ascorbic-acid and ascorbate-2-phosphate

We have devised a medium which supports the continuous growth of hepatocytes without losing their replicative potential and differentiation capacity for a longer period. The medium HCGM, contains four key substances in addition to foetal bovine serum. They are epidermal growth factor, nicotinamide, ascorbic acid 2-phosphate and dimethylsulphoxide. When a non-parenchymal cell fraction containing small hepatocytes and non-parenchymal cells was cultured in HCGM, small hepatocytes grew clonally and differentiated into cells expressing either mature hepatocyte marker proteins or biliary cell marker proteins. Thus, for the first time, we showed the presence of a small compartment of bipotent and highly replicative clonogenic hepatocytes in the rat adult liver. HCGM also supported the growth of stellate cells (Ito cells) which were in the original preparation, suggesting the important role of stellate cells for the successful cultivation of hepatocytes. Together, these results suggest that a microenvironment is produced as a result of cooperative interactions between hepatocytes and stellate cells: one which stimulates the growth and differentiation of clonogenic hepatocytes.

Topics: Animals; Ascorbic Acid; Cell Differentiation; Cell Division; Clone Cells; Culture Media; Dimethyl Sulfoxide; Epidermal Growth Factor; Liver; Niacinamide; Rats

The roles of extracellular matrix (ECM) components such as collagen, elastin, proteoglycan, and adhesive glycoprotein in the regulation of cell morphology, proliferation, and tissue formation were investigated. On a basement membrane gel, the perisinusoidal stellate cells (lipocytes, fat-storing cells, Ito cells) formed a mesh-like structure, proliferated slowly, and synthesized only a small amount of collagen. On polystyrene or type I collagen-coated culture dishes, the stellate cells spread well and extended cellular processes. The stellate cells proliferated better and synthesized more collagen on type I collagen-coated dishes than on polystyrene dishes. Co-cultures of hepatic parenchymal cells and fibroblasts formed a three-dimensional hepatic cord-like architecture in the medium supplemented with a long-acting vitamin C derivative, L-ascorbic acid 2-phosphate (Asc 2-P). Skin fibroblasts formed a three-dimensional dermis-like structure in the medium supplemented with Asc 2-P. Asc 2-P stimulated collagen synthesis of these cells. The stimulative effects of Asc 2-P on tissue formation were suppressed when collagen synthesis in these cells was inhibited. These data indicate that ECM can regulate cell morphology, proliferation and tissue formation. Regulation of cellular functions in other tissues such as mammary gland, thymus and prostate by ECM was also reviewed, and the molecular mechanisms of the regulation are discussed.

Topics: Adipocytes; Animals; Ascorbic Acid; Cell Division; Cells, Cultured; Extracellular Matrix Proteins; Fibroblasts; Humans; Liver; Skin

Ascorbic acid (vitamin C) is an antioxidant that is widely used in cosmetics in skincare products. Due to the excessive low stability of ascorbic acid in cosmetic formulations, the stabilized ascorbic acid derivative, magnesium ascorbyl phosphate (MAP) was formulated as vesicular carriers; ethosomes and niosomes. The aim was to deliver MAP at the intended site of action, the skin, for sufficient time with enhanced permeation to get an effective response. Ethosomes were formulated using a full 3

Topics: Administration, Cutaneous; Adult; Animals; Antineoplastic Agents; Ascorbic Acid; Chemistry, Pharmaceutical; Dose-Response Relationship, Drug; Drug Carriers; Drug Liberation; Drug Stability; Female; Gels; Humans; Liposomes; Male; Melanosis; Middle Aged; Neurocutaneous Syndromes; Rats; Surface Properties

Antioxidant containing cosmeceuticals are commonly prescribed products in treating wrinkles and revitalizing the skin. The aim of this study was the comparative evaluation of physicochemical stability and clinical anti-wrinkle efficacy of transdermal emulgel preparations of sodium ascorbyl phosphate (SAP) and ascorbic acid (AA) on human volunteers.. Emulgel preparations containing 5% of (SAP) and or (AA) were prepared. HPLC analysis was performed for stability evaluations. Clinical anti-wrinkle efficacy of the formulations was examined on human healthy volunteers in crow's feet area. Elasticity and digital images were recorded before and after treatment.. Formulations with added antioxidants and kept in the refrigerator exhibited better stability characteristics. Two-sided blind study and placebo-controlled study showed that both actives were effective in wrinkles depth reduction and also elasticity enhancement but statistically significant difference in the efficacy of the products was not observed.. Formulations containing (AA) and or (SAP) both improved elasticity and wrinkles of the skin almost by the same extent, and it is necessary to add antioxidant stabilizing agents to both preparations to reach a desired stability.

Topics: Administration, Cutaneous; Ascorbic Acid; Healthy Volunteers; Humans; Skin Aging

Little is known about the anti-pigmenting effects of whitening agents on solar lentigos (SLs), which comprise ~ 60% of hyperpigmented facial lesions of Asian subjects. Lotions with or without 6% L-ascorbate-2-phosphate trisodium salt (APS) [test lotion (TL) and placebo lotion (PL), respectively] were applied twice daily for 24 weeks in a double-blind half-face study of 27 Japanese females with SLs on both sides of their faces. Pigmentation scores were evaluated using a photo-scale and the skin colors were assessed using a color difference meter and a mexameter for SLs and the non-lesional surrounding skin (NLS). Although the pigmentation scores were not significantly different between the TL and PL-treated SLs after 24 weeks, the L values of TL-treated SLs and NLS increased significantly with a significantly higher △L value in SLs than in NLS. In contrast, the L values of PL-treated SLs and NLS remained unchanged after the treatment. The number of subjects with > 2.0 △L was 7 of 27 (TL) and 0 of 27 (PL) in SLs and 3 of 27 (TL) and 0 of 27 (PS) in NLS. In contrast, the melanin index in TL-treated SLs and NLS significantly decreased with a significantly higher △melanin index in SLs than in NLS. Similarly, the melanin index of PL-treated SLs and NLS were significantly decreased with a significantly higher △melanin index in SLs than in NLS. These findings strongly indicate that APS has a weak but significant anti-pigmenting effect on SLs and a significant whitening effect even on normally pigmented healthy skin.

Topics: Administration, Cutaneous; Ascorbic Acid; Asian People; Double-Blind Method; Female; Humans; Japan; Lentigo; Melanins; Melanocytes; Skin Lightening Preparations; Skin Pigmentation; Time Factors; Treatment Outcome

Topics: Antioxidants; Ascorbic Acid; Drug Stability; Drug Synergism; Emulsions; Face; Female; Healthy Volunteers; Humans; Sebum; Single-Blind Method; Skin; Skin Care

Reactive oxygen species might be associated with the onset and progression of gingival inflammation. The aim of this study is to investigate the effect of a dentifrice containing L-ascorbic acid 2-phosphate magnesium salt (APM), a long-acting ascorbic acid derivative with antioxidant properties, on gingival inflammation.. The clinical effects of APM were investigated in a multicenter, randomized, parallel-group, controlled clinical trial comprising 300 individuals with gingivitis. Half of the participants were given an APM-containing dentifrice and half were given a control dentifrice. The primary outcome was the gingival index (GI) at 3 months. Secondary outcomes included gingival redness as an indicator of the degree of local gingival inflammation, gingival bleeding as a measure of the gingivitis severity index, and total antioxidant activity of the saliva.. Under the intent-to-treat analysis, GI did not significantly differ between the groups (P = 0.12). However, under the per-protocol analysis, GI was significantly lower in the APM group (P = 0.01) than in the control group. In the APM group, gingival redness was significantly lower, and the difference from the baseline gingivitis severity index was significantly greater (P = 0.04 and P = 0.02, respectively). The total antioxidant activity of the saliva was significantly higher in the APM group (P = 0.03). The incidence of adverse events did not significantly differ between the groups (P > 0.15).. These findings indicate that the regular application of an APM-containing dentifrice could reduce gingival inflammation.

Topics: Adult; Antioxidants; Ascorbic Acid; Dentifrices; Double-Blind Method; Female; Follow-Up Studies; Gingival Hemorrhage; Gingivitis; Humans; Intention to Treat Analysis; Male; Middle Aged; Oxidation-Reduction; Periodontal Index; Safety; Saliva; Treatment Outcome; Young Adult

Melasma is an acquired disorder of hypermelanosis of great psychosocial concern. The treatments with various conventional therapies are often unsatisfactory. Lasers and light sources have been used to treat pigmented lesions, but in Asian skin with higher melanin content, such treatments may be challenging.. To determine the effectiveness of treating melasma with a combination of topical 5% magnesium ascorbyl phosphate (MAP) and fluorescent pulsed light (FPL).. Patients of skin types III-V with refractory melasma were treated for 12 weeks with topical application of 5% MAP and three sessions of FPL (570-950 nm) at 3, 6, and 9 weeks (fluence 12-14 J/cm(2) , pulse width 15 ms, and spot size 3 cm(2) ). They were followed up for another 12 weeks to assess the persistence of treatment benefit. Digital photographs of the patients were taken at each visit. Treatment efficacy was determined by calculating mean melasma area and severity index (MASI) at the beginning and then at weeks 6, 12, and 24. The subjective assessment was done by comparing pre-treatment and post-treatment photographs by an independent observer and self-assessment by patients using four-point scoring scale (1, poor, 2, fair, 3, good, and 4, excellent).. Sixty-five patients completed the study. The baseline mean MASI score of 14.80 decreased to 4.53 at the 12th week (end of treatment) and 6.35 at the 24th week (end of follow-up). The overall regression of mean MASI at these end-points was 69.3% and 57% (P < 0.01). The pre- and post-treatment photographic evaluation by independent observer and patients' self-assessment at the 12th week showed good to excellent response (scores 3 and 4) in 52.3% and 44.6% cases, respectively. No significant adverse effects of treatment were noted.. Combination of 5% MAP with FPL is effective, well tolerated, and safe in treating refractory melasma in Asian patients but for persistent improvement, maintenance treatments would be required.

Topics: Administration, Topical; Adolescent; Adult; Antineoplastic Agents; Ascorbic Acid; Asian People; Combined Modality Therapy; Female; Fluorescence; Follow-Up Studies; Humans; Male; Melanosis; Phototherapy; Treatment Outcome; Young Adult

Uneven skin pigmentation is a significant cosmetic concern, and the identification of topically applicable molecules to address this issue is of general interest. We report that the tetrapeptide PKEK (Pro-Lys-Glu-Lys) can exert skin whitening effects based on one in vitro and four double-blinded vehicle-controlled in vivo studies. (i) Treatment of human keratinocytes with PKEK significantly reduced UVB-stimulated mRNA expression of interleukin (IL)-6, IL-8 and TNF-α and, most importantly, proopiomelanocorticotropin (POMC), i.e. a gene encoding the pigmentation-inducing soluble mediator α- (α-MSH). (ii) PKEK treatment significantly inhibited UVB-induced upregulation of genes encoding for IL-1α, IL-6, IL-8, TNF-α as well as POMC and tyrosinase in 10 healthy volunteers pretreated with PKEK for 4 weeks once daily. (iii) In a study enrolling 39 Caucasian women, facial pigment spots significantly faded after 6 weeks when PKEK was combined with the skin whitener sodium ascorbyl phosphate (SAP), whereas PKEK or SAP alone led to less pronounced fading of the pigment spots. (iv) Addition of PKEK enhanced the skin whitening potency of a SAP-containing preparation if applied for 8 weeks to the back of hands of 19 Caucasians. (v) 27 Japanese women were treated on their faces twice daily with an SAP only or a PKEK+SAP-containing formulation for 8 weeks. Application of PKEK+SAP significantly reduced skin pigmentation by 26% and by 18% according to SCINEXA score. We demonstrate that PKEK has the capacity to reduce UVB-induced skin pigmentation and may be suited to serve as a skin tone-modulating agent in cosmetic products.

Topics: Adult; Aged; Ascorbic Acid; Asian People; Cells, Cultured; Colorimetry; Double-Blind Method; Female; Gene Expression; Humans; Interleukin-1beta; Interleukin-6; Interleukin-8; Keratinocytes; Male; Middle Aged; Monophenol Monooxygenase; Oligopeptides; Pro-Opiomelanocortin; Skin; Skin Aging; Skin Pigmentation; Treatment Outcome; Tumor Necrosis Factor-alpha; Ultraviolet Rays; White People

Antioxidants are becoming increasingly important in the treatment of skin disease. In addition to their known anti-inflammatory effects, antioxidants may act to prevent the oxidation of sebum which has been proposed to be comedogenic in acne patients. Sodium L-ascorbyl-2-phosphate (APS) is a stable vitamin C derivative and highly effective antioxidant that has demonstrated efficacy in acne in open label studies.. To evaluate the efficacy and safety of APS 5% lotion for the treatment of acne in a blinded controlled study.. A total of 50 subjects were randomized in a double-blind controlled trial to receive APS 5% lotion or vehicle for 12 weeks. Evaluation included an Investigator's Global Assessment Score, a Subjects' Global Assessment Score, lesion counts, cutaneous tolerability, and adverse events.. APS 5% lotion demonstrated statistically significant improvement when compared to vehicle in all of the parameters measured. The adverse event frequency and cutaneous tolerability profile for APS 5% lotion were similar to vehicle.. Adjunctive topical or oral agents and their impact on acne were not studied in this trial.. This study demonstrates that 5% sodium L-ascorbyl-2-phosphate is efficacious as monotherapy for the treatment of acne. APS 5% lotion offers a novel addition to our current acne armamentarium.

Topics: Acne Vulgaris; Administration, Cutaneous; Adolescent; Adult; Antioxidants; Ascorbic Acid; Dermatologic Agents; Double-Blind Method; Female; Humans; Male; Ointments; Phosphates; Severity of Illness Index; Treatment Outcome

Acne vulgaris impairs the appearance of an individual and causes psychological irritation. Inflammatory acne lesion is caused by multifactor incorporates in each step of acne pathogenesis. In an attempt to archive inflammatory lesion treatment with the promise of prevention of acne vulgaris, randomized and double-blind studies on the comparison of the efficacies of topical formulations containing 5% sodium ascorbyl phosphate (SAP) and 0.2% retinol, separately as well as in combination application, were conducted. The resulting data showed that SAP reduced the inflammatory lesion by 20.14% and 48.82% within 4 and 8 weeks respectively. Application of the formulation containing retinol slightly improved the treatment efficacy as the lesion reduced by 21.79% and 49.50% after 4 and 8 weeks respectively. The combination treatment significantly reduced the inflammatory lesion by 29.28% after 4 weeks and 63.10% after 8 weeks of application. The most effective treatment was by using the combination of 5% SAP and 0.2% retinol, which incorporated the synergistic effects on lipid peroxidation and sebaceous gland function in addition to the enhancement of SAP permeability by the desquamation of stratum corneum influenced by retinol, keratin plug removal and anti-inflammatory effect of retinol. This study promises for the development of cosmetic products to overcome aesthetic and psychological problems caused by acne vulgaris.

Topics: Acne Vulgaris; Administration, Topical; Adolescent; Adult; Ascorbic Acid; Double-Blind Method; Drug Therapy, Combination; Female; Humans; Male; Vitamin A; Young Adult

The aim of this study was to quantify and confirm the efficacy of cosmetic formulations for hyperpigmented spots over a wide area of the face using a high quality digital imaging system that we developed.. A total of 120 Japanese female volunteers aged 25-60 years with solar lentigines were treated for 6 months with a skin lightening moisturizer (SLM, thereafter) containing 3% magnesium ascorbyl phosphate on one side of the face and vehicle on the other side. During the course of the study, facial images were collected by the image analysis to measure facial skin colour and the total area of hyperpigmented spots. The evaluation was also conducted by visual grading. Measurements were made before and 1, 3, and 6 months after starting the application, and again 6 months after discontinuing the treatment. Three similar clinical studies using the same protocol were repeated for up to one-month to confirm the reproducibility of the results and to examine seasonal variation.. SLM significantly reduced the total area of hyperpigmented spots (P < 0.005) after one month of treatment compared to the vehicle, with no significant variation in facial skin colour tone in the areas outside the hyperpigmented spots. The results of the visual grading were consistent with those obtained by image analysis. The total area of hyperpigmented spots 6 months after discontinuing the treatment had returned to pre-treatment levels. The reproducibility of these clinical results was demonstrated in three follow-up studies.. A high-resolution digital imaging method, combined with a split-face clinical protocol is sensitive enough to prove that SLM readily reduces hyperpigmented spots, while maintaining normal facial skin colour.

Topics: Adult; Ascorbic Acid; Face; Female; Follow-Up Studies; Humans; Hyperpigmentation; Image Enhancement; Image Interpretation, Computer-Assisted; Japan; Middle Aged; Pharmaceutical Vehicles; Reproducibility of Results; Severity of Illness Index; Treatment Outcome

The coordination chemistry between phosphorylated molecules and metal ions has been reported, while few studies focus on its sensing capability. Herein, we report a colorimetric sensing strategy through the coordination chemistry between ascorbic acid 2-phosphate (AAP) and copper ions. The phosphate group-containing AAP can coordinate with copper ions to induce a visible color change from blue to green in a rapid way, which can be easily read by the naked eye or a smartphone based on the blue-to-green (B/G) ratio. This coordination chemistry provides a facile and convenient strategy for designing colorimetric assays. Alkaline phosphatase can catalyze the hydrolysis of AAP to ascorbic acid (AA), thus modulating the AAP/AA transformation and the AAP-mediated coordination, offering a straightforward way for monitoring the enzymatic activity. This colorimetric sensing strategy shows good performances in stability, sensitivity, cost, and scale-up production, holding great promise as a point-of-care technique for diagnostic applications.

Topics: Alkaline Phosphatase; Ascorbic Acid; Colorimetry; Copper; Ions

The epigenome delineates lineage-specific transcriptional programs and restricts cell plasticity to prevent non-physiological cell fate transitions. Although cell diversification fosters tumor evolution and therapy resistance, upstream mechanisms that regulate the stability and plasticity of the cancer epigenome remain elusive. Here we show that 2-hydroxyglutarate (2HG) not only suppresses DNA repair but also mediates the high-plasticity chromatin landscape. A combination of single-cell epigenomics and multi-omics approaches demonstrates that 2HG disarranges otherwise well-preserved stable nucleosome positioning and promotes cell-to-cell variability. 2HG induces loss of motif accessibility to the luminal-defining transcriptional factors FOXA1, FOXP1, and GATA3 and a shift from luminal to basal-like gene expression. Breast tumors with high 2HG exhibit enhanced heterogeneity with undifferentiated epigenomic signatures linked to adverse prognosis. Further, ascorbate-2-phosphate (A2P) eradicates heterogeneity and impairs growth of high 2HG-producing breast cancer cells. These findings suggest 2HG as a key determinant of cancer plasticity and provide a rational strategy to counteract tumor cell evolution.

Topics: Alcohol Oxidoreductases; Ascorbic Acid; Cell Differentiation; Cell Line, Tumor; Chromatin; DNA Repair; Epigenome; Forkhead Transcription Factors; Gene Expression; Gene Expression Regulation; Glutarates; Humans; Isocitrate Dehydrogenase; Neoplasms; Nucleosomes; Repressor Proteins

Diabetic cutaneous ulcers (DCU) are a complication of diabetes with diabetic foot ulcers being the most common, and the wounds are difficult to heal, increasing the risk of bacterial infection. Cell-based therapy utilizing mesenchymal stem cells (MSCs) is currently being investigated as a therapeutic avenue for both chronic diabetic ulcers and severe burns. Wharton's jelly mesenchymal stem cell (WJMSC) with PF-127 hydrogel and sodium ascorbyl phosphate (SAP) improved skin wound healing in mice. Whether this combination strategy is helpful to diabetic ulcers wound healing remains to be explored.. Firstly, the WJMSCs embedded in PF-127 and SAP combination were transplanted onto excisional cutaneous wound bed in type 2 diabetic Sprague Dawley (SD) rats. Two weeks after transplantation, the skin tissue was collected for histological and immunohistochemical analysis. Further, overexpressing-EGFP WJMSCs were performed to investigate cell engraftment in the diabetic cutaneous ulcer. The apoptosis of WJMSCs which encapsulated with combination of PF-127 and SAP was detected by TUNEL fluorescence assay and RT-PCR in vitro. And the mitochondrial damage induced by oxidative stress assessed by MitoTracker and CMH2DCFDA fluorescence assay.. In diabetic cutaneous wound rat model, PF-127 plus SAP-encapsulated WJMSCs transplantation promoted diabetic wound healing, indicating improving dermis regeneration and collagen deposition. In diabetic wound healing, less pro-inflammatory M1 macrophages, more anti-inflammatory M2 tissue-healing macrophages, and neovascularization were observed in PF-127 + SAP + WJMSCs group compared with other groups. SAP supplementation alleviated the apoptosis ratio of WJMSCs embedded in the PF-127 in vitro and promoted cell survival in vivo.. PF-127 plus SAP combination facilitates WJMSCs-mediated diabetic wound healing in rat through promoting cell survival, the macrophage transformation, and angiogenesis. Our findings may potentially provide a helpful therapeutic strategy for patients with diabetic cutaneous ulcer.

Topics: Animals; Ascorbic Acid; Diabetes Mellitus, Type 2; Diabetic Foot; Humans; Hydrogels; Mesenchymal Stem Cell Transplantation; Mesenchymal Stem Cells; Mice; Rats; Rats, Sprague-Dawley; Wharton Jelly; Wound Healing

Wound healing is a complex process which requires appropriate structural support for restoration of tissue continuity and function. Collagen can act as a template for cellular activities but poor physico-chemical properties necessitates the stabilization of collagen without impairing its structure and function. This study investigates the effect of magnesium ascorbyl phosphate (MAP) on collagen with reference to physico-chemical properties. Incorporation of MAP enhanced the rate of collagen fibrillation signifying increased interaction at reduced time interval. MAP did not induce any changes in the secondary structure of collagen while there was an increase in shear viscosity with increase in shear stress at different shear rate. MAP stabilized collagen film exhibited higher denaturation temperature and showed an increase in Young's Modulus when compared with that of collagen film. In vivo studies showed complete wound closure on day 16 in case of stabilized collagen film. Mechanical properties of healed skin revealed that MAP collagen film treated rat skin completely regained its properties similar to that of normal skin thereby making them a potential candidate for wound healing application.

Topics: Animals; Ascorbic Acid; Bandages; Collagen; Elastic Modulus; Female; HaCaT Cells; Humans; Mice; Protein Multimerization; Protein Stability; Rats; Rats, Wistar; Wound Healing

A two-dimensional (2D) Co-MOF nanosheet-based nanozyme was developed for colorimetric detection of disease-related biomolecules. The prepared 2D Co-MOFs exhibited ultrahigh peroxidase catalytic activity. 2D Co-MOFs can catalyze the oxidation of colorless 3,3',5,5'-tetramethylbenzidine (TMB) to the blue product oxTMB, accompanying an obvious change of absorption value at 652 nm. However, alkaline phosphatase can catalyze the hydrolysis of L-ascorbic acid-2-phosphate to produce ascorbic acid which can reduce the oxTMB to TMB, resulting in an obvious color fading. Therefore, by recording the change of absorption value at 652 nm, the 2D Co-MOF nanosheets were used to detect ascorbic acid (AA) and alkaline phosphatase (ALP). The limit of detection for AA and ALP was 0.47 μM and 0.33 U L

Topics: Alkaline Phosphatase; Ascorbic Acid; Benzidines; Catalysis; Chromogenic Compounds; Cobalt; Colorimetry; Humans; Limit of Detection; Metal-Organic Frameworks; Nanostructures; Oxidation-Reduction; Paper; Smartphone

Topics: Alkaline Phosphatase; Ascorbic Acid; Cobalt; Colorimetry; Fluorescent Dyes; Humans; Hydroxides; Limit of Detection; Metal Nanoparticles; Oxidation-Reduction; Phenylenediamines; Point-of-Care Testing; Smartphone

Photoelectrochemical (PEC) biosensors carried out the whole reaction process in the same solution, which would limit the sensitivity and selectivity of detection in the sensing system. Herein, we reported a promising new cathode-anode spatial division PEC platform based on the two-electrode synergistic enhancement strategy. With the photoanode and photocathode integrated in the same current circuit, the platform exhibited an increased photocurrent response, as well as an improved anti-interference ability led by separating the two electrodes spatially. In this proposal, red light-driven AgInS

Topics: Alkaline Phosphatase; Ascorbic Acid; Biosensing Techniques; DNA; Electrochemical Techniques; Electrodes; Enzymes, Immobilized; Gold; Humans; Indium; Lead; Light; Limit of Detection; Metal Nanoparticles; MicroRNAs; Photochemical Processes; Quantum Dots; Reproducibility of Results; Silver; Sulfides

Topics: Acid Phosphatase; Ascorbic Acid; Benzidines; Chlorides; Chromogenic Compounds; Colorimetry; Fluorescent Dyes; Gold Compounds; Humans; Limit of Detection; Oxidation-Reduction; Rhodamines; Smartphone; Spectrometry, Fluorescence

Growth factors produced by stem cells aid in the bone repair process. We investigated the ability of encapsulated rat adipose-derived stem cells (rASCs) treated with osteogenic media (OM) to produce growth factors, and determined the optimal combination of OM components that will lead to the production of both osteogenic and angiogenic factors. Our results demonstrate that microencapsulated stem cells were able to produce vascular endothelial growth factor (VEGF), fibroblast growth factor-2, and bone morphogenetic protein-2 (BMP2) necessary for bone regeneration. OM led to the reduction of angiogenic factors; however, the removal of dexamethasone restored angiogenic factor production. Additionally, we determined whether the effect of dexamethasone on VEGF and BMP2 varied among rat, rabbit, mouse, and humans. Dexamethasone led to a reduction in VEGF levels in ASCs derived from rats, mice, and humans, while this reduction was absent in rabbit ASCs (rbASCs). Human ASCs (hASCs) from donors of different race and sex showed a similar response to dexamethasone with secreted VEGF levels. BMP2 levels secreted by rbASCs, mouse ASCs (mASCs), and hASCs were independent of the media treatments, while rASCs responded differently in the surrounding media and within the microbeads. In conclusion, microencapsulated ASCs can be treated to produce osteogenic and angiogenic factors for tissue regeneration applications, but outcomes may vary with culture conditions.

Topics: Adipocytes; Angiogenesis Inducing Agents; Animals; Ascorbic Acid; Bone Morphogenetic Protein 2; Bone Regeneration; Cells, Cultured; Culture Media; Dexamethasone; Fibroblast Growth Factor 2; Gene Expression; Humans; Male; Mice; Mice, Inbred C57BL; Models, Animal; Osteogenesis; Rabbits; Rats; Rats, Sprague-Dawley; Stem Cells; Vascular Endothelial Growth Factor A

Ascorbic acid-2-phosphate (A2-P) is an oxidation-resistant derivative of ascorbic acid that has been widely employed in culturing adipose-derived stem cells (ASCs) for faster expansion and cell sheet formation. While high dose ascorbic acid is known to induce cellular apoptosis via metabolic stress and genotoxic effects, potential cytotoxic effects of A2-P at high concentrations has not been explored. In this study, the relationship between ASC seeding density and A2-P-induced cytotoxicity was investigated. Spheroid-derived ASCs with smaller cellular dimensions were generated to investigate the effect of cell-cell contact on the resistance to A2-P-induced cytotoxicity. Decreased viability of ASC, fibroblast, and spheroid-derived ASC was noted at higher A2-P concentration, and it could be reverted with high seeding density. Compared to control ASCs, spheroid-derived ASCs seeded at the same density exhibited decreased viability in the A2-P-supplemented medium. The expression of antioxidant enzymes (catalase, SOD1, and SOD2) was enhanced in ASCs at higher seeding densities. However, their enhanced expression in spheroid-derived ASCs was less evident. Furthermore, we found that co-administration of catalase or N-acetylcysteine nullified the observed cytotoxicity. Collectively, A2-P can induce ASC cytotoxicity at higher concentrations, which can be prevented by seeding ASCs at high density or co-administration of another antioxidant.

Topics: Adipose Tissue; Adult; Antineoplastic Agents; Apoptosis; Ascorbic Acid; Cell Count; Cell Differentiation; Cell Proliferation; Cells, Cultured; Humans; Middle Aged; Stem Cells

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Topics: Alkaline Phosphatase; Ascorbic Acid; Benzidines; Catalysis; Cerium; Colorimetry; Coloring Agents; Enzyme Assays; Limit of Detection; Metal Nanoparticles; Oxidation-Reduction; Palladium

Sodium ascorbyl phosphate is a hydrophilic derivative of ascorbic acid with better stability compared to the parent compound. However, sodium ascorbyl phosphate is not as stable in solution as it is in the solid state, and it has been found to degrade, with accompanying discoloration, under the influence of different conditions. Here, the degradation mechanism of sodium ascorbyl phosphate in the water-glycerol system was revealed and the thermal degradation kinetics was shown to follow second-order kinetics. A thermal degradation prediction model was established and successfully fitted to the experimental data. In addition, the stability of sodium ascorbyl phosphate in the water-glycerol system during storage was investigated under different conditions, including changes in concentration, temperature, pH, light and oxygen, and metal ions. Sodium ascorbyl phosphate content was quantitatively measured via HPLC, and the color and pH values of the sample were qualitatively measured using a spectrophotometer and a pH meter, respectively. It was found that temperature and pH are the most important factors affecting the stability of sodium ascorbyl phosphate.

Topics: Ascorbic Acid; Drug Stability; Drug Storage; Glycerol; Hydrogen-Ion Concentration; Kinetics; Temperature; Water

To provide scientific data for clinical practice in making strategies for accelerating corneal endothelial wound healing, we investigated the impact of UVA on the corneal endothelial wound healing process and the underlying mechanism using an. An. After scratching, the Ki-67. The migration of HCEnC plays a major role in the wound healing process of the established cell model, which is like the wound healing process

Topics: Actins; Ascorbic Acid; Cell Movement; Cell Proliferation; Cells, Cultured; Endothelium, Corneal; Humans; Ki-67 Antigen; Models, Biological; Reactive Oxygen Species; Ultraviolet Rays; Wound Healing

It is still a high challenge to develop a simple, sensitive and portable approach for bioassay in strong scattering medium. Herein, a photoacoustic (PA) device is developed for the detection of alkaline phosphatase (ALP) in serum with silver nanoparticles (AgNPs) as signal probe, without any requirements for expensive equipment, professional operation and pre-processing of real samples. ALP as an important disease marker could catalyze the breakdown of sodium L-ascorbyl-2-phosphate (AAP) into ascorbic acid (AA), thereby reducing Ag

Topics: Alkaline Phosphatase; Ascorbic Acid; Colorimetry; Humans; Light; Limit of Detection; Metal Nanoparticles; Photoacoustic Techniques; Reproducibility of Results; Silver; Silver Nitrate

Factors such as poor engraftment, retention, and survival of the transplanted stem cells are deemed to limit their therapeutic efficacy for wound regeneration. Hence, it is necessary to explore these issues in order to resolve them. In this study, we aim to investigate the role of Pluronic F-127 (PF-127) hydrogel plus antioxidant sodium ascorbyl phosphate (SAP) in enhancing Wharton's jelly mesenchymal stem cell (WJMSC)-mediated effectiveness on full-thickness skin wound healing in mice.. First, the cytotoxicity of PF-127 and the biological effect of SAP on the survival of WJMSCs were tested in vitro using cell viability and proliferation assays. Next, a cell suspension containing WJMSCs, PF-127, and SAP was topically administered onto an 8-mm diameter excisional full-thickness wound bed. Eight days after transplantation, the mice were sacrificed and the skin tissue was excised for histological and immunohistochemical analysis. Finally, in vivo distribution of transplanted WJMSCs was traced to investigate cell engraftment and the potential therapeutic mechanism.. PF-127 was found to be cytotoxic to WJMSCs while SAP significantly improved the survival of PF-127-embedded WJMSCs. When this combination was topically transplanted onto the wound bed, wound healing was facilitated and dermis regeneration was achieved on the 8th day after surgery, as evidenced by an increase in dermal thickness, newly developed hair follicles, and collagen fiber deposition accompanied by a reduction in scar width. Further, immunohistochemical analysis demonstrated a higher number of anti-inflammatory M2 macrophages, proliferating cells, and newly formed blood vessels in the WJMSCs/PF-127/SAP group relative to all other groups. In addition, in vivo tracking results revealed a highly enhanced engraftment of WJMSCs accumulated in the dermis in the WJMSCs/PF-127/SAP group.. SAP significantly improves the survival of WJMSCs in PF-127 encapsulation. Further, PF-127 plus SAP is an effective combination that enhances WJMSC engraftment in the dermis, which then promotes full-thickness wound healing through potential M2 macrophage formation and angiogenesis.

Topics: Animals; Ascorbic Acid; Hydrogels; Mesenchymal Stem Cells; Mice; Poloxamer; Wharton Jelly; Wound Healing

The advancement of autologous mesenchymal stem cell (MSC) therapy for the treatment of non-healing diabetic wounds is hampered by endogenous MSC dysfunction and limited viability of cells post-transplantation into the pathological wound environment. The development of effective strategies to restore the functional capabilities of these impaired MSCs prior to transplantation may be a key to their ultimate success as wound repair mediators. The current study therefore investigated whether antioxidant preconditioning [7.5 mM N-acetylcysteine (NAC) + 0.6 mM ascorbic 2-phosphate (AAP)] could restore the growth rate, migration ability and viability of impaired MSCs and whether this restored state is maintained in the presence of diabetic wound fluid (DWF). Healthy control (source: wild type, C57BL/6J mice) (n = 12) and impaired/diabetic MSCs (source: obese prediabetic, B6.Cg-Lepob/J mice) (n = 12) were isolated from the bone marrow of mice. Treatment groups post-isolation were as follow: (a) No treatment (baseline phenotype): MSCs expanded in standard growth media (SGM) (±8 days) and only exposed to growth media. (b) DWF (baseline response): MSCs expanded in SGM (±8 days) followed by exposure to DWF (24 hours, 48 hours, 96 hours). (c) Antioxidant preconditioning (preconditioned phenotype): MSCs expanded in the presence of NAC/AAP (±8 days). (d) Antioxidant preconditioning + DWF (preconditioned response): MSCs expanded in the presence of NAC/AAP (±8 days) followed by exposure to DWF (24 hours, 48 hours, 96 hours). The results demonstrated that expansion of MSCs (both healthy control and impaired diabetic) in the presence of combined NAC/AAP treatment improved ex vivo MSC viability and protected MSCs in the presence of DWF. Despite improved viability, AAP/NAC could however not rescue the reduced proliferation and migration capacity of impaired diabetic MSCs. The protective effect of NAC/AAP preconditioning against the toxicity of DWF could however be a potential strategy to improve cell number post-transplantation.

Topics: Acetylcysteine; Animals; Antioxidants; Ascorbic Acid; Case-Control Studies; Cell Movement; Cell Proliferation; Cell Survival; Diabetes Mellitus; Exudates and Transudates; Mesenchymal Stem Cell Transplantation; Mesenchymal Stem Cells; Mice; Mice, Obese; Osteogenesis; Prediabetic State; Transplantation, Autologous; Wounds and Injuries

A ratiometric fluorescent assay is fabricated for the evaluation of alkaline phosphatase (ALP) activity. This assay is composed of ionic liquid-functionalized carbon dots (IL-CDs) with blue fluorescence signal at 470 nm and 2,3-diaminophenazine (DAP) with yellow fluorescence signal at 570 nm. IL-CDs were synthesized via electrochemical method by using ionic liquid (1-butyl-3-methylimidazolium tetrafluoroborate) and ultrapure water as precursors. DAP is produced by the oxidation reaction between o-phenylenediamine and H

Topics: Alkaline Phosphatase; Armoracia; Ascorbic Acid; Carbon; Fluorescent Dyes; Horseradish Peroxidase; Humans; Hydrogen Peroxide; Imidazoles; Ionic Liquids; Limit of Detection; Phenylenediamines; Quantum Dots; Spectrometry, Fluorescence

Sodium ascorbyl phosphate (SAP) is a hydrophilic and stable L-ascorbic acid derivative, being converted by the cell phosphatases into free ascorbic acid (AA), which allows its sustained release in the medium. AA participates in the maintenance and healing of the periodontium. It presents a regulatory role of the osteoblastic activity, stimulating the deposition of collagen extracellular matrix followed by the induction of genes associated with the osteoblastic phenotype. It also acts in the elimination of reactive oxygen species, abundantly produced by defense cells in periodontal disease. The aim of this study was to evaluate the effect of SAP on osteoblast viability and differentiation.. Mouse preosteoblastic cells of the MC3T3-E1 strain were used. Cell viability was assessed by the trypan blue dye exclusion assay and the expression of genes related to osteoblast differentiation by quantitative PCR. Collagen I secretion was evaluated by ELISA, and mineralized matrix formation was assayed by Alizarin red S staining.. The results showed that SAP at concentrations from 50 to 500 µmol/L does not influence preosteoblast cell viability, but stimulates their differentiation, observed by the induction of RUNX2, COL1A1, and BGLAP2; by the higher secreted levels of collagen I; and also by the increase in the mineralization of the extracellular matrix in cells exposed to this agent at 200 or 400 µmol/L, compared with those not exposed.. By its stability and capacity to induce preosteoblastic cell differentiation, our results indicate that the incorporation of SAP into local release devices, membranes/scaffolds or biomaterials, could favor bone tissue formation and therefore periodontal healing.

Topics: Animals; Ascorbic Acid; Cell Differentiation; Mice; Osteoblasts; Osteogenesis

L-ascorbic acid 2-phosphate magnesium (APMg) salt is a vitamin C derivative frequently used as a raw material in cell and tissue therapy. APMg is not only used as a replacement of the unstable ascorbate, but also shows additional cell-biological functionalities. However, its unknown structural characteristics hamper the mechanistic elucidation of its biological role. Therefore, different techniques were applied for APMg structure characterization. Firstly, the stoichiometric composition was characterized by its solvent, ligand and magnesium content. No crystals of APMg could be obtained; however, a single crystal of APNa, the sodium salt of l-ascorbic acid 2-phosphate, was successfully obtained and its crystal structure was elucidated. FT-IR was applied to further clarify the structure of solid APMg. Finally, the structure of APMg in aqueous solution was explored by potentiometric titration as well as FT-IR.

Topics: Ascorbic Acid; Cations; Crystallization; Ligands; Magnesium; Molecular Structure; Solvents; Spectroscopy, Fourier Transform Infrared

Vitamin C (L-Ascorbic acid) has many favourable effects on the skin such as antioxidant, anti-aging and whitening effects. Its instability and low permeability limit its pharmaceutical use in cosmetic and dermatological products. Instead, Mg ascorbyl phosphate (MAP), an ascorbic acid derivative, has the same effect with higher stability is being used. In this work, a vesicular system, aspasomes, containing MAP was developed and evaluated. Aspasomes are multilayered vesicles formed by amphiphiles molecules, Ascorbyl palmitate (ASP), in combination with cholesterol and charged lipids for drug encapsulation. Here, we investigated the use of lecithin instead of the charged lipid dicetyl phosphate for aspasomes development. Nine formulations were prepared and evaluated for their entrapment efficiency, particle size, polydispersity index (PDI) and zeta potential. Their entrapment efficiency ranged from 33.00 ± 2.27 to 95.18 ± 1.06, while their particle size was from 373.34 ± 60.85 to 464.37 ± 93.46 nm with acceptable PDI (from 0.212 ± 0.068 to 0.351 ± 0.061) and zeta potential (from -37.52 ± 2.42 to -50.36 ± 1.82). Three formulations were selected and evaluated for their drug release, permeation and retention into skin. One formulation was selected to be formulated as aspasomal topical cream and gel. The aspasomal cream was found to have enhanced drug permeation and skin retention over the aspasomal gel as well as the aspasomes formulation. MAP aspasomal cream was evaluated clinically as an effective treatment for melasma against 15% trichloroacetic acid (TCA) and the results recorded that the aspasomal cream showed the greatest degree of improvement regarding the hemi-MASI scores with 35% of patients rating it as excellent treatment. The study showed that MAP aspasomal cream can be considered a novel treatment of melasma which is free of side effects. Its efficacy as a monotherapy is superior to that of chemical peeling using 15% TCA.

Topics: Administration, Cutaneous; Animals; Antineoplastic Agents; Ascorbic Acid; Biological Transport; Cholesterol; Drug Compounding; Drug Liberation; Drug Stability; Humans; Lecithins; Liposomes; Magnesium; Male; Melanosis; Rats, Wistar; Skin; Skin Absorption; Treatment Outcome

Topics: Acid Phosphatase; Ascorbic Acid; Biomimetic Materials; Biosensing Techniques; Catalysis; Colorimetry; Hydrogen Peroxide; Limit of Detection; Nanostructures; Oxidoreductases

Topics: Alkaline Phosphatase; alpha-Fetoproteins; Antibodies; Ascorbic Acid; Colorimetry; Coordination Complexes; Dehydroascorbic Acid; Enzyme-Linked Immunosorbent Assay; Humans; Iron; Limit of Detection; Oxidation-Reduction; Phenanthrolines; Reproducibility of Results

An innovative lyophilized dry powder formulation consisting of urea-crosslinked hyaluronic acid (HA-CL) and sodium ascorbyl phosphate (SAP) - LYO HA-CL - SAP- was prepared and characterized in vitro for physico-chemical and biological properties. The aim was to understand if LYO HA-CL - SAP could be used as adjuvant treatment for nasal inflammatory diseases. LYO HA-CL - SAP was suitable for nasal delivery and showed to be not toxic on human nasal septum carcinoma-derived cells (RPMI 2650 cells) at the investigated concentrations. It displayed porous, polygonal particles with unimodal, narrow size distribution, mean geometric diameter of 328.3 ± 27.5 µm, that is appropriate for nasal deposition with no respirable fraction and 88.7% of particles with aerodynamic diameter >14.1 µm. Additionally, the formulation showed wound healing ability on RPMI 2650 cells, and reduced interleukin-8 (IL-8) level in primary nasal epithelial cells pre-induced with lipopolysaccharide (LPS). Transport study across RPMI 2650 cells showed that HA-CL could act not only as carrier for SAP and active ingredient itself, but potentially also as mucoadhesive agent. In conclusion, these results suggest that HA-CL and SAP had anti-inflammatory activity and acted in combination to accelerate wound healing. Therefore, LYO HA-CL - SAP could be a potential adjuvant in nasal anti-inflammatory formulations.

Topics: Adjuvants, Immunologic; Administration, Intranasal; Adult; Anti-Inflammatory Agents; Ascorbic Acid; Cell Line; Cell Survival; Epithelial Cells; Humans; Hyaluronic Acid; Interleukin-8; Lipopolysaccharides; Nasal Mucosa; Powders; Urea; Wound Healing; Young Adult

The pathogenesis and progression of several lung disorders is propagated by inflammatory and oxidative processes, which can be controlled by adjunctive inhaled therapies. The present study aimed to develop an inhalable dry powder formulation consisting of co-spray-dried urea-crosslinked hyaluronic acid and sodium ascorbyl phosphate (SD HA-CL-SAP), a novel combination which was recently shown to possess anti-inflammatory, antioxidant, and wound healing properties. Native HA and SAP were co-spray dried (SD HA-SAP) and evaluated as control formulation. Yield (Y%) and encapsulation efficiency (EE%) were 67.0 ± 4.8% and 75.5 ± 7.2% for SD HA-SAP, 70.0 ± 1.5% and 66.5 ± 5.7% for SD HA-CL-SAP, respectively. Both formulations were shown to be suitable for lung delivery in terms of morphology, particle size (median volumetric diameter ∼ 3.4 μm), physical and thermal stability, in vitro aerosol performance - respirable fraction: 30.5 ± 0.7% for SD HA-SAP and 35.3 ± 0.3% for SD HA-CL-SAP. SAP release was investigated using Franz cells and air-interface Calu-3 cell model (>90% of SAP transported within 4 h). The innovative SD HA-CL-SAP formulation holds potential as inhalable dry powder for the treatment of inflammatory lung disorders.

Topics: Administration, Inhalation; Aerosols; Anti-Inflammatory Agents; Ascorbic Acid; Cell Line, Tumor; Chemistry, Pharmaceutical; Cross-Linking Reagents; Desiccation; Drug Combinations; Drug Compounding; Drug Stability; Dry Powder Inhalers; Humans; Hyaluronic Acid; Lung Diseases; Particle Size; Powders; Urea

An ultrasensitive fluorometric and colorimetric dual-mode assay is described for the determination of the activity of alkaline phosphatase (ALP). ALP catalyzes the decomposition of 2-phospho-L-ascorbic acid, and the ascorbic acid thus generated reduces silver ions. In the presence of gold nanoparticles, gold-silver nanoparticles (Au@Ag NPs) are formed. This is accompanied by a color change form pink to deep yellow. The Au@Ag NPs reduce the fluorescence of blue fluorescent graphene quantum dots due to spectral overlap. The changes of absorbance (measured at 410 and 520 nm) and fluorescence (measured at excitation/emission wavelengths of 346/415 nm) correlate well with the ALP activity in the 0.01-6 mU·mL

Topics: Adult; Alkaline Phosphatase; Ascorbic Acid; Colorimetry; Enzyme Assays; Gold; Graphite; Humans; Limit of Detection; Metal Nanoparticles; Oxidation-Reduction; Quantum Dots; Silver; Spectrometry, Fluorescence

The authors describe a fluorometric and resonance Rayleigh scattering dual-mode scheme for detection of the activity and inhibition of alkaline phosphatase (ALP). The method utilizes (a) CoOOH nanoflakes, which have high resonance Rayleigh scattering activity and can strongly adsorb ssDNA, and (b) Co(II)-dependent DNAzyme assisted signal amplification. ALP specifically catalyzes the hydrolysis of ascorbic acid-2-phosphate to produce ascorbic acid which reduces CoOOH nanoflakes to Co(II) ion. The Co(II)-dependent DNAzyme is then activated by Co(II) ion, and this results in the cleavage of a substrate labeled with both a fluorophore and a quencher. Following hydrolysis, fluorophore and quencher become separated and the fluorescence measured at excitation/emission wavelengths of 490/518 nm recovers, while the RRS signal at 405 nm decreases. The method works on the 0.2 to 2000 U L

Topics: Alkaline Phosphatase; Ascorbic Acid; Base Sequence; Biosensing Techniques; Cobalt; DNA, Catalytic; Enzyme Assays; Fluoresceins; Fluorescent Dyes; Humans; Limit of Detection; Metal Nanoparticles; Oxidation-Reduction; Oxides; Spectrometry, Fluorescence

Mycobacterium tuberculosis (Mtb) SapM is a secreted virulence factor critical for intracellular survival of the pathogen. The role of SapM in phagosome maturation arrest in host macrophages suggests its potential as a drug target to assist in the clearance of tuberculosis infection. However, the mechanism of action of SapM at the molecular level remains unknown. In this study, we provide new insights into the mechanism of catalysis, substrate specificity and inhibition of SapM, and we identify the critical residues for catalysis and substrate binding. Our findings demonstrate that SapM is an atypical monoester alkaline phosphatase, with a serine-based mechanism of catalysis probably metal-dependent. Particularly relevant to SapM function and pathogenesis, is its activity towards PI(4,5)P

Topics: Acid Phosphatase; Antitubercular Agents; Ascorbic Acid; Bacterial Proteins; Catalysis; Catalytic Domain; Humans; Inhibitory Concentration 50; Mycobacterium tuberculosis; Phosphatidylinositols; Substrate Specificity; THP-1 Cells; Virulence

Liposomes are an excellent candidate component for biosensors to transduce and amplify detection signals due to their outstanding ability in encapsulating signal marker compounds. However, the use of liposomes for photoelectrochemical (PEC) signal transduction has not yet been achieved due the lack of appropriate sensing strategy. Herein, we report on a novel liposomes-amplified PEC immunoassay (LAPIA) method for sensitive HIV-p24 antigen (p24) detection based on a split-type strategy. Initially, liposomes were encapsulated with alkaline phosphatase (ALP) in their hydrophilic chamber and conjugated with secondary antibody on the surface to form the ALP-encapsulated liposomes (ALP-Ls) based PEC signal label. Sandwiched immunoassay based on the ALP-Ls label was then carried out in microwell plate. Upon addition of tween 20, the ALP molecules were released and catalyzed the hydrolysis of ascorbic acid 2-phosphate (AA-p) to produce ascorbic acid (AA). The latter then donated electron to the graphene/g-C

Topics: Alkaline Phosphatase; Ascorbic Acid; Biosensing Techniques; Graphite; HIV; HIV Core Protein p24; HIV Infections; Humans; Immunoassay; Liposomes

Simple and fast detection of alkaline phosphatase (ALP) activity is of great importance for diagnostic and analytical applications. In this work, we report a turn-off approach for the real-time detection of ALP activity on the basis of the charge transfer induced fluorescence quenching of the Cu(BCDS)

Topics: Alkaline Phosphatase; Ascorbic Acid; Copper; Fluorescence; Humans; Phenanthrolines

In this paper, we developed a sensitive fluorescence biosensor for tyrosinase (TYR) and acid phosphatase (ACP) activity detection based on nitrogen-doped graphene quantum dots (N-GQDs). Tyrosine could be catalyzed by TYR to generate dopaquinone, which could efficiently quench the fluorescence of N-GQDs, and the degree of fluorescence quenching of N-GQDs was proportional to the concentration of TYR. In the presence of ACP, l-Ascorbic acid-2-phosphate (AAP) was hydrolyzed to generate ascorbic acid (AA), and dopaquinone was reduced to l-dopa, resulting in the fluorescence recovery of the quenched fluorescence by dopaquinone. Thus, a novel fluorescence biosensor for the detection of TYR and ACP activity based on N-GQDs was constructed. Under the optimized experimental conditions, the fluorescence intensity was linearly correlated with the concentration of TYR and ACP in the range of 0.43-3.85 U mL

Topics: Acid Phosphatase; Ascorbic Acid; Benzoquinones; Biosensing Techniques; Dihydroxyphenylalanine; Fluorescence; Graphite; Humans; Limit of Detection; Monophenol Monooxygenase; Nitrogen; Quantum Dots; Sensitivity and Specificity

Oligodendrocyte-formed myelin sheaths play important roles in the neuronal functions in the central nervous system. In demyelinating diseases, such as Multiple Sclerosis, the myelin sheaths are damaged and the remyelinating process is somehow hindered. Restoration of the myelin sheaths requires the differentiation of the oligodendrocyte precursor cells (OPCs) into mature oligodendrocytes (OLs). To discover small molecule compounds that might promote the OPC to OL differentiation, a high-throughput screening system is established and L-ascorbyl-2-phosphate (As-2P), a stable form of Vitamin C (Vc), is found to greatly enhance the OPC to OL differentiation. As-2P promotes gradual expression of OL lineage markers, including O4, CNPase and MBP, in a dose- and time-dependent manner. It also facilitates the formation of myelin sheaths in OPC-neuron co-culture. As-2P also promotes the repair of the myelin sheaths in vivo and provides significant therapeutic effect in a cuprizone-mediated demyelination animal model. Interestingly, As-2P's function in promoting OPC differentiation is not related to its antioxidant activity. And an intracellular rather than an extracellular mechanism might be involved. Considering the safe use of Vc as a dietary supplement for many years, it might also be used as an alternative medicine for CNS demyelinating diseases.

Topics: Animals; Antioxidants; Ascorbic Acid; Brain; Cell Differentiation; Coculture Techniques; Cuprizone; Demyelinating Diseases; Disease Models, Animal; Dose-Response Relationship, Drug; Female; Ganglia, Spinal; Mice; Mice, Inbred C57BL; Neurons; Neuroprotective Agents; Oligodendroglia; Remyelination; Time Factors

This in vitro study evaluated, for the first time, the safety and the biological activity of a novel urea-crosslinked hyaluronic acid component and sodium ascorbyl phosphate (HA-CL - SAP), singularly and/or in combination, intended for the treatment of inflammatory lung diseases. The aim was to understand if the combination HA-CL - SAP had an enhanced activity with respect to the combination native hyaluronic acid (HA) - SAP and the single SAP, HA and HA-CL components. Sample solutions displayed pH, osmolality and viscosity values suitable for lung delivery and showed to be not toxic on epithelial Calu-3 cells at the concentrations used in this study. The HA-CL - SAP displayed the most significant reduction in interleukin-6 (IL-6) and reactive oxygen species (ROS) levels, due to the combined action of HA-CL and SAP. Moreover, this combination showed improved cellular healing (wound closure) with respect to HA - SAP, SAP and HA, although at a lower rate than HA-CL alone. These preliminary results showed that the combination HA-CL - SAP could be suitable to reduce inflammation and oxidative stress in lung disorders like acute respiratory distress syndrome, asthma, emphysema and chronic obstructive pulmonary disease, where inflammation is prominent.

Topics: Anti-Inflammatory Agents; Antioxidants; Ascorbic Acid; Cell Line, Tumor; Cell Survival; Cross-Linking Reagents; Dose-Response Relationship, Drug; Drug Combinations; Drug Compounding; Electric Impedance; Epithelial Cells; Humans; Hyaluronic Acid; Hydrogen-Ion Concentration; Interleukin-6; Lung; Lung Diseases, Obstructive; Osmolar Concentration; Reactive Oxygen Species; Technology, Pharmaceutical; Urea; Viscosity

Loss of 5-hydroxymethylcytosine (5hmC) occurs frequently in a wide variety of tumours, including clear-cell renal cell carcinoma (ccRCC). It remains unknown, however, whether the restoration of 5hmC patterns in tumours could have therapeutic efficacy. Here, we used sodium L-ascorbate (vitamin C, AsANa) and the oxidation-resistant form L-ascorbic acid 2-phosphate sesquimagnesium (APM) for the restoration of 5hmC patterns in ccRCC cells. At physiological concentrations, both show anti-tumour efficacy during long-term treatment

Topics: 5-Methylcytosine; Animals; Ascorbic Acid; Carcinoma, Renal Cell; Cell Line, Tumor; Cell Proliferation; Dioxygenases; DNA-Binding Proteins; Enhancer Elements, Genetic; Humans; Kidney Neoplasms; Mice; Proto-Oncogene Proteins; Transcriptome; Xenograft Model Antitumor Assays

Scar formation remains a major clinical concern following tissue injuries such as skin wounds. Adipose-derived stem cell (ASC) sheets can be fabricated quickly through stimulation with l-ascorbate 2-phosphate and have valuable applications in tissue regeneration and wound healing. However, the antifibrotic capability of ASCs in cell sheet format has not been sufficiently investigated. We employed a murine model of healing-impaired cutaneous wounds and observed faster wound healing with ASC sheet treatment. Significantly more engrafted ASCs were observed in the wound tissue treated with ASC sheets at 14 days after wounding compared with dissociated cells. Moreover, no ASCs were found at day 28, which indicated a minimal risk of long-term side effects. The neoskin formed in the presence of ASC sheets exhibited a thickness comparable to normal skin and possessed a highly organized collagen structure. ASC sheets also suppressed macrophage infiltration and modulated TNF-α and TGF-β1 expression in vivo. Examination of fibroblasts cultured in ASC-conditioned medium indicated an anti-scarring effect of the ASC sheets evidenced by the downregulation of TGF-β1 and α-SMA in fibroblasts, which was likely mediated through the increased secretion of hepatocyte growth factor. Moreover, ASC sheets secreted significantly more C1q/TNF-related protein-3, which inhibited the C-C motif ligand 2 release by macrophages in vitro and subsequently reduced the chemotaxis of unstimulated macrophages. This mechanism may account for the observed decrease in recruitment of macrophages into the wound tissue. We conclude that ASC sheets possess the necessary paracrine factors to improve skin wound healing with a superior neoskin quality.. Adipose-derived stem cell (ASC) sheets exhibit great potential for tissue regeneration. In this study, we investigated whether ASC sheets can ameliorate skin wound healing with reduced scar formation, and faster wound healing was observed when applying ASC sheets in an impaired wound healing model of mice. The neoskin formed in the presence of ASC sheets exhibited a thickness comparable to normal skin with a more organized collagen structure. In vitro experiments suggested that the anti-scarring effect of the ASC sheets was partly mediated through increased secretion of hepatocyte growth factor. Moreover, ASC sheets secreted significantly more C1q/TNF-related protein-3, which may account for the decreased recruitment of macrophages into the wound tissue. Therefore, ASC sheets possess the necessary paracrine factors to improve skin wound healing with less scarring, thus representing a desirable method of topical wound treatment.

Topics: Adipocytes; Adult; Animals; Ascorbic Acid; Chemotaxis; Cicatrix; Female; Fibrosis; Humans; Macrophages; Mice; Mice, Nude; Middle Aged; Regeneration; Regenerative Medicine; Skin; Stem Cell Transplantation; Stem Cells; Wound Healing

Hand-foot skin reaction is recognized as one of the most common adverse events related to multiple tyrosine kinase inhibitors, but an effective prevention method has not been identified. The chief aim of this study was to find a mechanism-based preventive method for the skin toxicity induced by sorafenib using vitamin C derivatives. The effects of ascorbyl-2-phosphate magnesium (P-VC-Mg) on the molecular and pathological changes induced by sorafenib were investigated in human keratinocyte HaCaT cells. The cell growth inhibition and apoptotic effects of sorafenib were attenuated by P-VC-Mg. Moreover, P-VC-Mg inhibited the decrease of signal transducer and activator of transcription 3 (STAT3) phosphorylation and the expression of apoptosis suppressors treated by sorafenib. HaCaT cells transfected with the STAT3 dominant-negative form (STAT3DN) and STAT3 small interfering RNA (siRNA) combined with P-VC-Mg did not exhibit the attenuation of cell growth inhibition. Interestingly, after exposure to sorafenib in a three dimensional (3D) skin model assay, the basal layer was significantly thickened and the granular and spinous layers became thinner. In contrast, after exposure to sorafenib with P-VC-Mg, the thickness of the basal, granular, and spinous layers was similar to that of the control image. These findings suggest that P-VC-Mg attenuates sorafenib-induced apoptosis and pathological changes in human keratinocyte cells and in the 3D skin model mediated by the maintenance of STAT3 activity.

Topics: Antineoplastic Agents; Apoptosis; Ascorbic Acid; Cell Line; Hand-Foot Syndrome; Humans; Keratinocytes; Magnesium; Niacinamide; Phenylurea Compounds; Phosphorylation; Reactive Oxygen Species; RNA, Small Interfering; Signal Transduction; Skin; Sorafenib; STAT3 Transcription Factor

The gingival epithelium is a first line of defense against bacterial challenge. E-cadherin (E-cad) plays an important role in cell-cell adhesion as a barrier in the epithelium. Recently, a decrease in the expression of E-cad has been observed in inflamed gingival tissue. The aims of this study were to clarify the changes in E-cad expression and barrier function in human gingival epithelial cells stimulated with Porphyromonas gingivalis-lipopolysaccharide (P. gingivalis-LPS) and to evaluate the influence of these changes on the inflammatory reaction. Furthermore, to clarify the mechanism of the E-cad changes induced by P. gingivalis-LPS, we focused on reactive oxygen species (ROS) that are reported to induce a decrease in E-cad expression.. Human gingival epithelial cells were incubated in Humedia-KG2 in the presence or absence of P. gingivalis-LPS and antioxidants to analyze ROS involvement in P. gingivalis-LPS-induced E-cad changes. E-cad protein expression was analyzed by immunofluorescence staining. To investigate barrier function and inflammatory changes, we performed transport and cytokine assays using gingival epithelial cells and macrophages co-culture model in transwell plates. Medium containing 10 μg/mL P. gingivalis-LPS (transport substance) was added to the upper compartment, which harvested gingival epithelial cells, and medium without P. gingivalis-LPS was added to the lower compartment, which harvested macrophages. In the transport assay, P. gingivalis-LPS penetration was analyzed using the Limulus amebocyte lysate test. In the cytokine assay, we examined the change in tumor necrosis factor-α (TNF-α) production from the macrophages in the lower compartment using enzyme-linked immunosorbent assay.. Expression of E-cad in human gingival epithelial cells was decreased by P. gingivalis-LPS, and the decrease in E-cad accelerated the penetration of P. gingivalis-LPS through the monolayer. In addition, the concentration of TNF-α was higher under the E-cad reduced monolayer. Antioxidants, particularly vitamin E and l-ascorbic acid 2-phosphate magnesium salt, inhibited the decrease in E-cad expression, penetration of P. gingivalis-LPS and increase in TNF-α.. These results suggest that the decrease in E-cad caused by P. gingivalis-LPS leads to destruction of the epithelial barrier function in human gingival epithelial cells, and finally accelerates the inflammatory reaction under the barrier. Antioxidants, particularly vitamin E and l-ascorbic acid 2-phosphate magnesium salt, may restore the impaired function by scavenging ROS, which are related to the decrease in E-cad expression by P. gingivalis-LPS.

Topics: Antioxidants; Ascorbic Acid; Cadherins; Cell Line; Cytokines; Epithelium; Fluorescent Antibody Technique; Gingiva; Humans; Lipopolysaccharides; Oxidative Stress; Porphyromonas gingivalis; Real-Time Polymerase Chain Reaction; Vitamin E

Osteogenically differentiated cell sheet techniques using mesenchymal stem cells (MSCs) are available to stimulate bone regeneration. The advantage of the cell sheet technique is delivering live cells effectively into the focal region. We developed a novel osteogenic cell sheet technique by adding gelatin to osteogenic cell medium. Gelatin-induced osteogenic cell sheets (GCSs) were compared to conventional osteogenic cell sheets (OCSs). Undifferentiated MSCs (UCs) were used as a control. The morphology of these cell sheets was evaluated microscopically and histologically. The time-dependent cell proliferation rate was estimated by DNA quantification. The expression of osteogenic gene markers and the number of calcium depositions were assessed by quantitative real-time polymerase chain reaction and Alizarin red S (ARS) staining, respectively. GCSs were thicker and stronger than OCSs. GCSs showed a significantly higher cell proliferation rate compared to OCSs (p < 0.05). GCSs exhibited significantly higher upregulation of BMP-7 mRNA compared to OCSs (p < 0.05). Both GCSs and OCSs showed negative ARS reactivity on day 10, but only GCSs showed positive ARS reactivity on day 21. With this technique, we observed active cell proliferation with abundant ECM and upregulation of osteogenic bone markers, and our results suggest that GCSs could be promising for therapeutic applications in bone regeneration.

Topics: Animals; Ascorbic Acid; Bone Morphogenetic Protein 7; Cell Differentiation; Cell Proliferation; Cells, Cultured; Dogs; Extracellular Matrix; Gelatin; Mesenchymal Stem Cells; Osteogenesis; Real-Time Polymerase Chain Reaction

Topics: Alkaline Phosphatase; Animals; Ascorbic Acid; Biosensing Techniques; Carbon; Catalysis; Goats; Gold; Humans; Immunoconjugates; Immunoenzyme Techniques; Limit of Detection; Metal Nanoparticles; Nanotubes, Carbon; Oligopeptides; Silver

Accumulation of hypoxia inducible factor-1 alpha (HIF-1α) in malignant tissue is known to contribute to oncogenic progression and is inversely associated with patient survival. Ascorbic acid (AA) depletion in malignant tissue may contribute to aberrant normoxic activity of HIF-1α. While AA supplementation has been shown to attenuate HIF-1α function in malignant melanoma, the use of dehydroascorbic acid (DHA) as a therapeutic means to increase intracellular AA and modulate HIF-1α function is yet to be evaluated. Here we compared the ability of AA and DHA to increase intracellular vitamin C content and decrease the malignant potential of human melanoma by reducing the activity of HIF-1α.. HIF-1α protein accumulation was evaluated by western blot and transcriptional activity was evaluated by reporter gene assay using a HIF-1 HRE-luciferase plasmid. Protein expressions and subcellular localizations of vitamin C transporters were evaluated by western blot and confocal imaging. Intracellular vitamin C content following AA, ascorbate 2-phosphate (A2P), or DHA supplementation was determined using a vitamin C assay. Malignant potential was accessed using a 3D spheroid Matrigel invasion assay. Data was analyzed by One or Two-way ANOVA with Tukey's multiple comparisons test as appropriate with p<0.05 considered significant.. Melanoma cells expressed both sodium dependent vitamin C (SVCT) and glucose (GLUT) transporters for AA and DHA transport respectively, however advanced melanomas responded favorably to AA, but not DHA. Physiological glucose conditions significantly impaired intracellular vitamin C accumulation following DHA treatment. Consequently, A2P and AA, but not DHA treated cells demonstrated lower HIF-1α protein expression and activity, and reduced malignant potential. The ability of AA to regulate HIF-1α was dependent on SVCT2 function and SVCT2 was not significantly inhibited at pH representative of the tumor microenvironment.. The use of ascorbic acid as an adjuvant cancer therapy remains under investigated. While AA and A2P were capable of modulating HIF-1α protein accumulation/activity, DHA supplementation resulted in minimal intracellular vitamin C activity with decreased ability to inhibit HIF-1α activity and malignant potential in advanced melanoma. Restoring AA dependent regulation of HIF-1α in malignant cells may prove beneficial in reducing chemotherapy resistance and improving treatment outcomes.

Topics: Ascorbic Acid; Biological Transport; Cell Line; Cell Line, Tumor; Dehydroascorbic Acid; Glucose; Humans; Hypoxia-Inducible Factor 1, alpha Subunit; Melanoma; Sodium; Transcription, Genetic; Tumor Microenvironment

Stability of hydrophilic and lipophilic vitamin C derivatives for quenching synergistic antioxidant activities and to treat oxidative related diseases is a major issue. This study was aimed to encapsulate hydrophilic and lipophilic vitamin C derivatives (ascorbyl palmitate and sodium ascorbyl phosphate) as functional ingredients in a newly formulated multiple emulsion of the W//W type to attain the synergistic antioxidant effects and the resultant system's long term physical and chemical stability. Several multiple emulsions using the same concentration of emulsifiers but different concentrations of ascorbyl palmitate and sodium ascorbyl phosphate were developed. Three finally selected multiple emulsions (ME₁, ME₂ and ME₃) were evaluated for physical stability in terms of rheology, microscopy, conductivity, pH, and organoleptic characteristics under different storage conditions for 3 months. Chemical stability was determined by HPLC on Sykam GmbH HPLC system (Germany), equipped with a variable UV detector. Results showed that at accelerated storage conditions all the three multiple emulsions had shear thinning behavior of varying shear stress with no influence of location of functional ingredients in a carrier system. Conductivity values increased and pH values remained within the skin pH range for 3 months. Microscopic analysis showed an increase in globule size with the passage of time, especially at higher temperatures while decreased at low temperatures. Centrifugation test did not cause phase separation till the 45th day, but little effects after 2 months. Chemical stability analysis by HPLC at the end of 3 months showed that ascorbyl palmitate and sodium ascorbyl phosphate were almost stable in all multiple emulsions with no influence of their location in a carrier system. Multiple emulsions were found a stable carrier for hydrophilic and lipophilic vitamin C derivatives to enhance their desired effects. Considering that many topical formulations contain simple vitamin C it is suggested that present study may contribute to the development of more stable formulations with a combination of vitamin C derivatives to enhance their cosmetic benefits.

Topics: Ascorbic Acid; Chromatography, High Pressure Liquid; Cosmetics; Drug Stability; Emulsions; Hydrogen-Ion Concentration

A plasmonic colorimetric strategy was designed for sensitive detection of biomolecules through enzyme guided silver nanoparticles (AgNPs) growth on gold nanostars (AuNS). The growth of AgNPs on AuNS led to a substantial blue shift of the localized surface plasmon resonance (LSPR) peak and the color change of AuNS from blue to dark blue, purple and ultimately orange. Both the LSPR blueshift wavelength and the color of detection solution containing AuNS, Ag(+) and ascorbic acid 2-phosphate (AAP) depend on the amount of enzyme that catalyzed the dephosphorylation of AAP to reduce Ag(+) on AuNS surface. Thus this strategy could be used for LSPR and naked-eye detections of both the enzyme such as alkaline phosphatase (ALP) and other biomolecules involved in biorecognition events using ALP as a tag. The LSPR detection method for ALP showed a linear range from 1.0 pM to 25 nM with a detection limit of 0.5 pM. Using DNA as a mode target molecule, this technique showed a detection range from 10 fM to 50 pM DNA with a detection limit of 2.6 fM through the convenient combination with hybridization chain reaction amplification. The proposed plasmonic colorimetric strategy could be extended as a general analytical platform for design of immunosensors and aptasensors with ALP as a label.

Topics: Alkaline Phosphatase; Ascorbic Acid; Biosensing Techniques; Colorimetry; DNA; Gold; Limit of Detection; Metal Nanoparticles; Silver; Surface Plasmon Resonance

In this Letter, on the basis of the CdS quantum dots functionalized TiO2 nanotubes electrode, we proposed a simultaneous photoelectrochemical (PEC) immunoassay of dual cardiac markers using specific enzyme tags of alkaline phosphatase (ALP) and acetylcholine esterase (AChE). ALP and AChE were integrated into the PEC system through the sandwich immunobinding and could specifically catalyze the hydrolysis of ascorbic acid 2-phosphate (AAP) or the acetylthiocholine (ATC) to in situ generate ascorbic acid (AA) or thiocholine (TC) for sacrificial electron donating. These two enzymes were thus used to differentiate the signals of two cardiac targets in connection with the sandwich immunorecognition and PEC responses to the corresponding electron donors. This strategy demonstrates a proof of principle for the successful integration of dual enzyme tags with PEC immunoassay that can potentially provide a general format for multiplexed PEC bioanalysis.

Topics: Acetylcholinesterase; Acetylthiocholine; Alkaline Phosphatase; Antibodies, Immobilized; Ascorbic Acid; C-Reactive Protein; Chemistry Techniques, Analytical; Electrochemical Techniques; Electrodes; Humans; Immunoassay; Nanotubes; Quantum Dots; Titanium; Troponin I

Advanced glycosylation end products (AGEs) are endogenous inflammatory mediators that induce apoptosis of mesenchymal stem cells. A potential mechanism includes increased generation of reactive oxygen species (ROS). MicroRNA-223 (miR-223) is implicated in the regulation of cell growth and apoptosis in several cell types. Here, we tested the hypothesis that antioxidants N-acetylcysteine (NAC) and ascorbic acid 2-phosphate (AAP) inhibit AGE-induced apoptosis via a microRNA-dependent mechanism in human adipose tissue-derived stem cells (ADSCs). Results showed that AGE-HSA enhanced apoptosis and caspase-3 activity in ADSCs. AGE-HSA also increased ROS generation and upregulated the expression of miR-223. Interestingly, reductions in ROS generation and apoptosis, and upregulation of miR-223 were found in ADSCs treated with antioxidants NAC and AAP. Furthermore, miR-223 mimics blocked antioxidant inhibition of AGE-induced apoptosis and ROS generation. Knockdown of miR-223 amplified the protective effects of antioxidants on apoptosis induced by AGE-HSA. miR-223 acted by targeting fibroblast growth factor receptor 2. These results indicate that NAC and AAP suppress AGE-HSA-induced apoptosis of ADSCs, possibly through downregulation of miR-223.

Topics: Acetylcysteine; Adipose Tissue; Antioxidants; Apoptosis; Ascorbic Acid; Caspase 3; Cell Proliferation; Gene Expression Regulation, Developmental; Glycation End Products, Advanced; Humans; Mesenchymal Stem Cells; MicroRNAs; Reactive Oxygen Species; Serum Albumin; Serum Albumin, Human

The Tixel is a novel device based on a thermo-mechanical ablation technology that combines a sophisticated motion and a temperature control. The fractional technology is used to transfer a very precise thermal energy to the skin thereby creating an array of microchannels, accompanying by no signs of pain or inconvenience. This study aimed to evaluate the effect of the Tixel on the skin permeability of three hydrophilic molecular models: verapamil hydrochloride, diclofenac sodium, and magnesium ascorbyl phosphate. Tixel's gold-platted stainless steel tip heated to a temperature of 400°C was applied on skin for 8ms or 9ms at a protrusion of 400μm (the distance in which the tip protrudes beyond the distance gauge). The experiments were carried out partly in vivo in humans using a fluorescent dye and a confocal microscopy and partly in vitro using porcine skin and a Franz diffusion cell system. The results obtained in this study have shown that (a) no significant collateral damage to the skin tissue and no necrosis or dermal coagulation have been noted, (b) the microchannels remained open and endured for at least 6h, and (c) the skin permeability of hydrophilic molecules, which poorly penetrate the lipophilic stratum corneum barrier, was significantly enhanced by using Tixel's pretreatment.

Topics: Animals; Ascorbic Acid; Diclofenac; Drug Delivery Systems; Hot Temperature; Humans; Injections, Intradermal; Phenolsulfonphthalein; Skin; Skin Absorption; Swine; Verapamil

Activin A is a growth factor released by mature osteoblasts that has a critical effect on bone formation. We investigated the effect of bone morphogenetic protein (BMP)-4 on activin A gene expression during in vitro osteogenic differentiation of mouse embryonic stem (ES) cells. Embryoid bodies were cultured in retinoic acid (RA) for three days and then without RA for two days. Seeded cells received osteogenic medium with β-glycerophosphate, L-ascorbic acid 2-phosphate and dexamethasone during 19 days, with or without BMP-4. Six independent experiments were carried out. Real-time PCR was used to detect gene expression of activin A, Oct-4, Nanog, osteocalcin, RUNX2 and bone alkaline phosphatase. Immunofluorescence was used to co-localize activin A with the undifferentiation marker stage-specific embryonic antigen 1. Cells treated with BMP-4 had an increased gene expression of activin A, osteocalcin and bone alkaline phosphatase (p < 0.05). In conclusion, BMP-4 increases activin A gene expression during mouse ES cell differentiation into bone precursors.

Topics: Activins; Animals; Ascorbic Acid; Bone Morphogenetic Protein 4; Cell Differentiation; Culture Media; Dexamethasone; DNA Primers; Fibroblasts; Gene Expression Regulation, Developmental; Glycerophosphates; Mice; Microscopy, Fluorescence; Mouse Embryonic Stem Cells; Osteogenesis; Real-Time Polymerase Chain Reaction; RNA, Messenger; Tretinoin

Zinc oxide (ZnO) appears as a promising preservative for pharmaceutical or cosmetic formulations. The other ingredients of the formulations may have specific interactions with ZnO that alter its antimicrobial properties. The influence of common formulation excipients on the antimicrobial efficacy of ZnO has been investigated in simple model systems and in typical topical products containing a complex formulation. A wide variety of formulation excipients have been investigated for their interactions with ZnO: antioxidants, chelating agents, electrolytes, titanium dioxide pigment. The antimicrobial activity of ZnO against Escherichia coli was partially inhibited by NaCl and MgSO4 salts. A synergistic influence of uncoated titanium dioxide has been observed. The interference effects of antioxidants and chelating agents were quite specific. The interactions of these substances with ZnO particles and with the soluble species released by ZnO were discussed so as to reach scientific guidelines for the choice of the ingredients. The preservative efficacy of ZnO was assessed by challenge testing in three different formulations: an oil-in-water emulsion; a water-in-oil emulsion and a dry powder. The addition of ZnO in complex formulations significantly improved the microbiological quality of the products, in spite of the presence of other ingredients that modulate the antimicrobial activity.

Topics: Administration, Topical; Anti-Infective Agents; Antioxidants; Ascorbic Acid; Aspergillus; Butylated Hydroxytoluene; Candida albicans; Chelating Agents; Edetic Acid; Escherichia coli; Excipients; Magnesium Sulfate; Preservatives, Pharmaceutical; Pseudomonas aeruginosa; Sodium Chloride; Staphylococcus aureus; Titanium; Zinc Oxide

This paper presents a chronoamperometric method to determine tungsten in water using screen-printed carbon electrodes modified with gold nanoparticles and cross linked alkaline phosphatase immobilized in the working electrode. Enzymatic activity over 2-phospho-l-ascorbic acid trisodium salt, used as substrate, was affected by tungsten ions, which resulted in a decrease of chronoamperometric current, when a potential of 200 mV was applied on 10 mM of substrate in a Tris HCl buffer pH 8.00 and 0.36 M of KCl. Calibration curves for the electrochemical method validation, give a reproducibility of 5.2% (n = 3), a repeatability of 9.4% (n = 3) and a detection limit of 0.29 ± 0.01 µM. Enriched tap water, purified laboratory water and bottled drinking water, with a certified tungsten reference solution traceable to NIST, gave a recovery of 97.1%, 99.1% and 99.1% respectively (n = 4 in each case) and a dynamic range from 0.6 to 30 µM. This study was performed by means of a Lineweaver-Burk plot, showing a mixed kinetic inhibition.

Topics: Alkaline Phosphatase; Ascorbic Acid; Biosensing Techniques; Carbon; Enzymes, Immobilized; Gold; Limit of Detection; Metal Nanoparticles; Solutions; Tungsten; Water

l-ascorbic acid 2-phosphate (Asc-2P) acts as an antioxidant and a stimulator of hepatocyte growth factor (HGF) production. Previously, we reported that depletion of growth factors such as fibroblast growth factor (FGF)-2, epidermal growth factor (EGF), FGF-4 and HGF during serial passage could induce autophagy, senescence and down-regulation of stemness (proliferation via FGF-2/-4 and differentiation via HGF). In this study, we investigated the proliferation and differentiation potential of BMSCs by FGF-2 and Asc-2P. Co-treatment with FGF-2 and Asc-2P induced optimal proliferation of BMSCs and increased the accumulation rate of BMSC numbers during a 2-month culture period. Moreover, differentiation potential was maintained by co-treatment with FGF-2 and Asc-2P via HGF expression. Adipogenic differentiation potential by FGF-2 and Asc-2P was dramatically suppressed by c-Met inhibitors (SU11274). These data suggest that co-treatment with FGF-2 and Asc-2P would be beneficial in obtaining BMSCs that possess "stemness" during long-term culture.

Topics: Adipocytes; Adult; Ascorbic Acid; Autophagy; Bone Marrow Cells; Cell Differentiation; Cell Proliferation; Cells, Cultured; Cellular Senescence; Fibroblast Growth Factor 2; Healthy Volunteers; Hepatocyte Growth Factor; Humans; Mesenchymal Stem Cells; Reactive Oxygen Species; Young Adult

We report the first in vitro enzymatic synthesis of paramagnetic and antiferromagnetic nanoparticles toward magnetic ELISA reporting. With our procedure, alkaline phosphatase catalyzes the dephosphorylation of l-ascorbic-2-phosphate, which then serves as a reducing agent for salts of iron, gadolinium, and holmium, forming magnetic precipitates of Fe45±14Gd5±2O50±15 and Fe42±4Ho6±4O52±5. The nanoparticles were found to be paramagnetic at 300 K and antiferromagnetic under 25 K. Although weakly magnetic at 300 K, the room-temperature magnetization of the nanoparticles found here is considerably greater than that of analogous chemically-synthesized LnxFeyOz (Ln = Gd, Ho) samples reported previously. At 5 K, the nanoparticles showed a significantly higher saturation magnetization of 45 and 30 emu/g for Fe45±14Gd5±2O50±15 and Fe42±4Ho6±4O52±5, respectively. Our approach of enzymatically synthesizing magnetic labels reduces the cost and avoids diffusional mass-transfer limitations associated with pre-synthesized magnetic reporter particles, while retaining the advantages of magnetic sensing.

Topics: Alkaline Phosphatase; Ascorbic Acid; Enzyme-Linked Immunosorbent Assay; Gadolinium; Holmium; Iron; Magnetite Nanoparticles

Acne is the most common inflammatory skin disease. Interleukin-1 (IL-1) is at the beginning of the cytokine signaling cascade and may be involved in the pathogenesis of this disorder. It activates redox-sensitive transcription factors, which induce IL-6 and IL-8 expression. Interestingly, L-ascorbyl-2-phosphate (APS) was shown to have beneficial effects in patients with acne vulgaris. The mechanism of action of this agent remains unknown. Here, we investigated if APS attenuates IL-1β- or TNF-α-mediated IL-6 and IL-8 expression in SZ95 sebocytes, whereas TNF-α was used as control. We also explored NF-κB activation which is known to orchestrate IL-1β- and TNF-α-mediated cytokine expression in many cell types. Both IL-1β and TNF-α increased IL-6 and IL-8 mRNA expression in SZ95 sebocytes. However, only IL-1β induced IL-6 and IL-8 secretion. IL-1β but not TNF-α activated NF-κB canonical signaling as demonstrated by Iκ-Bα phosphorylation and degradation as well as by nuclear accumulation of NF-κB/p65. Concomitant treatment of SZ95 sebocytes with APS attenuated the effect of IL-1β and TNF-α on IL-6 and IL-8 gene expression as well as on IL-1β-mediated NF-κB signaling. In contrast, APS failed to reduce IL-1β-mediated IL-6 and IL-8 secretion, presumably by maintained IL-1β-mediated p38 activation, which is known to control IL-8 secretion. Our findings shed light into the impact of IL-1β on the inflammatory cytokine response and its molecular mechanisms in human sebocytes. Our data further suggest that the beneficial effect of APS in acne patients involves attenuation of NF-κB signaling but not reduction of IL-6 or IL-8 secretion.

Topics: Ascorbic Acid; Cell Line; Cell Survival; Gene Expression Regulation; Humans; Interleukin-1beta; Interleukin-6; Interleukin-8; NF-kappa B; RNA, Messenger; Signal Transduction; Tumor Necrosis Factor-alpha

Human mesenchymal stem cells (hMSCs) contribute to ischemic tissue repair, regeneration, and possess ability to self-renew. However, poor viability of transplanted hMSCs within ischemic tissues has limited its therapeutic efficiency. Therefore, it is urgent to explore new method to improve the viability of the grafted cells. By using a systematic analysis, we reveal the mechanism of synergistic protection of N-acetylcysteine (NAC) and ascorbic acid 2-phosphate (AAP) on hMSCs that were under H2O2-induced oxidative stress. The combined treatment of NAC and AAP (NAC/AAP) reduces reactive oxygen species (ROS) generation, stabilizes mitochondrial membrane potential and decreases mitochondrial fission/fragmentation due to oxidative stress. Mitochondrial fission/fragmentation is a major prologue of mitoptosis. NAC/AAP prevents apoptotic cell death via decreasing the activation of BAX, increasing the expression of BCL2, and reducing cytochrome c release from mitochondria that might lead to the activation of caspase cascade. Stabilization of mitochondria also prevents the release of AIF, and its nuclear translocation which may activate necroptosis via H2AX pathway. The decreasing of mitoptosis is further studied by MicroP image analysis, and is associated with decreased activation of Drp1. In conclusion, NAC/AAP protects mitochondria from H2O2-induced oxidative stress and rescues hMSCs from mitoptosis, necroptosis and apoptosis.

Topics: Acetylcysteine; Apoptosis; Apoptosis Inducing Factor; Ascorbic Acid; bcl-2-Associated X Protein; Cell Proliferation; Cells, Cultured; Cytochromes c; Drug Synergism; Dynamins; GTP Phosphohydrolases; Histones; Humans; Hydrogen Peroxide; Mesenchymal Stem Cells; Microtubule-Associated Proteins; Mitochondria; Mitochondrial Proteins; Necrosis; Oxidative Stress; Protective Agents; Proto-Oncogene Proteins c-bcl-2

To evaluate the effect of 3% phosphate ascorbyl gel (PA) in different times onto the microshear bond strength of composite resin (CR) to bovine enamel treated with 35% hydrogen peroxide (HP).. Thirty enamel blocks of bovine incisors were made and divided into 5 groups (n = 6) with three specimens per group (n = 18), according to treatment: G1= No bleaching + CR; G2 = HP + CR after 15d; G3 = HP + CR after 24 hours; G4 = HP + PA (15 min) + CR after 24 hours; G5 = HP + PA (2 hours) + CR after 24 hours. The resin cylinders were made by Tygon matrices. Microshear bond strength test was performed using universal testing machine with a 50N load at a speed of 0.5 mm/min. Fracture modes were assessed by a stereomicroscope 40 ×. Microshear bond strength values were submitted to the analysis of variance (ANOVA) one-way and Tukey test (p < 0.05).. G1 had significant results when compared to G3 and G5 (p < 0.01). However, G2, G3, G4 and G5 have showed no significant differences among groups (p > 0.05). Failure modes were categorized into adhesive (90%) and mixed (10%).. The use of 3% phosphate ascorbyl gel for 15 minutes was able to improve bond strength of composite resin to bleached bovine enamel, but when 3% phosphate ascorbyl gel was applied during 40 minutes it negatively interfered in the adhesion of the resin to bleached bovine enamel.

Topics: Animals; Antioxidants; Ascorbic Acid; Cattle; Composite Resins; Dental Bonding; Dental Enamel; Dental Materials; Dental Stress Analysis; Hydrogen Peroxide; Light-Curing of Dental Adhesives; Materials Testing; Resin Cements; Shear Strength; Stress, Mechanical; Surface Properties; Temperature; Time Factors; Tooth Bleaching Agents; Water

Tenocytes represent a valuable source of cells for the purposes of tendon tissue engineering and regenerative medicine and as such, should possess a high degree of tenogenic differentiation prior to their use in vivo in order to achieve maximal efficacy. In the current report, we identify an efficient means by which to maintain differentiated tenocytes in vitro by employing the hanging drop technique in combination with defined growth media supplements. Equine tenocytes retained a more differentiated state when cultured as scaffold-free microtissue spheroids in low serum-containing medium supplemented with L-ascorbic acid 2-phosphate, insulin and transforming growth factor (TGF)-β1. This was made evident by significant increases in the expression levels of pro-tenogenic markers collagen type I (COL1A2), collagen type III (COL3A1), scleraxis (SCX) and tenomodulin (TNMD), as well as by enhanced levels of collagen type I and tenomodulin protein. Furthermore, tenocytes cultured under these conditions demonstrated a typical spindle-like morphology and when embedded in collagen gels, became highly aligned with respect to the orientation of the collagen structure following their migration out from the microtissue spheroids. Our findings therefore provide evidence to support the use of a biomimetic microtissue approach to culturing tenocytes and that in combination with the defined growth media described, can improve their differentiation status and functional repopulation of collagen matrix.

Topics: Animals; Ascorbic Acid; Biomimetics; Cell Differentiation; Cells, Cultured; Collagen; Culture Media; Horses; Intercellular Signaling Peptides and Proteins; Regeneration; Spheroids, Cellular; Tendons; Tissue Engineering; Tissue Scaffolds; Transforming Growth Factor beta1

The rate of a bimolecular reaction between a fluorophore and a freely diffusing molecule in the solvent depends on the accessibility of the fluorophore for collision with the molecule. We previously reported that the observation of blinking, caused by the formation of R6G in the excited triplet state ((3) R6G*) and its quenching reaction with O2 , allowed us to monitor the DNA conformational changes between a duplex and a hairpin. However, the small molecular size of O2 hampered sensitive monitoring of the microenvironment changes around R6G. In this study, we control redox blinking by adding a reductant ascorbic acid 2-phosphate (VcP), which converts (3) R6G* into the radical anion form R6G(.-) , and by adding a bulky oxidant FeDTPA. The bimolecular electron-transfer rate between R6G(.-) and bulky FeDTPA was more strongly affected by microenvironment changes around R6G, compared with that between (3) R6G* and the smaller O2 . This allowed us to monitor subtle DNA conformational changes caused by a single different nucleotide.

Topics: Ascorbic Acid; DNA; Electron Transport; Nucleic Acid Conformation; Oxidation-Reduction; Oxygen

Hypoxia inducible factor-1 alpha (HIF-1α) is thought to play a role in melanoma carcinogenesis. Posttranslational regulation of HIF-1α is dependent on Prolyl hydroxylase (PHD 1-3) and Factor Inhibiting HIF (FIH) hydroxylase enzymes, which require ascorbic acid as a co-factor for optimal function. Depleted intra-tumoral ascorbic acid may thus play a role in the loss of HIF-1α regulation in melanoma. These studies assess the ability of ascorbic acid to reduce HIF-1α protein and transcriptional activity in metastatic melanoma and reduce its invasive potential.. HIF-1α protein was evaluated by western blot, while transcriptional activity was measured by HIF-1 HRE-luciferase reporter gene activity. Melanoma cells were treated with ascorbic acid (AA) and ascorbate 2-phosphate (A2P) to assess their ability to reduce HIF-1α accumulation and activity. siRNA was used to deplete cellular PHD2 in order to evaluate this effect on AA's ability to lower HIF-1α levels. A2P's effect on invasive activity was measured by the Matrigel invasion assay. Data was analyzed by One-way ANOVA with Tukey's multiple comparisons test, or Student-T test as appropriate, with p < .05 considered significant.. Supplementation with both AA and A2P antagonized normoxic as well as cobalt chloride- and PHD inhibitor ethyl 3, 4-dihydroxybenzoate induced HIF-1α protein stabilization and transcriptional activity. Knockdown of the PHD2 isoform with siRNA did not impede the ability of AA to reduce normoxic HIF-1α protein. Additionally, reducing HIF-1α levels with A2P resulted in a significant reduction in the ability of the melanoma cells to invade through Matrigel.. These studies suggest a positive role for AA in regulating HIF-1α in melanoma by demonstrating that supplementation with either AA, or its oxidation-resistant analog A2P, effectively reduces HIF-1α protein and transcriptional activity in metastatic melanoma cells. Our data, while supporting the function of AA as a necessary cofactor for PHD and likely FIH activity, also suggests a potential non-PHD/FIH role for AA in HIF-1α regulation by its continued ability to reduce HIF-1α in the presence of PHD inhibition. The use of the oxidation-resistant AA analog, A2P, to reduce the ability of HIF-1α to promote malignant progression in melanoma cells and enhance their response to therapy warrants further investigation.

Topics: Ascorbic Acid; Cell Hypoxia; Cell Line, Tumor; Gene Expression; Gene Expression Regulation, Neoplastic; Genes, Reporter; Humans; Hypoxia-Inducible Factor 1, alpha Subunit; Melanoma; Neoplasm Metastasis; Protein Stability; Transcription, Genetic

In this work, a photoelectrochemical (PEC) biosensor was fabricated for sensitive and specific detection of microRNA based on Bi2S3 nanorods and enzymatic signal amplification. Using the catalytic effect of alkaline phosphatase on l-ascorbic acid 2-phosphate trisodium salt (AAP), ascorbic acid (AA) was in situ generated and used as electron donor. Based on this, a signal-on protocol was successively achieved for microRNAs detection due to the dependence of photocurrent response on the concentration of electron donor of AA. The results demonstrated that the photocurrent response enhanced with increasing the hybridized concentration of microRNA. Under the amplification of the immunogold labeled streptavidin (SA-AuNPs), a low detection limit of 1.67 fM was obtained. The fabricated biosensor showed good detection stability and specificity, and it could discriminate only one-base mismatched microRNA sequence. Moreover, the down-regulated expression of microRNA-21 in DF-1 chicken fibroblast cells infected with subgroup J avian leukemia virus (ALVs) was confirmed by the developed method, indicating that microRNA-21 might be a new biomarker for avian leukemia. This work opens a different perspective for microRNAs detection and early diagnose of avian leukemia.

Topics: Alkaline Phosphatase; Animals; Ascorbic Acid; Biosensing Techniques; Chickens; Electrons; Leukemia; Limit of Detection; MicroRNAs; Nanotubes

This study was designed to investigate the potential of a wound dressing composed of hyaluronic acid (HA) and collagen (Col) spongy sheet containing epidermal growth factor (EGF) and vitamin C derivative (VC). High-molecular-weight HA aqueous solution, hydrolyzed low-molecular-weight HA aqueous solution and heat-denatured Col aqueous solution were mixed, followed by freeze-drying to obtain a spongy sheet. Cross-linkage between Col molecules was induced by UV irradiation of the spongy sheet (C-wound dressing). In a similar manner, three types of spongy sheet containing EGF (EGF-wound dressing), containing VC (VC-wound dressing) or containing EGF and VC (EGF·VC-wound dressing) were prepared by freeze-drying the mixed solution containing the specified components. Cytokine production by fibroblasts was assessed in a wound surface model using a fibroblast-incorporating Col gel sheet (cultured dermal substitute; CDS). CDS was elevated to the air-medium interface, onto which each wound dressing was placed and cultured for 7 days. Fibroblasts in CDS covered with EGF-wound dressing released 3.6 times more VEGF and 3.0 times more HGF, as compared with the C-wound dressing. Fibroblasts in CDS covered with EGF·VC-wound dressing released 4.2 times more VEGF and 6.0 times more HGF, as compared with the C-wound dressing. The efficacy of these wound dressings was evaluated in animal tests using diabetic mice. Each wound dressing was applied to a full-thickness skin defect on the dorsal area measuring 1.5 × 2.0 cm. After 1 week of application, wound conditions were evaluated histologically. The EGF·VC-wound dressing more effectively promoted granulation tissue formation associated with angiogenesis, as compared with other wound dressings.

Topics: Animals; Ascorbic Acid; Bandages; Cell Line; Collagen; Drug Evaluation, Preclinical; Drug Synergism; Epidermal Growth Factor; Fibroblasts; Hepatocyte Growth Factor; Humans; Hyaluronic Acid; Male; Mice; Vascular Endothelial Growth Factor A; Wound Healing

Cell sheet technology has emerged as an important tissue engineering approach. Adipose-derived stem cells (ASCs) have valuable applications in regenerative medicine, but their stemness and differentiation capabilities in the cell sheet format have not been well investigated. In this study, we found that l-ascorbate 2-phosphate (A2-P), a stable form of ascorbic acid, significantly enhanced ASC proliferation and induced ASC sheet fabrication in 7 days with abundant extracellular matrix deposition. Importantly, A2-P treatment significantly enhanced expression of pluripotent markers Sox-2, Oct-4 and Nanog, but treating ASCs with antioxidants other than A2-P revealed no stemness enhancement. Moreover, ASC treatment with A2-P and a collagen synthesis inhibitor, L-2-azetidine carboxylic acid or cis-4-hydroxy-d-proline, significantly inhibited the A2-P-enhanced expression of stemness markers. These findings demonstrated that A2-P enhances stemness of ASCs through collagen synthesis and cell sheet formation. We also showed that A2-P-stimulated collagen synthesis in ASCs may be mediated through ERK1/2 pathway. By culturing the ASC sheets in proper induction media, ASC transdifferentiation capabilities into neuron and hepatocyte-like cells were significantly enhanced after cell sheet formation, while adipogenic and osteogenic differentiation capacities were still maintained. Using a murine model of healing-impaired cutaneous wound, faster wound healing was noted in the group that received ASC sheet treatment, and we observed significantly more engrafted ASCs with evidence of differentiation toward endothelial and epidermal lineages in the cutaneous wound tissue. Therefore, A2-P-mediated ASC sheet formation enhanced ASC stemness and transdifferentiation capabilities, thereby representing a promising approach for applications in regenerative medicine.

Topics: Adipose Tissue; Adult; Animals; Antioxidants; Ascorbic Acid; Biomarkers; Cell Culture Techniques; Cell Proliferation; Cell Transdifferentiation; Collagen; Disease Models, Animal; Female; Humans; Mice; Mice, Nude; Middle Aged; Neurogenesis; Phenotype; Stem Cells; Wound Healing

In peripheral blood hematopoietic stem cell transplantation (PBHSCT) , the mobilization and circulating of bone marrow hematopoietic stem cells in blood with higher oxygen concentration all increase reactive oxygen species(ROS) production, which has negative effect on the biological function of BMHSC. In order to investigate the protective effect of antioxidant on hematopoietic stem cells (HSC), the ascorbic acid 2-phosphate (AA2P), an ascorbic acid derivative of vitamin C, was added in HSC culturing by imitating oxygen conditions which BMHSC experienced in peripheral blood stem cell transplantation. The protective effect of above-mentioned culture methods on the biologic functions of BMHSC was evaluated by vitro amplification assay, committed division assay, reactive oxygen species (ROS) measurement, CD34(+) HSC engraftment. The results showed that the ROS level in HSC from in vitro cultures was much higher than that freshly separated BMHSC, and the amplified AC133(+)CD34(+) HSC, BFU-E, CFU-GM, CFU-GEMM colonies, migration rate and severe combined immunodeficiency (SCID)-repopulating cells (SRC) were all much more than HSC cultured without AA2P. It is concluded that antioxidant intervention may be an effective methods for protecting the biological function of PBHSC and improving the therapeutic effect of PBHSCT.

Topics: Antigens, CD34; Antioxidants; Ascorbic Acid; Cells, Cultured; Hematopoietic Stem Cells; Humans; Reactive Oxygen Species

This study was conducted to investigate the effects of dietary ascorbic acid (AA) on transcriptional expression patterns of antioxidant proteins, heat shock proteins (HSP) and nuclear factor kappa B (NF-κB) in the hepatopancreas of Pacific abalone Haliotis discus hannai Ino (initial average length: 84.36 ± 0.24 mm) using real-time quantitative PCR assays. L-ascorbyl-2-molyphosphate (LAMP) was added to the basal diet to formulate four experimental diets containing 0.0, 70.3, 829.8 and 4967.5 mg AA equivalent kg(-1) diets, respectively. Each diet was fed to triplicate groups of adult abalone in acrylic tanks (200 L) in a flow-through seawater system. Each tank was stocked with 15 abalone. Animals were fed once daily (17:00) to apparent satiation for 24 weeks. The results showed that the dietary AA (70.3 mg kg(-1)) could significantly up-regulate the expression levels of Cu/Zn superoxide dismutase (SOD), catalase (CAT), glutathione-S-transferase (GST), feritin (FT) and heat shock protein 26 (HSP26) in the hepatopancreas of abalone in this treatment compared to the controls. However, the expression levels of Mn-SOD, glutathione peroxidase (GPX), thioredoxin peroxidase (TPx), selenium-binding protein (SEBP), HSP70 and HSP90 were significantly down-regulated. Compared with those in the group with 70.3 mg kg(-1) dietary AA, the expression levels of CAT, GST and HSP26 were decreased in abalone fed with very high dietary AA (4967.5 mg kg(-1)). In addition, significant up-regulations of expression levels of Mn-SOD, GPX, TPx, SEBP, FT, HSP70, HSP90 and NF-κB were observed in abalone fed with apparently excessive dietary AA (829.8 and 4967.5 mg kg(-1)) as compared to those fed 70.3 mg kg(-1) dietary AA. These findings showed that dietary AA influenced the expression levels of antioxidant proteins, heat shock proteins and NF-κB in the hepatopancreas of abalone at transcriptional level. Levels of dietary AA that appeared adequate (70.3 mg kg(-1)) reduced the oxidative stress by influencing gene expression of antioxidant proteins, but excessive dietary AA (829.8 and 4967.5 mg kg(-1)) induced oxidative stress in Pacific abalone H. discus hannai.

Topics: Animals; Ascorbic Acid; Catalase; Dietary Supplements; Dose-Response Relationship, Drug; Ferritins; Gastropoda; Gene Expression Regulation; Glutathione Transferase; Heat-Shock Proteins; Hepatopancreas; NF-kappa B; Oxidative Stress; Real-Time Polymerase Chain Reaction; Superoxide Dismutase

Nanostructure-based visual assay has been developed for determination of enzymatic activity, but most involve in poor visible color resolution and are not suitable for routine utilization. Herein, we designed a high-resolution colorimetric protocol based on gold/silver core/shell nanorod for visual readout of alkaline phosphatase (ALP) activity by using bare-eyes. The method relied on enzymatic reaction-assisted silver deposition on gold nanorod to generate significant color change, which was strongly dependent on ALP activity. Upon target ALP introduction into the substrate, the ascorbic acid 2-phosphate was hydrolyzed to form ascorbic acid, and then, the generated ascorbic acid reduced silver ion to metal silver and coated on the gold nanorod, thereby resulting in the blue shift of longitudinal localized surface plasmon resonance peak of gold nanorod accompanying a perceptible color change from red to orange to yellow to green to cyan to blue and to violet. Under optimal conditions, the designed method exhibited the wide linear range 5-100 mU mL(-1) ALP with a detection limit of 3.3 mU mL(-1). Moreover, it could be used for the semiquantitative detection of ALP from 20 to 500 mU mL(-1) by using the bare-eyes. The coefficients of variation for intra- and interassay were below 3.5% and 6.2%, respectively. Finally, this method was validated for the analysis of real-life serum samples, giving results matched well with those from the 4-nitrophenyl phosphate disodium salt hexahydrate (pNPP)-based standard method. In addition, the system could even be utilized in the enzyme-linked immunosorbent assay (ELISA) to detect IgG at picomol concentration. With the merits of simplification, low cost, user-friendliness, and sensitive readout, the gold nanorod-based colorimetric assay has the potential to be utilized by the public and opens a new horizon for bioassays.

Topics: Alkaline Phosphatase; Ascorbic Acid; Colorimetry; Gold; Humans; Immunoassay; Nanotubes; Reference Standards; Silver; Spectrophotometry, Ultraviolet

A variety of different growth factors, most notably bone morphogenetic proteins (BMPs), have been shown to stimulate the osteogenic differentiation of mesenchymal stromal cells (MSCs) in vitro. Yet, due to the lack of comparative studies it remains unclear which protocol is the most effective in the induction of osteogenesis in MSC cultures. The aim of this study was to compare the most potent growth factors in regard to their osteoinductive potential. Human MSCs were cultured for 10 days in the presence of BMP-2, BMP-6, BMP-9 + IGF-2 and BMP-2, -6, -9 (day 1 + 2: 50 ng/ml; days 3-6: 100 ng/ml; days 7-10: 200 ng/ml). The formation of the osteoblast phenotype was assessed by quantification of osteoblast-related marker genes using reverse transcription polymerase chain reaction (RT-PCR) and alkaline phosphatase (ALP) staining. Matrix mineralization was assessed by alizarin red S and von Kossa staining. Statistical analysis was carried out using the one-way analysis of variance (ANOVA) followed by Scheffe's post hoc procedure. Among the tested growth factors the combination of BMP-2 + BMP-6 + BMP-9 most effectively induced the upregulation of collagen type I, collagen type V, osteocalcin, alkaline phosphatase, RUNX2, BMP-2, osteonectin and DLX5 (p < 0.01) and resulted in a consistent matrix mineralization. The findings suggest the combined addition of BMP-2, BMP-6 and BMP-9 to the osteoinductive culture medium containing dexamethasone, β-glycerophosphate and ascorbate-2-phosphate produces more potent osteoblast differentiation of human MSCs in vitro.

Topics: Alkaline Phosphatase; Ascorbic Acid; Bone Morphogenetic Protein 2; Bone Morphogenetic Protein 6; Calcification, Physiologic; Cell Culture Techniques; Cell Differentiation; Cells, Cultured; Collagen Type I; Collagen Type V; Core Binding Factor Alpha 1 Subunit; Culture Media; Dexamethasone; Glycerophosphates; Growth Differentiation Factor 2; Growth Differentiation Factors; Homeodomain Proteins; Humans; Insulin-Like Growth Factor II; Intercellular Signaling Peptides and Proteins; Mesenchymal Stem Cells; Osteoblasts; Osteocalcin; Osteogenesis; Osteonectin; Phenotype; Transcription Factors

Cells are exposed to several types of integrin stimuli, which generate responses generally referred to as "integrin signals", but the specific responses to different integrin stimuli are poorly defined. In this study, signals induced by integrin ligation during cell attachment, mechanical force from intracellular contraction, or cell stretching by external force were compared. The elevated phosphorylation levels of several proteins during the early phase of cell attachment and spreading of fibroblast cell lines were not affected by inhibition of ROCK and myosin II activity, i.e. the reactions occurred independently of intracellular contractile force acting on the adhesion sites. The contraction-independent phosphorylation sites included ERK1/2 T202/Y204, AKT S473, p130CAS Y410, and cofilin S3. In contrast to cell attachment, cyclic stretching of the adherent cells induced a robust phosphorylation only of ERK1/2 and the phosphorylation levels of the other investigated proteins were not or only moderately affected by stretching. No major differences between signaling via α5β1 or αvβ3 integrins were detected. The importance of mitochondrial ROS for the integrin-induced signaling pathways was investigated using rotenone, a specific inhibitor of complex I in the respiratory chain. While rotenone only moderately reduced ATP levels and hardly affected the signals induced by cyclic cell stretching, it abolished the activation of AKT and reduced the actin polymerization rate in response to attachment in both cell lines. In contrast, scavenging of extracellular ROS with catalase or the vitamin C analog Asc-2P did not significantly influence the attachment-derived signaling, but caused a selective and pronounced enhancement of ERK1/2 phosphorylation in response to stretching. In conclusion, the results showed that "integrin signals" are composed of separate sets of reactions triggered by different types of integrin stimulation. Mitochondrial ROS and extracellular ROS had specific and distinct effects on the integrin signals induced by cell attachment and mechanical stretching.

Topics: Ascorbic Acid; Biomechanical Phenomena; Catalase; Cell Adhesion; Cell Line; Humans; Integrins; Intracellular Space; Ligands; Mechanical Phenomena; Mitochondria; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Proto-Oncogene Proteins c-akt; Reactive Oxygen Species; Signal Transduction

The objective of this study was to investigate the effects of two lasers (Er:YAG and CO2) in enhancing skin permeation of three vitamin C derivatives, L-ascorbic acid 2-phosphate sesquimagnesium salt (MAP-1), magnesium L-ascorbic acid-2-phosphate (MAP-2), and 2-phospho-L-ascorbic acid trisodium salt (SAP). Dorsal skin of 1-week-old pathogen-free pigs was used for this in vitro study. Changes in permeation in laser-treated skin treated by the lasers were examined by confocal scanning electron microscopy. Transdermal flux of vitamin C derivatives was examined with a Franz diffusion cell. Fluxes of MAP-1, MAP-2, and SAP across Er:YAG laser-treated skin were 15-27-fold, 48-123-fold, and 22-56-fold higher, respectively, than their fluxes across intact skin. The fluxes of MAP-1, MAP-2, and SAP across CO2 laser-treated skin were 28-36-fold, 116-156-fold, and 79-102-fold higher, respectively, than their fluxes across intact skin. Optimal fluency for the Er:YAG laser was 3.8 J/cm(2) for MAP-1 and 5 J/cm(2) for MAP-2 and SAP. Optimal fluency for the CO2 laser was 5 W for all three derivatives. In conclusion, optimal fluency for all derivatives was 5 W for the CO2 laser and 3.8 to 5 J/cm(2) for the Er:YAG laser.

Topics: Administration, Cutaneous; Animals; Ascorbic Acid; In Vitro Techniques; Lasers, Gas; Lasers, Solid-State; Microscopy, Confocal; Microscopy, Electron, Scanning; Permeability; Skin; Swine

The aim of this study was to evaluate inhibitory effects of L-ascorbic acid-2-O-phosphate-Na(2) (APS), a pro-vitamin C, combined with hyperthermia on adipogenic differentiation of mouse stromal cells, OP9.. OP9 preadipocytes were differentiated with serum replacement, administered with APS, and simultaneously treated with hyperthermia using a capacitive-resistive electric transfer (CRet) apparatus, which was conducted repeatedly twice a day. After 2 days, intracellular lipid droplets were stained with Oil Red O, then observed by microscopy and assessed spectrophotometrically.. After stimulation by serum replacement for 2 days, lipid droplets were accumulated surrounding nucleus of OP9 cells. When APS of 0.15-0.6 mM was administered without hyperthermia, the amount of lipid droplets was markedly suppressed to 50.5%∼-11.3% versus the undifferentiated control, and diminished huge aggregates of lipid droplets. In OP9 cells treated by hyperthermia at 42°C for 0.5 min, 1 min or 3 min in the absence of APS, adipogenesis was suppressed abruptly in a time-dependent manner to 95.4%, 18.7% or -5.5%, respectively. Whereas, the percentage of adipogenesis was 96.8% in OP9 cells treated by mild hyperthermia alone at 41°C for 1 min. The simultaneous application of APS and hyperthermia at 41°C for 1 min markedly suppressed the accumulation of lipid droplets to 25.7%∼-66.2%. By scanning electron microscopy (SEM) observation, the surface of OP9 cells treated with APS and hyperthermia appeared to have the morphological property of undifferentiated OP9 cells.. Combined treatment of APS and mild hyperthermia suppresses adipogenesis in OP9 cells, particularly in lipid droplets accumulation during spontaneous differentiation of OP9 preadipocytes.

Topics: Adipocytes; Adipogenesis; Animals; Antineoplastic Agents; Antioxidants; Ascorbic Acid; Cell Line; Hyperthermia, Induced; Lipid Metabolism; Mice; Stromal Cells

Recent endeavors to use stem cells as trophic factor production sources have the potential to translate into viable therapies for damaged or diseased musculoskeletal tissues. Adipose stem cells (ASCs) can be differentiated into chondrocytes using the chondrogenic medium (CM), but it is unknown if this approach can optimize ASC growth factor secretion for cartilage regeneration by increasing the chondrogenic factor production, while decreasing angiogenic and hypertrophic factor production. The objective of this study was to determine the effects the CM and its components have on growth factor production from ASCs to promote cartilage regeneration. ASCs isolated from male Sprague-Dawley rats and cultured in monolayer or alginate microbeads were treated with either the growth medium (GM) or the CM for 5 days. In subsequent studies, ASC monolayers were treated with either the GM supplemented with different combinations of 50 μg/mL ascorbic acid-2-phosphate (AA2P), 100 nM dexamethasone (Dex), 10 ng/mL transforming growth factor (TGF)-β1, and 100 ng/mL bone morphogenetic protein (BMP)-6 or with the CM excluding different combinations of AA2P, Dex, TGF-β1, and BMP-6. mRNA levels and growth factor production were quantified at 8 and 24 h after the last media change, respectively. The CM increased chondrogenic factor secretion (TGF-β2, TGF-β3, and insulin-like growth factor [IGF]-I) and decreased angiogenic factor production (the vascular endothelial growth factor [VEGF]-A, the fibroblast growth factor [FGF]-2). Microencapsulation in the GM increased production of the chondrogenic (IGF-I, TGF-β2) and angiogenic (VEGF-A) factors. AA2P increased secretion of chondrogenic factors (IGF-I, TGF-β2), and decreased angiogenic factor (VEGF-A) secretion, in addition to decreasing mRNA levels for factors associated with chondrocyte hypertrophy (FGF-18). Dex increased mRNA levels for hypertrophic factors (BMP-2, FGF-18) and decreased angiogenic factor secretion (VEGF-A). TGF-β1 increased angiogenic factor production (FGF-2, VEGF-A) and decreased chondrogenic factor mRNA levels (IGF-I, PTHrP). BMP-6 increased hypertrophic mRNA levels (FGF-18) and chondrogenic factor production (TGF-β2). When ASC microbeads preconditioned with the CM were implanted in a focal cartilage defect and immobilized within an RGD-conjugated hydrogel, tissue infiltration from the edges of the defect and perichondrium was observed. These results show that differentiation media components have distinct ef

Topics: Adipose Tissue; Animals; Ascorbic Acid; Bone Morphogenetic Protein 6; Cartilage; Cell Differentiation; Chondrocytes; Chondrogenesis; Culture Media; Dexamethasone; Humans; Intercellular Signaling Peptides and Proteins; Male; Microspheres; Rats; Rats, Sprague-Dawley; Regeneration; RNA, Messenger; Signal Transduction; Stem Cells; Transforming Growth Factor beta1

Cell-based tissue engineering strategies have shown tremendous promise for the repair of bone mass deficiencies, but the efficient and appropriate induction of stem cells down osteogenic pathways remains a significant roadblock to the effective implementation of cell-based therapies. When grown in culture, human Mesenchymal Stromal/Stem Cells (hMSCs) remain multipotent, requiring specific exogenous signals to induce osteogenic differentiation. hMSCs used in transplantations, therefore, must be presented with local signals, often provided by the host's own tissues, to be directed down bone-related lineages. This process is relatively inefficient and remains difficult to control. In an effort to enhance osteogenesis, hMSCs were transfected with specific miRNA mimics and inhibitors that had originally identified for their ability to increase Alkaline Phosphatase (ALP) activity. Transfection with miRNA reagents had the effect of sensitizing hMSCs to soluble osteogenic factors, resulting in a rapid and robust induction of bone-related markers, including ALP activity and calcium deposition. Synthetic 3D tissue constructs prepared with miRNA-transfected hMSCs demonstrated similar responses to soluble osteogenic signals, suggesting that controlling miRNA activity in hMSCs can be an effective tool for enhancing the induction of osteogenesis for tissue engineering purposes.

Topics: Alkaline Phosphatase; Ascorbic Acid; Biomarkers; Calcium; Cell Differentiation; Cells, Cultured; Collagen; Culture Media; Dexamethasone; Humans; Mesenchymal Stem Cells; MicroRNAs; Osteogenesis; Tissue Engineering; Tissue Scaffolds; Transfection

L-Ascorbic acid 2-phosphate magnesium salt (APM) is an L-ascorbic acid (AsA) derivative developed to improve AsA stability and display effective biochemical characteristics. This study aimed to investigate the effects of APM on the functions and properties of human gingival fibroblasts with respect to the prevention of periodontal disease in comparison with those of AsA.. Human gingival fibroblasts were incubated in the presence or absence of APM or L-ascorbic acid sodium salt (AsANa). Intracellular AsA was analysed by HPLC. Collagen synthesis was measured by ELISA and real-time RT-PCR. Intracellular reactive oxygen species (ROS) induced by hydrogen peroxide (H(2)O(2)) were quantified using a fluorescence reagent, and cell damage was estimated with calcein acetoxymethyl ester. Furthermore, intracellular ROS induced by tumor necrosis factor-α (TNF-α) were quantified, and expression of TNF-α-induced interleukin-8 expression, which increases due to inflammatory reactions, was measured by ELISA and real-time RT-PCR.. APM remarkably and continuously enhanced intracellular AsA and promoted type 1 collagen synthesis and mRNA expression. Furthermore, APM decreased cell damage through the suppression of H(2)O(2)-induced intracellular ROS and inhibited interleukin-8 production through the suppression of TNF-α-induced intracellular ROS. These effects of APM were superior to those of AsANa.. These results suggest that APM is more effective than AsANa in terms of intake, collagen synthesis, decreasing cell damage and inhibiting interleukin-8 expression in human gingival fibroblasts. This suggests that local application of APM can help to prevent periodontal disease.

Topics: Anti-Inflammatory Agents; Antioxidants; Ascorbic Acid; Cell Line; Cell Proliferation; Cell Survival; Cells, Cultured; Chromatography, High Pressure Liquid; Collagen Type I; Fibroblasts; Fluoresceins; Fluorescent Dyes; Free Radical Scavengers; Gingiva; Humans; Hydrogen Peroxide; Interleukin-8; Reactive Oxygen Species; Tumor Necrosis Factor-alpha

In pH 8.9 Tris-HCl buffer solutions, alkaline phosphatase (ALP) catalyzed the hydrolysis of ascorbic acid 2-phosphate (AAP) substrate to form ascorbic acid. Then H(3)PO(4) was added to stop the enzymatic reaction and HAuCl(4) was used to react with ascorbic acid to generate gold nanoparticles that exhibited a resonance scattering (RS) peak at 600 nm. Under the selected conditions, when the activity of ALP increased, the formed ascorbic acid and gold nanoparticles also increased. Thus, the RS intensity at 600 nm enhanced linearly. The linear range was 0.06-22 U/L, with a detection limit of 0.03 U/L. The ALP in serum was analyzed, and the results were in agreement with those of the fluorescence method.

Topics: Alkaline Phosphatase; Ascorbic Acid; Biological Assay; Gold; Hydrogen-Ion Concentration; Metal Nanoparticles; Phosphates

Reprogramming of somatic cells to induced pluripotent stem cells (iPSC) provides an important cell source to derive patient-specific cells for potential therapeutic applications. However, it is not yet clear whether reprogramming through pluripotency allows the production of differentiated cells with improved functional properties that may be beneficial in regenerative therapies. To address this, we compared the production and assembly of extracellular matrix (ECM) by iPSC-derived fibroblasts to that of the parental, dermal fibroblasts (BJ), from which these iPSC were initially reprogrammed, and to fibroblasts differentiated from human embryonic stem cells (hESC). iPSC- and hESC-derived fibroblasts demonstrated stable expression of surface markers characteristic of stromal fibroblasts during prolonged culture and showed an elevated growth potential when compared to the parental BJ fibroblasts. We found that in the presence of L: -ascorbic acid-2-phosphate, iPSC- and hESC-derived fibroblasts increased their expression of collagen genes, secretion of soluble collagen, and extracellular deposition of type I collagen to a significantly greater degree than that seen in the parental BJ fibroblasts. Under culture conditions that enabled the self-assembly of a 3D stromal tissue, iPSC- and hESC-derived fibroblasts generated a well organized, ECM that was enriched in type III collagen. By characterizing the functional properties of iPSC-derived fibroblasts compared to their parental fibroblasts, we demonstrate that these cells represent a promising, alternative source of fibroblasts to advance future regenerative therapies.

Topics: Ascorbic Acid; Biomarkers; Cell Culture Techniques; Cell Differentiation; Cells, Cultured; Collagen; Embryonic Stem Cells; Extracellular Matrix; Extracellular Matrix Proteins; Fibroblasts; Humans; Induced Pluripotent Stem Cells

Topics: Antibodies, Monoclonal; Antibodies, Monoclonal, Humanized; Antineoplastic Agents; Ascorbic Acid; Cells, Cultured; Cetuximab; Cytokines; Drug Eruptions; ErbB Receptors; Humans; Keratinocytes; Sebaceous Glands

The aim of this study was to investigate whether magnesium ascorbyl phosphate (MAP) and coenzyme Q10 (CoQ10) can protect keratinocytes against ultraviolet (UV)A irradiation by increasing the levels of glutathione (GSH). The cell survival fraction was 89.9% when the keratinocytes were irradiated with UVA at a dose of 4 J/cm2. The cell survival fractions were 48.4, 9.1 and 4.8%, at doses of 8, 16 and 32 J/cm2, respectively. MAP was added to the cells prior to UVA irradiation at a dose of 8 J/cm2 and then the cell viability was assayed. The cell survival fractions were 51.6, 55.5, 64.8 and 76.7%, when MAP was added at concentrations of 125, 250, 500 µM and 1 mM, respectively. The results showed that MAP is capable of protecting keratinocytes against UVA irradiation. The cell survival fractions were 77.2, 89.4 and 90.1%, when CoQ10 was added at concentrations of 2.5, 5 and 10 µM, respectively. The results revealed that CoQ10 is capable of protecting keratinocytes against UVA irradiation. At the same time, the levels of GSH within cells were detected. The level of GSH within cells was 0.3 mmol/g protein when the keratinocytes were irradiated with UVA at a dose of 8 J/cm2. We measured the levels of GSH within the cells after MAP or CoQ10 was added prior to UVA irradiation at a dose of 8 J/cm2. The levels of GSH within the cells were 0.344, 0.388, 0.456 and 0.5 mmol/g protein, when MAP was added at concentrations of 125, 250, 500 µM and 1 mM, respectively. The levels of GSH within the cells were 0.328, 0.35 and 0.394 mmol/g protein, when CoQ10 was added at concentrations of 2.5, 5 and 10 µM, respectively. These results imply that MAP and CoQ10 can protect the keratinocytes against UVA irradiation, possibly via increasing the levels of GSH.

Topics: Antioxidants; Ascorbic Acid; Cell Death; Cell Line; Cell Survival; Dose-Response Relationship, Drug; Glutathione; Humans; Keratinocytes; Radiation-Protective Agents; Ubiquinone; Ultraviolet Rays

The present work describes the influence of both vitamin C (VC) and mechanical stimulation on development of the extracellular matrix (ECM) and improvement in mechanical properties of a chondrocyte-agarose construct in a regenerating tissue disease model of hyaline cartilage. We used primary bovine chondrocytes and two types of VC, ascorbic acid (AsA) as an acidic form and ascorbic acid 2-phosphate (A2P) as a non-acidic form, and applied uniaxial compressive strain to the tissue model using a purpose-built bioreactor. When added to the medium in free-swelling culture conditions, A2P downregulated development of ECM and suppressed improvement of the tangent modulus more than AsA. By contrast, application of mechanical stimulation to the construct both increased the tangent modulus more than the free-swelling group containing A2P and enhanced the ECM network of inner tissue to levels nearly as high as the free-swelling group containing AsA. Thus, mechanical stimulation and strain appears to enhance the supply of nutrients and improve the synthesis of ECM via mechanotransduction pathways of chondrocytes. Therefore, we suggest that mechanical stimulation is necessary for homogenous development of ECM in a cell-associated construct with a view to implantation of a large-sized articular cartilage defect.

Topics: Animals; Ascorbic Acid; Cartilage, Articular; Cattle; Cell Survival; Chondrocytes; Extracellular Matrix; Mechanotransduction, Cellular; Regeneration; Stress, Mechanical

Mesenchymal stem cells (MSCs) have been shown to improve the outcome of acute renal injury models; but whether MSCs can delay renal failure in chronic kidney disease (CKD) remains unclear. In the present study, the were cultured in media containing various concentrations of basic fibroblast growth factor, epidermal growth factor and ascorbic acid 2-phosphate to investigate whether hepatocyte growth factor (HGF) secretion could be increased by the stimulation of these growth factors. Then, TGF-β1-treated renal interstitial fibroblast (NRK-49F), renal proximal tubular cells (NRK-52E) and podocytes were co-cultured with conditioned MSCs in the absence or presence of ascorbic acid 2-phosphate to quantify the protective effects of conditioned MSCs on renal cells. Moreover, male Sprague-Dawley rats were treated with 1 × 10(6) conditioned MSCs immediately after 5/6 nephrectomy and every other week through the tail vein for 14 weeks. It was found that basic fibroblast growth factor, epidermal growth factor and ascorbic acid 2-phosphate promoted HGF secretion in MSCs. Besides, conditioned MSCs were found to be protective against TGF-β1 induced epithelial-to-mesenchymal transition of NRK-52E and activation of NRK-49F cells. Furthermore, conditioned MSCs protected podocytes from TGF-β1-induced loss of synaptopodin, fibronectin induction, cell death and apoptosis. Rats transplanted with conditioned human MSCs had a significantly increase in creatinine clearance rate, decrease in glomerulosclerosis, interstitial fibrosis and increase in CD4(+)CD25(+)Foxp3(+) regulatory T cells counts in splenocytes. Together, our studies indicated that conditioned MSCs preserve renal function by their anti-fibrotic and anti-inflammatory effects. Transplantation of conditioned MSCs may be useful in treating CKD.

Topics: Animals; Apoptosis; Ascorbic Acid; CD4-Positive T-Lymphocytes; Cells, Cultured; Coculture Techniques; Creatinine; Disease Progression; Epidermal Growth Factor; Epithelial-Mesenchymal Transition; Female; Fibroblast Growth Factor 2; Fibronectins; Fibrosis; Glomerulosclerosis, Focal Segmental; Hepatocyte Growth Factor; Humans; Kidney; Kidney Tubules, Proximal; Lymphocyte Count; Male; Mesenchymal Stem Cell Transplantation; Mesenchymal Stem Cells; Microfilament Proteins; Middle Aged; Nephrectomy; Podocytes; Rats; Rats, Sprague-Dawley; Renal Insufficiency, Chronic; Transforming Growth Factor beta1; Young Adult

To investigate the mechanisms by which L-ascorbic acid 2-phosphate (Asc-2P) increases the proliferation of human corneal endothelial cells (HCECs).. Growth of cultured HCECs was examined in the presence of various antioxidants, including Asc-2P, retinyl acetate (vitamin A), reduced glutathione, oxidized glutathione, carnosine, and sodium alpha-tocopherol phosphate (a water-soluble vitamin E derivative). Synthesis of type I, III, and IV collagen by HCECs cultured with or without Asc-2P was evaluated by measuring cell lysates and conditioned medium with Western blotting, immunocytochemistry, or enzyme-linked immunosorbent assay (ELISA). The gene expression profiles of HCECs cultured with or without Asc-2P were compared by microarray analysis to determine critical proliferative factors, and the proliferative response of these cells to selected factors was tested.. Among the antioxidants tested, only Asc-2P promoted the growth of HCECs. Asc-2P did not promote deposition of type I, III, or IV collagen. Microarray analysis revealed that several cytokines were potently upregulated by Asc-2P, but among them, only hepatocyte growth factor (HGF) stimulated HCEC growth. ELISA revealed the upregulation of HGF protein production by Asc-2P, while the stimulatory effect of Asc-2P was abolished by an anti-HGF neutralizing antibody or PHA-665752 (a specific inhibitor of the HGF receptor, c-Met).. Asc-2P increases the proliferation of cultured HCECs through upregulation of HGF production via an HGF/c-Met autocrine loop.

Topics: Antineoplastic Agents; Ascorbic Acid; Blotting, Western; Cell Proliferation; Cells, Cultured; Endothelium, Corneal; Enzyme-Linked Immunosorbent Assay; Gene Expression Regulation, Developmental; Hepatocyte Growth Factor; Humans; RNA; Tissue Array Analysis

Magnesium l-ascorbic acid 2-phosphate (MAAP) and α-tocopherol acetate (α-TAc), as the stable vitamin C and vitamin E derivative, respectively, are often applied to skin care products for reducing UV damage. The encapsulation of MAAP (0.5%, g/mL) and α-TAc (5%, g/mL) together within the polyacrylonitrile (PAN) nanofibers was demonstrated using a coaxial electrospinning technique. The structure and morphology characterizations of the core-shell fibers MAAP/α-TAc-PAN were investigated by SEM, FTIR and XRD. As a negative control, the blend nanofibers MAAP/α-TAc/PAN were prepared from a normal electrospinning method. The results from SEM indicated that the morphology and diameter of the nanofibers were influenced by concentration of spinning solution, the polymer component of the shell, the carrying agent of the core and the fabricating methods, and the core-shell nanofibers obtained at the concentration of 8% had finer and uniform structure with the average diameters of 200 ± 15nm. From in vitro release studies it could be seen that both different fiber specimens showed a gradual increase in the amount of α-TAc or MAAP released from the nanofibers. Furthermore, α-TAc and MAAP released from the blend nanofibers showed the burst release at the maximum release of ∼15% and ∼40% during the first 6h, respectively, but their release amount from the core-shell nanofibers was only 10-12% during the initial part of the process. These results showed that core-shell nanofibers alleviated the initial burst release and gave better sustainability compared to that of the blend nanofibers. The present study would provide a basis for further optimization of processing conditions to obtain desired structured core-shell nanofibers and release kinetics for practical applications in dermal tissue.

Topics: Acrylic Resins; alpha-Tocopherol; Ascorbic Acid; Drug Delivery Systems; Lactic Acid; Light; Nanofibers; Polyglycolic Acid; Polylactic Acid-Polyglycolic Acid Copolymer; Protective Agents; Solutions; Spectroscopy, Fourier Transform Infrared; X-Ray Diffraction

Here a highly sensitive electrochemical method is described for the detection of point mutation in DNA. Polymerization extension reaction is applied to specifically initiate enzymatic electrochemical amplification to improve the sensitivity and enhance the performance of point mutation detection. In this work, 5'-thiolated DNA probe sequences complementary to the wild target DNA are assembled on the gold electrode. In the presence of wild target DNA, the probe is extended by DNA polymerase over the free segment of target as the template. After washing with NaOH solution, the target DNA is removed while the elongated probe sequence remains on the sensing surface. Via hybridizing to the designed biotin-labeled detection probe, the extended sequence is capable of capturing detection probe. After introducing streptavidin-conjugated alkaline phosphatase (SA-ALP), the specific binding between streptavidin and biotin mediates a catalytic reaction of ascorbic acid 2-phosphate (AA-P) substrate to produce a reducing agent ascorbic acid (AA). Then the silver ions in solution are reduced by AA, leading to the deposition of silver metal onto the electrode surface. The amount of deposited silver which is determined by the amount of wild target can be quantified by the linear sweep voltammetry (LSV). The present approach proved to be capable of detecting the wild target DNA down to a detection limit of 1.0×10(-14) M in a wide target concentration range and identifying -28 site (A to G) of the β-thalassemia gene, demonstrating that this scheme offers a highly sensitive and specific approach for point mutation detection.

Topics: Alkaline Phosphatase; Ascorbic Acid; Biosensing Techniques; DNA; DNA Probes; Electrochemical Techniques; Electrodes; Magnesium; Oxidation-Reduction; Point Mutation; Polymerization; Sensitivity and Specificity; Silver; Streptavidin

An enzyme electrode was prepared with acid phosphatase (ACP) for development of a new electric power generation system using ascorbic acid 2-phosphate (AA2P) as a fuel. The properties of the electrode were investigated with respect to biocatalytic dephosphorylation of AA2P and electrochemical oxidation of resulting ascorbic acid (AA). The enzyme electrode was fabricated by immobilization of ACP through amide linkage onto a self-assembled monolayer of 3-mercaptopropionic acid on a gold electrode. AA2P was not oxidized on a bare gold electrode in the potential sweep range from -0.1 to +0.5 V vs. Ag/AgCl. However, the enzyme electrode gave an oxidation current in citric buffer solution of pH 5 containing 10 mM of AA2P. The oxidation current began to increase at +0.2V, and reached to 5.0 μA cm(-2) at +0.5 V. The potential +0.2 V corresponded to the onset of oxidation of ascorbic acid (AA). These results suggest that the oxidation current observed with the enzyme electrode is due to AA resulting from dephosphorylation of AA2P. The oxidation current increased with increasing concentration of AA2P and almost leveled off at around the concentration of 5mM. Thus the enzyme electrode brought about biocatalytic conversion of AA2P to AA, followed by electrochemical oxidation of the AA. The oxidation current is likely to be controlled by the biocatalytic reaction.

Topics: Acid Phosphatase; Ascorbic Acid; Bioelectric Energy Sources; Biosensing Techniques; Electrochemical Techniques; Oxidation-Reduction; Triticum

Herein, an ultrasensitive solid-state tris(2,2'-bipyridyl) ruthenium(II) (Ru(bpy)(3)(2+)) electrochemiluminescence (ECL) aptasensor using in-situ produced ascorbic acid as coreactant was successfully constructed for detection of thrombin. Firstly, the composite of Ru(bpy)(3)(2+) and platinum nanoparticles (Ru-PtNPs) were immobilized onto Nafion coated glass carbon electrode, followed by successive adsorption of streptavidin-alkaine phosphatase conjugate (SA-ALP) and biotinylated anti-thrombin aptamer to successfully construct an ECL aptasensor for thrombin determination. In our design, Pt nanoparticles in Ru(bpy)(3)(2+)-Nafion film successfully inhibited the migration of Ru(bpy)(3)(2+) into the electrochemically hydrophobic region of Nafion and facilitated the electron transfer between Ru(bpy)(3)(2+) and electrode surface. Furthermore, ALP on the electrode surface could catalyze hydrolysis of ascorbic acid 2-phosphate to in-situ produce ascorbic acid, which co-reacted with Ru(bpy)(3)(2+) to obtain quite fast, stable and greatly amplified ECL signal. The experimental results indicated that the aptasensor exhibited good response for thrombin with excellent sensitivity, selectivity and stability. A linear range of 1 × 10(-15)-1 × 10(-8) M with an ultralow detection limit of 0.33 fM (S/N=3) was obtained. Thus, this procedure has great promise for detection of thrombin present at ultra-trace levels during early stage of diseases.

Topics: Alkaline Phosphatase; Aptamers, Nucleotide; Ascorbic Acid; Biosensing Techniques; Enzymes, Immobilized; Luminescent Measurements; Nanoparticles; Organometallic Compounds; Platinum; Sensitivity and Specificity; Thrombin

Vitamin C derivatives (VCDs) have been widely used as the alternative and stable sources of vitamin C, and accordingly exhibit many new applications, such as anti-tumor and central nervous system drug delivery. In this study, their Ru(bpy)(3)(2+) electrochemiluminescence (ECL) properties have been investigated for the first time using well-known ascorbyl phosphate and ascorbyl palmitate as representative VCDs. Ascorbyl phosphate and ascorbyl palmitate are VCDs with different substituted positions. Both of them increase Ru(bpy)(3)(2+) ECL, indicating that other VCDs may also enhance Ru(bpy)(3)(2+) ECL signal. The calibration plot for ascorbyl phosphate is linear from 3×10(-6) to 1.0×10(-3) M with a detection limit of 1.4×10(-6) M at a signal-to-noise ratio of 3. The relative standard deviation is 3.6% for six replicate measurements of 0.01mM ascorbyl 2-phosphate solution. The proposed method is about one order of magnitude more sensitive than electrochemical and UV-vis methods for the determination of ascorbyl phosphate, and is used successfully for the determination of ascorbyl phosphate in whitening and moisturising body wash.

Topics: 2,2'-Dipyridyl; Ascorbic Acid; Carbon; Coordination Complexes; Electrochemical Techniques; Electrodes; Luminescent Measurements

To explore an alternative culture method for human corneal endothelial cells (HCECs) and to examine the effect of l-ascorbic acid 2-phosphate (Asc-2P) on the growth of these cells.. The influence of various mitogens, extracellular matrices (ECMs), and Asc-2P on growth of cultured HCECs was examined. HCECs were obtained from donors ranging in age from 12 to 74 years, and primary cultures and subcultures were performed with or without Asc-2P. Expanded HCECs were characterized with immunostaining and reverse transcription polymerase chain reaction (RT-PCR) and evaluated for generation of 8-hydroxy-2-deoxyguanosine (8-OHdG) with immunostaining and an enzyme-linked immunosorbent assay (ELISA).. Culture with Asc-2P and bFGF on atelocollagen promoted the proliferation of HCECs in both primary cultures and subcultures as efficiently as conventional culture using ECM derived from bovine corneal endothelial cells. Zonula occludens-1, N-cadherin, connexin 43, and Na+/K+-ATPase were localized at plasma membranes of cultured HCECs. mRNAs of the voltage-dependent anion channels (VDAC2 and VDAC3), sodium bicarbonate cotransporter member 4 (SLC4A4), and chloride channel proteins (CLCN2 and CLCN3) were detected by RT-PCR. During multiple passages, cultures without Asc-2P showed a decrease in growth and irregular cell morphology, whereas cultures with Asc-2P sustained cell growth and maintained the characteristic polygonal morphology. ELISA for 8-OHdG showed that the levels in mitochondrial DNA significantly decreased when HCECs were subcultured with Asc-2P.. Combination of Asc-2P and bFGF on atelocollagen allows successful culture for HCECs. Asc-2P extends the lifespan of cultured HCECs, partly due to protection against oxidative DNA damage.

Topics: Adult; Aged; Antineoplastic Agents; Ascorbic Acid; Cell Division; Cellular Senescence; Child; DNA Damage; Epithelial Cells; Epithelium, Corneal; Fibroblast Growth Factor 2; Humans; Middle Aged; Oxidative Stress; Primary Cell Culture

To assess the effect of dietary ascorbate on lipid metabolism, 1-year black sea bream (Acanthopagrus schlegelii) were reared on a casein-based purified diet and an ascorbate fortified diet (1,100 mg of L: -ascorbyl-2- monophosphate-Mg/kg diet). The fortified ascorbate was effectively incorporated into the fish body and elevated muscle carnitine content. Fortifications of dietary ascorbate depressed activities of glucose-6-phosphate dehydrogenase and NADP-isocitrate dehydrogenase as lipogenic enzymes in the hepatopancreas and intraperitoneal fat body. Starvation after feeding experiment activated carnitine palmitoyltransferase as a lipolysis enzyme in the hepatopancreas in both control and vitamin C(VC) groups, while the lipolysis activity was significantly higher in VC group. These results confirmed that dietary ascorbate depressed lipogenesis and activated lipolysis, i.e., influenced the lipid metabolism of black sea bream.

Topics: Animals; Ascorbic Acid; Body Weights and Measures; Carnitine; Food, Fortified; Glucosephosphate Dehydrogenase; Hepatopancreas; Isocitrate Dehydrogenase; Lipid Metabolism; Lipogenesis; Lipolysis; Muscle, Skeletal; Proteins; Sea Bream

It is generally accepted that the addition of vitamin C to cell culture medium improves cell growth. However, once added, the vitamin C concentration declines rapidly. This situation differs from the in vivo environment where the endothelium is constantly supplied with ascorbate from the blood. With a focus on intracellular vitamin C, we simulated constant supply of ascorbate by the hourly addition of freshly prepared medium containing 75 microM ascorbate and subsequently compared it with more practical regimens using combinations of ascorbate and 2-phosphoascorbate. We found that a single supplement of ascorbate and 2-phosphoascorbate adequately maintains intracellular vitamin C at physiological levels for up to 72 h.

Topics: Ascorbic Acid; Cell Culture Techniques; Cells, Cultured; Endothelial Cells; Humans; Time Factors

Intracellular vitamin C acts to protect cells against oxidative stress by intercepting reactive oxygen species (ROS) and minimising DNA damage. However, rapid increases in intracellular vitamin C may induce ROS with subsequent DNA damage priming DNA repair processes. Herein, we examine the potential of vitamin C and the derivative ascorbate-2-phosphate (2-AP) to induce a nucleotide excision repair (NER) response to DNA damage in a model of peripheral blood mononuclear cells. Exposure of cells to elevated levels of vitamin C induced ROS activity, resulting in increased levels of deoxycytidine glyoxal (gdC) and 8-oxo-2'-deoxyguanosine (8-oxodG) adducts in DNA; a stress response was also induced by 2-AP, but was delayed in comparison to vitamin C. Evidence of gdC repair was also apparent. Measurement of cyclobutane thymine-thymine dimers (T < >T) in DNA and culture supernatant were included as a positive marker for NER activity; this was evidenced by a reduction in DNA and increases in culture supernatant levels of T < >T for vitamin C-treated cells. Genomics analysis fully supported these findings confirming that 2-AP, in particular, induced genes associated with stress response, cell cycle arrest, DNA repair and apoptosis, and additionally provided evidence for the involvement of vitamin C in the mobilisation of intracellular catalytic Fe.

Topics: 8-Hydroxy-2'-Deoxyguanosine; Apoptosis; Ascorbic Acid; Cell Cycle; Cell Line, Tumor; Deoxyguanosine; DNA; DNA Damage; DNA Repair; Gene Expression; Genetic Markers; Genomics; Glyoxal; Humans; Iron; Leukocytes, Mononuclear; Models, Biological; Pyrimidine Dimers; Reactive Oxygen Species; Vitamins

We have previously reported a hydrogel template approach for the construction of centimeter-sized three-dimensional (3D) engineered tissues composed of cultured cells and extracellular matrices (ECM) produced by the cells. However, the interior of the engineered tissues was low in cell density; thus, it was pouched and non-dense morphologies. In this study, we developed thick and highly cell-incorporated engineered tissues by using basic fibroblast growth factor (bFGF) and ascorbic acid 2-phosphate (Asc 2-P). bFGF was loaded into freeze-dried poly(gamma-glutamic acid) hydrogels with disulfide cross-links (gamma-PGA-SS gels) as a template to enhance cell growth. After prescribed times of cell culture, the scaffolds were decomposed by adding a biocompatible reductant, cysteine, and cell proliferation and collagen production were investigated. The loading of bFGF into the scaffolds enhanced cellular invasion and proliferation. Meanwhile, the addition of Asc 2-P to the culture medium induced collagen production from the adhered fibroblasts. However, Asc 2-P did not affect cellular invasion into the template gamma-PGA-SS gels. The volume and weight of the obtained tissues using bFGF after 28 days of culture were 1.3- and 1.4-fold higher than those of control tissues. The thick and highly cell-incorporated 3D-engineered tissues can be useful as a novel cell implantation material for tissue engineering.

Topics: Ascorbic Acid; Cell Count; Cell Proliferation; Cells, Cultured; Collagen; Extracellular Matrix; Fibroblast Growth Factor 2; Fibroblasts; Hydrogel, Polyethylene Glycol Dimethacrylate; Polyglutamic Acid; Time Factors; Tissue Engineering

Recent studies suggested that dihydrotestosterone (DHT)-driven alteration in the autocrine and paracrine factors may be a key to androgen-potentiated balding. Also, we recently claimed that DHT-inducible dickkopf-1 (DKK-1) is one of the key factors involved in the androgen-potentiated balding. Here, we investigated whether L-ascorbic acid 2-phosphate (Asc 2-P), a derivative of L-ascorbic acid, could attenuate DHT-induced DKK-1 expression in dermal papilla cells (DPCs) from balding scalp. We observed that DHT-induced DKK-1 mRNA expression was attenuated in the presence of Asc 2-P as examined by RT-PCR analysis. In addition, we found that DHT-induced activation of luciferase reporter activity was significantly repressed when Asc 2-P was added together with DHT. Moreover, Asc 2-P repressed DHT-induced DKK-1 protein expression as examined by enzyme-linked immunosorbent assay (ELISA). Although there will be many hurdles to apply our finding to actual remedies, these results suggest that it would be worthy to evaluate Asc 2-P or its derivatives for the treatment and prevention of androgen-driven balding.

Topics: Alopecia; Ascorbic Acid; Cells, Cultured; Dihydrotestosterone; Epithelial Cells; Gene Expression Regulation; Growth Substances; Hair Follicle; Humans; Intercellular Signaling Peptides and Proteins; Male; Promoter Regions, Genetic; Receptors, Androgen

The skin is a protective barrier against external insults and any lesion must be rapidly and efficiently repaired. Dermal fibroblasts are the major source of extracellular connective tissue matrix and play an important role in wound healing. Vitamin C is an important water-soluble free radical scavenger and an essential cofactor for collagen synthesis by dermal fibroblasts and, consequently, may contribute to the maintenance of healthy skin. Using microarray analysis, we investigated the effects of long-term exposure to a stable vitamin C derivative, ascorbic acid 2-phosphate (AA2P), in contact-inhibited populations of primary human dermal fibroblasts. Compared with "scorbutic" cells, cells exposed to AA2P increased the expression of genes associated with DNA replication and repair and with the G(2)/M phase of the cell cycle. Consistent with the gene expression changes, AA2P increased the mitogenic stimulation of quiescent fibroblasts by serum factors and cell motility in the context of wound healing. Furthermore, AA2P-treated fibroblasts showed faster repair of oxidatively damaged DNA bases. We propose that vitamin C may protect the skin by promoting fibroblast proliferation, migration, and replication-associated base excision repair of potentially mutagenic DNA lesions, and we discuss the putative involvement of hypoxia-inducible transcription factor-1 and collagen receptor-related signaling pathways.

Topics: Ascorbic Acid; Cell Cycle Proteins; Cell Division; Cell Movement; Cell Proliferation; Cells, Cultured; Contact Inhibition; Cytoprotection; Dermis; DNA Repair; DNA Replication; Fibroblasts; G2 Phase; Gene Expression Profiling; Gene Expression Regulation; Humans; Microarray Analysis; Reverse Transcriptase Polymerase Chain Reaction; Time Factors; Wound Healing

Articular cartilage has only very limited potential for self-repair and regeneration. For this reason, various tissue engineering approaches have been developed to generate cartilage tissue in vitro. Usually, most strategies require ascorbate supplementation to promote matrix formation by isolated chondrocytes. In this study, we evaluate and compare the effect of different ascorbate forms and concentrations on in vitro cartilage formation in porcine chondrocyte high-density pellet cultures. l-ascorbate, sodium l-ascorbate, and l-ascorbate-2-phosphate were administered in 100 microM, 200 microM, and 400 microM in the culture medium over 16 days. Pellet thickness increased independently from the supplemented ascorbate form and concentration. Hydroxyproline content increased as well, but here, medium concentration of AsAP and low concentration of AsA showed a more pronounced effect. Proteoglycan and collagen formation were evaluated histologically and could be proven in all supplemented cultures. Non-supplemented cultures, however, showed no stable matrix formation at all. Effects on the gene expression pattern of cartilage marker genes (type I and type II collagen, aggrecan, and cartilage oligomeric matrix protein (COMP)) were studied by real-time RT-PCR and compared to non-supplemented control cultures. Expression level of cartilage marker genes was elevated in all cultures showing that dedifferentiation of chondrocytes could be prevented. Again, all supplementations caused a similar effect except for low concentration of AsA, which resulted in an even higher expression level of all marker genes. Besides that, we could not detect a pronounced difference between ascorbate and its derivates as well as between the different concentrations.

Topics: Animals; Ascorbic Acid; Cartilage, Articular; Cell Dedifferentiation; Cells, Cultured; Chondrocytes; Chondrogenesis; Gene Expression Profiling; Hydroxyproline; Swine; Tissue Engineering

We have reported on a novel enzyme immunoassay method for the detection of protein using biocatalytic silver nanoparticles as an enhanced substrate based on surface-enhanced Raman scattering (SERS). First, ascorbic acid was converted from ascorbic acid 2-phosphate by alkaline phosphatase immobilized on polystyrene microwells after a typical sandwich immunoreaction. Then Ag(I) ions were reduced to silver nanoparticles by the obtained ascorbic acid, which would result in a SERS signal when Raman dyes were absorbed. Using human IgG as a model protein, a wide linear dynamic range (1 to 100 ng ml(-1)) was reached with a low detection limit (0.02 ng ml(-1)) under the optimized assay conditions. Moreover, the production of an enhanced substrate was chosen as the signaling element in this method, which demonstrates a new way for SERS-based quantitative detection. These results suggest that the application of SERS enhanced by biocatalytic production of metal nanopaticles holds a promising potential for a sensitive immunoassay.

Topics: Alkaline Phosphatase; Ascorbic Acid; Biocatalysis; Enzymes, Immobilized; Humans; Immunoenzyme Techniques; Immunoglobulin G; Metal Nanoparticles; Polystyrenes; Reproducibility of Results; Sensitivity and Specificity; Silver; Spectrum Analysis, Raman; Surface Properties

l-Ascorbic acid 2-phosphate (Asc 2-P), a derivative of l-ascorbic acid, promotes elongation of hair shafts in cultured human hair follicles and induces hair growth in mice.. To investigate whether the promotion of hair growth by Asc 2-P is mediated by insulin-like growth factor-1 (IGF-1) and, if so, to investigate the mechanism of the Asc 2-P-induced IGF-1 expression.. Dermal papilla (DP) cells were cultured and IGF-1 level was measured by reverse transcription-polymerase chain reaction and enzyme-linked immunosorbent assay after Asc 2-P treatment in the absence or presence of LY294002, a phosphatidylinositol 3-kinase (PI3K) inhibitor. Also, hair shaft elongation in cultured human scalp hair follicles and proliferation of cocultured keratinocytes were examined after Asc 2-P treatment in the absence or presence of neutralizing antibody against IGF-1. In addition, keratinocyte proliferation in cultured hair follicles after Asc 2-P treatment in the absence or presence of LY294002 was examined by Ki-67 immunostaining.. IGF-1 mRNA in DP cells was upregulated and IGF-1 protein in the conditioned medium of DP cells was significantly increased after treatment with Asc 2-P. Immunohistochemical staining showed that IGF-1 staining is increased in the DP of cultured human hair follicles by Asc 2-P. The neutralizing antibody against IGF-1 significantly suppressed the Asc 2-P-mediated elongation of hair shafts in hair follicle organ culture and significantly attenuated Asc 2-P-induced growth of cocultured keratinocytes. LY294002 significantly attenuated Asc 2-P-inducible IGF-1 expression and proliferation of follicular keratinocytes in cultured hair follicles.. These data show that Asc 2-P-inducible IGF-1 from DP cells promotes proliferation of follicular keratinocytes and stimulates hair follicle growth in vitro via PI3K.

Topics: Animals; Ascorbic Acid; Cell Proliferation; Cells, Cultured; Dermis; Hair; Hair Follicle; Humans; Insulin-Like Growth Factor I; Male; Mice; Phosphatidylinositol 3-Kinases

A novel amphiphilic vitamin C (VC) derivative, disodium isostearyl 2-O-L-ascorbyl phosphate (VCP-IS-2Na), which possesses a C(18) alkyl chain attached to a stable ascorbate derivative, sodium L-ascorbic acid 2-phosphate (VCP-Na), was evaluated as a topical prodrug of VC with transdermal activity in human living skin equivalent (LSE) models. The permeation assay used was EPI-606X in the Franz-type diffusion cell system. VCP-IS-2Na exhibited much better permeability than VC and VCP-Na. The accumulation assays applied were EPI-200X and LSE-high by the dynamic system. The increased skin accumulation of VCP-IS-2Na was superior to that of VCP-Na and VC. VCP-IS-2Na that is susceptible to enzymatic hydrolysis by esterase and/or phosphatase released VC in the skin. Measurement of the metabolites that permeated and accumulated from the skin model suggested that VCP-IS-2Na was mainly metabolized via VCP-Na to VC in EPI-606X and EPI-200X, while it was mainly metabolized directly to VC in TESTSKIN LSE-high. Thus, these characteristics indicate that the novel VC derivative, VCP-IS-2Na, may be advantageous as a readily available source of VC for skin care applications.

Topics: Administration, Cutaneous; Ascorbic Acid; Chromatography, High Pressure Liquid; Humans; In Vitro Techniques; Models, Biological; Prodrugs; Skin Absorption; Vitamins

The purpose of this study was to characterize a tissue-engineered construct (TEC) generated with human synovial mesenchymal stem cells (MSCs). MSCs were cultured in medium with ascorbic acid 2-phosphate (Asc-2P) and were subsequently detached from the substratum. The detached cell/matrix complex spontaneously contracted to develop a basic TEC. The volume of the TEC assessed by varying initial cell density showed that it was proportional to initial cell densities up to 4 x 10(5) cells/cm(2). Assessment of the mechanical properties of TEC using a custom device showed that the load at failure and stiffness of the constructs significantly increased with time of culture in the presence of Asc-2P, while in the absence of Asc-2P, the constructs were mechanically weak. Thus, the basic TEC possesses sufficiently self-supporting mechanical properties in spite of not containing artificial scaffolding. TEC further cultured in chondrogenic media exhibited positive alcian blue staining with elevated expression of chondrogenic marker genes. Based on these findings, such human TEC may be a promising method to promote cartilage repair for future clinical application.

Topics: Adult; Ascorbic Acid; Biomechanical Phenomena; Bone Morphogenetic Protein 2; Cell Differentiation; Chondrogenesis; Collagen; Culture Media; Extracellular Matrix; Humans; Male; Materials Testing; Mesenchymal Stem Cells; Synovial Membrane; Tissue Engineering; Tissue Scaffolds

The primary goal of periodontal treatment is regeneration of the periodontium. Current theories suggest that the periodontal ligament (PDL) cells have the capacity to participate in restoring connective and mineralized tissues, when appropriately triggered. We evaluated whether human PDL cell sheets could reconstruct periodontal tissue.. To obtain the cell sheet, human PDL cells were cultured on temperature-responsive culture dishes with or without osteogenic differentiation medium. The cell sheets were transplanted on periodontal fenestration defects of immunodeficient rats. Forty rats were divided in two groups: in one group, cell sheets cultured with control medium were transplanted and in the other, cell sheets cultured with osteogenic differentiation medium were transplanted. The defects were analysed histologically and histomorphologically after healing.. Most of the experimental group exhibited a new cementum-like layer and new attachment of collagen fibres to the layer. Histomorphological analyses indicated significant periodontal regeneration. The control group revealed dense extracellular matrix and fibre formation, but an obvious cementum layer was not observed.. Transplanted PDL cell sheets cultured with osteogenic differentiation medium induced periodontal regeneration containing an obvious cementum layer and Sharpey's fibres. Thus, the method could be feasible as a new therapeutic approach for periodontal regeneration.

Topics: Actins; Animals; Ascorbic Acid; Cell Culture Techniques; Cell Differentiation; Cells, Cultured; Cementogenesis; Culture Media, Conditioned; Dexamethasone; Glycerophosphates; Humans; Integrin-Binding Sialoprotein; Osteoblasts; Osteopontin; Periodontal Ligament; Rats; Rats, Nude; Regeneration; Sialoglycoproteins; Tooth Root

Transdermal formulation of L-ascorbic acid 2-phosphate magnesium salt (A2P) was prepared using multilamellar vesicles (MLV). A2P was either physically mixed with or entrapped into three different MLVs of neutral, cationic, and anionic liposome vesicles. For the preparation of neutral MLVs, phosphatidylcholine (PC) and cholesterol (CH) were used. For cationic and anionic MLVs, dioleoyl-trimethylammonium-propane and dimyristoyl glycerophosphate were added as surface charge inducers, respectively, in addition to PC and CH. Particle size of the three A2P-loaded MLVs was submicron, and polydispersity index revealed homogenous distribution of the prepared MLVs except neutral ones. Skin penetration study with hairless mouse skin showed that both physical mixtures of A2P with empty MLVs and A2P-loaded MLVs increased penetration of the drug compared to aqueous A2P solution. During the penetration, however, significant amount of the drug was metabolized into L-ascorbic acid, which has no beneficial effect on stimulation of hair growth. Out of the physical mixtures and A2P-loaded MLVs tested, physical mixture of A2P with empty cationic MLV resulted in the greatest skin penetration and retention in hairless mouse skin.

Topics: Aniline Compounds; Animals; Ascorbic Acid; Chemistry, Physical; Cholesterol; Chromatography, High Pressure Liquid; Electrochemistry; Fatty Acids, Monounsaturated; Lecithins; Male; Membranes, Artificial; Mice; Mice, Hairless; Particle Size; Phosphatidylcholines; Quaternary Ammonium Compounds; Skin Absorption

Topics: Ascorbic Acid; Connective Tissue Growth Factor; Cysteine-Rich Protein 61; Hair Follicle; Humans; Immediate-Early Proteins; Intercellular Signaling Peptides and Proteins; Signal Transduction; Transcriptional Activation

Human mesenchymal stem cells (hMSCs) isolated from bone marrow aspirates were cultured on silk scaffolds in rotating bioreactors for three weeks with either chondrogenic or osteogenic medium supplements to engineer cartilage- or bone-like tissue constructs. Osteochondral composites formed from these cartilage and bone constructs were cultured for an additional three weeks in culture medium that was supplemented with chondrogenic factors, supplemented with osteogenic factors or unsupplemented. Progression of cartilage and bone formation and the integration between the two regions were assessed by medical imaging (magnetic resonance imaging and micro-computerized tomography imaging), and by biochemical, histological and mechanical assays. During composite culture (three to six weeks), bone-like tissue formation progressed in all three media to a markedly larger extent than cartilage-like tissue formation. The integration of the constructs was most enhanced in composites cultured in chondrogenic medium. The results suggest that tissue composites with well-mineralized regions and substantially less developed cartilage regions can be generated in vitro by culturing hMSCs on silk scaffolds in bioreactors, that hMSCs have markedly higher capacity for producing engineered bone than engineered cartilage, and that chondrogenic factors play major roles at early stages of bone formation by hMSCs and in the integration of the two tissue constructs into a tissue composite.

Topics: Analysis of Variance; Ascorbic Acid; Bioreactors; Bone Morphogenetic Protein 2; Bone Morphogenetic Proteins; Cell Culture Techniques; Chondrogenesis; Dexamethasone; Glycerophosphates; Humans; Immunohistochemistry; Insulin; Magnetic Resonance Imaging; Mesenchymal Stem Cells; Osteogenesis; Silk; Tissue Engineering; Transforming Growth Factor beta; Transforming Growth Factor beta1

Porcine mesenchymal stem cells have been isolated previously from bone marrow but not from adipose tissue. In the present study a new cell-culture method, using a low-calcium medium supplemented with N-acetyl-L-cysteine and L-ascorbic acid 2-phosphate (the PM2 medium) was developed to grow pASCs (porcine adipose-tissue-derived stem cells). The pASCs developed using the new medium showed a high growth rate and a high proliferation potential, as measured by a cumulative population doubling level (55) that was significantly higher than those reported for ASCs in the literature. These pASCs lacked gap-junctional intercellular communication and were capable of differentiation into three mesodermal lineages (i.e. adipocytes, osteoblasts and chondrocytes) and an ectodermal lineage (i.e. neural cells). Surprisingly, osteogenic ability, but not adipogenesis, was found to increase dramatically with increasing passages. The high proliferative and differentiation potential of these pASCs should facilitate the development of a large-animal model to study the use of ASCs in regenerative and reparative medicine.

Topics: Acetylcysteine; Adipocytes; Adipose Tissue; Animals; Ascorbic Acid; Cell Culture Techniques; Cell Differentiation; Cell Proliferation; Cell Transdifferentiation; Cells, Cultured; Chondrocytes; Culture Media; Female; Gap Junctions; Multipotent Stem Cells; Neurons; Osteoblasts; Swine

Adipose tissue serves as a source of adipokines and cytokines with both local and systemic actions in health and disease. In this study, we examine the hypothesis that multipotent human adipose-derived stem cells (ASCs), capable of differentiating along the adipocyte, chondrocyte, and osteoblast pathways, contribute to adipose tissue-derived cytokine secretion. Following exposure to basic fibroblast growth factor (bFGF) or epidermal growth factor (EGF), the ASCs significantly increase their secretion of hepatocyte growth factor (HGF), a cytokine implicated in hematopoiesis, vasculogenesis, and mammary epithelial duct formation. Ascorbic acid synergizes with these inductive factors, further increasing HGF levels. Following exposure to lipopolysaccharide, ASCs increase their secretion of both hematopoietic (granulocyte/monocyte, granulocyte, and macrophage colony stimulating factors, interleukin 7) and proinflammatory (interleukins 6, 8, and 11, tumor necrosis factor alpha) cytokines based on ELISA and RT-PCR. In co-cultures established with umbilical cord blood-derived CD34(+) cells, the ASCs support long-term hematopoiesis in vitro. Furthermore, in short-term 12-day co-cultures, the ASC maintain and expand the numbers of both myeloid and lymphoid progenitors. These observations are consistent with the functionality of the secreted cytokines and confirm recent reports by other laboratories concerning the hematopoietic supportive capability of ASCs. We conclude that the ASCs display cytokine secretory properties similar to those reported for bone marrow-derived mesenchymal stem cells (MSCs).

Topics: Adipocytes; Adipose Tissue; Adult; Adult Stem Cells; Angiogenic Proteins; Ascorbic Acid; Cell Differentiation; Cell Proliferation; Cells, Cultured; Coculture Techniques; Cytokines; Endothelial Cells; Epidermal Growth Factor; Female; Fibroblast Growth Factor 2; Granulocyte Colony-Stimulating Factor; Granulocyte-Macrophage Colony-Stimulating Factor; Hematopoiesis; Hematopoietic Stem Cells; Hepatocyte Growth Factor; Humans; Inflammation Mediators; Interleukin-11; Interleukin-6; Interleukin-7; Interleukin-8; Lipopolysaccharides; Middle Aged; Multipotent Stem Cells; Paracrine Communication; RNA, Messenger; Time Factors; Tumor Necrosis Factor-alpha

Inhibitory effects of 2-O-substituted ascorbic acid derivatives, ascorbic acid 2-glucoside (AA-2G), ascorbic acid 2-phosphate (AA-2P), and ascorbic acid 2-sulfate (AA-2S), on 2,2'-azobis(2-amidinopropane) dihydrochloride (AAPH)-induced oxidative hemolysis of sheep erythrocytes were studied and were compared with those of ascorbic acid (AA) and other antioxidants. The order of the inhibition efficiency was AA-2S> or =Trolox=uric acid> or =AA-2P> or =AA-2G=AA>glutathione. Although the reactivity of the AA derivatives against AAPH-derived peroxyl radical (ROO(*)) was much lower than that of AA, the derivatives exerted equal or more potent protective effects on AAPH-induced hemolysis and membrane protein oxidation. In addition, the AA derivatives were found to react per se with ROO(*), not via AA as an intermediate. These findings suggest that secondary reactions between the AA derivative radical and ROO(*) play a part in hemolysis inhibition. Delayed addition of the AA derivatives after AAPH-induced oxidation of erythrocytes had already proceeded showed weaker inhibition of hemolysis compared to that of AA. These results suggest that the AA derivatives per se act as biologically effective antioxidants under moderate oxidative stress and that AA-2G and AA-2P may be able to act under severe oxidative stress after enzymatic conversion to AA in vivo.

Topics: Amidines; Animals; Antioxidants; Ascorbic Acid; Erythrocyte Membrane; Free Radicals; Hemolysis; Sheep; Time Factors

A previous study showed that high doses of methamphetamine induce self-injurious behavior (SIB) in rodents. Furthermore, the combination of methamphetamine and morphine increased lethality in mice. We recently surmised that the rise in SIB and mortality induced by methamphetamine and/or morphine may be related to oxidative stress. The present study was designed to determine whether an antioxidant could inhibit SIB or mortality directly induced by methamphetamine and/or morphine. The SIB induced by 20mg/kg of methamphetamine was abolished by the administration of Na L-ascorbyl-2-phosphate (APS: 300 mg/kg), but not Na DL-alpha-tocopheryl phosphate (TPNa: 200mg/kg). In contrast, APS (300 mg/kg) and TPNa (200mg/kg) each significantly attenuated the lethality induced by methamphetamine and morphine. The present study showed that the signal intensity of superoxide adduct was increased by 20mg/kg of methamphetamine in the heart and lungs, and methamphetamine plus morphine tended to increase superoxide adduct in all of the tissues measured by ESR spin trap methods. Adduct signal induced in brain by methamphetamine administration increased in significance, but in mouse administrated methamphetamine plus morphine. There are differential effects of administration of methamphetamine and coadministration of methamphetamine plus morphine on adduct signal. These results suggest that APS and TPNa are effective for reducing methamphetamine-induced toxicity and/or toxicological behavior. While APS and TPNa each affected methamphetamine- and/or morphine-induced toxicology and/or toxicological behavior, indicating that both drugs have antioxidative effects, their effects differed.

Topics: alpha-Tocopherol; Animals; Antioxidants; Ascorbic Acid; Central Nervous System Stimulants; Dopamine; Electron Spin Resonance Spectroscopy; Free Radicals; Iron-Binding Proteins; Male; Methamphetamine; Mice; Mice, Inbred BALB C; Molecular Structure; Morphine; Neurotoxicity Syndromes; Self-Injurious Behavior

Chronic morphine-induced withdrawal syndrome after morphine cessation remains a severe obstacle in the clinical treatment of morphine. Previous studies have shown that nitric oxide synthetase (NOS) inhibitors may have therapeutic potential in morphine withdrawal in humans. The mechanisms that underlie expression of morphine-induced withdrawal syndrome are, however, not yet fully understood. Therefore, this study was designed to determine the mechanism of the expression of morphine-induced withdrawal syndrome in mice. Morphine-dependent mice showed marked body weight loss and several withdrawal signs after naloxone challenge. Pretreatment with a NOS inhibitor, such as N-nitro-L-arginine methyl ester (L-NAME) or 7-nitroindazole, but not aminoguanidine, significantly attenuated the expression of morphine-induced withdrawal syndrome. Furthermore, mepacrine (a phospholipase A2 inhibitor) significantly attenuated the morphine-induced withdrawal syndrome in a manner that was different than that with a NOS inhibitor. These results suggest that nNOS and phospholipase A2, which might increase free radicals, play an important role in the expression of morphine-induced withdrawal syndrome. On the contrary, free radical scavengers (including fullerenes, ascorbate-2-phosphate, and DL-alpha-tocopheryl phosphate) attenuated the expression of the morphine-induced withdrawal syndrome. These results indicate that free radicals play an important role in the expression of physical dependence on morphine, and fullerenes could be a potential clinical tool in the relief of morphine withdrawal syndrome.

Topics: alpha-Tocopherol; Animals; Ascorbic Acid; Enzyme Inhibitors; Free Radical Scavengers; Hydroxyl Radical; Male; Mice; Naloxone; NG-Nitroarginine Methyl Ester; Nitric Oxide Synthase; Opioid-Related Disorders; Phospholipase A2 Inhibitors; Substance Withdrawal Syndrome; Superoxides

With the aim to produce ascorbic acid-2-phosphate (AsA-2-P) from L-ascorbic acid (AsA, Vitamin C), nine bacteria conferring the ability to transform AsA to AsA-2-P were isolated from soil samples alongside known strains from culture collections. Most isolates were classified to the genus Brevundimonas by 16S phylogenetic analysis. Among them, Brevundimonas diminuta KACC 10306 was selected as the experimental strain because of its the highest productivity of AsA-2-P. The optimum set of conditions for the AsA-2-P production from AsA using resting cells as the source of the enzyme was also investigated. The optimum cultivation time was 16 h and the cell concentration was 120 g/l (wet weight). The optimum concentrations of AsA and pyrophosphate were 550 mM and 450 mM, respectively. The most effective buffer was 50 mM sodium formate. The optimum pH was 4.5 and temperature was 40 degrees C. Under the above conditions, 27.5 g/l of AsA-2-P was produced from AsA after 36 h of incubation, which corresponded to a 19.7% conversion efficiency based on the initial concentration of AsA.

Topics: Ascorbic Acid; Buffers; Caulobacteraceae; DNA, Bacterial; DNA, Ribosomal; Genes, rRNA; Hydrogen-Ion Concentration; Molecular Sequence Data; Phylogeny; RNA, Bacterial; RNA, Ribosomal, 16S; Sequence Analysis, DNA; Sequence Homology, Nucleic Acid; Soil Microbiology; Temperature; Time Factors

Mortality of mouse keratinocytes Pam212 that were irradiated with ultraviolet-B (UVB) was shown to be repressed by pre-irradiated administration with L-ascorbic acid (Asc) or more markedly with Asc-2-O-phosphate (Asc2P), but not with dehydroascorbic acid (DehAsc) or Asc-2-O-alpha-glucoside (Asc2G), although not repressed by post-irradiated administration. The cytoprotection by Asc2P was restricted against UVB below 5-20 mJ/cm2, and exhibited markedly by administration for a period over 2 h, which may be caused by intracellular Asc that was accumulated via dephosphorylation of Asc2P and was increased, 6-24 h after, to levels above twice as abundant as those of Asc-administration. Pre-irradiated Asc2P-administration slightly repressed a DNA ladder-like electrophoretic pattern for UVB-irradiated keratinocytes, containing the histone-bound DNA fragments as shown by ELISA assay, and appreciably repressed the DNA-3'OH cleavage terminals as shown by terminal deoxyribonucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) stain. Thus, prevention of UVB-induced cell death by Asc2P was shown to occur concurrently with inhibition of DNA cleavages and enrichment of intracellular Asc.

Topics: Animals; Apoptosis; Ascorbic Acid; Cells, Cultured; Cytoprotection; DNA; Enzyme-Linked Immunosorbent Assay; Epidermal Cells; Epidermis; In Situ Nick-End Labeling; Keratinocytes; Mice; Phosphorylation; Skin; Ultraviolet Rays

Topics: Animals; Ascorbic Acid; Cell Differentiation; Cell Proliferation; Cells, Cultured; Epithelial Cells; Hair; Hair Follicle; Humans; Insulin-Like Growth Factor I; Keratinocytes; Mice; Mice, Inbred C57BL; RNA, Messenger

Ascorbic acid has been reported to promote the differentiation of embryonic stem (ES) cells into cardiomyocytes; however, the specific functions of ascorbic acid have not been defined. A stable form of ascorbic acid, namely, l-ascorbic acid 2-phosphate (A2-P), significantly enhanced cardiac differentiation; this was assessed by spontaneous beating of cardiomyocytes and expression of cardiac-specific markers obtained from mouse ES cells. This effect of ascorbic acid was observed only when A2-P was present during the early phase of differentiation. Treatment with two types of collagen synthesis inhibitors, l-2-azetidine carboxylic acid and cis-4-hydroxy-d-proline, significantly inhibited the A2-P-enhanced cardiac differentiation, whereas treatment with the antioxidant N-acetyl cysteine showed no effect. These findings demonstrated that ascorbic acid enhances differentiation of ES cells into cardiomyocytes through collagen synthesis and suggest its potential in the modification of cardiac differentiation of ES cells.

Topics: Animals; Antioxidants; Ascorbic Acid; Biomarkers; Cell Differentiation; Cell Line; Collagen; Gene Expression Regulation; Heart; Mice; Myocytes, Cardiac; RNA, Messenger; Stem Cells

Topics: Ascorbic Acid; Cell Line; Chondroitin Sulfate Proteoglycans; Chromones; Enzyme Inhibitors; Hair Follicle; Humans; In Vitro Techniques; Lectins, C-Type; Morpholines; Phosphoinositide-3 Kinase Inhibitors; Protein Isoforms; Versicans

Long-term exposure of the skin to UV-A and UV-B radiation causes degenerative effect which can be decreased by scavenging reactive photochemical intermediates with antioxidants. In this study sodium ascorbyl phosphate (SAP), a very effective oxygen species scavenger, was encapsulated into liposomes in order to improve its penetration through the stratum corneum into the deeper layers of the skin. Two types of multilamellar vesicles were prepared, one from non-hydrogenated and the other from hydrogenated soybean lecithin, together with cholesterol, by the thin films method. They were characterized for size, polydispersity index, and zeta potential. In vitro diffusion of SAP and ex vivo penetration experiments were performed on pig ear epidermis membrane in a Franz diffusion cell. The size and zeta potential of liposomes containing SAP are significantly greater than those of empty liposomes. The upper limit of SAP entrapment efficiency was 8-10% in both types of liposomes. The stability of SAP in liposome formulations is much more influenced by storage temperature than by liposome composition. SAP penetrated through epidermis membrane significantly better from liposome dispersions than from water solution. The amount penetrating is much more influenced by the concentration of SAP in the formulation than by the lipid composition of liposomes. The SAP that penetrates through the epidermis reflects the active compound available to prevent or slow down the complex process of photodamage in the skin.

Topics: Administration, Cutaneous; Animals; Ascorbic Acid; Diffusion; Dose-Response Relationship, Drug; Drug Stability; Liposomes; Skin Absorption; Sunscreening Agents; Swine

Human mesenchymal stem cells (MSCs) have been isolated from bone marrow and other adult tissues and are potentially useful for tissue engineering. Adipose tissue has several clear advantages as a starting material for harvesting stem cells, as it is abundant and relatively easy to procure. However, existing methods to expand adipose-derived MSCs are less than optimal. Here we describe a new cell culture method that accelerates greatly the growth rate and prolongs the lifespan of adipose MSCs. This was accomplished by using a growth medium with low calcium and supplemented with N-acetyl-L-cysteine and L-ascorbic acid-2-phosphate. Cells produced early in these cultures displayed characteristics similar to those previously reported for multipotential stem cells, including a high frequency of anchorage- independent growth in soft agar, lack of gap junctional intercellular communication in a cell type with serpiginous morphology, and the expression of Oct-4. Furthermore, these cells could readily be induced to differentiate into adipocytes, osteoblasts, and chondrocytes. Thus, modification of growth medium by reduction of calcium and addition of antioxidants greatly enhanced the growth rate and extended the lifespan of adipose-derived multipotential human MSCs.

Topics: Acetylcysteine; Adipocytes; Adipose Tissue; Antioxidants; Ascorbic Acid; Calcium; Cell Culture Techniques; Cell Differentiation; Cell Proliferation; Cellular Senescence; Chondrocytes; Culture Media; Humans; Mesenchymal Stem Cells; Osteoblasts

An unsolved problem with stem cell-based engineering of bone tissue is how to provide a microenvironment that promotes the osteogenic differentiation of multipotent stem cells. Previously, we fabricated porous poly(D,L-lactide-co-glycolide) (PLGA) scaffolds that released biologically active dexamethasone (Dex) and ascorbate-2-phosphate (AsP), and that acted as osteogenic scaffolds. To determine whether these osteogenic scaffolds can be used for bone formation in vivo, we seeded multipotent human marrow stromal cells (hMSCs) onto the scaffolds and implanted them subcutaneously into athymic mice. Higher alkaline phosphatase expression was observed in hMSCs in the osteogenic scaffolds compared with that of hMSCs in control scaffolds. Furthermore, there was more calcium deposition and stronger von Kossa staining in the osteogenic scaffolds, which suggested that there was enhanced mineralized bone formation. We failed to detect cartilage in the osteogenic scaffolds (negative Safranin O staining), which implied that there was intramembranous ossification. This is the first study to demonstrate the successful formation of mineralized bone tissue in vivo by hMSCs in PLGA scaffolds that release Dex and AsP.

Topics: Alkaline Phosphatase; Animals; Anti-Inflammatory Agents; Ascorbic Acid; Biocompatible Materials; Bone and Bones; Bone Development; Bone Marrow Cells; Calcium; Cartilage; Cells, Cultured; Coloring Agents; Dexamethasone; Guided Tissue Regeneration; Humans; In Situ Hybridization; Lactic Acid; Mice; Mice, Inbred BALB C; Osteogenesis; Phenazines; Polyglycolic Acid; Polylactic Acid-Polyglycolic Acid Copolymer; Polymers; Stromal Cells; Time Factors; Tissue Engineering; Up-Regulation

In vivo cells exist in a three-dimensional environment generated and maintained by multiple cell-cell and cell-matrix interactions. Proteoglycans, like decorin, affect these complex interactions. Thus, we sought to investigate the role of decorin in a three-dimensional environment where the matrix was generated over time by decorin-deficient fibroblasts in the presence of L-ascorbic acid 2-phosphate. The cells were viable and proliferated in response to FGF2. Decorin was incorporated in the matrix and caused a approximately 2 nm shift in the average diameter of the collagen fibrils, and the range and distribution of the fibrils became narrower and more uniform. Although there were no appreciable changes in collagen composition, we found that exogenous decorin induced the de novo synthesis of collagen I and V and cross-linked beta(I). In the early phases of the three-dimensional culture, decorin reduced apoptosis. However, following the establishment of a three-dimensional matrix, the cells did not require decorin for their survival.

Topics: Animals; Apoptosis; Ascorbic Acid; Caspase 3; Caspase 8; Caspases; Cell Communication; Cell Line; Cell Proliferation; Cell Survival; Cells, Cultured; Collagen; Collagen Type I; Collagen Type V; Decorin; Extracellular Matrix Proteins; Fibroblasts; Humans; Mice; Mice, Transgenic; Microscopy, Electron; Pepsin A; Phenotype; Proteoglycans

To examine age-related efficacy of bone morphogenetic protein (BMP)-2, ascorbate, and dexamethasone as osteogenic inducers in canine marrow-derived stromal cells (MSCs).. Samples of femoral bone marrow obtained from 15 skeletally immature (< 1 year old) and 4 skeletally mature (> 1.5 years old) dogs.. First-passage canine MSC cultures were treated with 100 microg of ascorbate phosphate/mL, 10(-7)M dexamethasone, 100 ng of BMP-2/mL, or a combination of these osteoinducers. On day 6, cultures were harvested for quantitation of alkaline phosphatase (ALP) activity and isolation of RNA to prepare cDNA for real-time polymerase chain reaction analyses of osteoblast markers.. Early markers of osteogenesis were induced in canine MSCs by BMP-2 but not dexamethasone. In young dogs, the combination of BMP-2 and ascorbate yielded the highest ALP mRNA concentrations and activity. This combination also induced significant increases in mRNA for osteopontin and runt-domain transcription factor 2. In comparison to MSCs from immature dogs, those from mature dogs had diminished ALP activity in response to BMP and ascorbate. Results for cultures treated with 3,4-dehydroproline suggested that ascorbate-induced production of extracellular matrix was important for maximal BMP-2 response in canine MSCs.. BMP-2 was capable of inducing markers of osteogenesis in short-term cultures of canine MSCs. In MSCs obtained from skeletally immature dogs, ascorbate was required for maximal effects of BMP These results define optimal conditions for stem cell osteogenesis in dogs and will facilitate development of stem cell-based treatments for dogs with fractures.

Topics: Age Factors; Alkaline Phosphatase; Animals; Ascorbic Acid; Bone Morphogenetic Protein 2; Bone Morphogenetic Proteins; Dexamethasone; DNA Primers; Dogs; Mesenchymal Stem Cells; Osteogenesis; Reverse Transcriptase Polymerase Chain Reaction; Transforming Growth Factor beta

We investigated the implications of induced osteogenic differentiation on gene delivery in multipotent rat marrow stromal cells (MSCs). Prior to genetic manipulation cells were cultured with or without osteogenic supplements (5x10(-8) M dexamethasone, 160 microM l-ascorbic acid 2-phosphate, and 10 mM beta-glycerophosphate). Comparison of liposome, retroviral, and adenoviral vectors demonstrated that all three vectors could mediate gene delivery to primary rat MSCs. When these vectors were applied in the absence or presence of osteogenic supplements, we found that MSCs differentiated prior to transduction with adenovirus type 5 vectors produced a 300% increase in transgene expression compared to MSCs that were not exposed to osteogenic supplements. This differentiation effect appeared specific to adenoviral mediated gene delivery, since there was minimal increase in retroviral gene delivery and no increase in liposome gene delivery when MSCs were treated with osteogenic supplements. In addition, we also determined this increase in transgene production to occur at a higher concentration of dexamethasone (5x10(-8) M) in the culture medium of MSCs prior to adenoviral transduction. We found that this increased transgene production could be extended to the osteogenic protein, human bone morphogenetic protein 2 (hBMP-2). When delivered by an adenoviral vector, hBMP-2 transgene production could be increased from 1.4 ng/10(5) cells/3 days to 4.3 ng/10(5) cells/3 days by culture of MSCs with osteogenic supplements prior to transduction. These results indicate that the utility of MSCs as a therapeutic protein delivery mechanism through genetic manipulation can be enhanced by pre-culture of these cells with dexamethasone.

Topics: Adenoviridae; Alkaline Phosphatase; Animals; Ascorbic Acid; Bone Marrow Cells; Bone Morphogenetic Protein 2; Bone Morphogenetic Proteins; Cell Differentiation; Dexamethasone; Dose-Response Relationship, Drug; Drug Combinations; Gene Expression; Gene Transfer Techniques; Genetic Vectors; Glycerophosphates; Luciferases; Osteoblasts; Rats; Rats, Wistar; Stromal Cells; Transforming Growth Factor beta

Maximum activity for phosphorylating C(2)-OH of the ascorbic acid was observed at the time of 16 h incubation from the culture of Flavobacterium devorans ATCC 10829. The enzyme was purified 1.178-fold, via ammonium sulfate fractionation, Fast Q anion exchange, and phenyl agarose chromatography. Gel chromatography and SDS-polyacrylamide electrophoresis experiments showed that the enzyme is a tetramer with subunit MW of 29 kDa. Among available second substrates, pyrophosphate showed the highest activity. Optimum temperature and pH were 45 degrees C and 5.5, respectively. The enzyme was chemically modified only by diethylpyrocarbonate and 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDC), indicating that histidine and carboxylate are in the active site. pH studies showed that two histidines are involved in the binding of the substrates and a carboxylate in catalysis. Therefore, the chemical mechanism of the enzyme is likely that two histidines bind to pyrophosphate and carboxylate, respectively, and a carboxylate acts as a general base.

Topics: Ascorbic Acid; Enzyme Activation; Enzyme Inhibitors; Flavobacterium; Hydrogen-Ion Concentration; Metals; Models, Chemical; Molecular Weight; Phosphoric Monoester Hydrolases; Phosphotransferases; Substrate Specificity; Temperature; Time Factors

In order to investigate the effect of ascorbic acid (AsA) and ascorbic acid 2-phosphate (Asc 2-P), a long-acting vitamin C derivative, on the growth and differentiation of human osteoblast-like cells, we supplemented the culture medium of MG-63 cells with various concentrations (0.25 to 1 mM) of these factors. Asc 2-P significantly stimulated nascent cell growth at all concentrations in the presence of fetal bovine serum (FBS). On the other hand, AsA showed a growth repressive effect depending on its concentration, and that of FBS. Asc 2-P also increased expression of osteoblast differentiation markers, such as collagen synthesis and alkaline phosphatase (ALP) activity. These stimulative activities of Asc 2-P were attenuated by inhibitors of collagen synthesis, indicating that these effects were dependent on collagen synthesis. Electron micrographs of the cells showed the formation of a three-dimensional tissue-like structure endowed with a mature extracellular matrix in the presence of Asc 2-P.

Topics: Alkaline Phosphatase; Ascorbic Acid; Azetidinecarboxylic Acid; Cell Differentiation; Cell Division; Cells, Cultured; Collagen; Humans; Microscopy, Electron, Transmission; Osteoblasts

Radiation generates reactive oxygen species (ROS) that interact with cellular molecules, including DNA, lipids, and proteins. To know how ROS contribute to the induction of genetic instability, we examined the effect of the anti-ROS condition, using both ascorbic acid phosphate (APM) treatment or a low oxygen condition, on the induction of delayed reproductive cell death and delayed chromosome aberrations. The primary surviving colonies of mouse m5S-derived cl. 2011-14 cells irradiated with 6 Gy of X-rays were replated and allowed to form secondary colonies. The anti-ROS treatments were applied to either preirradiation culture or postirradiation cultures for primary or secondary colony formation. Both anti-ROS conditions relieved X-ray-induced acute cell killing to a similar extent. These anti-ROS conditions also relieved genetic instability when those conditions were applied during primary colony formation. However, no effect was observed when the conditions were applied during preirradiation culture and secondary colony formation. We also demonstrated that the amounts of ROS in X-ray-irradiated cells rapidly increase and then decrease at 6 hr postirradiation, and the levels of ROS then gradually decrease to a baseline within 2 weeks. The APM treatment kept the ROS production at a lower level than an untreated control. These results suggest that the cause of genetic instability might be fixed by ROS during a 2-week postirradiation period.

Topics: Animals; Apoptosis; Ascorbic Acid; Cell Hypoxia; Cell Line; Cell Survival; Chromosome Aberrations; Chromosomes, Human, Pair 11; Dose-Response Relationship, Drug; Dose-Response Relationship, Radiation; Genomic Instability; Humans; Mice; Oxygen; Reactive Oxygen Species; X-Rays

The cellular life-span of cultivated human skin epidermis keratinocytes NHEK-F was shown to be extended up to 150% of population doubling levels (PDLs) by repetitive addition with two autooxidation-resistant derivatives of ascorbic acid (Asc), Asc-2-O-phosphate (Asc2P), and Asc-2-O-alpha-glucoside (Asc2G), respectively, but to be not extended with Asc itself. In contrast, hydrogen peroxide (H(2)O(2)) as dilute as 20 microM which was non-cytotoxic to the keratinocytes, or at 60 microM being marginally cytotoxic achieved the cellular longevity, unexpectedly, up to 160 and 120% of PDLs, respectively, being regarded as a hormesis-like stimulatory effect. The lifespan-extended cells that were administered with Asc2P, Asc2G, or 20 microM H(2)O(2) were prevented from senescence-induced symptoms such as PDL-dependent enlargement of a cell size of 14.7 microm finally up to 17.4 microm upon Hayflick's limit-called loss of proliferation ability as estimated with a channelizer, and retained young cell morphological aspects such as thick and compact shape and intense attachment to the culture substratum even upon advanced PDLs, whereas other non-extended cells looked like thin or fibrous shape and large size upon lower PDLs. The PDL-dependent shortening of telomeric DNA of 11.5 kb finally down to 9.12-8.10 kb upon Hayflick's limit was observed in common for each additive-given cells, but was decelerated in the following order: 20 microM H(2)O(2) > Asc2P = Asc2G > 60 microM H(2)O(2) > Asc = no additive, being in accord with the order of cell longevity. Intracellular reactive oxygen species (ROS) was diminished by Asc2P, Asc2G or 20 microM H(2)O(2), but not significantly by Asc or 60 microM H(2)O(2) as estimated by fluorometry using the redox indicator dye CDCFH. There was no appreciable difference among NHEK keratinocytes that were administered with or without diverse additives in terms of telomerase activity per cell, which was 1.40 x 10(4)-4.48 x 10(4) times lower for the keratinocytes than for HeLa cells which were examined as the typical tumor cells. Thus longevity of the keratinocytes was suggested to be achieved by slowdown of age-dependent shortening of telomeric DNA rather than by telomerase; telomeres may suffer from less DNA lesions due to the continuous and thorough repression of intracellular ROS, which was realized either by pro-vitamin C such as Asc2P or Asc2G that exerted an antioxidant ability more persistent than Asc itself or by 20 microM H(2)O(2) whi

Topics: Ascorbic Acid; Cell Proliferation; Cell Size; Cells, Cultured; Cellular Senescence; Flow Cytometry; Humans; Hydrogen Peroxide; Keratinocytes; Oxidative Stress; Skin; Telomerase; Telomere

Vascular endotheliocytes BAE-2 underwent the gradually proceeding cell death until 48 h after reoxygenation (Reox) following 3 h anoxia (Anox), but protected by pre-Anox administration with L-ascorbic acid (Asc)-2-O-phosphate (Asc2P), an autooxidation-resistant Asc derivative, but not by Asc itself. This cytoprotection with Asc2P was achieved in a glucose (Glc)-lacking buffer more advantageously than in a Glc-containing buffer where less efficiency had been demonstrated for Asc entry into BAE-2 cells than in a Glc-lacking buffer. Superoxide anion radicals were detected explosively in the extracellular space at 2-5 min after Reox following the Anox treatment of HUVE endotheliocytes, and were thereafter retained at levels as high as approximately one-half of the maximum level until 60 min after Reox, as shown by cytochrome c reduction assay. Superoxide anions at 3 and 60 min after Reox were suppressed by pre-Anox administration with Asc2P, but not with Asc or dehydro-Asc, and were not suppressed by post-Anox administration with Asc2P; the cytoprotection may need the intracellular accumulation of the ROS-scavenging effector Asc that is converted from Asc2P until 3 min after Reox. The ROS-generator tert-butylhydroperoxide (t-BuOOH) also induced both the diminished cell viability and nuclear DNA strand cleavages of BAE-2 endotheliocytes, which were also protected dose-dependently with Asc2P. The cytoprotection was attributed to reduction of intracellular ROS including hydroperoxide and hydrogen peroxide with Asc2P as shown by fluorometry with the redox indicator CDCFH-DA. Thus Anox/Reox-induced cell death can be prevented by Asc2P that suppresses ROS-generation immediately after Reox following Anox more efficiently in the intracellular sphere rather than in the extracellular space.

Topics: Animals; Antioxidants; Ascorbic Acid; Cattle; Cell Death; Cell Hypoxia; Endothelial Cells; Endothelium, Vascular; Humans; Oxidative Stress; Reactive Oxygen Species; tert-Butylhydroperoxide; Umbilical Veins

Topics: Arbutin; Ascorbic Acid; Chromatography, High Pressure Liquid; Cosmetics; Pyrones

Effects of dietary ascorbyl-2-phosphate on immune function after a 210-km trip were measured in 18 Holstein heifers. After transport on d 0, 10 g of ascorbyl-2-phosphate each were added to the diets of 10 heifers, whereas eight heifers were fed a control diet. Plasma cortisol concentrations increased by an average of 25.6 microgram/ml on d 0 following transport, but by d 7 after transport had decreased to pretransport levels. Average daily gain was lower in heifers fed ascorbyl-2-phosphate from d 28 to 49 d after transport, but did not differ over the entire study. Feeding ascorbyl-2-phosphate maintained plasma ascorbate concentrations on d 7 post-transport, which decreased in control heifers. Plasma keyhole limpet hemocyanin antibody titers were significantly higher in control heifers from d 7 to 49. Mononuclear leukocyte proliferation responses were decreased on d 0 in lymphocytes stimulated by mitogens, with pokeweed mitogen-stimulated cells showing less of a response than cells stimulated by the other mitogens. In the absence of mitogens, dietary ascorbyl-2-phosphate increased basal 3H-methyl thymidine incorporation by cultured lymphocytes. Across diets and mitogens, lymphocytes treated with cortisol showed decreased 3H-methyl thymidine incorporation. Transportation acted as a stressor, as evidenced by the increased plasma cortisol levels at d 0 immediately after transport, but immunological effects were not apparent by d 7. Feeding ascorbyl-2-phosphate maintained plasma ascorbate concentrations on d 7, but had negative effects on immune responses posttransport.

Topics: Animals; Ascorbic Acid; Body Weight; Cattle; Cell Division; Cells, Cultured; Diet; Female; Hydrocortisone; Immunity; Immunoglobulin G; Leukocytes, Mononuclear; Lymphocytes; Mitogens; Transportation; Weight Gain

Vitamin C (ascorbic acid) is a non-enzymatic antioxidant important in protecting the lung against oxidative damage and is decreased in lung lining fluid of horses with airway inflammation. To examine possible therapeutic regimens in a species with ascorbate-synthesising capacity, we studied the effects of oral supplementation of two forms of ascorbic acid, (each equivalent to 20 mg ascorbic acid per kg body weight) on the pulmonary and systemic antioxidant status of six healthy ponies in a 3 x 3 Latin square design. Two weeks supplementation with ascorbyl palmitate significantly increased mean plasma ascorbic acid concentrations compared to control (29 +/- 5 and 18 +/- 7 micromol/l, respectively; p < 0.05). Calcium ascorbyl-2-monophosphate, a more stable form of ascorbic acid, also increased mean plasma ascorbic acid concentrations, but not significantly (23 +/- 1 micromol/l; p = 0.07). The concentration of ascorbic acid in bronchoalveolar lavage fluid increased in five out of six ponies following supplementation with either ascorbyl palmitate or calcium ascorbyl-2-monophosphate compared with control (30 +/- 10, 25 +/- 4 and 18 +/- 8 micromol/l, respectively; p < 0.01). Neither supplement altered the concentration of glutathione, uric acid or alpha-tocopherol in plasma or bronchoalveolar lavage fluid. In conclusion, the concentration of lung lining fluid ascorbic acid is increased following ascorbic acid supplementation (20 mg/kg body weight) in an ascorbate-synthesising species.

Topics: Animals; Antioxidants; Ascorbic Acid; Bronchoalveolar Lavage Fluid; Calcium; Chromatography, High Pressure Liquid; Glutathione; Horses; Lung; Time Factors; Trachea

The purpose of this research was to develop porous poly(D,L-lactide-co-glycolide) (PLGA) scaffolds from which ascorbate-2-phosphate (AsAP) and dexamethasone (Dex) are continuously released for a month for osteogenesis of mesenchymal stem cells for bone tissue engineering. Porous PLGA matrices containing AsAP and Dex were prepared by solvent casting/particulate leaching method. In vitro release and water uptake studies were performed in Dulbecco's phosphate buffered saline at 37 degrees C and 15 rpm. Drug loading and release rates were determined by high performance liquid chromatography. Release studies of Dex and AsAP showed that, after an initial burst release lasting 4 and 9 days, respectively, release rates followed zero order kinetics with high correlation coefficients at least until 35 days. Incorporation of AsAP into the scaffolds increased the release rates of Dex and AsAP, and the scaffold water uptake. When mesenchymal stem cells (MSCs) were cultured in the AsAP and Dex containing scaffolds in vitro, the amount of mineralization was significantly higher than in control scaffolds. In conclusion, AsAP and Dex were incorporated into porous PLGA scaffolds and continuously released over a month and osteogenesis of MSCs was increased by culture in these scaffolds.

Topics: Animals; Antineoplastic Agents, Hormonal; Ascorbic Acid; Biocompatible Materials; Cells, Cultured; Dexamethasone; Drug Carriers; Implants, Experimental; Lactic Acid; Materials Testing; Mesenchymal Stem Cells; Osteogenesis; Particle Size; Polyglycolic Acid; Polylactic Acid-Polyglycolic Acid Copolymer; Polymers; Porosity; Rabbits; Tissue Engineering; Water

Our previous study showed that tumor invasion of human fibrosarcoma cells HT-1080 is hardly inhibited by ascorbic acid itself (Asc), but inhibited by 2-O-phosphorylated Asc-6-O-palmitylester (Asc2P6Plm) more markedly than 2-O-phosphorylated Asc or Asc-6-O-palmitylester, and that the inhibitory effect may be attributed to an increase in intracellular Asc derived from Asc2P6Plm. In the present study, the mechanism underlying the inhibitory effect of Asc2P6Plm on tumor invasion was analyzed. Migratory ability of the tumor cells was shown to be inhibited in a dose-dependent manner by either treatment with Asc2P6Plm at 50-300 micro M for 1 h or at 10-50 micro M for 18 h as assessed by cell sheet scratching assay. Hydroxyl radicals in homogenates of Asc2P6Plm-treated HT-1080 cells were markedly diminished relative to those of non-treated cells as evaluated by electron spin resonance method using the spin trapping agent DMPO. This may be closely related to attenuation of intracellular gross reactive oxygen species by Asc2P6Plm as was shown with the redox indicator CDCFH-DA. Actin was localized in the vicinity of the cell membrane abundantly in non-treated cells, but was diminished in a time-dependent manner in Asc2P6Plm-treated cells together with disappearance of pseudopods as shown with the actin-directed agent NBD-phallacidin and by immunocytochemical stain. The cell adhesion-controling molecule RhoA was increased time-dependently in the cytoplasm of Asc2P6Plm-treated cells as shown by Western blots. Thus the inhibition of tumor invasion by Asc2P6Plm was shown to be attributed to decrease in both the cell migratory ability and the actin localization near the cell membrane, which may result from an increase in cytoplasmic RhoA and reduction of intracellular ROS that is achieved by enrichment of intracellular Asc derived from Asc2P6Plm.

Topics: Actins; Ascorbic Acid; Blotting, Western; Cell Line, Tumor; Cell Movement; Cyclic N-Oxides; Dose-Response Relationship, Drug; Electron Spin Resonance Spectroscopy; Humans; Hydroxyl Radical; Immunohistochemistry; Microscopy, Fluorescence; Neoplasm Invasiveness; Neoplasms; Reactive Oxygen Species; rhoA GTP-Binding Protein; Spin Labels; Time Factors

In muscle cells, reactive oxygen species (ROS) are continually generated. It is believed that these molecules have a well-established role as physiological modulators of skeletal muscle functions, ranging from development to metabolism and from blood flow to contractile functions. Moreover, ROS may contribute to the development of muscle fatigue, inflammation, and degeneration, and may be implicated in many muscle diseases. The aim of the present study was to verify the role of short or prolonged exposure to oxidative stress, generated by different concentrations of H(2)O(2), on growth, chromosomal aberrations, and apoptosis induced in cultured L6C5 rat muscle cells used as model for myoblasts. Our results indicate that, in L6C5 cells, reactive oxygen intermediates (ROI) can activate distinct cell pathways leading to cell growth induction and development of resistant phenotype, or to chromosomal aberrations, cell cycle arrest, or cell death. The positive vs. negative effects of H(2)O(2)-altered redox potential in myoblasts are strictly related to the intensity of oxidative stress, likely depending on the types and number of cellular targets involved. Among these, DNA molecules appear to be very sensitive to breakage by H(2)O(2), although DNA damage is not directly responsible for ROI-induced apoptosis in L6C5 rat myoblasts.

Topics: Animals; Antimetabolites, Antineoplastic; Antioxidants; Apoptosis; Ascorbic Acid; Bleomycin; Catalase; Cell Division; Cells, Cultured; Chromosome Aberrations; DNA; Dose-Response Relationship, Drug; Free Radicals; Hydrogen Peroxide; Mitosis; Muscle Cells; Oxidation-Reduction; Oxidative Stress; Phenotype; Rats; Reactive Oxygen Species; Superoxide Dismutase; Time Factors

The objective of this study was to evaluate the ability of lasers and microdermabrasion, both of which are skin resurfacing modalities, to enhance and control the in vitro skin permeation and deposition of vitamin C. The topical delivery of magnesium ascorbyl phosphate, the pro-drug of vitamin C, was also examined in this study. All resurfacing techniques evaluated produced significant increases in the topical delivery of vitamin C across and/or into the skin. The erbium:yttrium-aluminum-garnet (Er:YAG) laser showed the greatest enhancement of skin permeation of vitamin C among the modalities tested. The laser fluence and spot size were found to play important parts in controlling drug absorption. An excellent correlation was observed in the Er:YAG laser fluence and transepidermal water loss, which is an estimation of skin disruption. Permeation of magnesium ascorbyl phosphate was not enhanced by the Er:YAG laser. The CO2 laser at a lower fluence promoted vitamin C permeation with no ablation of the stratum corneum or epidermal layers. Further enhancement was observed with the CO2 laser at higher fluences, which was accompanied by a prominent ablation effect. Microdermabrasion ablated the stratum corneum layers with minimal disruption of the skin barrier properties according to transepidermal water loss levels. The flux and skin deposition of vitamin C across microdermabrasion-treated skin was approximately 20-fold higher than that across intact skin. The techniques used in this study may be useful for basic and clinical investigations of enhancement of topical vitamin C delivery.

Topics: Administration, Topical; Animals; Ascorbic Acid; Dermabrasion; Female; Lasers; Mice; Mice, Inbred BALB C; Mice, Nude; Skin; Skin Absorption

Using a culture system that facilitates osteogenic differentiation of bone marrow-derived human mesenchymal stem cells, we analyzed gene-expression profiles during the mineralization process by means of a cDNA microarray system consisting of 23,040 genes. We compared expression profiles of the cells at days 3, 15, and 27 of incubation in media containing either a combination of 0.1 microM dexamethasone, 0.05 mM ascorbic acid-2-phosphate, and 10 mM beta-glycerophosphate, dexamethasone only, ascorbic acid-2-phosphate plus beta-glycerophosphate, or medium without any of these osteogenic supplements. Histochemical analysis revealed osteogenic differentiation of cells incubated in the presence of all three agents, but not in the other cultures. Comparison of the expression profiles disclosed transcriptional stimulation of 55 genes and repression of 82 genes among more than 20,000 examined. A set of differentially expressed genes we report here should contribute to a better understanding of the process of mineralization in the matrix surrounding human mesenchymal stem cells.

Topics: Antineoplastic Agents; Antineoplastic Agents, Hormonal; Ascorbic Acid; Cell Differentiation; Dexamethasone; DNA, Complementary; Down-Regulation; Glycerophosphates; Humans; Mesoderm; Oligonucleotide Array Sequence Analysis; Reverse Transcriptase Polymerase Chain Reaction; Stem Cells; Time Factors; Transcription, Genetic; Up-Regulation

Whitening effects of cosmetics have been evaluated with change of skin reflectance of the colorimeter. However, skin color implies Hue (H) and Saturation (S) as well as skin reflectance (Value). We have developed a new evaluation method of change of skin color using computer analysis of the video-captured digital image. We have also investigated whitening effects of a new whitening cosmetic essence, 'Concentre anti-tache nuit' (CAN; Parfums Christian Dior, France), which contained 3% magnesium l-ascorbyl 2-phosphate, on skin color of the face in 15 healthy Japanese females. Measurement was performed after 6 weeks application. Whitening effects were also evaluated with the combination of observation and photographs. CAN showed significant improvement in Saturation of the forehead and left cheek and Value of the forehead. CAN showed greater whitening effects both on the forehead and left cheek in eight (53%) of 15 subjects with the combination of observation and photographs. These results indicated that CAN may have a whitening effect on skin of the face after 6 weeks application. This method may be useful to evaluate the whitening effect of cosmetics and color change of cutaneous disorders.

Topics: Adolescent; Adult; Ascorbic Acid; Cosmetics; Face; Female; Humans; Image Processing, Computer-Assisted; Japan; Middle Aged; Oligodeoxyribonucleotides, Antisense; Skin Pigmentation; Solutions; Vitamin A

Sepsis is associated with oxidative stress and impaired glutamatergic transmission in brain. We investigated whether sepsis impairs accumulation of the antioxidant, ascorbate, and uptake of glutamate by astrocytes. Bacterial endotoxin (Escherichia coli lipopolysaccharide, LPS) and the inflammatory cytokine, interferon-gamma (IFNgamma), were applied to primary astrocyte cultures to model sepsis. In the absence of ascorbate, the combination of LPS and IFNgamma (LPS + IFNgammay) up-regulated inducible nitric oxide synthase (iNOS) and decreased the initial rate of glutamate uptake by 50% within 24 h. Cell viability and facilitated glucose transport activity were not affected at 24 h. Pre-treatment with ascorbate-2-O-phosphate increased intracellular ascorbate concentration and attenuated the induction of iNOS and inhibition of glutamate uptake caused by LPS + IFNgamma. Subsequent experiments examined the mechanisms by which cells accumulate ascorbate. LPS + IFNy decreased slightly the initial rate of uptake of ascorbate and inhibited markedly the rate with which intracellular dehydroascorbic acid (DHAA) was reduced to ascorbate. We conclude that septic insult impairs astrocytic clearance of DHAA from the extracellular fluid and decreases intracellular ascorbate concentration. Furthermore, sepsis induces iNOS and inhibits glutamate uptake by astrocytes through mechanisms that can be modulated by intracellular ascorbate. These results indicate treatments that increase intracellular ascorbate concentration may be beneficial for patients at risk for neurologic complication in sepsis.

Topics: Animals; Ascorbic Acid; Astrocytes; Biological Transport; Cell Survival; Cells, Cultured; Dehydroascorbic Acid; Enzyme Induction; Enzyme Inhibitors; Extracellular Space; Glial Fibrillary Acidic Protein; Glucose; Glutamic Acid; Interferon-gamma; Intracellular Fluid; Lipopolysaccharides; Nitric Oxide Synthase; Nitric Oxide Synthase Type II; Oxidation-Reduction; Rats; Rats, Wistar; Sepsis; Up-Regulation

Our previous report showed that human fetal lung fibroblasts secreted non-disulfide-bonded, non-helical collagenous polypeptides of alpha1(IV) and alpha2(IV) chains depending on culture conditions [Connective Tissue (1999) 31, 161-168]. The secretion of non-helical collagenous polypeptides is unexpected from the current consensus that such polypeptides are not secreted under physiological conditions. The absence of interchain disulfide bonds among alpha1(IV) and alpha2(IV) chains was always correlated with the absence of triple-helical structure of the type IV collagen. The finding corresponds with the fact that the interchain disulfide bonds are formed at or close to the completion of the type IV collagen triple-helix formation. The present report shows that ascorbate is the primary factor for the triple-helix formation of the type IV collagen. When human mesangial cells were cultured with ascorbate, only the triple-helical type IV collagen was secreted. However, when the cells were cultured without ascorbate, the non-helical alpha1(IV) and alpha2(IV) chains were secreted. Relative amounts of the secreted products were unchanged with or without ascorbate, suggesting that ascorbate is required for the step of the triple-helix formation. The ascorbate-dependency of the triple-helix formation of the type IV collagen was observed in all the human cells examined. The non-helical alpha1(IV) chain produced by the ascorbate-free culture contained about 80% less hydroxyproline than the alpha1(IV) chain from the triple-helical type IV collagen. The evidence for the non-association of the non-helical alpha1(IV) and alpha2(IV) chains in the conditioned medium was obtained by an anti-alpha1(IV) antibody-coupled affinity column chromatography for the conditioned medium. Although all the non-helical alpha1(IV) chains were found in the bound fraction, all the non-helical alpha2(IV) chains were recovered in the flow-through fraction. The present findings suggest that ascorbate plays a key role in the trimerization step of three alpha chains and/or in the subsequent triple-helix formation of the type IV collagen.

Topics: Antibodies, Monoclonal; Aorta; Ascorbic Acid; Cells, Cultured; Chromatography, Affinity; Collagen; Endothelium, Vascular; Glomerular Mesangium; Humans; Hydroxylation; Hydroxyproline; Immunoblotting; Muscle, Smooth, Vascular; Oxidation-Reduction; Peptide Fragments; Proline; Protein Structure, Quaternary; Protein Structure, Secondary; Umbilical Cord

Ascorbyl palmitate and sodium ascorbyl phosphate are derivatives of ascorbic acid, which differ in stability and hydro-lipophilic properties. They are widely used in cosmetic and pharmaceutical preparations. In the present work the stability of both derivatives was studied in microemulsions for topical use as carrier systems. The microemulsions were of both o/w and w/o types and composed of the same ingredients. The stability of the less stable derivative ascorbyl palmitate was tested under different conditions to evaluate the influence of initial concentration, location in microemulsion, dissolved oxygen and storage conditions. High concentrations of ascorbyl palmitate reduced the extent of its degradation. The location of ascorbyl palmitate in the microemulsion and oxygen dissolved in the system together significantly influence the stability of the compound. Light accelerated the degradation of ascorbyl palmitate. In contrast, sodium ascorbyl phosphate was stable in both types of microemulsions. Sodium ascorbyl phosphate is shown to be convenient as an active ingredient in topical preparations. In the case of ascorbyl palmitate, long-term stability in selected microemulsions was not adequate. To formulate an optimal carrier system for this ingredient other factors influencing the stability have to be considered.

Topics: Administration, Topical; Ascorbic Acid; Chemistry, Pharmaceutical; Chromatography, High Pressure Liquid; Drug Carriers; Drug Stability; Emulsions

There is an increasing recognition of the damaging role played by oxygen radicals in mediating necrotic neuronal injury. As such, it becomes important to understand the transport mechanisms that help maintain appropriate levels of small molecule antioxidants such as ascorbate in the brain. It has long been known that the transport of dehydroascorbate (DHA) into a variety of cell types is accomplished through the Glut-1 glucose transporter. In this paper, we characterize interactions among the transports of ascorbate, DHA and glucose in hippocampal cultures. We find: (a) sodium-dependent transport of ascorbate in mixed neuronal/glial, pure glial, and neuron-enriched hippocampal cultures; in contrast, we observed no such transport of DHA; (b) such ascorbate transport appeared to be independent of the glucose transporter, in that glucose did not compete for such transport, and overexpression of the Glut-1 glucose transporter did not alter ascorbate uptake; (c) in contrast, ascorbate, at concentrations ranging from 1 to 20 mM inhibited 2-dexogyglucose transport in mixed, glial and enriched neuronal hippocampal cultures; (d) potentially, ascorbate, by acting as an electron donor, could impair the function of molecules involve in the transport or metabolism of glucose. We observed mild inhibition of glucose transport by one unrelated electron donor (glutathione). Moreover, transport was also inhibited by an ascorbate analog which is not an electron donor. Thus, we conclude that ascorbate transport in hippocampal neurons and glia occurs independent of the glucose transporter but that, nevertheless, ascorbate, at concentrations generally thought to be supraphysiological, has the potential for disrupting glucose transport.

Topics: Animals; Ascorbic Acid; Brain Injuries; Carbon Radioisotopes; Cells, Cultured; Dehydroascorbic Acid; Deoxyglucose; Drug Interactions; Fetus; Glucose; Glucose Transporter Type 1; Glutathione; Hippocampus; Monosaccharide Transport Proteins; Nerve Degeneration; Neuroglia; Neurons; Oxidative Stress; Rats

During wound healing and inflammation, fibroblasts express elevated alkaline phosphatase (ALP), but are not in contact with collagen fibrils in the fibronectin (FN)-rich granulation tissue. We hypothesized that the extracellular matrix (ECM) environment might influence the induction of ALP in fibroblasts. Here we tested this hypothesis by studying the ALP-inductive response of normal human gingival fibroblasts to ascorbic acid (AsA). AsA induced ALP activity and protein in cells in conventional monolayer culture. This induction was inhibited by blocking-antibodies to the FN receptor alpha 5 beta 1 integrin and by the proline analog 3,4-dehydroproline (DHP). DHP prevented cells from arranging FN fibrils into a pericellular network and reduced the activity of cell spreading on FN. Plating of cells on FN facilitated the up-regulation by AsA of ALP expression, but did not substitute for AsA. In contrast, AsA did not cause ALP induction in cells cultured on and in polymerized type I collagen gels. Collagen fibrils inhibited the up-regulation by AsA of ALP expression in cells plated on FN. These results indicate that the ECM regulates the induction of ALP expression by AsA in fibroblasts: FN enables them to express ALP in response to AsA through interaction with integrin alpha 5 beta 1, whereas type I collagen fibrils cause the suppression of ALP expression and overcome FN.

Topics: Adult; Alkaline Phosphatase; Antibodies; Ascorbic Acid; Cells, Cultured; Collagen; Extracellular Matrix; Fibroblasts; Fibronectins; Humans; Middle Aged; Proline; Receptors, Fibronectin; Up-Regulation

Recent studies demonstrate that collagen IV selectively promotes the repair of physiological processes in sublethally injured renal proximal tubular cells (RPTC). We sought to further define the mechanisms of cell repair by measuring the effects of toxicant injury and stimulation of repair by L-ascorbic acid-2-phosphate (AscP), exogenous collagen IV, or function-stimulating integrin antibodies on the expression and subcellular localization of collagen-binding integrins (CBI) in RPTC. Expression of CBI subunits alpha1, alpha2, and beta1 in RPTC was not altered on day 1 after sublethal injury by S-(1,2-dichlorovinyl)-L-cysteine (DCVC). On day 6, expression of alpha1 and beta1 subunits remained unchanged, whereas a 2.2-fold increase in alpha2 expression was evident in injured RPTC. CBI localization in control RPTC was limited exclusively to the basal membrane. On day 1 after injury, RPTC exhibited a marked inhibition of active Na(+) transport and a loss of cell polarity characterized by a decrease in basal CBI localization and the appearance of CBI on the apical membrane. On day 6 after injury, RPTC still exhibited marked inhibition of active Na(+) transport and localization of CBI to the apical membrane. However, DCVC-injured RPTC cultured in pharmacological concentrations of AscP (500 microM) or exogenous collagen IV (50 microg/ml) exhibited an increase in active Na(+) transport, relocalization of CBI to the basal membrane, and the disappearance of CBI from the apical membrane on day 6. Function-stimulating antibodies to CBI beta1 did not promote basal relocalization of CBI despite stimulating the repair of Na(+)/K(+)-ATPase activity on day 6 after injury. These data demonstrate that DCVC disrupts integrin localization and that physiological repair stimulated by AscP or collagen IV is associated with the basal relocalization of CBI in DCVC-injured RPTC. These data also suggest that CBI-mediated repair of physiological functions may occur independently of integrin relocalization.

Topics: Animals; Antibodies; Ascorbic Acid; Collagen Type IV; Female; Integrins; Kidney; Kidney Tubules, Proximal; Rabbits; Receptors, Collagen; Sodium-Potassium-Exchanging ATPase; Subcellular Fractions

Tumor metastasis and invasion were shown to be inhibited by the 2-O-phosphorylated form (Asc2P) of L-ascorbic acid (Asc); intact Asc did not inhibit tumor invasion when added once, but appreciably inhibited it upon repeated addition. The anti-metastatic effect is attributable to a marked enrichment of intracellular Asc by Asc2P, subsequently dephosphorylated. Asc2P scavenged most of the intracellular reactive oxygen species (ROSin), and notably inhibited production of matrix metalloproteases and cell motility. ROSin was decreased by Asc2P more markedly than by Asc added once. Thus, involvement of ROSin in tumor invasion and a potent anti-metastatic therapy by ROSin-decreasing agents are suggested.

Topics: Animals; Antineoplastic Agents; Ascorbic Acid; Cell Movement; Extracellular Matrix; Female; Fibrosarcoma; Free Radical Scavengers; Humans; Melanoma, Experimental; Mice; Mice, Inbred C57BL; Neoplasm Invasiveness; Neoplasm Metastasis; Oxidative Stress; Reactive Oxygen Species

Invasion of rat fibroblastic cells Rat-1 through Matrigel was shown to be promoted by transfection with tax gene of human T-cell leukemia virus type 1. We found that an oxidation-resistant type of vitamin C (Asc), Asc-2-O-phosphate (Asc2P), inhibited the invasion of the tax-transfected Rat-1 cells (W4 cells). Intracellular Asc (Ascin), after enzymatic dephosphorylation of administered Asc2P, was more abundant in W4 cells than in Rat-1 cells, and the ratio of dehydroascorbic acid versus Asc was increased in W4 cells but scarcely in Rat-1 cells, according to the enhanced level of intracellular reactive oxygen species (ROSin) in W4 cells. Asc2P notably repressed the increases in both ROSin and secretion of matrix metalloproteases (MMPs), but did not affect Tax protein expression in tax-transfectants. NF-kappa B activation, as evidenced by its translocation to the nucleus in W4 cells, was also repressed by Asc2P. Thus, the tax-promoted invasion together with the enhanced production of MMPs occurred with NF-kappa B activation and the increase in ROSin, both of which were effectively reduced by Asc2P. These findings indicate the therapeutic efficacy of Ascin-enriching agents for the prevention against tumor invasion in which ROSin plays a major role.

Topics: Animals; Ascorbic Acid; Basement Membrane; Cell Line; Cell Movement; Fibroblasts; Gene Products, tax; Genes, pX; Humans; Neoplasm Invasiveness; NF-kappa B; Oxidative Stress; Phenotype; Rats; Transfection

The present study succeeded for the first time in cultivating for more than 2 months human normal hepatocytes which showed a high growth potential and expressed their differentiated phenotypes. Constituents of culture medium were critical for this culture, and the medium optimized for their growth contained fresh human serum, fetal bovine serum, Swiss 3T3-cell conditioned medium, L-ascorbic acid 2-phosphate, epidermal growth factor, nicotinamide, and dimethyl sulfoxide. Hepatocytes steadily replicated and formed colonies which continued to increase in size up to around 35 days. The number of hepatocytes in the most replicative colonies increased 17-fold during 31 days. Cells in colonies expressed normal differentiated hepatocytic phenotypes for as long as 35 days. These hepatocytes retained normal liver functions at least for 70 days such as to secrete albumin, and to metabolize lidocaine and D-galactose.

Topics: 3T3 Cells; Adult; Aged; Albumins; Animals; Ascorbic Acid; Biomarkers; Blood Proteins; Cell Culture Techniques; Cell Differentiation; Cell Division; Cell Size; Cell Survival; Cells, Cultured; Culture Media, Conditioned; Dimethyl Sulfoxide; DNA; Epidermal Growth Factor; Female; Galactose; Humans; Lidocaine; Liver; Male; Mice; Middle Aged; Niacinamide; Time Factors

We have investigated the enzymatic reduction and accumulation of vitamin C in HaCaT epithelial cells. The subcellular localization and the activities of ascorbyl free radical reductase and dehydroascorbate reductase showed that mitochondrial, microsomal and plasma membranes fractions express high levels of ascorbyl free radical reductase activity, whereas dehydroascorbate reductase activity was found at low levels only in the post microsomal supernatant. We have also investigated cell proliferation and vitamin C accumulation induced by ascorbic acid 2-phosphate. This derivative caused no inhibition of cell growth, was uptaken from the extracellular medium and accumulated as ascorbic acid in mM concentrations. These results show that HaCaT cells possess very efficient systems to maintain high levels of both intracellular and extracellular ascorbic acid. The regeneration and uptake of ascorbic acid from extracellular medium contributes to the intracellular antioxidant capacity, as evaluated by 2',7'-dihydrodichlorofluorescein staining. Consequently, cells became more resistant to free radical generation and cell death induced by UV-B irradiation.

Topics: Antioxidants; Apoptosis; Ascorbic Acid; Cell Division; Cell Line; Free Radicals; Humans; Keratinocytes; Kinetics; NADH, NADPH Oxidoreductases; Oxidoreductases; Subcellular Fractions; Ultraviolet Rays

Staining of collagens by Sirius Red, a standard histological procedure, was applied to quantify collagen synthesis in human osteoblast-like cell cultures in situ. After morphological analysis of the deposited material, the stain was dissolved and its optical density determined spectrophotometrically using a microtiter plate assay system. The method was sensitive with a detection limit for collagen synthesized by 3000 normal human periosteal cells. The assay is easy to perform and specific with respect to different extracellular materials, for example, collagen types I and III were well stained, collagen type IV and laminin exhibited only low staining, and fibronectin, chondroitin sulfate, dermatan sulfate, and amyloid beta were negative. A major advantage of the method is the combination of identification of collagen-producing cells in situ with subsequent spectrophotometric quantification of the dissolved stain. Thus it is possible to obtain information on cell morphology, active sites of collagen deposition in a cell culture, microscopic detection of high-and low-producer cells prior to dissolution and quantification of the deposited material. In this regard the assay is superior to either radioactive labeling, hydroxyproline determination, or Sirius Red-based colorimetric assays with cell lysates. Since the quantification is based on microtiter plate reading, the method can be recommended for the screening of large quantities of samples.

Topics: Adolescent; Ascorbic Acid; Azo Compounds; Collagen; Colorimetry; Coloring Agents; Dexamethasone; Drug Interactions; Humans; Male; Osteoblasts; Sensitivity and Specificity; Staining and Labeling; Transforming Growth Factor beta; Tumor Cells, Cultured

The protective effect of sodium-L-ascorbyl-2 phosphate (As-2P), a stable form of ascorbic acid (AsA), against photodamage induced by a single dose of UVB exposure (290-320 nm, Max 312 nm) was investigated using cultured mouse skin. When the cultured skin was treated with various As-2P concentrations, the cutaneous AsA level increased in proportion to the As-2P concentration. After 3 h of incubation, the AsA level in the cultured skin treated with 2, 20 and 100 mM As-2P increased 1.03-, 2.17- and 6.27-fold, respectively, compared with that of the control skin. These results suggest that As-2P was transported into the cultured mouse skin where it was converted to AsA. After 3 h, the cutaneous AsA level in irradiated (20 kJ/m2) skin was depleted to a half of that in the control skin. However, the level in skin pretreated with 20 mM As-2P was maintained within normal limits, even after 24 h. Pretreatment with 20 mM As-2P significantly prevented such photodamage as sunburn cell formation, DNA fragmentation and lipid peroxidation, which were caused by a single dose of UVB irradiation. These results suggest that the protective effect of 20 mM As-2P on UVB-induced cutaneous damage is due to the maintenance of a normal As level by conversion of As-2P to As in skin tissue.

Topics: Animals; Ascorbic Acid; Cells, Cultured; DNA Fragmentation; Female; Lipid Peroxidation; Mice; Mice, Hairless; Radiation-Protective Agents; Skin; Ultraviolet Rays

The application of mechanical loads to bone cells in vitro has been found to generate variable responses, which may in part be due to the source of the cell used and the characteristics of the strain applied. The aim of this study was to establish a system for applying well-defined physiological levels of mechanical strain to a well-defined population of human osteoblast-like cells. Human bone-derived cells obtained from the greater trochanter of the femur during total hip arthroplasty for osteoarthritis were cultured in the presence of 10 nmol/L dexamethasone and 100 mumol/L L-ascorbate-2-phosphate. Replicates of cells from each patient were loaded on separate occasions using controlled cyclical strains of 4000 microstrain (mu epsilon) or less. Strain gauges recorded reliable, reproducible strains between 1000 and 6000 mu epsilon. To establish reproducibility, sequential explant cultures derived from two patients were studied. A consistent increase (p < 0.05) in proliferation between replicates and explants derived from one patient subjected to 1600 mu epsilon on separate occasions was observed. Cells derived from sequential explants of the second patient showed no consistent increase in proliferation between replicates and explants. Three of six patients showed a significant increase (p < 0.05) in PGE2 production after 5 h in response to stretch (4000 mu epsilon) in all replicates on separate occasions, whereas, in the other three populations of cells, no increase in PGE2 was measured in any of the replicates. These results show that the application of highly controlled strains causes a significant effect on human bone cells, but only in a proportion of subjects. The response is consistent between sequential explants derived from the same patient. The implications of this study are that human osteoblast-like cells do respond to physiological strain in vitro, although some cells are more strain sensitive than others.

Topics: Anti-Inflammatory Agents; Arthroplasty, Replacement, Hip; Ascorbic Acid; Cell Division; Cells, Cultured; Dexamethasone; Dinoprostone; Femur; Humans; Osteoarthritis; Osteoblasts; Reproducibility of Results; Stress, Mechanical

The effects of stable vitamin C, magnesium-L-ascorbyl-2-phosphate (MAP), administered after acute and chronic exposure to UVB irradiation were investigated using hairless mice. Intraperitoneal administration of 100 mg/kg of MAP immediately after acute exposure to 15 kJ/m2 of UVB significantly prevented increases of UVB-induced lipid peroxidation in skin and sialic acid in serum, an inflammation marker. Administration of 50 mg/kg of MAP immediately after each exposure significantly delayed skin tumor formation and hyperplasia induced by chronic exposure to 2 kJ/m2 of UVB. Intraperitoneal administration of 200 mg/kg of MAP produced an increase in ascorbic acid (As) levels in the serum, liver and skin within 15 min. Serum As levels quickly returned to normal, but hepatic and cutaneous levels remained elevated before returning to normal after 24 h, suggesting that MAP was converted to As in the serum and in those tissues. Ultraviolet B-induced hydroxyl radical generation in murine skin homogenates was scavenged by As-Na addition, which was directly detected by electron spin resonance (ESR). These results suggest that postadministration of MAP delays progression of skin damage induced by UVB irradiation. It is presumed that MAP, once converted to As, exhibits such inhibitory effects by scavenging hydroxyl and lipid radicals generated as a direct or indirect result of UVB exposure.

Topics: Animals; Ascorbic Acid; Lipid Peroxidation; Mice; Mice, Hairless; N-Acetylneuraminic Acid; Skin; Thiobarbituric Acid Reactive Substances; Time Factors; Ultraviolet Rays

We conducted two experiments to evaluate the efficacy of a stable source of vitamin C for improving performance and iron status in early-weaned pigs. A preparation of L-ascorbyl-2-polyphosphate (Rovimix Stay-C 25, Roche Vitamins, Ames, IA and Bramus, NJ), which supplies 25% ascorbic acid activity in a stable form, served as the vitamin C source and was incorporated at dietary vitamin C levels of 0, 75, or 150 ppm. In Exp. 1, 72 pigs (14 +/- 2 d of age and 4.98 kg BW) were blocked based on initial BW and penned in groups of three (eight pens per treatment) in an off-site nursery for 42 d. Phase 1 lasted from d 0 to 14, Phase 2 from d 14 to 28, and Phase 3 from d 28 to 42 after weaning. Daily gain and gain:feed ratio (G/F) increased during Phase 1 (quadratic, P < .1 and P < .05, respectively), Phase 3 (linear, P < .1 and P < .01, respectively), and for the overall 42-d experiment (linear, P < .05 and P < .1, respectively) in response to increasing dietary vitamin C. At 14 d after weaning, plasma vitamin C increased (linear, P < .05) with increasing dietary vitamin C, but plasma iron, hemoglobin, and hematocrit were not influenced by dietary vitamin C. In Exp. 2, 120 pigs (20 +/- 3 d of age and 7.2 kg BW) were blocked based on initial BW and penned in groups of five (eight pens per treatment) in a conventional nursery system for 31 d. Phase 1 consisted of d 0 to 7, Phase 2 from d 7 to 17, and Phase 3 from d 17 to 31 after weaning. During the period from d 0 to 17 after weaning, ADG and G/F were improved (linear, P < .1) with increasing dietary vitamin C. At d 17 after weaning, plasma vitamin C and serum iron increased (linear, P < .05), but unbound iron-binding capacity and total iron-binding capacity decreased (linear, P < .05 and P < .1, respectively) with increasing dietary vitamin C. These results suggest that dietary vitamin C is needed during the first 42 d after weaning when pigs are weaned as early as 12 d of age and reared in an off-site nursery and during the first 17 d after weaning when pigs are weaned as early as 17 d of age and reared in a conventional nursery system. L-Ascorbyl-2-polyphosphate at a supplemental level of 75 ppm was adequate to meet the dietary vitamin C requirement of early-weaned pigs. Vitamin C supplementation with a stable product will improve performance in young pigs during the high-stress postweaning period and may be particularly beneficial to pigs weaned at a very early age.

Topics: Animals; Ascorbic Acid; Diet; Dose-Response Relationship, Drug; Drug Stability; Eating; Female; Iron; Male; Random Allocation; Swine; Weaning; Weight Gain

Telomeres in eukaryotic somatic cells are destined to the age-dependent shortening, which has not been demonstrated to correlate to direct lesion of telomeric DNA by reactive oxygen intermediates (ROI); still less explicable is the inhibitory effect of ROI-scavenging on telomere shortening. Here, we succeeded in artificial slowdown of age-dependent telomere shortening to 52-62% of the untreated control, in human vascular endothelial cells, by addition of the oxidation-resistant type of ascorbic acid (Asc), Asc-2-O-phosphate (Asc2P), which concurrently achieved both extension of cellular life-span and prevention of cell size enlargement indicative of cellular senescence. The results are attributable to a 3.9-fold more marked enrichment of intracellular Asc (Asc(in)) by addition of Asc2P, subsequently dephosphorylated before or during transmembrane influx, than by addition of Asc itself, and also attributed to diminution of intracellular ROI to 53% of the control level by Asc2P; telomerase activity was at a trace level and underwent an age-dependent decline, which was significantly decelerated by Asc2P. Thus, age-dependent telomere-shortening can be decelerated by suppression of intracellular oxidative stress and/or by telomerase retention, both of which are achieved by enriched Asc(in) but not by extracellular Asc overwhelmingly more abundant than Asc(in).

Topics: Ascorbic Acid; Cells, Cultured; Cellular Senescence; Endothelium, Vascular; Humans; Intracellular Fluid; Oxidative Stress; Telomerase; Telomere; Umbilical Veins

Clinical and experimental studies demonstrate that injured anterior cruciate ligaments (ACL) do not usually heal and that autografts used to repair the ACL rapidly weaken in the early period and take a long time to regain strength. The aim of this study was to develop an in vitro culture system in which environmental and biochemical factors influencing the proliferation and matrix synthesis of cells derived from human anterior cruciate ligaments can be studied. Primary cultures of human ACL cells were obtained by outgrowth from explants of normal ACL obtained at knee replacement for osteoarthritis in Dulbecco's minimum essential medium (DMEM). The effects of the additives 100 microm L-ascorbic acid-2-phosphate (Asc-2-P) and 10 n m dexamethasone (dex) on proliferation and collagen synthesis were assessed after 4, 8 and 12 days in culture. Ligament cells were grown at 0, 5, 10 and 21%p O(2)in the presence of 100 microm asc-2-P and 10 n m dex. DNA content was assessed using the Hoechst dye method and collagen synthesis by the incorporation of 5 mCi/ml [(3)H]proline after 3, 6 and 12 days in culture. At 21%p O(2), the presence of asc-2-P and dex induced significantly greater (P< 0.01, ANOVA) cell proliferation than with either additives alone. Greatest percentage collagen to total protein synthesis was observed when cells were grown in the presence of asc-2-P only. Least proliferation and percentage collagen to total protein synthesis was seen when both additives were omitted. Greatest cell proliferation was seen when cells were grown in 10%p O(2)and 5%p O(2)was associated with increased collagen synthesis. These results suggest that it is possible to study the effects of environmental and biochemical factors on human ACL healing in vitro. Our data suggest oxygen can influence certain biosynthetic activities of ACL cells. Low oxygen tensions lead to an increase in collagen production by ACL cells. However early responses to injury require extensive cell proliferation which may be activated at higher p O(2). Variation of p O(2)in ligaments during healing may therefore be an important modulator of successful repair.

Topics: Anterior Cruciate Ligament; Ascorbic Acid; Cell Division; Cells, Cultured; Collagen; Dexamethasone; Humans; Oxygen; Transplantation, Autologous; Wound Healing

The addition of L-ascorbic acid 2-phosphate (Asc 2-P), which is active and stable under a conventional culture condition, could render dermal fibroblasts to the organization of a dermis-like structure on a plastic dish without any prior treatment. The cell layer was composed of multilayered fibroblasts surrounded by dense extracellular matrices. Confocal microscopic examination disclosed that the fibroblasts in the upper layer were spindle-shaped and those in the lower layer were polygonal. Electron microscopic examination revealed the accumulation of mature collagen fibrils in the intercellular space. These morphological observations suggest that the cell layer may resemble the dermis-like structure. Biochemical analyses revealed that the hydroxyproline content of the cell layer increased in a time-dependent manner, while the monolayer culture system without. Asc 2-P yielded no measurable amount of hydroxyproline. On sodium dodecylsulphate-polyacrylamide gel electrophoresis, neutral insoluble collagens extracted from the cell layer showed the identical electrophoretic pattern to those from the human dermis. In addition, these bands were completely digested by bacterial collagenase. This novel culture system could provide a simple tool with which to investigate the collagen metabolism by fibroblasts under more physiological conditions.

Topics: Adolescent; Ascorbic Acid; Cell Culture Techniques; Collagen; Electrophoresis, Polyacrylamide Gel; Fibroblasts; Humans; Hydroxyproline; Male; Skin

The stability of ascorbic acid, ascorbyl palmitate and magnesium ascorbyl phosphate (VC-PMG) in both standard solutions and topical formulations was investigated by direct RP-HPLC analysis after sample dilution with a suitable aqueous-organic solvent mixture. The results showed that, whereas the two vitamin C derivatives were more stable than ascorbic acid, the ascorbyl esters showed significant differences. Esterification with palmitic acid in 6 position did not prevent hydrolysis of the molecule, either in solution or in emulsion; only the special preparation of products with high viscoelastic properties was able to reduce the typical behaviour of this compound. Conversely, the introduction of the phosphoric group in 2 position protected the molecule from break-up of the enediol system, confirming VC-PMG as a very stable derivative of vitamin C that may be easily used in various types of cosmetic products.

Topics: Ascorbic Acid; Chromatography, High Pressure Liquid; Cosmetics; Drug Stability; Emulsions; Molecular Structure; Reference Standards; Solutions; Spectrophotometry, Ultraviolet

Bovine aortic endothelial BAE-2 cells exposed to the peroxidizing agent, tert-butylhydroperoxide (t-BuOOH) or 2,4-nonadienal (NDE), suffered from disruption of cell membrane integrity and from reduction of mitochondrial dehydrogenase activity as assessed by fluorometry using ethidium homodimer and photometry using WST-1, respectively. The cells were protected from t-BuOOH-induced injury more markedly by L-ascorbic acid-2-O-phosphate (Asc2P) stably masked at the 2,3-enediol moiety, which is responsible for the antioxidant ability of L-ascorbic acid (Asc), than by Asc itself. In contrast, NDE-induced membrane disruption but not mitochondrial dysfunction was prevented by Asc2P, whereas Asc exhibited no prevention against both types of injury. The amount of intracellular Asc was 7.2- to 9.0-fold larger in Asc2P-administered BAE-2 cells, where the intact from Asc2P was not detected, than in Asc-administered cells as assessed by HPLC of cell extract with detection by coulometric ECD and UV. During transmembrane influx into the cell, Asc2P was concentrated as highly as 70- to 90-fold relative to the extracellular Asc2P concentration, whereas Asc was 8- to 13-fold concentrated as estimated based on an intracellular water content of 0.59 pL/cell determined by [14C]PEG/gas chromatography. Thus, Asc2P but not Asc is highly concentrated in the aqueous phase of the cell after prompt dephosphorylation, and may thereby render the cell more resistant to t-BuOOH-peroxidation assumedly via scavenging of intracellular reactive oxygen species than to peroxidation with the less hydrophilic agent NDE.

Topics: Animals; Aorta; Ascorbic Acid; Cattle; Cell Death; Cell Membrane; Cells, Cultured; Endothelium, Vascular; Oxidants; Oxidation-Reduction; Phosphorylation

An inhibitory effect of ascorbic acid (AsA) on melanogenesis has been described. However, AsA is quickly oxidized and decomposed in aqueous solution and thus is not generally useful as a depigmenting agent.. Our purpose was to examine the effect on pigmentation of magnesium-L-ascorbyl-2-phosphate (VC-PMG), a stable derivative of AsA.. Percutaneous absorption of VC-PMG was examined in dermatomed human skin, and its effect on melanin production by mammalian tyrosinase and human melanoma cells in culture was also measured. A 10% VC-PMG cream was applied to the patients.. VC-PMG suppressed melanin formation by tyrosinase and melanoma cells. In situ experiments demonstrated that VC-PMG cream was absorbed into the epidermis and that 1.6% remained 48 hours after application. The lightening effect was significant in 19 of 34 patients with chloasma or senile freckles and in 3 of 25 patients with normal skin.. VC-PMG is effective in reducing skin hyperpigmentation in some patients.

Topics: Ascorbic Acid; Female; Humans; In Vitro Techniques; Melanins; Melanoma, Experimental; Melanosis; Monophenol Monooxygenase; Skin Absorption; Skin Neoplasms; Tumor Cells, Cultured

The present study succeeded in cultivating normal adult rat hepatocytes for at least 85 days without losing their replicative potential and differentiation capacity. Small pieces of hepatocyte aggregates (clusters) were prepared from the primary culture of hepatocytes and used as starting material for the growth experiment. Some of the hepatocytes started to proliferate at 3 days when the clusters were cultured in Dulbecco's modified Eagle's medium containing 10% fetal bovine serum, 10 ng/ml epidermal growth factor, 10 mmol/L nicotinamide, 0.2 mmol/L L-ascorbic acid 2-phosphate, and 1% dimethylsulfoxide. Clusters continued to grow and formed colonies. All the cells covering colonies expressed normal hepatocyte-specific proteins. The number of albumin-expressing cells in the most replicative colonies increased sixfold during 32 days. Most of the cells were mononucleate and small in size and some of them expressed immature hepatocyte markers such as alpha-fetoprotein. Electron microscopy of cells in colonies revealed the presence of peroxisomes in the cytoplasm and desmosomes, tight junctions, and bile canaliculus-like structures between the cells. Depletion of one of the additives inhibited the growth of hepatocytes. The culture medium used also supported the growth of stellate cells (Ito cells) that had contaminated the original preparation in small numbers and seems to cooperatively stimulate a proliferative population of hepatocytes.

Topics: Albumins; alpha-Fetoproteins; Animals; Ascorbic Acid; Cell Aggregation; Cell Culture Techniques; Cell Division; Culture Media; Dimethyl Sulfoxide; Epidermal Growth Factor; Immunohistochemistry; Liver; Male; Microscopy, Electron; Niacinamide; Phenotype; Rats; Rats, Inbred F344; Time Factors

The protective effect of magnesium-L-ascorbyl-2-phosphate (MAP) on cutaneous photodamage such as lipid peroxidation and inflammation induced by ultraviolet B (UVB) exposure (290-320 nm, max. 312 nm) was investigated using hairless mice. When MAP was administered intraperitoneally to mice at a dose of 100 mg of ascorbic acid (AS) per kg body weight base immediately before irradiation (15 kj/m2), the expected increases in thiobarbituric acid reactive substance (TBARS) formation in skin and serum sialic acid, indices of lipid peroxidation and inflammatory reaction, respectively, were significantly reduced. However, the expected decrease in the level of cutaneous AS was unchanged. Similar results were observed for animals given 100 mg of AS-Na per kg body weight before UVB irradiation. When MAP was administered intracutaneously immediately before irradiation, the expected UVB-induced increases in TBARS and sialic acid were again significantly prevented. Ascorbic acid-Na had a less protective effect than intracutaneous MAP administration. The cutaneous AS level was significantly higher in the MAP-treated mice than in the controls, and the UVB-induced decrease in tissue AS was prevented by intracutaneous MAP administration. These results suggest that MAP protects against UVB irradiation-induced lipid peroxidation and inflammation in cutaneous tissue, regardless of the drug administration route. We found, in an in vitro experiment, that MAP was converted to AS as it crossed the epidermis, but that AS-Na did not pass through the epidermis. Furthermore, MAP was also converted to AS in serum. These results suggest that the protective effect of MAP on UVB-induced cutaneous damage is due to conversion of MAP to AS.

Topics: Animals; Ascorbic Acid; Female; Lipid Peroxidation; Mice; Mice, Hairless; Photobiology; Radiation-Protective Agents; Skin; Ultraviolet Rays

L-Ascorbic-2-phosphate magnesium salt (APM) in fish tissues was determined by high-performance liquid chromatography. APM extracted with 5% metaphosphoric acid was separated by a LiChrospher 100 RP18 column within 20 min. The detection limit was 0.1 microgram/g tissue.

Topics: Animals; Ascorbic Acid; Carps; Chromatography, High Pressure Liquid; Fishes; Liver

The addition of L-ascorbic acid 2-phosphate (AscP) to primary cultures of rabbit renal proximal tubular cells (RPTC) grown under improved culture conditions resulted in an extended growth phase and increased cellular density (1.3-fold increase in monolayer DNA and protein contents). AscP reduced glycolysis, increased net lactate consumption by 38%, and stimulated net glucose production by 47%. Basal O2 consumption increased by 39% in RPTC grown in the presence of AscP and was equivalent to that in freshly isolated proximal tubules. AscP increased ouabain-sensitive O2 consumption (81%) and Na(+)-K(+)-ATPase activity (2.5-fold), which suggested increased active Na+ transport. Addition of AscP increased Na(+)-dependent glucose uptake by 43% and brush-border enzyme marker activities by 46%. It is concluded that supplementation of media with AscP further improves RPTC culture conditions by promotion of cellular growth and stimulation of in vivo-like respiration, lactate utilization, and net glucose synthesis. These changes are accompanied by an increase in brush-border enzyme activities and stimulation of active Na+ transport and Na(+)-dependent glucose transport, which demonstrate an improved expression of brush-border membrane functions in RPTC.

Topics: Animals; Ascorbic Acid; Cell Division; Cells, Cultured; Female; Kidney Tubules; Oxygen; Oxygen Consumption; Rabbits

Ascorbic acid is an essential nutrient in rainbow trout diets and has been shown to play an important role in fish reproduction. Recommended dietary levels are based on immature fish, and the specific requirements for brood stock are unknown. To establish the optimum dietary level for mature rainbow trout, six graded levels of ascorbyl-2-monophosphate were fed to groups of female fish over a period of 10 mo until spawning. Increasing dietary levels of ascorbyl monophosphate resulted in significantly increased ascorbic acid concentrations in liver, kidney, ovaries, and ovulated eggs. Liver and egg concentrations were saturable at 109.3 and 266.6 micrograms ascorbic acid/g tissue, respectively. Tissue saturation levels of 83.7% and 91.2%, respectively, were reached at the highest dietary level (870 mg ascorbyl monophosphate/kg diet) tested. Both fecundity and embryo survival increased significantly with dietary ascorbyl monophosphate levels. The results indicated that the present National Research Council recommended dietary level of 50 mg ascorbic acid/kg diet for rainbow trout is inadequate for brood stock fish. An amount 8 times higher is necessary to optimize tissue ascorbic acid levels and achieve maximum reproductive success.

Topics: Animals; Ascorbic Acid; Diet; Female; Kidney; Liver; Nutritional Requirements; Oncorhynchus mykiss; Oocytes; Ovary; Reproduction

Small hepatocytes existed in the supernatant following low-speed centrifugation of the cell suspension after collagenase liver perfusion. The cells proliferated for more than 2 months and formed colonies in the Dulbecco's modified Eagle's medium supplemented with 10 mM nicotinamide, 10% fetal bovine serum, 1 mM ascorbic 2-phosphate, and 10 ng/ml epidermal growth factor. One small cell finally proliferated to several hundred cells. In addition, some cells in the colonies were shown to differentiate into mature hepatocytes that had a large cytoplasm and sometimes two nuclei. The secretion of albumin in the medium by the hepatocytes increased with time in culture, and the cells possessed connexin 32 in their cell membrane and many peroxisomes. Thus, the small hepatocytes may be "committed progenitor cells" which can further differentiate into mature hepatocytes.

Topics: Animals; Ascorbic Acid; Cell Differentiation; Cell Division; Cell Separation; Cells, Cultured; Collagenases; Epidermal Growth Factor; Liver; Male; Niacinamide; Rats; Rats, Sprague-Dawley; Serum Albumin

Pancreatic islets from young normal and scorbutic male guinea pigs were examined for their ability to release insulin when stimulated with elevated D-glucose. Islets from normal guinea pigs released insulin in a D-glucose-dependent manner showing a rapid initial secretion phase and three secondary secretion waves during a 120-min period. Islets from scorbutic guinea pigs failed to release insulin during the immediate period, and only delayed and decreased responses were observed over the 40-60 min after D-glucose elevation. Insulin release from scorbutic islets was greatly elevated if 5 mM L-ascorbic acid 2-phosphate was supplemented in the perifusion medium during the last 60 min of perifusion. When 5 mM L-ascorbic acid 2-phosphate was added to the perifusion medium concurrently with elevation of medium D-glucose, islets from scorbutic guinea pigs released insulin as rapidly as control guinea pig islets and to a somewhat greater extent. L-Ascorbic acid 2-phosphate without elevated D-glucose had no effect on insulin release by islets from normal or scorbutic guinea pigs. The pancreas from scorbutic guinea pigs contained 2.4 times more insulin than that from control guinea pigs, suggesting that the decreased insulin release from the scorbutic islets was not due to decreased insulin synthesis but due to abnormal insulin secretion.

Topics: Animals; Ascorbic Acid; Ascorbic Acid Deficiency; Biological Transport; Glucose; Glutathione; Glutathione Disulfide; Guinea Pigs; In Vitro Techniques; Insulin; Insulin Secretion; Islets of Langerhans; Male; Perfusion

Antiviral activity of L-ascorbic acid-2-phosphate (ASC-2P), a long-acting derivative of L-ascorbic acid, against several human cytomegalovirus (CMV) strains was examined in cultures of human foreskin fibroblasts (HFF) and endothelial cells (EC). ASC-2P at concentrations ranging from 0.2 to 2 mM had no effect on the number of cells expressing 72 kDa CMV immediate early antigen (IEA) while it inhibited expression of 68 kDa late antigen (LA) in infected cultures of both cell types (30% and 55% reduction for EC and HFF, respectively). In HFF cells, virus yield was reduced up to 4-fold, when ASC-2P was added after CMV infection. Antiviral effects were significantly increased in cultures pretreated with ASC-2P. In HFF and EC pretreated for three subcultures (18 days) with 0.2 mM ASC-2P, a significant reduction of cells expressing IEA (75% and 80% reduction in EC and HFF, respectively) and LA (92% and 90% reduction for EC and HFF, respectively) was observed. Pretreatment for three subcultures with ASC-2P inhibited virus yield 50- to 100-fold in EC and 100- to 1000-fold in HFF. The continuous presence of ASC-2P was not required for its antiviral activity. A significantly higher reduction of virus replication with ganciclovir and foscarnet was obtained in ASC-2P pretreated cells than in untreated controls. The results showed that ASC-2P provides L-ascorbic acid with long-lasting antiviral activity against CMV. ASC-2P may be of benefit for the adjunctive treatment of CMV infection.

Topics: Antigens, Viral; Antiviral Agents; Ascorbic Acid; Cells, Cultured; Cytomegalovirus; Endothelium; Fibroblasts; Foscarnet; Ganciclovir; Humans; Virus Replication

Human or mouse epidermal keratinocytes NHEK or Pam212 was less susceptible to ultraviolet (UV)-B irradiation than mouse neuroblastoma NAs1 cells in culture, undergoing apoptosis-like cell death as shown by cell fragmentation and cell membrane integrity disruption. UV susceptibility was appreciably reduced by the reactive oxygen species (ROS)-scavenger L-ascorbic acid-2-phosphate (Asc2P) endowed with long-lasting functions but not by L-ascorbic acid (Asc) for each cell type. DehydroAsc reduced UV susceptibility of Pam212 or NAs1 established cell lines but not of normal diploid NHEK cells destined to be thereafter submitted to cellular senescence. The susceptibility reduction may not be ascribed to extracellular Asc2P or DehAsc, which was removed by aspirating and/or rinsing upon irradiation after the intracellular channelyzer analysis and dead cell-specific DNA-intercalator ethidium homodimer/fluorometry, respectively. Thus, the three cell types differed in UV susceptibility partly because of their different ROS-scavenging abilities, which may be potently promoted by Asc2P or dehydroAsc but not Asc.

Topics: Animals; Ascorbic Acid; Cell Membrane; Dehydroascorbic Acid; Free Radical Scavengers; Humans; Keratinocytes; Mice; Neuroblastoma; Tumor Cells, Cultured; Ultraviolet Rays

In developing chick embryos, hydrocortisone induces cataract formation following a decrease in lens glutathione content but an increase in lipid peroxide content in lens, blood and liver. The preventive effects of ascorbic acid 2-O-alpha-glucoside (AA-2G) on these parameters were compared on cataract formation with those of ascorbic acid (AsA) and ascorbic acid 2-O-phosphate (AA-2P). In these tissues, AA-2G inhibited a decrease in glutathione content and an increase in lipid peroxide content more effectively than either AsA or AA-2P. Various tissues including lens and liver have alpha-glucosidase activity, strongly suggesting that AsA is enzymatically liberated from AA-2G in these tissues. In summary, these results suggest that AA-2G exerts a potent anti-cataract activity via a reduction in oxidative damage through AsA release.

Topics: alpha-Glucosidases; Animals; Ascorbic Acid; Cataract; Chick Embryo; Glutathione; Hydrocortisone; Lens, Crystalline; Lipid Peroxides; Liver

We performed the quantitative and qualitative analysis on the main disaccharide units of glycosaminoglycans produced by human dermal fibroblasts in the 3-dimensional culture supplemented with L-ascorbic acid 2-phosphate (Asc 2-p) comparing with the monolayer culture system. The addition of Asc 2-p rendered fibroblasts to the organization of the dermis-like 3-dimensional structure in vitro without any pre-treatments with the plastic dish. Main disaccharide units were analyzed using HPLC after 1-phenyl-3-methyl-5-pyrasolone (PMP) labeling. The addition of Asc 2-p significantly increased the total amount of main disaccharide units and, furthermore, the composition revealed it to be more similar to that of the dermis. This 3-dimensional culture may offer a simple and useful system to investigate the glycosaminoglycan metabolism of human dermal fibroblasts in vitro.

Topics: Ascorbic Acid; Cells, Cultured; Chromatography, High Pressure Liquid; Disaccharides; Fibroblasts; Glycosaminoglycans; Histological Techniques; Humans; Skin

L-Ascorbic acid 2-phosphate (Asc-P: a stable ascorbic acid derivative) markedly stimulated synthesis of marker proteins for osteoblastic differentiation such as alkaline phosphatase and osteocalcin in a murine osteoblastic cell line, MC3T3-E1, suggesting that Asc-P could promote osteoblastic differentiation. L-Azetidine 2-carboxylate (AzC) diminished the stimulatory effects of Asc-P on the synthesis of alkaline phosphatase and osteocalcin due to its inhibitory effects on mature collagen secretion. Growing cells on the dishes coated with type I collagen resulted in an increased expression of osteoblastic phenotypes even in the presence of AzC. Coating with fibronectin, however, failed to promote differentiation. These results suggest that the promotion of cell differentiation caused by Asc-P is mediated by the accumulation of mature collagen.

Topics: Alkaline Phosphatase; Animals; Ascorbic Acid; Azetidinecarboxylic Acid; Cell Differentiation; Cell Division; Cell Line; Collagen; Mice; Osteoblasts; Osteocalcin; RNA, Messenger

Two experiments were conducted, one with weanling pigs (n = 288) and the second with grower-finisher swine (n = 216), to evaluate the efficacy of dietary vitamin C on various performance and serum measurements. Magnesium-L-ascorbyl-2-phosphate (46% L-ascorbic acid) served as the vitamin C source and was incorporated at dietary levels of 0, 50, or 500 ppm in both experiments. Pigs were allotted by sex, weight, and litter to randomized complete block designs. The nursery trial was conducted at four time periods and contained 12 replicates, whereas the grower-finisher experiment was over four time periods and contained nine replicates. Blood samples were collected initially from nine randomly selected pigs in both experiments, and from each pig within each pen at 2 and 5 wk postweaning, and at the 4- and 8-wk period in the grower-finisher trial. A killed Salmonella typhinurium bacterin was injected i.m. into starter pigs at 2 wk postweaning and at wk 4 and 6 in grower-finisher pigs. Hemagglutination titers were evaluated at 5 wk with the nursery pigs and at the 8-wk period with the grower-finisher swine. At the end of the grower-finisher trial, liver and kidney tissue were analyzed for ascorbate. Starter pigs grew faster (P < .05) and had improved gain:feed ratios (P < .05) when vitamin C was provided during the first 2 wk postweaning, but not during the latter 3-wk period. There was no improvement in pig gain or feed efficiency to vitamin C supplementation during any phase of the grower-finisher period.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Animal Feed; Animals; Antibodies, Bacterial; Ascorbic Acid; Dehydroascorbic Acid; Drug Stability; Eating; Female; Glutathione Peroxidase; Male; Random Allocation; Salmonella typhimurium; Swine; Weaning; Weight Gain

Duplicate lots of 150 Atlantic salmon (Salmo salar), average weight 0.5 g, were fed NRC diet H-440 base containing L-ascorbic acid (C1) or L-ascorbyl-2-sulfate (C2S); or L-ascorbyl-2-monophosphate (C2MP): at 0 or 100 mg C1; 50, 100, 300 mg C2S; or 50, 100 mg C2MP per kg dry diet in 12 degrees C freshwater tanks. After 12 weeks, negative controls (no vitamin C) exhibited reduced growth, scoliosis, lordosis, and petechial hemorrhages typical of fish scurvy. All other lots grew normally. Four 100-fish lots of scorbutic salmon, average weight 3.3 g, were placed on recovery diets of 0, 50, or 300 mg C2S, or 100 mg C2MP per kg dry diet. After 5 weeks, fish fed either level of C2S intake had recovered and resumed growth. Negative controls continued to develop acute scurvy. The 41 survivors in this no-vitamin-C group all had advanced scurvy, whereas all fish in both C2S-fed recovery groups appeared normal. Tissue assays for C vitamers disclosed normal levels of C1 and C2S in the recovery groups. All other test treatment lots containing C1, C2S, or C2MP had fish with normal appearance and no significant differences in growth response for the 17-week test period. C2S at 50 mg or more per kg diet as the sole vitamin C source promoted normal growth in young Atlantic salmon for more than 20-fold increase in weight.

Topics: Animals; Ascorbic Acid; Body Weight; Fish Diseases; Liver; Salmon; Scurvy

Hormones and growth factors are important regulators of myogenic cell differentiation, but little is known about the effect of vitamins on muscle differentiation and development. We recently showed that L-ascorbic acid 2-phosphate, a stable form of vitamin C, increased the expression of muscle-specific glucose and ion transporters. We now show the effect of L-ascorbic acid 2-phosphate on the kinetics of myogenin expression at both the mRNA and protein levels during differentiation of L6 muscle cells. At the fully differentiated stage, control and L-ascorbic acid 2-phosphate treated cultures showed the same degree of cell fusion, but L-ascorbic acid 2-phosphate treated myotubes had a larger diameter than control myotubes. During L6 cell differentiation, the amount of both myogenin mRNA and protein reached a maximal level on day 4 before full myotube formation and then declined. L-ascorbic acid 2-phosphate treated cells expressed a higher amount of myogenin at both the mRNA and protein levels on day 4 compared to untreated cultures. Ethyl-3,4-dihydroxybenzoate, an inhibitor of collagen synthesis, prevented expression of myogenin mRNA and protein in both the control and L-ascorbic acid 2-phosphate treated cells. These results demonstrate that vitamin C can promote muscle differentiation likely through the increase of myogenin expression in myogenic cells, which may in turn regulate muscle differentiation in vivo.

Topics: Animals; Ascorbic Acid; Cell Differentiation; Cells, Cultured; Collagen; Gene Expression; In Vitro Techniques; Muscles; Myogenin; Rats; RNA, Messenger

The effects of supplemental ascorbyl-2-polyphosphate (APP) on adrenocortical function and underlying fearfulness in broiler chickens were assessed in a number of test situations. Chicks pretreated for a minimum of 24 h with APP (1,000 ppm equivalents of L-ascorbic acid) in their drinking water or with no APP (tap water controls; CON) had blood samples taken immediately following water treatment and again after exposure to a capture and cooping stressor for 10 min. First, although the cooping stressor markedly increased plasma corticosterone concentrations, pretreatment with APP failed to attenuate this adrenocortical response. Second, APP-treated chicks showed less freezing and vocalized sooner in an open field (novel environment) than did controls. They also showed nonsignificant tendencies toward accelerated and enhanced ambulation. Third, supplementation with APP reduced the duration of the birds' tonic immobility fear reactions. Collectively, these behavioral effects are indicative of dampened fear. The apparent reduction of nonspecific, underlying fearfulness by APP treatment may have important implications for poultry welfare and performance.

Topics: Animals; Ascorbic Acid; Chickens; Corticosterone; Fear; Random Allocation

Morphology, proliferation, and collagen synthesis of perisinusoidal stellate cells (fat-storing cells, lipocytes, Ito cells) varied according to the nature of the substratum. On a basement membrane gel, the stellate cells formed a mesh-like structure and proliferated slowly. On non-coated polystyrene and type I collagen-coated culture dishes, the cells spread well and extended cellular processes. The cell proliferation and collagen synthesis were more prominent on culture dishes coated with type I collagen than on polystyrene dishes. On each extracellular substrate, proliferation and collagen synthesis were stimulated by a long-acting vitamin C derivative, L-ascorbic acid 2-phosphate. These results indicate that morphology, proliferation, and collagen metabolism of the stellate cells are regulated by extracellular matrix and modulated further by L-ascorbic acid 2-phosphate.

Topics: Animals; Ascorbic Acid; Basement Membrane; Cell Division; Cell Size; Collagen; DNA; Extracellular Matrix; In Vitro Techniques; Liver; Male; Polystyrenes; Rats; Rats, Wistar; Retinoids

Broiler chick diets and drinking water were supplemented with two sources of vitamin C: crystalline L-ascorbic acid (AsA) or L-ascorbyl-2-polyphosphate (APP) to provide 0, 25, 50, 100, 200, 400, 800, 1,600, and 3,200 ppm (mg/kg) AsA. The bioavailability of APP relative to AsA, as estimated by the change in plasma AsA concentration, was evaluated during 24-h periods of supplementation. When provided in the feed, no differences in dietary AsA content were attributed to vitamin source. In contrast, APP administration at 25 and 50 ppm, resulted in higher (P < .001) AsA values in drinking water when compared with AsA supplementation. Plasma AsA values were elevated (P < .05) above baseline when either AsA or APP were supplemented in the feed or water at a level of 400 ppm or greater. Plasma AsA concentrations, following supplementation of the diets, were higher (P < .05) in AsA-treated (800 ppm) chicks when compared with APP-supplemented chicks. During water supplementation, AsA (800 ppm) and APP (3,200 ppm) administration resulted in higher plasma AsA values when compared with their alternate vitamin source. At all other levels of water supplementation, no differences in plasma AsA were associated with vitamin source. The absence of a consistent difference in plasma AsA, relative to vitamin source, suggests that the isolated differences observed may be due to chance. It was concluded that APP was of similar bioavailability to that of AsA, as estimated by the ability to elevate plasma AsA concentrations in broiler chicks.

Topics: Animals; Ascorbic Acid; Biological Availability; Chickens; Drinking; Eating; Feeding Behavior; Female; Food, Fortified

Confluent bovine aortic endothelial cells (BAECs) formed a cobblestone-shaped cell monolayer when cultured on a collagen gel in a 1:1 mixture of Dulbecco's modified Eagle's medium and Ham's-F12 supplemented with 5% newborn calf serum. Within a few days, however, they lost cell-cell attachment and became fibroblastic. When BAECs were cultured in the same culture medium but further supplemented with either 10 micrograms/ml malotilate or 1 mM phosphoascorbic acid, the monolayer organization and the cobblestone-like cell morphology were maintained for more than 2 weeks although many sprout cells were observed underneath the monolayer. In contrast, if both malotilate and phosphoascorbic acid were present in the culture medium, a tight monolayer without underlying sprout cells was maintained for at least 4 months and the cells expressed factor VIII-related antigens and massively internalized acetylated low density lipoprotein. By electron microscopy, we observed well-developed gap and adherence junctions, Golgi apparatuses and vesicles many of which were open to the outside by fusing with either the apical or the basal surface, indicating high metabolic activity of the cells cultured for weeks in the same dish. Although malotilate-treated BAEC monolayers secreted increased levels of prostacyclin (PGI2), the drug did not appear to directly affect the PGI2 production pathway since the similarly increased PGI2 production was noted in tight monolayers formed without the use of malotilate. Our results indicate that malotilate and phosphoascorbic acid together preserve differentiated phenotypes in cultured endothelial cells.

Topics: Animals; Aorta; Ascorbic Acid; Cattle; Culture Media; Endothelium, Vascular; Factor VIII; Lipoproteins, LDL; Malonates; Phenotype

The effect of ascorbic acid 2-O-alpha-glucoside (AA-2G) on hydrocortisone (HC)-induced lens opacity in developing chick embryo was examined and compared with those of ascorbic acid (AsA) and ascorbic acid 2-phosphate (AA-2P). The opacity was dose-dependently inhibited by a single administration of 10 or 20 mumol/egg of AA-2G and by three repeated administrations of 1, 3 or 10 mumol/egg of AA-2G. AA-2G was the most effective among the three compounds. Glucose did not enhance the preventive effect of AsA against HC-induced opacity, and neither dehydro ascorbic acid nor glucose also prevented HC-induced cataract. In the histological study, we observed many small vacuoles in the nuclear region of the opaque lens treated with HC. AA-2G inhibited the formation of such vacuoles, an effect closely correlated with the prevention of cataract formation.

Topics: Animals; Ascorbic Acid; Cataract; Chick Embryo; Dose-Response Relationship, Drug; Hydrocortisone; Lens, Crystalline; Vacuoles

L-Ascorbic acid 2-phosphate (Asc 2-P), a long-acting vitamin C derivative, stimulated transcription of genes for pro alpha 1(I) and pro alpha 2(I) collagen in normal human skin fibroblasts after 8 h of treatment in the absence or in the presence of cycloheximide, indicating Asc 2-P stimulates transcription of type I collagen genes in the absence of protein synthesis. The transcriptional rate in these cells reached the maximum value after 40 h of treatment, and at that time it was three to four times higher than that of the control cells cultured in the absence of Asc 2-P. Steady-state levels of mRNAs for pro alpha 1(I) and pro alpha 2(I) chains were also increased to be three to four times higher than the control levels by treatment of the cells with Asc 2-P for 72 h. When the fibroblasts obtained from a patient with Ehlers-Danlos syndrome were treated with Asc 2-P, the derivative also stimulated transcription of the gene for pro alpha 1(I) chain and accumulation of mRNA for pro alpha 1(I) chain. On the other hand, Asc 2-P failed to stimulate transcription of the pro alpha 2(I) gene or an increase in mRNA for pro alpha 2(I) chain. Sodium ascorbate showed effects quite similar to those of Asc 2-P, when fibroblasts obtained from a normal control or the patient were cultured for 16 h with it. These results indicate the existence of cis-regulatory elements responsible for transcriptional activation by Asc 2-P or ascorbic acid in pro alpha 1(I) and pro alpha 2(I) genes of normal fibroblasts. These data also suggest some defect(s) of these elements in the pro alpha 2(I) gene of the patient with Ehlers-Danlos syndrome.

Topics: Ascorbic Acid; Blotting, Southern; Cell Division; Cells, Cultured; Collagen; Cycloheximide; DNA; Ehlers-Danlos Syndrome; Fibroblasts; Humans; RNA, Messenger; Skin; Time Factors; Transcription, Genetic

We examined the effect of L-ascorbic acid 2-phosphate (P-Asc) on the healing of alkali-burned corneas in rabbits. Round filter paper containing 1 N NaOH was applied to the central cornea for 60 or 120 s to produce the alkali burn. Animals were treated with topical saline, 10% ascorbate, or 6.5% P-Asc applied on the cornea. The corneas were then examined histologically. Burned stroma showed no toluidine blue staining, indicating a loss of glycosaminoglycan. In the 60-s burn group, P-Asc reduced the size of the unstained area as compared with the control. Transmission electron microscopy showed basal lamina under new epithelia in the corneas treated with ascorbate or P-Asc, but not in controls. These observations support the theory that P-Asc may have a therapeutic role in the repair of corneal alkali burns.

Topics: Administration, Topical; Animals; Ascorbic Acid; Basement Membrane; Burns, Chemical; Cornea; Corneal Injuries; Corneal Stroma; Eye Burns; Female; Male; Ophthalmic Solutions; Rabbits; Sodium Hydroxide; Wound Healing

Ascorbic acid has been shown to stimulate collagen synthesis in dermal fibroblasts by increasing the rate of transcription of collagen genes. Experiments involving the use of ascorbic acid require daily supplementation due to the instability of the molecule in aqueous solutions. In order to provide a more stable alternative to ascorbic acid, two salts of ascorbyl-2-phosphate, having a greater chemical stability than ascorbic acid, were tested for their ability to stimulate collagen synthesis in monolayer fibroblast cultures. The concentration and time dependence of their activities were compared with ascorbic acid. The magnesium salt of ascorbyl-2-phosphate was found to be equivalent to ascorbic acid in stimulating collagen synthesis in these assays, while the sodium salt required at least a tenfold greater concentration to produce the same effect as ascorbic acid. Solutions of either ascorbic acid or the ascorbyl-2-phosphate analogs (at 10 mM) in phosphate-buffered saline (PBS) were relatively stable as shown by their decay rates and their ability to stimulate collagen synthesis even after nine days in solution prior to testing their effects on cultured cells. Ascorbic acid was unstable at neutral pH compared to solutions of either sodium or magnesium ascorbyl-2-phosphate. These data support the use of magnesium ascorbyl-2-phosphate in experiments where stability of ascorbic acid is a concern, e.g. in long-term cultures or in in vivo studies.

Topics: Ascorbic Acid; Cells, Cultured; Chemistry, Pharmaceutical; Collagen; Female; Fibroblasts; Humans; Infant, Newborn; Kinetics; Magnesium; Pregnancy; Skin; Sodium; Stimulation, Chemical

The effect of ascorbate and L-ascorbic acid 2-phosphate (P-Asc), a stable derivative of ascorbate, on the growth of cultured rabbit keratocytes was examined. In a 12-day culture, P-Asc (0.05-0.1 mM) and ascorbate (0.05 mM) enhanced the growth of the cells, whereas 0.1-1.0 mM ascorbate was cytotoxic to the cells. An increase in the concentration of P-Asc up to 1.0 mM resulted in no further change in the cell growth. The effect of 0.1 mM P-Asc was observed after the confluency had been achieved in the culture. The lower toxicity of P-Asc may be due to its stability. The presence of both 0.1 mM P-Asc and mouse epidermal growth factor in the medium stimulated cell growth to a higher degree than each factor alone. The results suggested that these two factors enhance the growth of keratocytes in a different manner.

Topics: Animals; Ascorbic Acid; Cell Count; Cell Division; Cell Survival; Cells, Cultured; Connective Tissue; Cornea; DNA; DNA Replication; Epidermal Growth Factor; Male; Rabbits

Topics: Adipose Tissue; Animals; Ascorbic Acid; Basement Membrane; Capillaries; Cell Differentiation; Collagen; Endothelium, Vascular

The activity of alkaline phosphatase (ALPase) was significantly enhanced in a human osteoblast cell line, HuO-3N1, when it was cultured in the presence of L-ascorbic acid 2-phosphate (AsA-P; a stable ascorbic acid derivative). With AsA-P in the culture, the level of ALPase activity increased approximately 3-fold without any effect on either the morphology or growth rate. This increase was dependent on the AsA-P concentration in the range of 0.2-2 mM and required at least 48 h incubation with AsA-P. The ALPase mRNA level, however, remained rather constant irrespective of the enzyme activity. Removal of AsA-P from the precultured medium decreased the stimulatory effect of ascorbic acid on the ALPase activity, indicating that the effect was reversible. Dexamethasone, an inducer for osteoblastic differentiation, enhanced the level of ALPase activity irreversibly, in parallel with the increase in the level of its mRNA. The enhancement of the ALPase activity by ascorbic acid in this cell line appeared to be independent of cell differentiation.

Topics: Alkaline Phosphatase; Ascorbic Acid; Blotting, Northern; Bone and Bones; Cell Division; Cell Line; Dexamethasone; Humans; Isoenzymes; Osteoblasts; RNA, Messenger

I studied the effect of L-ascorbic acid 2-phosphate (P-Asc), a long-acting derivative of L-ascorbic acid, on the fine structures of cultured rabbit keratocytes. The results showed that cells cultured with 0.1 mM P-Asc for 30 days were more markedly multilayered than those grown without P-Asc. Dilation of the cisternae of the endoplasmic reticulum in the control cells indicated an accumulation of protein (probably procollagen). In addition, many lysosome-like structures, which may degrade underhydroxylated procollagen, were observed in the cytoplasm of the control cells. A slight increment of free ribosomes was also found within the control cells. P-Asc enhanced the multilayerization of cultured keratocytes. We conclude that the changes seen in the cytoplasm are due to the effect of P-Asc acting as vitamin C.

Topics: Animals; Ascorbic Acid; Cells, Cultured; Connective Tissue; Corneal Stroma; Extracellular Matrix; Male; Organelles; Rabbits

We studied the effect of L-ascorbic acid 2-phosphate (P-Asc), a long-acting phosphate derivative of L-ascorbic acid, on the production and secretion of type I and type III collagen peptides in cultured rabbit keratocytes using immunohistochemistry and enzyme immunoassay. P-Asc enhanced production and secretion of these collagen peptides. Our observations support a therapeutic role for P-Asc in the repair of corneal stromal damage such as caused by corneal chemical burn.

Topics: Animals; Antibodies, Monoclonal; Ascorbic Acid; Binding, Competitive; Cells, Cultured; Collagen; Cornea; Enzyme-Linked Immunosorbent Assay; Immunohistochemistry; Mice; Mice, Inbred BALB C; Procollagen; Rabbits

1. Ascorbyl-2-monophosphate was enzymatically formed in the reaction mixture of L-ascorbic acid, pyrophosphate and the homogenate of rainbow trout Oncorhynchus mykiss liver. 2. The liver had the highest activity among the liver, spleen, kidney, stomach, pyloric caeca and intestine. 3. Pyrophosphate, triphosphate, ADP and ATP were good substrates as phosphoryl donors, but phosphoric acid and AMP were poor. 4. The optimum pH and temperature of AP-forming activity in the liver were around 5.0 and 30 degrees C, respectively. 5. The Km values for ascorbic acid and pyrophosphate were 370 and 83 mM, respectively.

Topics: Animals; Ascorbic Acid; Chromatography, High Pressure Liquid; Chromatography, Thin Layer; Hydrogen-Ion Concentration; Kinetics; Liver; Magnetic Resonance Spectroscopy; Mass Spectrometry; Phosphoric Monoester Hydrolases; Salmon; Spectrophotometry, Ultraviolet; Temperature

Channel catfish (Ictalurus punctatus) fingerlings (13 g average initial weight) were fed semipurified diets supplemented with 0, 0.06, 0.12, 0.24 and 0.72 mmol/kg (0, 11, 22, 44 or 132 mg/kg) of ascorbic acid molar equivalent supplied by either L-ascorbic acid, L-ascorbyl-2-monophosphate (Mg salt) (AAP), or L-ascorbyl-2-sulfate (K salt) (AAS). After 14 wk, weight gains were equal for all fish fed diets containing L-ascorbic acid or AAP; however, growth rates were less for fish fed AAS at all dietary levels and for fish fed the ascorbic acid-free diet (control). There were no gross signs of vitamin C deficiency in any of the fish fed L-ascorbic acid or AAP, whereas spinal deformities were found in the controls and in fish fed all but the highest concentration of AAS. The percentage of spinal deformities decreased as dietary levels of AAS increased. Reduced bone collagen content and histopathology in liver and gill tissues also indicated ascorbic acid deficiency in the controls and in fish fed all but the highest concentration of AAS. Limited histopathology was found in fish fed the lowest level of L-ascorbic acid but not in those fed the lowest level of AAP. Regression analysis of weight gain data showed that the vitamin activity of ascorbic acid from AAS was only 5.2% of that from L-ascorbic acid for growth. This study indicates that AAP has equimolar activity to L-ascorbic acid as a vitamin C source for channel catfish and that AAS has vitamin activity for this species but at a much lower level than the other compounds.

Topics: Administration, Oral; Animals; Anticholesteremic Agents; Ascorbic Acid; Ascorbic Acid Deficiency; Ictaluridae; Liver

We examined the effect of L-ascorbic acid 2-phosphate (P-Asc), a long-acting phosphate derivative of L-ascorbic acid, on intracellular distribution and production of type I collagen in cultured rabbit keratocytes by an immunohistochemistry and enzyme immunoassay. Exposure of 0.1 mM P-Asc for 10 hours decreased a type I collagen immunoreactivity of the cytoplasm as stained in fine granular materials. 0.1 mM P-Asc induced increase of type I collagen level in the medium. The results suggested that 0.1 mM P-Asc might increase the biosynthesis and secretion of type I collagen in keratocytes and have a therapeutic effect on corneal stromal damage.

Topics: Animals; Ascorbic Acid; Cells, Cultured; Collagen; Cornea; Enzyme-Linked Immunosorbent Assay; Immunohistochemistry; Rabbits

We examined the effect of L-ascorbic acid 2-phosphate (P-Asc) on the proliferation of cultured rabbit keratocytes. P-Asc is a phosphate derivative of L-ascorbic acid and has more prolonged vitamin C activity in solution than does L-ascorbic acid. The proliferation of cultured keratocytes was promoted by the presence of P-Asc in culture medium. Transmission electron microscopic observations revealed that cells were more multi-layered after culture in the presence of P-Asc (0.1 mM) for 30 days than were those cultured in the absence of P-Asc. The effect of P-Asc was abrogated by L-azetidine 2-carboxylic acid, which is an analogue of proline that inhibits the production and secretion of collagen. Our observations support a therapeutic role for P-Asc in the repair of corneal stromal damage such as that caused by a corneal chemical burn.

Topics: Animals; Ascorbic Acid; Azetidinecarboxylic Acid; Cell Division; Cells, Cultured; Corneal Stroma; Female; Keratinocytes; Male; Rabbits

Recombinant human epidermal growth factor (EGF, 2-10 ng/ml) stimulated growth and production of non-collagenous proteins, but inhibited production of collagen by 60% in cultured human skin fibroblasts. Type analysis of the collagen produced indicated that inhibition of the collagen production observed was mainly a reflection of a reduction in type I collagen. The accumulation of pro alpha 1(I) and pro alpha 2(I) mRNAs and the transcriptional activity of these genes were determined in human skin fibroblasts in order to investigate site(s) of regulation of type I collagen production by human EGF in the absence and presence of L-ascorbic acid 2-phosphate (Asc 2-P), a long-acting vitamin C derivative. Human EGF (10 ng/ml) used alone reduced the steady state levels of mRNAs for pro alpha 1(I) and pro alpha 2(I) chains and transcriptional activity of these genes in vitro by 45%. Asc 2-P (0.2 mM) alone, on the other hand, raised production of type I collagen and the steady state levels of mRNAs for pro alpha 1(I) and pro alpha 2(I) collagen chains as well as stimulated transcriptional activity of these genes. Human EGF attenuated these stimulative effects of Asc 2-P. These results indicate that human EGF regulates type I collagen synthesis at the transcriptional level in cultured fibroblasts in the presence and absence of Asc 2-P. The possibility that human EGF plays a role as a regulator of type I collagen genes in vivo was discussed.

Topics: Ascorbic Acid; Cells, Cultured; Collagen; DNA; Electrophoresis, Polyacrylamide Gel; Epidermal Growth Factor; Fibroblasts; Humans; Nucleic Acid Hybridization; RNA, Messenger; Skin; Transcription, Genetic

We examined the effect of L-ascorbic acid 2-phosphate (P-Asc) on the proliferation of cultured rabbit keratocytes. P-Asc is a derivative of L-ascorbic acid and it yields more prolonged effects of vitamin C in solution than L-ascorbic acid. The proliferation of cultured rabbit keratocytes was promoted by the presence of P-Asc in culture medium for 10, 20, and 30 days. Transmission electron microscopic observations revealed that the cells were more multilayered in the presence of P-Asc (0.1mM) for thirty days than those in the absence of P-Asc. Moreover, this effect of P-Asc was attenuated by azetidine 2-carboxylic acid which is an inhibitor of collagen synthesis. Hence, it is suggested that the promotive effect of P-Asc on the growth of cultured keratocytes is related to the synthesis of collagen. Based on our observations, P-Asc may have a therapeutic effect on corneal stromal damages such as a corneal chemical burn and surgical trauma.

Topics: Animals; Ascorbic Acid; Cell Division; Cells, Cultured; Corneal Stroma; Rabbits; Stimulation, Chemical

Ascorbic acid (AsA) plays an important role in the formation of collagen in the periodontal ligaments. However, the biochemical role of AsA on the cell metabolism of periodontal ligaments remains undeveloped. This study attempts to explore the effect of AsA and L-Ascorbic acid-2-phosphate (AsA-P) on the cell proliferation and differentiation, and the activity of cell attachment and spreading of conditioned medium (CM) prepared from human fibroblasts derived from the periodontal ligaments of deciduous teeth (HPLF-Y) and permanent teeth (HPLF) according to the explanation methods described by Saito et al. The effect of AsA and AsA-P on DNA synthesis was observed as 15 to 20 percent enhancement in comparison with that of the control for both HPLF-Y and HPLF with exception of AsA-P for HPLF at the 7 day of cell culture. The ALPase activity of HPLF-Y and HPLF at confluent phase was stimulated significantly by the presence of AsA and AsA-P. By morphological observation, the cell spreading activity of the CM of HPLF-Y exposed with AsA-P was higher than that of exposed with AsA. An induction of the cell attachment factors from HPLF-Y is enhanced intensively by the treatment of AsA-P. Therefore, AsA-P may regulate intensively the proliferation and differentiation of HPLF-Y in terms of DNA synthesis and ALPase activity including the enhanced secretion of attachment and spreading factors.

Topics: Alkaline Phosphatase; Ascorbic Acid; Cell Adhesion; Cell Differentiation; Collagen; DNA; Fibroblasts; Humans; Periodontal Ligament; Tooth, Deciduous

We showed that the synthesis and secretion of type IV collagen, entactin, and laminin were enhanced when adipose conversion of 3T3-L1 cells at confluence was stimulated by hormones (Y. Aratani and Y. Kitagawa (1988) J. Biol. Chem. 263, 16163-16169). Ascorbic acid phosphate (Asc-P) stimulated the synthesis and secretion of type IV collagen and other collagens from both 3T3-L1 preadipocytes and adipocytes. The synthesis and secretion of laminin and entactin were not affected by Asc-P. The continuous addition of Asc-P stimulated cell growth and increased cell density at confluence 1.3-fold. Concomitantly, Asc-P remarkably accelerated the emergence of lipoprotein lipase, glycerophosphate dehydrogenase, and Oil Red O-stainable lipid droplets. These findings suggest an important role for type IV collagen in adipocyte differentiation.

Topics: Adipose Tissue; Animals; Ascorbic Acid; Cell Differentiation; Cell Division; Cells, Cultured; Collagen; Fibronectins; Kinetics; Laminin; Methionine; Mice

Proliferation of human skin fibroblasts was stimulated significantly by the presence of L-ascorbic acid 2-phosphate (Asc 2-P). The presence of Asc 2-P (0.1-1.0 mM) in the culture medium for 3 weeks enhanced the relative rate of collagen synthesis to total protein synthesis 2-fold as well as cell growth 4-fold. Coexistence of L-azetidine 2-carboxylic acid (AzC), an inhibitor of collagen synthesis, attenuated both effects of Asc 2-P in a dose-dependent manner. Supplementation of the medium with Asc 2-P also accelerated procollagen processing to collagen and deposition of collagen in the cell layer. Among the acidic glycosaminoglycans (GAG), another major component of extracellular matrix (ECM), deposition of sulfated forms was increased by the additive. Electron microscopic observations showed multilayered, rough endoplasmic reticulum-rich cells surrounded by dense ECM. These results indicate that Asc 2-P is useful in culture systems as a long-acting vitamin C derivative and also that it promotes reorganization of a three-dimensional tissuelike substance from skin fibroblasts in culture by stimulating collagen accumulation in the fibroblasts.

Topics: Ascorbic Acid; Cell Division; Cells, Cultured; Collagen; Dose-Response Relationship, Drug; Extracellular Matrix; Fibroblasts; Glycosaminoglycans; Humans; Microscopy, Electron; Skin

Topics: Adjuvants, Pharmaceutic; Ascorbic Acid; Blood Preservation; Diphosphoglyceric Acids; Erythrocytes; Oxalates

A high-performance liquid chromatographic procedure was developed to assay for adenine, hypoxanthine, and ascorbate-2-phosphate in both red blood cells and plasma. The samples were diluted and filtered through Centriflo filter cones to remove most of the blood proteins before injection onto the column. The application of the technique to an experimental blood preservation study is illustrated.

Topics: Adenine; Ascorbic Acid; Chromatography, High Pressure Liquid; Erythrocytes; Humans; Hypoxanthines; Plasma; Spectrophotometry, Ultraviolet

Topics: Ascorbic Acid; Drug Stability; Food, Fortified

Topics: Adult; Animals; Ascorbic Acid; Food, Fortified; Guinea Pigs; Humans; Male

Topics: Animals; Arylsulfatases; Ascorbic Acid; Binding Sites; Binding, Competitive; Cattle; Kinetics; Liver; Organophosphorus Compounds; Phosphates; Protein Binding; Sulfatases