats; Rats, Sprague-Dawley; Rats, Wistar; Tissue Distribution; Vitamin K; Vitamin K 2

The role of vitamin K in osteocalcin accumulation in the extracellular matrix of normal human osteoblasts in culture was investigated by using a human intact osteocalcin-specific assay system. Human osteoblasts produced osteocalcin by treatment with 10(-9) M 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) for 20 days in culture. With the addition of vitamin K2 (1.5-5.0 microM), osteocalcin accumulation in the extracellular matrix of the osteoblasts was increased, but the osteocalcin content in the conditioned medium decreased, in comparison with that treated with 10-9 M 1,25(OH)2D3 alone. The enhancement of osteocalcin accumulation induced by vitamin K2 was dependent on the duration of the treatment. The vitamin K2 plus 1,25(OH)2D3-induced osteocalcin accumulation was blocked by the addition of warfarin 2 days before the vitamin treatment. At that time, warfarin significantly reduced the mineralization by osteoblasts in vitro. Osteocalcin accumulated in the extracellular matrix was almost completely precipitated by a low concentration of hydroxyapatite, 10 mg/ml. Moreover, the gamma-carboxyglutamic acid (Gla)-containing osteocalcin level was increased by the vitamin K2 plus 1,25(OH)2D3 treatment. These results proved that vitamin K2 increased Gla-containing osteocalcin, which accumulated osteocalcin in the extracellular matrix, and facilitated mineralization in vitro. Vitamin K2 also enhanced the 1,25(OH)2D3-induced osteocalcin mRNA level, but vitamin K2 alone did not show osteocalcin mRNA expression. We thus demonstrated that vitamin K2 enhanced not only the accumulation of Gla osteocalcin, but also the osteocalcin production induced by 1,25(OH)2D3 in human osteoblasts in culture.

Topics: Bone Density; Calcitriol; Carboxylic Acids; Cells, Cultured; Extracellular Matrix; Humans; Osteoblasts; Osteocalcin; Vitamin K; Vitamin K 2; Warfarin

The distributions of phylloquinone (PK) and menaquinone-4 (MK-4) in various tissues were assessed after the oral administration of phylloquinone. Wistar rats were fed a vitamin-K-deficient diet for nine days, fasted for 24 h and then given phylloquinone orally at 4 mg/kg body weight. Rats were sacrificed 0, 6, 12 and 24 h after the administration, and an analysis was made of the vitamin K analogues in the plasma, liver, brain, testis, kidney and spleen. The phylloquinone concentration in plasma and the tissues reached a peak 6 h after the oral administration of phylloquinone. By contrast, the concentration of MK-4 peaked in the liver, plasma, kidney and spleen at 12 h, and in brain and testis at 24 h. This data suggests that the ingested phylloquinone was probably converted into MK-4 within the tissues themselves, rather than via hepatic metabolism. The evidence for this is that, after phylloquinone administration, (i) in each of the tissues, the MK-4 concentration increased much more slowly than that of phylloquinone, and (ii) the MK-4 concentration in the plasma and liver reached only much lower levels than those seen in other tissues.

Topics: Administration, Oral; Animals; Antifibrinolytic Agents; Brain; Chromatography, High Pressure Liquid; Fluorescence; Hemostatics; Kidney; Liver; Male; Rats; Rats, Wistar; Specific Pathogen-Free Organisms; Spleen; Testis; Time Factors; Tissue Distribution; Vitamin K; Vitamin K 1; Vitamin K 2

Geranylgeraniol, a polyprenylalcohol composing the side chain of vitamin K2 (VK2), was previously reported to be a potent inducer of apoptosis in tumor cell lines (Ohzumi H et al, J Biochem 1995; 117: 11-13). We examined the apoptosis-inducing ability of VK2 (menaquinone 3 (MK3), MK4 and MK5) and its derivatives such as phytonadione (VK1), as well as polyprenylalcohols with side chains of various lengths including farnesol (C15-OH; FO), geranylgeraniol (C20-OH; GGO), and geranylfarnesol (C25-OH; GFO) toward leukemia cells in vitro. MK3, MK4, MK5 and GFO (at 10 microM) showed a potent apoptosis-inducing activity for all freshly isolated leukemia cells tested and for leukemia cell lines such as NB4, an acute promyelocytic leukemia (APL)-derived cell line and MDS92, a cell line derived from a patient with myelodysplastic syndrome, although there were some differences depending on the cells tested. In contrast, VK1 showed no effect on any of the leukemia cells. The combination of MK5 plus all-trans retinoic acid (ATRA) resulted in enhanced induction of apoptosis in both freshly isolated APL cells and NB4 cells as compared to each reagent alone. These data suggest the possibility of using VK2 and its derivatives for the treatment of myelogenous leukemias, including APL.

Topics: Apoptosis; Bone Marrow; Diterpenes; Drug Synergism; Farnesol; Flow Cytometry; Gefarnate; Humans; Leukemia; Leukemia, Myelogenous, Chronic, BCR-ABL Positive; Leukemia, Myeloid; Leukemia, Promyelocytic, Acute; Molecular Structure; Myelodysplastic Syndromes; Structure-Activity Relationship; Tretinoin; Tumor Cells, Cultured; Vitamin K; Vitamin K 1; Vitamin K 2

A method is described for the determination of phylloquinone and menaquinones following enzymatic digestion, extraction and a single-stage HPLC technique utilizing post-column reduction with zinc and fluorescence detection. The technique is applicable to both routine compliance control of phylloquinone supplemented infant formula powders (30-150 micrograms per 100 g) and fundamental studies of the K vitamins at endogenous levels in fluid milks (0-5.0 micrograms per 100 g). Analytical figures of merit include a detection limit of 30 micrograms on-column, recoveries greater than 98% for both K1 and MK4, an RSDR of 2.35% (K1) and 2.32% (MK4) and a regression correlation of 0.9932 for a wide range of infant formulas when compared against an alternative HPLC-UV technique. MK4 and 2',3'-dihydrophylloquinone, both with undefined bioactivity, were detected at measurable levels in a range of infant formulas. Although the higher menaquinones were found to be essentially absent in the milk of several species, the significant presence of MK4 relative to K1 has been confirmed in all milks examined, with both dominant forms correlated during early lactation in the cow. These observations suggest an as yet unrecognized physiological function for MK4 in infant nutrition.

Topics: Animals; Chromatography, High Pressure Liquid; Hemostatics; Humans; Infant; Infant Food; Milk; Vitamin K; Vitamin K 1; Vitamin K 2

Vitamin K is involved in the biosynthesis of a number of blood coagulation factors and bone proteins. It has been suggested that the vitamin K requirement of bone tissue is higher than that of the liver. Here we report that in rats very high doses of vitamin K affected neither the blood coagulation characteristics nor the blood platelet aggregation rate. This was observed for both phylloquinone and menaquinone-4. Both vitamers were also tested for their effects on the arterial thrombosis tendency in the rat aorta loop model. The mean obstruction times were prolonged at a high intake of menaquinone-4 (250 mg/kg body weight/day), and shortened after a similarly high phylloquinone regimen. Since (a) both vitamers only differ in their aliphatic side chains; and (b) a similar trend was observed after administration of phytol and geranylgeraniol, we conclude that the modulation of the arterial thrombosis tendency is accomplished by the side chain of vitamin K.

Topics: Animals; Blood Coagulation; Diet; Dietary Fats, Unsaturated; Disease Models, Animal; Disease Susceptibility; Diterpenes; Dose-Response Relationship, Drug; Male; Phytol; Platelet Aggregation; Rats; Rats, Wistar; Thrombosis; Vitamin K; Vitamin K 1; Vitamin K 2; Vitamin K Deficiency

Vitamin K is an essential factor for synthesis of plasma clotting proteins and for site-specific carboxylation of bone Gla protein and other bone matrix proteins. Low vitamin K has been associated with reduced bone mineral density. Warfarin therapy, which inhibits vitamin K-dependent blood-clotting, has been demonstrated to reduce the risk of stroke in nonrheumatic atrial fibrillation. We evaluated vitamin K and bone mineral density in nonrheumatic atrial fibrillation patients who had long-term warfarin therapy after an ischemic stroke.. Sera were collected from 64 patients with non-rheumatic atrial fibrillation and ischemic stroke who had been treated with warfarin, 63 stroke patients without warfarin, and 39 control subjects. All stroke patients in both groups had hemiplegia. Sera were assayed for vitamins K1 and K2, bone Gla protein, and 25-hydroxyvitamin D. Bone mineral density was determined in both second metacarpals.. Serum vitamin K1 concentrations (ng/mL) were lower in treated patients (.234 +/- .177 ng/mL) than in untreated patients (.329 +/- .284) or controls (.553 +/- .164). Bone Gla protein was lower in treated patients' sera (1.241 +/- .799 ng/mL) than in untreated patients (4.476 +/- 3.226). Concentrations of 25-hydroxyvitamin D were lower in both patient groups. Bone mineral density was lower on both sides in treated patients than in untreated patients (P < .0001). Vitamin K1 and bone Gla protein were significantly related to bone mineral density bilaterally in treated but not in untreated patients.. Bone mineral density was significantly lower in stroke patients with long-term warfarin treatment than in untreated patients. Both warfarin-induced reduction in vitamin K function and lowered vitamin K1 concentrations are probable causes of this osteopenia.

Topics: Administration, Oral; Aged; Anticoagulants; Antifibrinolytic Agents; Atrial Fibrillation; Bone Density; Cerebral Infarction; Female; Humans; Male; Middle Aged; Regression Analysis; Vitamin K; Vitamin K 1; Vitamin K 2

Gamma-Carboxyglutamic acid (Gla)-containing protein, synthesized in the presence of vitamin K, has been found in atherogenic plaques, but the pharmacological effect of vitamin K on atherosclerosis is unclear. We examined whether vitamin K2 (menatetrenone) could affect the progression of both atherosclerosis and hypercoagulability in hypercholesterolemic rabbits. Vitamin K2 in daily doses of 1, 10 and 100 mg/kg was given with a 0.5% cholesterol diet for 10 weeks to 8 rabbits each. The plasma levels of total-cholesterol in the vitamin K2-treated groups were clearly lower than that of the hypercholesterolemic control group. The excessive dose of vitamin K2, even at the high dose of 100 mg/kg/day for 10 weeks, did not accelerate the progression of atherosclerosis and did not promote the coagulative tendency in the rabbits. In contrast, the vitamin K2 treatment (1 to 10 mg/kg/day) suppressed the progression of atherosclerotic plaques, intima-thickening and pulmonary atherosclerosis, the increase of ester-cholesterol deposition in the aorta, and both the elevation in plasma factor X level and increase in Hepaplastin test value in the rabbits. These results indicate that the pharmacological dose of vitamin K2 prevents both the progression of atherosclerosis and the coagulative tendency by reducing the total-cholesterol, lipid peroxidation and factor X activity in plasma, and the ester-cholesterol deposition in the aorta in hypercholesterolemic rabbits.

Topics: Animals; Aorta; Arteriosclerosis; Blood Coagulation; Hemostatics; Hypercholesterolemia; Lipid Peroxides; Male; Rabbits; Vitamin K; Vitamin K 2

Filter occlusion during intravenous infusion of injectable menatetrenone in an electrolyte fluid was examined. The menatetrenone concentration and its emulsion droplet size were not changed by in-line filtration. However, particle counts in the admixed solution and examination of scanning electron micrographs of the surface of the filter membrane indicated that particle formation occurred immediately after the admixture of menatetrenone injection with Hicaliq No. 2, but not with 5% glucose injection. This may be due to the interaction of excess lecithin in the emulsion with the electrolyte. Filter occlusion also occurred with the admixture of menatetrenone-free emulsion and Hicaliq No. 2, but not with 5% glucose injection. These findings indicate that injectable menatetrenone should not be admixed with electrolyte fluids.

Topics: Drug Therapy, Combination; Equipment Failure; Flow Injection Analysis; Glucose; Hemostatics; Infusions, Intravenous; Particle Size; Ultrafiltration; Vitamin K; Vitamin K 2

We measured the vitamin K status in postmortem human tissues (brain, heart, kidney, liver, lung, pancreas) to see if there is a tissue-specific distribution pattern. Phylloquinone (K1) was recovered in all tissues with relatively high levels in liver, heart and pancreas (medians, 10.6 (4.8), 9.3 (4.2), 28.4 (12.8) pmol(ng)/g wet weight tissue); low levels (< 2 pmol/g) were found in brain, kidney and lung. Menaquinone-4 (MK-4) was recovered from most of the tissues; its levels exceeded the K1 levels in brain and kidney (median, 2.8 ng/g) and equalled K1 in pancreas. Liver, heart and lung were low in MK-4. The higher menaquinones, MK-6-11, were recovered in the liver samples (n 6), traces of MK-6-9 were found in some of the heart and pancreas samples. The results show that in man there are tissue-specific, vitamin-K distribution patterns comparable to those in the rat. Furthermore, the accumulation of vitamin K in heart, brain and pancreas suggests a hitherto unrecognized physiological function of this vitamin.

Topics: Adult; Aged; Aged, 80 and over; Autopsy; Brain Chemistry; Female; Humans; Kidney; Liver; Lung; Male; Middle Aged; Myocardium; Organ Specificity; Pancreas; Tissue Distribution; Vitamin K; Vitamin K 1; Vitamin K 2

The human vitamin K requirement is not known precisely, but the minimal requirement is often assumed to be between 0.5 and 1 x 10(-6) g/kg body weight. In the present study we addressed the question to what extent circulating vitamin K concentrations are influenced by the form in which the vitamer is consumed. The experimental group consisted of five healthy volunteers who received phylloquinone after an overnight fast. On the first day of three successive weeks the participants consumed 1 mg (2.2 mumol) phylloquinone, either in the form of a pharmaceutical preparation (Konakion), or in the form of spinach + butter, or as spinach without added fat. Circulating phylloquinone levels after spinach with and without butter were substantially lower (7.5- and 24.3-fold respectively) than those after taking the pharmaceutical concentrate. Moreover, the absorption of phylloquinone from the vegetables was 1.5 times slower than from Konakion. In a second experiment in the same five volunteers it was shown that relatively high amounts of menaquinone-4 enter the circulation after the consumption of butter enriched with this vitamer. It is concluded that the bioavailability of membrane-bound phylloquinone is extremely poor and may depend on other food components, notably fat. The bioavailability of dietary vitamin K (phylloquinone + menaquinones) is lower than generally assumed, and depends on the form in which the vitamin is ingested. These new insights may lead to a revision of the recommended daily intake for vitamin K.

Topics: Adult; Biological Availability; Butter; Female; Food; Hemostatics; Humans; Intestinal Absorption; Male; Middle Aged; Spinacia oleracea; Vitamin K; Vitamin K 1; Vitamin K 2

The effect of vitamin K on mineralization by human periosteal osteoblasts was investigated in the absence and presence of 1alpha, 25 dihydroxyvitamin D3 (1,25(OH)2D3). Vitamin K1 and K2, but not vitamin K3, at 2.5 microM enhanced in vitro mineralization when cells were cultured with vitamin K for 20 days after reaching confluence in vitro. Vitamin K2 (2-methyl-3-all-trans-tetraphenyl-1, 4-naphthoquinone : menatetrenone) was the most potent of these vitamin K analogs; it slightly inhibited alkaline phosphatase (ALP) activity. Human osteoblasts were mineralized and showed the enhanced ALP activity on treatment with 10(-9) M of 1,25(OH)2D3 for 20 or 25 days after confluence. Vitamin K2 promoted the 1,25(OH)2D3-induced mineralization, but slightly inhibited the 1,25(OH)2D3-induced ALP activity. Moreover, vitamin K2 enhanced the 1,25(OH)2D3-induced osteocalcin accumulation in the cells and the extracellular matrix (cell layer), but inhibited the osteocalcin content in the medium produced by the 1,25(OH)2D3 treatment. However, vitamin K2 alone did not induce osteocalcin production in the human osteoblasts. On Northern blot analysis, osteocalcin mRNA expression on 1, 25(OH)2D3-treated cells was enhanced by vitamin K2 treatment, but vitamin K2 alone did not induce osteocalcin mRNA expression. Warfarin blocked both the 1,25(OH)2D3-induced osteocalcin production and the accumulation in the cell layer, and also blocked the 1, 25(OH)2D3 plus vitamin K2-induced osteocalcin production and the accumulation in the cell layer. The 1,25(OH)2D3-induced mineralization promoted by vitamin K2 was probably due to the enhanced accumulation of osteocalcin induced by vitamin K2 in the cell layer. However, we concluded that the mineralization induced by vitamin K2 alone was due to the accumulation of osteocalcin in bovine serum on the cell layer, since osteocalcin extracted from the cell layer was not identified by specific antiserum against human osteocalcin, which does not cross-react with bovine osteocalcin. These results suggest that the mechanism underlying the mineralization induced by vitamin K2 in the presence of 1,25(OH)2D3 was different from that of vitamin K2 alone, and that osteocalcin plays an important role in mineralization by osteoblasts in vitro.

Topics: Adult; Alkaline Phosphatase; Animals; Calcification, Physiologic; Calcitriol; Cattle; Cells, Cultured; Humans; Osteoblasts; Osteocalcin; Periosteum; RNA, Messenger; Vitamin K; Vitamin K 2; Warfarin

To study the metabolism of K Vitamins (VK) in the liver, two types of natural VK, phylloquinone (K1) and menaquinone-4 (MK-4), were administered to male Wistar rats orally (P.O.), intravenously (I.V.) and intraperitoneally (I.P.). Blood and a small portion of the liver (and ascites by I.P.) were collected 8 times up to 72 h (P.O.) or 24 h (I.V. and I.P.). A modified assay procedure followed by high performance liquid chromatography (HPLC) was developed to detect VK from small amounts of liver tissue. After oral administration of both K1 and MK-4 (10 mumol/kg-P.O.), their concentrations in the liver increased from 1 h then reached a maximum at 6 h (10 nmol/g v.s. 0.35 nmol/g). After intravenous or intraperitoneal administration of K1 and MK-4 (0.5 mumol/kg-I.V. and I.P.), MK-4 concentrations in the liver reached a maximum faster than those of K1 (1.2 nmol/g -3 h vs. 1.3 nmol/g -0.5 h I.V. and 0.97 nmol/g -6 h vs. 0.47 nmol/g -1 h I.P.). MK-4 also increased in the liver from 6 h to 12 h (0.11 nmol/g -12 h) after oral administration of K1 (P.O.). These results indicate that K1 stays in plasma and liver longer than MK-4 and orally administered K1 might be transformed partially into MK-4 in the liver.

Topics: Animals; Ascites; Injections, Intraperitoneal; Injections, Intravenous; Kinetics; Liver; Male; Rats; Rats, Wistar; Vitamin K; Vitamin K 1; Vitamin K 2

Rats were made vitamin K-deficient by feeding them a diet devoid of vitamin K and by rigorously preventing coprophagy. After one week, circulating prothrombin concentrations were between 5 and 10% of initial values, and various amounts of phylloquinone, menaquinone-4, and menaquinone-9 were given in a single dose either subcutaneously, orally, or colorectally. The relative 'vitamin K activities' of these compounds were assessed by comparing their ability to support prothrombin synthesis after subcutaneous injection. Intestinal and colonic absorption were deduced from the difference between subcutaneous and either oral or colorectal administration of the vitamers. It is concluded that the colonic absorption of all three forms of vitamin K is extremely poor, suggesting that physiological menaquinones in the colon do not contribute substantially to vitamin K status in rats. Furthermore, the stimulation of prothrombin synthesis by menaquinone-9 lasted much longer than that by the two other K-vitamers, resulting in a substantially higher 'vitamin K activity' of menaquinone-9.

Topics: Animals; Biological Availability; Hemostatics; Intestinal Absorption; Male; Prothrombin; Rats; Vitamin K; Vitamin K 1; Vitamin K 2; Vitamin K Deficiency

To clarify the mechanism of absorption and metabolism of K vitamins (VK), we administered phylloquinone (K1) and menaquinone-4 (MK-4) orally to male Wistar rats whose portal and femoral veins were cannulated. Blood was collected 9 times up to 120 min later, and 50 microliters plasma was used for VK analysis by high performance liquid chromatography (HPLC) using a modification of our previous method. There were no significant differences between portal and femoral venous VK concentrations throughout all the experiments. When K1 and MK-4 were administered simultaneously, K1 appeared in plasma at 15 min, and MK-4 at 10 min. The concentration of MK-4 was significantly higher than that of K1 up to 60 min. In contrast to the single administration, the MK-4 concentration was reduced after 60 min. After K1 single administration, the MK-4 was not detected in either portal or femoral venous plasma up to 120 min. These results suggested that (1) the main absorption route of both VK may be the extra-portal pathway, (2) MK-4 is absorbed faster than K1 (3) some interactions may exist between K1 and MK-4 and (4) the intestinal cells might not be the major site to transform K1 into MK-4.

Topics: Administration, Oral; Animals; Antifibrinolytic Agents; Blood Specimen Collection; Chromatography, High Pressure Liquid; Femoral Vein; Hemostatics; Male; Portal Vein; Rats; Rats, Wistar; Time Factors; Vitamin K; Vitamin K 1; Vitamin K 2

Although the effects of vitamin K2 and vitamin K1 on bone metabolism have been reported, the difference between them has not been investigated. We now show the effects of menatetrenone, one of the vitamin K2 homologues, and vitamin K1 on bone resorption. Menatetrenone at greater than 3 x 10(-6) M significantly inhibited the calcium release from mouse calvaria induced by 3 x 10(-10) M of 1,25(OH)2D3 or 10(-7) M of prostaglandin E2, and it also inhibited osteoclast-like multinucleated cell (MNC) formation induced by 10(-8) M of 1,25(OH)2D3 in co-culture of spleen cells and stromal cells at the same concentrations. In contrast, the same doses of vitamin K1 had no effects on bone resorption and MNC formation in these in vitro systems. The inhibitory effect of menatetrenone on the calcium release from calvaria was not affected by the addition of 3 x 10(-5) M of warfarin, an inhibitor of vitamin K cycle. The same concentration of geranylgeraniol, the side-chain component of menatetrenone at the 3-position of the naphthoquinone, inhibited tartrate-resistant acid phosphatase (TRACP) activity and MNC formation to the same degree as menatetrenone. Phytol, the side-chain component of vitamin K1, did not affect TRACP activity at all doses tested, but weakly inhibited MNC formation. Moreover, multi-isoprenyl alcohols of two to seven units, except geranylgeraniol which contains four units, did not effect MNC formation. These findings suggest that the inhibitory effect of menatetrenone on bone resorption is not due to gamma-carboxylation and that the side chain of menatetrenone may play an important role in this inhibitory effect.

Topics: Acid Phosphatase; Analysis of Variance; Animals; Bone Resorption; Calcitriol; Calcium; Cells, Cultured; Dinoprostone; Diterpenes; Giant Cells; Male; Mice; Mice, Inbred ICR; Organ Culture Techniques; Osteoclasts; Phytol; Spleen; Stromal Cells; Structure-Activity Relationship; Vitamin K; Vitamin K 1; Vitamin K 2; Warfarin

[14C]Menaquinone-4 was administered orally once daily at a dose of 4 mg/kg for ten days to female rats of different ages to determine its blood and tissue distribution with particular attention to its distribution in bone. Animals aged 10 and 30 months were either ovariectomized or sham-operated as a control, and young rats aged 7 weeks were used as untreated controls. Blood concentrations of radioactivity at 24h after each dose during repeated administration increased daily and approached a steady rate by the seventh dose. Higher concentrations of radioactivity in blood (plasma) were observed in older animals than in the younger ones, but there was little difference between ovariectomized rats (OVX rats) and sham-operated rats (Sham rats). In tissue samples collected at 1.5 h after administration, the liver, adipose tissue, spleen and adrenals showed higher concentrations of radioactivity than the other organs and the plasma. IN bone tissues, the bone marrow (BM) and cancellous tissue (CT) of the femur showed radioactivity concentrations which were higher than that in the plasma, and these increased during repeated administration. Finally, at 24 h after the last dose, the concentrations of radioactivity in bone tissues of older animals (BM, 5,807.2 ng eq/g; CT, 5,264.8 ng eq/g in OVX rats aged 10 months and BM, 11,479.3 ng eq/g; CT, 4,023.0 ng eq/g in OVX rats aged 30 months) were several times higher than those in younger animals (BM, 2,771.6 ng eq/g; CT, 890.2 ng eq/g in 7-week-old untreated rats). The values in OVX rats were also higher than those in Sham rats. Furthermore, micro autoradiography studies of femur sections from OVX rats indicated that [14C] Menaquinone-4 localized in cancellous tissue where bone is known to be actively remodelled. The concentrations of radioactivity in cancellous tissue and bone marrow of OVX rats aged 10 and 30 months were comparable to the pharmacologically effective concentrations of Menaquinone-4 (10(-6)-10(-5) M) in in vitro studies on bone formation. These findings suggest that orally administered Menaquinone-4 distributes specifically into the bone tissues of ovariectomized rats and this is consistent with its effect as a therapeutic agent for osteoporosis.

Topics: Aging; Animals; Autoradiography; Bone and Bones; Bone Marrow; Carbon Radioisotopes; Drug Stability; Female; Kinetics; Osteoporosis; Ovariectomy; Rats; Rats, Inbred F344; Tissue Distribution; Vitamin K; Vitamin K 2

An important marker for hepatocellular carcinoma is the presence of des-gamma-carboxy (abnormal) prothrombin. However, the molecular basis for the reduced carboxylation of prothrombin is unknown.. Two groups of patients were defined according to the absence (Group I, n = 7) or presence (Group II, n = 8) of des-gamma-carboxy prothrombin. The enzymatic activity of gamma-carboxylase and the total microsomal prothrombin concentration were determined in all tumors. The kinetic parameters for the synthetic peptide Phe-Leu-Glu-Glu-Leu (FLEEL) were measured in eight tumors. The gamma-carboxylase mRNA expression was evaluated by Northern blot analysis in 12 of 15 tumors. In addition, the total vitamin K content (K1, K1 epoxide, and menaquinones 4-10) in 10 tumors was investigated by high performance liquid chromatography.. Concentrations of menaquinones 4-10 were normal in the nontumorous part of the liver but significantly decreased (P = 0.02) in all the tumors (Groups I and II). This decrease was more severe in Group II (P = 0.02). The tumors in Group I had normal or increased gamma-carboxylase activity and increased mRNA expression (P < 0.02) as compared with their nontumorous counterparts. The tumors in Group II were heterogeneous. Five tumors displayed low gamma-carboxylase activity, associated with low mRNA expression in two, whereas two others had high gamma-carboxylase activity and mRNA expression. The concentration of FLEEL at half-maximal velocity was normal in all the tumors examined (Groups I and II), and a relation was found between the level of expression of gamma-carboxylase and the maximal velocity for FLEEL carboxylation in the tumors in Group II (r = 0.98; P < 0.01). The microsomal content of normal prothrombin was within normal limits in all tumors (Groups I and II).. Tumor vitamin K content has a critical role in the synthesis of des-gamma-carboxy prothrombin. Furthermore, the gamma-carboxylase defect, which is observed in some secreting tumors, is the result of the defective gene expression of a normal enzyme and not the consequence of the presence of a competitive inhibitor. It is possible that a 75% reduction in gamma-carboxylase gene expression could take a part in the secretion of des-gamma-carboxy prothrombin, but this mechanism is not predominant.

Topics: alpha-Fetoproteins; Biomarkers; Carbon-Carbon Ligases; Carcinoma, Hepatocellular; Factor V; Gene Expression Regulation, Neoplastic; Humans; Ligases; Liver; Liver Neoplasms; Microsomes, Liver; Protein Precursors; Prothrombin; RNA; RNA, Neoplasm; Vitamin K; Vitamin K 1; Vitamin K 2

The effects of menatetrenone, a vitamin K2 homologue, on osteoclast-like cell formation in mouse bone marrow culture were investigated. After 7 days of incubation, menatetrenone at 10(-6) M, 3 x 10(-6) M and 10(-5) M dose dependently inhibited the tartrate-resistant acid phosphatase-positive multinucleated cell formation induced by 1,25-dihydroxyvitamin D3 (1,25(OH)2D3). The addition of menatetrenone for the last 3 days of the 7-day incubation period was required to inhibit formation of multinucleated cells in response to 1,25(OH)2D3. Moreover, the addition of 1,25(OH)2D3 for the last 3 days was essential for multinucleated cell formation, and this activity was markedly inhibited by the simultaneous addition of menatetrenone. The inhibitory effects of menatetrenone on multinucleated cell formation may contribute to its ameliorative action on bone loss in vivo, and may indicate a new mechanism of vitamin K2 activity in bone metabolism.

Topics: Acid Phosphatase; Animals; Bone Marrow; Bone Marrow Cells; Calcitriol; Cell Differentiation; Cells, Cultured; Giant Cells; Hemostatics; Mice; Osteoclasts; Vitamin K; Vitamin K 2

To evaluate whether the vitamin D3 level in the plasma influences the inhibitory effect of vitamin K2 on bone loss, vitamin K2 (25 mg/kg/day) was administered to ovariectomized (OVX) rats fed a diet containing vitamin D3 (V.D.(+)) or a diet deficient in vitamin D3 (V.D.(-)). After 3 months of treatment, the plasma 25-OH-vitamin D3 (25-OH-D3) level in the V.D(-)-sham group was about 1/3 of that in the V.D(+)-sham group. The plasma calcium level and alkaline phosphatase activity were also significantly lower in the V.D(-)-sham group than in the V.D(+)-sham group. In the V.D(+) group, the plasma 25-OH-D3 level in the vitamin K2 group was about 1.5 times higher than that in the OVX-control group. Ovariectomy resulted in a significant decrease in bone density, bone mineral content (BMC) and bone mineral density (BMD) of femurs in both the V.D(+) and V.D(-) groups. In the V.D(-) group, vitamin K2 had no marked effect on the bone loss. In the V.D(+) group, the bone density and BMD in the mid portion of the femur were significantly increased by vitamin K2 treatment. These findings suggest that the effect of vitamin K2 on bone loss is affected by the vitamin D3 level in the plasma.

Topics: Animals; Bone Density; Bone Resorption; Cholecalciferol; Drug Synergism; Female; Femur; Ovariectomy; Rats; Rats, Inbred F344; Vitamin K; Vitamin K 2

The present study was undertaken to determine whether there is selective tissue distribution of vitamin K in the rat and whether this distribution mirrors the distribution of tissue vitamin K metabolism. The effects of feeding a vitamin K-free diet followed by resupplementation with phylloquinone (K1) were studied. K1 was recovered in all tissues. In K1-supplemented rats, most tissues accumulated K1 relative to plasma K1 with the highest levels in liver, heart, bone, and cartilaginous tissue (sternum). Low K1 levels were found in the brain. In the K1-free rats, relatively high K1 levels were still found in heart, pancreas, bone and sternum. Surprisingly, menaquinone-4 (MK-4) was detected in all tissues, with low levels in plasma and liver, and much higher levels in pancreas, salivary gland and sternum. MK-4 levels exceeded K1 levels in brain, pancreas, salivary gland and sternum. Supplementation with K1, orally and by intravenous infusion, caused MK-4 levels to rise. Some accumulation of K1 and MK-4 in the mitochondrial fraction was found for kidney, pancreas and salivary gland. In the liver the higher menaquinones (MK-6-9) accumulated in the mitochondria. The results indicate that: (1) there is selective tissue distribution of K1 and MK-4, (2) dietary K1 is a source of MK-4. The results also suggest there may be an as yet unrecognized physiological function for vitamin K (MK-4).

Topics: Animals; Diet; Male; Prothrombin Time; Rats; Rats, Wistar; Tissue Distribution; Vitamin K; Vitamin K 1; Vitamin K 2

The effects of menatetrenone (2-methyl-3-tetraprenyl-1,4-naphthoquinone, MK-4) on calcium balance were studied in male Sprague-Dawley rats. Experiment 1: Rats in metabolic cages that were fed a vitamin K-deficient diet and injected daily with latamoxef (100 mg/kg, i.p.) were either treated or untreated with MK-4 for 7 days. Daily food intake, urine volume and feces weight were determined, and calcium concentration in these samples was measured. Calcium balance was calculated as the difference between calcium intake and urinary and fecal calcium excretion. Cumulative calcium balance in the vitamin K-deficient group treated with latamoxef was lower than that in normal rats; this balance was significantly improved by MK-4 (1 and 10 mg/kg, s.c.) administered for 7 days. Experiment 2: Rats were fed a vitamin K-deficient diet containing 4.6% sodium chloride for 6 weeks. MK-4 was administered as a dietary supplement. Forty-eight-hour calcium balance, determined once a week, was significantly reduced compared with that of normal rats after 3 and 5 weeks; the balance was restored dose-dependently by MK-4 administration (1 and 10 mg/kg). Experiment 3: Rats were subjected to the same experimental conditions as experiment 2 for 6 weeks, and intestinal calcium transport was determined using an everted gut-sac technique. Calcium transport was reduced by the high sodium, vitamin K-deficient diet, and this reduction was restored by MK-4 administration (10 mg/kg). These results suggest that MK-4 improves the reduced calcium balance by increasing intestinal calcium absorption in these rats.

Topics: Alkaline Phosphatase; Animals; Blood Coagulation; Calcium; Diet; Hemostatics; Intestinal Absorption; Intestinal Mucosa; Intestines; Kidney Function Tests; Male; Phosphates; Rats; Rats, Sprague-Dawley; Sodium; Vitamin K; Vitamin K 2; Vitamin K Deficiency

Coagulopathy manifest by elevation of the prothrombin time (PT) in patients receiving broad spectrum antimicrobials indirectly suggests a role for intestinal microflora synthesized menaquinone (MK) in the maintenance of normal coagulation. Nonetheless, no direct evidence is available to support this contention.. Our objective was therefore to provide evidence that bacterially produced MK may be absorbed by the distal small bowel of humans.. Using a cell harvester, Staphylococcus aureus (ATCC 29213) was grown in 12-L batches, harvested, and extracted by high performance liquid chromatography (HPLC) to obtain 8 mg of pure MK. Four normal volunteers were placed on a diet severely restricted in vitamin K1 (median 32-40 U/day), and were given warfarin to maintain an International Normalized Ratio of approximately 2.0. On the 10th day of warfarin administration, naso-ileal intubation was performed and 1.5 mg of MK was delivered into the ileum. PT, factor VII, II and serum vitamin K1 levels were monitored throughout the study.. Mean serum vitamin K1 levels were reduced to 30% of the pre-diet value at the time of MK administration. Within 24 h of ileal MK administration, there was a significant (p < 0.05) increase in the factor VII level of 0.28 +/- 0.10 U/ml (mean +/- SEM) and a significant decrease of 2.5 (+/- 0.1) s in the PT, whereas in the control phase (during which no MK was administered), there were no significant changes in the PT or factor VII at corresponding time intervals.. These data provide direct evidence for the absorption of vitamin K2 from the distal small bowel, supporting a definite role for bacterially synthesized vitamin K2 in contributing to the human nutritional requirements of this vitamin.

Topics: Adult; Bacteria; Factor VII; Humans; Ileum; Male; Nutritional Requirements; Prothrombin; Prothrombin Time; Staphylococcus aureus; Vitamin K; Vitamin K 1; Vitamin K 2

A simple and sensitive assay method for a pharmacokinetic study of Menaquinone-4 in dogs was established using HPLC with fluorescence detection following extraction with organic solvent. The quantification limit of this method was 1 ng/ml of plasma. A new oily solution formulation of Menaquinone-4 was administered orally to nonfasted dogs at doses of 0.4, 4 and 40 mg/kg. The plasma concentrations reached maximum levels at 1 to 1.5 h after dosing, and then decreased slowly. AUC values up to 24 h after administration were almost dose-proportional. Menaquinone-4 was also administered to dogs in soft-capsules, for comparison with a conventional hard-capsule oral formulation and an intravenous lecithin formulation. The mean AUC for oral dosing in the soft-capsule formulation was 13.5% of that for intravenous dosing in lecithin, and was 4.6 times higher than that for oral dosing in hard-capsules. Additional dosing in fasted dogs indicated that the AUC in pre-fed dogs was about 4 times higher, suggesting that feeding before giving Menaquinone-4 raises the bioavailability. Overall Menaquinone-4 was absorbed rapidly after administration in non-fasted dogs and dose-proportional bioavailability was obtained among the doses of 0.4 to 40 mg/kg. Higher plasma concentrations were observed after administration in the soft-capsule formulation rather than in the hard-capsule formulation. These findings suggest that the soft-capsule formulation would show a good pharmacokinetic profile for elderly patients with osteoporosis.

Topics: Administration, Oral; Animals; Biological Availability; Capsules; Chromatography, High Pressure Liquid; Dogs; Dose-Response Relationship, Drug; Eating; Vitamin K; Vitamin K 2

Prostaglandin (PG) E2, a potent bone-resorbing agent, is synthesized in osteoblast-like cells. Since vitamin K reportedly plays an important role in bone metabolism, we investigated the effects of vitamin K2 (menatetrenone) on PGE2 production by human osteoblast-like periosteal cells. In cells incubated with menatetrenone (1 microgram/mL = 2.25 x 10(-6) M) for 2 days, PGE2 production was reduced to 50% of that in untreated control cells. This inhibition was dose and time dependent for up to 10 micrograms/mL and 20 days, respectively, and involved two major steps. In one of these menatetrenone at doses of 0.5-10 micrograms/mL dose dependently inhibited the calcium ionophore A23187-induced release of arachidonic acid (AA) from membrane phospholipids, and in the other the conversion of AA to PG was inhibited, as evidenced by the PG-synthesizing activity in the homogenates of menatetrenone-treated cells with AA being lower than that in untreated cells. The inhibitory effect was almost identical to that for PG production. The PG synthesizing activity in cell homogenates was inhibited only by a high concentration of menatetrenone (10 micrograms/mL) when this was added directly. Menatetrenone (1 microgram/mL) also inhibited 52% of the purified PGH synthase activity from a ram seminal vesicle. This study shows that menatetrenone inhibited PGE2 release from cells by inhibiting both PG production steps, AA release from the membrane and PG synthesizing activity with AA. Inhibition of PGE2 production by menatetrenone might be important in improving bone metabolism.

Topics: Adult; Arachidonic Acid; Cells, Cultured; Cyclooxygenase Inhibitors; Dinoprostone; Dose-Response Relationship, Drug; Fibroblasts; Humans; Middle Aged; Osteoblasts; Periosteum; Phospholipases; Prostaglandin-Endoperoxide Synthases; Vitamin K; Vitamin K 2

Rats were made vitamin K-deficient by feeding them a 1:1 (w/w) mixture of a commercial vitamin K-depleted diet and boiled white rice. After one week of treatment the rats had developed severe vitamin K deficiency, resulting in Thrombotest values of 5-10% of the initial values. In this experimental system the efficacy of phylloquinone (K1) was compared with that of menaquinone-4 (MK-4) by measuring the extent to which the Thrombotest was normalized after the administration of varying doses of the respective vitamins. Oral administration of the vitamins showed that the efficacy of K1 was at least two-fold higher than that of MK-4. As comparable results were obtained after subcutaneous administration of the vitamins, we conclude that after oral administration the intestinal absorption had been quick and nearly complete. A less pronounced effect of K1 and MK-4 was found after colorectal administration. For both forms of vitamin K relatively high amounts (well above the physiological concentration) were required before significant effects on the Thrombotest could be observed. Therefore these data demonstrate the importance of sufficient dietary vitamin K consumption in rats. The efficacy of other menaquinones may be investigated in the same experimental animal model system.

Topics: Administration, Oral; Animals; Blood Coagulation Factors; Injections, Subcutaneous; Male; Rats; Rats, Inbred Lew; Rectum; Vitamin K; Vitamin K 1; Vitamin K 2; Vitamin K Deficiency

The effects of menatetrenone, a vitamin K2 homologue, on bone loss induced by ovariectomy in rats were studied in 3 experiments. Menatetrenone was given as a dietary supplement. In experiment 1, at 2 weeks postovariectomy, menatetrenone (10 mg/kg/day given for 2 weeks) inhibited the decrease in bone density of the femoral metaphysis induced by the ovariectomy. In experiment 2, menatetrenone (3 or 30 mg/kg/day given for 6 months) inhibited the decrease in bone strength of the femur and the decrease in calcium and hydroxyproline content of the femoral diaphysis at 6 months postovariectomy. In experiment 3, menatetrenone treatment, at 30 or 100 mg/kg/day for 6 months, protected against the decrease in bone strength and calcium and hydroxyproline content in the bone loss model induced by ovariectomy and calcium-deficient diet. These findings suggest that menatetrenone protects against the bone loss induced by ovariectomy.

Topics: Animals; Bone Density; Calcium, Dietary; Female; Femur; Osteoporosis; Ovariectomy; Rats; Rats, Inbred F344; Rats, Sprague-Dawley; Vitamin K; Vitamin K 2

We studied the effect of menatetrenone, a vitamin K2 homolog, on bone resorption stimulated by interleukin-1 alpha (IL-1 alpha), prostaglandin E2 (PGE2), parathyroid hormone (PTH), and 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3]. Bone-resorbing activity was assessed by measurement of calcium and hydroxyproline in the media and calvariae. IL-1 alpha (0.1-100 U/ml), 1,25-(OH)2D3 (10(-10)-10(-7) M), PGE2 (10(-9)-10(-6) M), and PTH (3 x 10(-8)-3 x 10(-7) M) dose dependently increased the levels of calcium and hydroxyproline in the medium. Indomethacin (10(-6) M) completely inhibited bone resorption induced by IL-1 alpha and partially inhibited bone resorption induced by 1,25-(OH)2D3. However, indomethacin did not affect the action of PGE2 or PTH. Menatetrenone (3 x 10(-6)-3 x 10(-5) M) inhibited the bone resorption induced by IL-1 alpha (2 U/ml), PGE2 (10(-7) M), PTH (3 x 10(-7) M), and 1,25-(OH)2D3 (3 x 10(-10) M) in a dose-dependent manner. Menatetrenone also inhibited the PGE2 production stimulated by IL-1 alpha. These results indicate that menatetrenone may inhibit bone resorption through at least two different mechanisms; one possibly is an inhibitory effect on prostaglandin production.

Topics: Animals; Bone and Bones; Bone Resorption; Calcitriol; Calcium; Dinoprostone; Hydroxyproline; Indomethacin; Interleukin-1; Mice; Mice, Inbred ICR; Organ Culture Techniques; Parathyroid Hormone; Vitamin K; Vitamin K 2

Topics: Detergents; Fermentation; Flavobacterium; Hemostatics; Mutation; Vitamin K; Vitamin K 2

Topics: Blood Coagulation; Hemostatics; Humans; Liver; Liver Diseases; Liver Neoplasms; Postoperative Period; Vitamin K; Vitamin K 1; Vitamin K 2

Topics: Administration, Oral; Animals; Germ-Free Life; Intestines; Male; Mice; Mice, Inbred ICR; Vitamin K; Vitamin K 1; Vitamin K 2; Vitamin K Deficiency

The effect of menaquinone-4 (MK-4, vitamin K2) was studied on des-gamma-carboxy prothrombin (DCP or PIVKA-II) levels in three subjects with vitamin K deficiency and five patients with hepatocellular carcinoma (HCC) with positive DCP. The half-life of DCP in HCC patients after intravenous MK-4 administration (50 mg daily for 14 days) was determined to be 60 hours, identical to that found in vitamin K-deficient subjects who received MK-4. When a single dose of MK-4 (10 mg) was given intravenously to three patients with HCC and elevated DCP, the levels decreased with a reduction rate identical to that in vitamin K-deficient subjects for the first 1 to 3 days, followed by an increase reaching the previous level in 7 to 10 days. Changes in plasma coagulant activity were compared between subjects with vitamin K deficiency and those with HCC before and after a single dose of MK-4 (10 mg). The activity increased in DCP-positive patients with HCC as in vitamin K-deficient subjects who received the same single dose of MK-4. The increase was greater in HCC patients with higher DCP levels. These results suggest that the level of plasma DCP in patients with HCC responded to vitamin K with the same sensitivity as that in vitamin K-deficient subjects. When patients with HCC underwent effective tumor therapy (resection or arterial embolization), the reduction rate (slope of DCP decline) was found to be identical to that in vitamin K-deficient subjects given with MK-4. In patients with less effective therapy, the reduction rate was smaller, or there was an increase in DCP. These observations strongly suggest that sequential measurements of the DCP reduction rate after treatment for HCC are useful for assessing therapeutic effects.

Topics: Aged; Biomarkers; Carcinoma, Hepatocellular; Embolization, Therapeutic; Female; Half-Life; Humans; Liver Neoplasms; Male; Middle Aged; Protein Precursors; Prothrombin; Vitamin K; Vitamin K 2; Vitamin K Deficiency

The metabolic basis for the high vitamin K requirement of chicks compared with rats was investigated. When chicks and rats were fed the same diet, containing 500 micrograms phylloquinone/kg, the total amounts of phylloquinone and its epoxide metabolite found in the liver and plasma were similar in both species. However, phylloquinone 2,3-epoxide was present in high concentrations in chick liver and serum but not in rat liver and serum. This metabolite of the vitamin is normally reduced by a hepatic vitamin K epoxide reductase. The activity of this enzyme in chicks was approximately 10% of that in rats, and the inability of chicks to effectively recycle the epoxide of vitamin K seems to be the major factor in its high requirement. Other species differences in vitamin K metabolism were observed. Much higher concentrations of bacterial menaquinones were present in rat feces compared with chick feces, but neither species had appreciable hepatic concentrations of menaquinones. Chicks, but not rats, were found to have a liver concentration of menaquinone-4 that exceeded that of phylloquinone. This vitamer was present even when its recognized precursor, menadione, was not present in the diet, and the data indicate that chicks convert phylloquinone to menaquinone-4 under the conditions of these experiments. The mechanism of this conversion was not established.

Topics: Alkylation; Animals; Chickens; Diet; Epoxy Compounds; Feces; Liver; Male; Nutritional Requirements; Rats; Vitamin K; Vitamin K 1; Vitamin K 2

A human osteosarcoma cell line, HOS TE85 cells, and a mouse osteoblastic cell line, MC3T3-E1 cells, were cultured for 3 days in a medium containing various concentrations of menaquinone-4 (vitamin K2). As a result, the proliferation of HOS cells was suppressed by vitamin K2 in a dose dependent manner up to 56% of control by 10(-7)M of vitamin K2 and that of MC3T3-E1 cells was suppressed to 84% of control by 10(-6)M of vitamin K2. Vitamin K2 increased alkaline phosphatase activity in both kinds of cells. Warfarin counteracted the effect of vitamin K2 on osteoblastic cell proliferation. Our results show that vitamin K2 modulates proliferation and function of osteoblastic cells by some mechanisms including gamma-carboxylation system.

Topics: Animals; Cell Division; Dose-Response Relationship, Drug; Humans; Mice; Osteoblasts; Osteosarcoma; Tumor Cells, Cultured; Vitamin K; Vitamin K 2; Warfarin

The colonic absorption of menaquinones was examined in rats by the in situ loop method. The overall disappearance of [14C]menaquinone-4 from the colonal loop was approximately 6% at 3 h and much slower than that of menadione. After administration of [14C]menaquinone-4 into the jejunal loop with bile, approximately 17% of unchanged menaquinone-4 was recovered in the lymph after 6 h, but none was found when the administration had been into the colonal loop. Portal absorption of menaquinone-4 from the colon was detected and the unchanged form (approximately 23% of the absorbed radioactivity) was identified in the mesenteric venous blood. When menaquinone-9 was administered into the colon, almost all was recovered from the colonal loop. No transfer of menaquinone-9 from the colon into the lymph or blood was observed at 6 h after dosing. The present observations indicate that only a part of bacterially produced menaquinones is absorbed from the colon via the portal pathway, but the absorption rates of menaquinones decrease markedly with an increase in the number of isoprenoid units.

Topics: Animals; Bacteria; Bile; Colon; Intestinal Absorption; Intestine, Large; Jejunum; Liver; Lymph; Male; Rats; Rats, Inbred Strains; Tissue Distribution; Vitamin K; Vitamin K 2

Liver and serum concentrations of vitamin K active compounds were measured in two groups of (deficient and normal) broilers after administration of phylloquinone 1 mg/kg. Assays were performed by HPLC after extraction and purification of these compounds. The only menaquinone found in the chicken was menaquinone-4. In the deficient group, the chickens exhibited hepatic concentrations of vitamin K1, vitamin K1 epoxide and menaquinone-4 markedly lower than those of the control group. After administration of phylloquinone, vitamin K and vitamin K epoxide levels fell sharply. There is no hepatic storage of vitamin K comparable to that of vitamin A. However, while menaquinone levels were found to be stable in the control group, they rose significantly in the deficient group after vitamin K injection. The question is: is there a transformation of vitamin K into menaquinone and/or is there a preferential utilization of one of the vitamin K active compounds?

Topics: Animals; Chickens; Diet; Female; Injections, Intravenous; Liver; Vitamin K; Vitamin K 1; Vitamin K 2; Vitamin K Deficiency

The correlation between the dose of menatetrenone and the incidence of post-laparotomy peritoneal adhesion in Ryan's model was investigated with the use of rats. In the menatetrenone treated group, the menatetrenone was intramuscularly given in a dosage of 10 mg immediately after closure of the abdominal wound and every 24 hours for two days. In this group, the incidence of ceco-colonic adhesion was 54% (20/37), whereas the incidence in non-treated group was 26% (10/39) (p less than 0.012). Especially in cases with an air-drying time of 1-2 minutes, the difference between incidences of ceco-colonic adhesion in the menatetrenone and that of the non-treated group was high. The former incidence was 61% (17/28) and that of the latter was 21% (6/29) (p less than 0.01). In addition, the incidence of peritoneal adhesion was proportionally dose-dependent to the menatetrenone. In our clinical retrospective study, the incidence of post-gastrectomy adhesive ileus increased with menatetrenone treatment to a significant degree. It is concluded that prophylactic administration of a large dose of menatetrenone should be avoided, because the incidence of post-laparotomy peritoneal adhesion could be increased.

Topics: Animals; Hemostatics; Intestinal Diseases; Intestinal Obstruction; Male; Peritoneal Diseases; Postoperative Complications; Rats; Rats, Inbred Strains; Tissue Adhesions; Vitamin K; Vitamin K 2

We measured menaquinone-4 (MK-4) and MK-4 epoxide concentrations in plasma and liver tissue after intravenous injection of 200 micrograms/kg MK-4 in 42 patients who underwent hepatectomy. They were classified into normal (N; n = 10), chronic hepatitis (CH; n = 12), and liver cirrhosis (LC; n = 20) groups, on the basis of the diagnosis given by the pathologist after examining resected liver specimens. The plasma MK-4 epoxide concentration reached maximum level (Cmax) 60 min after MK-4 injection. The Cmax in groups LC and CH were 85.9 and 126.3 nmol/l, respectively, which is significantly reduced compared with that of group N (184.4 nmol/l) (p less than 0.01 and p less than 0.05, respectively). The MK-4 concentrations in liver tissues of 24 patients 60 min after MK-4 injection were 2.77 in group N, 3.79 in group CH, and 3.83 nmol/g in group LC, and the MK-4 epoxide concentrations were 4.01, 3.09, and 2.62 nmol/g in the respective groups. Consequently, the ratio of MK-4 epoxide to total MK-4 (MK-4 + MK-4 epoxide) in groups CH and LC was significantly lower than in group N (p less than 0.01). It is concluded that the Cmax of MK-4 epoxide after MK-4 injection may serve as an indicator of liver function and that the low ratio of MK-4 epoxide to total MK-4 in the liver shows impairment in vitamin K metabolism.

Topics: Carcinoma, Hepatocellular; Chronic Disease; Female; Hepatectomy; Hepatitis; Humans; Liver Cirrhosis; Liver Neoplasms; Male; Regression Analysis; Vitamin K; Vitamin K 2

We studied whether the administration of vitamin K to mothers could increase the concentration of vitamin K in breast milk and prevent idiopathic vitamin K deficient bleeding in breast-feeding infants. Sixty puerperal women were divided into three groups, the control group, Menaquinone-4 (MK-4) administered group and vitamin K1 administered group. We measured the concentrations of vitamin K1, MK-4 and MK-7 in maternal plasma and breast milk on the fourth day after delivery. In the MK-4 group, the concentrations of MK-4(2.13 ng/ml in plasma, 49.3 ng/ml in milk) were significantly higher than in the control group (0.28 ng/ml, 1.51 ng/ml). In the vitamin K1 group, the concentrations of vitamin K1 (49.0 ng/ml in plasma, 71.6 ng/ml in milk) were significantly higher than in the control group (1.17 ng/ml, 2.41 ng/ml). The concentration rates (milk/plasma ratio) of vitamin K1, MK-4 and MK-7 were 2.52, 5.43 and 0.52 in the control group, 1.60, 40.2 and 0.67 in the MK-4 group and 1.65, 10.8 and 0.71 in the vitamin K1 group, respectively. The concentration rate of MK-4 was higher than that of vitamin K1 and was increased by MK-4 administration. After delivery, the daily concentration of MK-4 in milk was increased from 1.69 ng/ml on the first day to 49.3 ng/ml on the fourth day in the MK-4 group. These results indicate that MK-4 is accumulated and concentrated into breast milk, and continuous MK-4 administration can increase the concentration of vitamin K in milk, preventing idiopathic vitamin K deficient bleeding in infants.

Topics: Female; Humans; Infant; Milk, Human; Postpartum Period; Pregnancy; Vitamin K; Vitamin K 1; Vitamin K 2; Vitamin K Deficiency

Four metabolites of menaquinone-4 [MQ-4] were isolated from rat urine, bile and liver. From rat urine following intravenous or oral administration of [14C]MQ-4, two major metabolites were isolated and their aglycones were identified as 2-methyl-3-(5'-carboxy-3'-methyl-2'-pentyl)-1,4-naphthoquinone [K acid 1] and 3-(3'-carboxybutyl)-2-methyl-1,4-naphthoquinone [K acid 2]. The aglycone of a third minor metabolite isolated from bile was tentatively identified as 2-methyl-3-(15'-carboxy-3',7',11'-trimethyl-2',6',10', 14'-hexadecatetranyl)-1,4-naphthoquinone [MQ-4-COOH]. The structures of the three aglycones, which were excreted into the urine or bile mainly as glucuronide conjugates, indicated that oxidative degradation of the alkyl side chain of MQ-4 had occurred by omega- and beta-oxidation. In addition, 2,3-epoxy-MQ-4 was identified in the liver of rats which were pretreated with warfarin and then dosed with [14C]MQ-4.

Topics: Animals; Bile; Chemical Phenomena; Chemistry, Physical; Gas Chromatography-Mass Spectrometry; Hydrolysis; Liver; Male; Methylation; Rats; Rats, Inbred Strains; Scintillation Counting; Vitamin K; Vitamin K 2; Warfarin

Following intravenous administration to rats of all-trans [14C]menaquinone-4 solubilized with purified soybean lecithin [L] or with HCO-60 [H], we examined the effect of the solubilizers on the distribution and excretion of menaquinone-4 [MQ-4]. The level of radioactivity in the liver after dosing with L was about 2 times higher than in dosing with H, and a similar result was obtained in the hepatic microsomal fraction, a target of MQ-4. The rate and amount of biliary excretion of radioactivity after dosing with L were greater than in dosing with H. In addition, the uptake of [14C]MQ-4 by the isolated perfused rat liver was greater with L than H, consistent with the in vivo observation. Further, upon incubation of L or H with hepatic microsomes, the MQ-4 metabolizing enzyme was more highly active toward L than H. These results show that L is more easily transported to the target region, and more rapidly metabolized and excreted into the bile than H, suggesting that the lecithin-solubilized preparation of MQ-4 may be more effective clinically.

Topics: Animals; Bile; Castor Oil; In Vitro Techniques; Liver; Male; Microsomes, Liver; Particle Size; Perfusion; Phosphatidylcholines; Rats; Rats, Inbred Strains; Scintillation Counting; Surface-Active Agents; Tissue Distribution; Vitamin K; Vitamin K 2

The transfer of menaquinone-4 (vitamin K2(20] to the fetus and milk was studied in pregnant and lactating rats, respectively, after oral administration (4 mg/kg) of [3'-14C]menaquinone-4. Intestinal absorption of menaquinone-4 was rapid and the highest level of radioactivity in each tissue except guts of fetal rats was observed at 4h after dosing. The level in the fetal homogenate was low. At that time, the concentration of menaquinone-4 in the fetal liver was 84 ng/g, corresponding to 9% of the value found in the placenta. Therefore, we conclude that the transfer of menaquinone-4 to the developing rat fetus is restricted by the blood-placenta barrier, but that a sufficient amount of menaquinone-4 (more than the essential amount of vitamin K to ensure full carboxylation) can be transferred into the fetal liver. It was also observed that the radioactivity was transferred to milk after oral administration to lactating rats. Milk/blood concentration ratios at 6 and 24h after dosing were 13.8 and 65.1, respectively. The elimination half-life of radioactivity in milk was about 17h. Eighty-four percent of milk of radioactivity was due to menaquinone-4. These results suggest that the prophylactic maternal oral administration of menaquinone-4 may be efficacious for a prophylaxis of neonatal and infantile vitamin K deficiency.

Topics: Animals; Animals, Newborn; Biological Transport; Female; Fetus; Mammary Glands, Animal; Maternal-Fetal Exchange; Milk; Placenta; Pregnancy; Rats; Tissue Distribution; Vitamin K; Vitamin K 2; Whole-Body Irradiation

A highly sensitive method for measuring endogenous vitamin K1, menaquinone-4 (which is one of the K2 vitamins) and vitamin K1 2,3-epoxide in human plasma was developed, based on high-performance liquid chromatography with coulometric reduction and fluorimetric detection, following extraction from plasma and purification on a Sep-Pak silica cartridge. The detection limits of vitamin K1, menaquinone-4 and vitamin K1 2,3-epoxide were 5, 5 and 8 pg per injection for the standard substances and 30, 30 and 50 pg/ml in human plasma, respectively.

Topics: Adult; Chromatography, High Pressure Liquid; Female; Humans; Male; Middle Aged; Oxidation-Reduction; Perchlorates; Sex Factors; Sodium Compounds; Spectrometry, Fluorescence; Vitamin K; Vitamin K 1; Vitamin K 2

Topics: Adolescent; Adult; Aged; Female; Hemostatics; Humans; Infusions, Parenteral; Male; Middle Aged; Surgery, Oral; Vitamin K; Vitamin K 2

Topics: Animals; Antifibrinolytic Agents; Chick Embryo; Chickens; Eggs; Female; Metabolism; Naphthoquinones; Research; Vitamin K; Vitamin K 1; Vitamin K 2