hydroxylysine and galactosylhydroxylysine

Hydroxylysine and its glycosylated forms, galactosylhydroxylysine and glucosylgalactosylhydroxylysine, are post-translational modifications unique to collagenous sequences. They are found in collagens and in many proteins having a collagenous domain in their structure. Since the last published reviews, significant new data have accumulated regarding these modifications. One of the lysyl hydroxylase isoforms, lysyl hydroxylase 3 (LH3), has been shown to possess three catalytic activities required sequentially to produce hydroxylysine and its glycosylated forms, that is, the lysyl hydroxylase (LH), galactosyltransferase (GT), and glucosyltransferase (GGT) activities. Studies on mouse models have revealed the importance of these different activities of LH3 in vivo. LH3 is the main molecule responsible for GGT activity in mouse embryos. A lack of this activity causes intracellular accumulation of type IV collagen, which disrupts the formation of basement membranes (BMs) during mouse embryogenesis and leads to embryonic lethality. The specific inactivation of the LH activity of LH3 causes minor alterations in the structure of the BM and collagen fibril organization, but does not affect the lifespan of mutated mice. Recent data from zebrafish demonstrate that growth cone migration depends critically on the LH3 glycosyltransferase domain. LH3 is located in the ER loosely associated with the membranes, but, unlike the other isoforms, LH3 is also found in the extracellular space in some tissues. LH3 is able to adjust the amount of hydroxylysine and hydroxylysine-linked carbohydrates of extracellular proteins in their native conformation, suggesting that it may have a role in matrix remodeling.

Topics: Amino Acid Sequence; Animals; Basement Membrane; Catalytic Domain; Collagen; Embryonic Development; Endoplasmic Reticulum; Extracellular Space; Galactosyltransferases; Glucosyltransferases; Glycosylation; Humans; Hydroxylysine; Mice; Molecular Sequence Data; Procollagen-Lysine, 2-Oxoglutarate 5-Dioxygenase; Protein Processing, Post-Translational; Sequence Homology, Amino Acid; Substrate Specificity

Bone remodelling is a process by which bone grows and turns over. This process involves a series of highly regulated steps that depend on the interaction of two cell lineages, the osteoclasts and the osteoblasts. Information on metabolic activity of bone tissue are achieved with the determination, in blood and in urine, of biochemical products derived from the activity of this cells. The ability to determine bone turnover with biochemical markers has been enhanced considerably in recent years with the development of new assays for more sensitive and specific markers. These new markers can now replace the outdated and non-specific markers of bone remodeling such as serum total alkaline phosphatase (ALP) and urinary hydroxyproline (Hyp) determination. Biochemical markers of bone turnover can be classified according to the process that underlie in markers of bone formation, products of the osteoblast activity [bone ALP, osteocalcin (OC), procollagene I C- and N-terminal propeptides] and markers of bone resorption, products of the osteocalst activity [pyridinuim crosslinks, collagen I C- and N-terminal telopeptides (CTX-I and NTX-I), tartrate resistent acid phosphatase (TRACP) isoform 5b]. The interpretation of laboratory results should always include the consideration of potential sources of variability. Variation in the results of biochemical markers of bone metabolism can compromise their ability to characterize disorders of bone metabolism. Variation can be categorized into pre-analytical, analytical and biological sources. However, the determination of biochemical markers of bone turnover offers many advantages in clinical practice, since they are non-invasive, can be repeated often, and major changes occur in a short time.

Topics: Acid Phosphatase; Alkaline Phosphatase; Biomarkers; Bone and Bones; Bone Remodeling; Bone Resorption; Humans; Hydroxylysine; Hydroxyproline; Isoenzymes; Osteoblasts; Osteocalcin; Osteoclasts; Osteogenesis; Procollagen; Tartrate-Resistant Acid Phosphatase

Analytical and biological variability of three bone markers, deoxypyridinoline (DPD), CrossLaps (CTx) and galactosylhydroxylysine (GHYL) were compared. From 14 healthy subjects (six women, eight men; age 29-44 years) recruited from our laboratory staff, two sets of samples of early morning urine were obtained - four samples taken weekly for 4 weeks (all subjects) and three samples taken monthly for 3 months from five subjects. Data were expressed as the ratio to creatinine concentration. All the methods met the analytical goals (CV(A)< or =1/2CV(I(within-subject))) DPD 0.06, CTx 0.05 and GHYL 0.07 with CV(I(within-subject)) being 0.22, 0.19 and 0.38, respectively. The reference values were of limited usefulness particularly for CTx and GHYL, the index of individuality (II) being 0.50 and 0.48 respectively. As the index of heterogeneity (IH) was not significant, being 0.23 for CTx, 0.28 for DPD and 0.46 for GHYL, which are all <1.71 (1+2S.D.), within-subject variances can be used to calculate the reference change value (RCV): 0.58 for DPD, 0.54 for CTx and 1. 08 for GHYL. Moreover, we found constant variations in DPD and CTx, week to week and month to month. Our findings suggest that DPD and CTx provide more reliable results than GHYL, showing a lower within-subject variation, a lower and time-constant RCV allowing reliable monitoring without regard for timing.

Topics: Adult; Algorithms; Amino Acids; Biomarkers; Bone and Bones; Collagen; Collagen Type I; Female; Humans; Hydroxylysine; Immunoassay; Male; Peptides

Serum-based biochemical markers of bone resorption may provide better clinical information than urinary markers because direct comparison with serum markers of bone formation is possible and because the within-subject variability of serum markers may be lower. We describe a method for the measurement of free beta-1-galactosyl-O-hydroxylysine (Gal-Hyl) in serum.. The assay used preliminary ultrafiltration of serum, dansylation, and separation by reversed-phase HPLC with fluorescence detection. Healthy subjects were recruited from population-based studies of bone turnover.. The within-run (n = 15) and between-run (n = 15) CVs were 7% and 14%, respectively, at a mean value of 48 nmol/L. In women and pubertal girls, serum free Gal-Hyl correlated with urine free Gal-Hyl (r = 0.84; P <0.001). Serum Gal-Hyl was higher during puberty and increased after menopause. The fractional renal clearance of free Gal-Hyl relative to that of creatinine was 0.90 (95% confidence interval, 0.82-0.98). Serum free Gal-Hyl decreased by 36% (SE = 4%) in 14 patients with mild Paget disease treated with an oral bisphosphonate, and this decrease was significantly (P <0. 001) greater than that seen for either serum tartrate-resistant acid phosphatase (9%; SE = 4%) or serum C-terminal telopeptide of collagen I (19%; SE = 8%).. Serum free Gal-Hyl may be useful as a serum marker of bone resorption.

Topics: Adolescent; Adult; Age Factors; Aged; Aged, 80 and over; Biomarkers; Bone Resorption; Child; Diphosphonates; Female; Humans; Hydroxylysine; Menopause; Middle Aged; Osteitis Deformans; Puberty

There is increasing awareness that growth hormone (GH) replacement therapy is also essential in adult patients with growth hormone deficiency (GHD). There are little data available on the dose requirements for replacement therapy in this age group. In childhood, the growth response to GH therapy can serve as an indicator for correct replacement dose. Because this indicator does not exist in adults, we analyzed growth factors and biochemical markers of bone metabolism by specific radioimmunoassays in a group (n = 12) of adult patients (age, 20.0-31.6 years) with GHD with childhood onset before and after a 4-week treatment period (daily, s.c.) with recombinant, human GH at different doses (0.125, 0.25 and 0.5 IU/kg body weight/week). Comparing the basal levels to those on low-dose GH (0.125 IU/kg/week) and on a high dose (0.5 IU/kg/week), the following results were obtained. Insulin-like growth factor-I (IGF-I) in serum: basal, 68.6 +/- 37 ng/ml; low dose, 176.9 +/- 65 ng/ml (p < or = 0.05); high dose, 380.6 +/- 200 ng/ml (p < or = 0.01). IGF-binding protein-3 in serum: basal, 2.13 +/- 0.58 mg/l; low dose, 3.23 +/- 0.84 mg/l (p < or = 0.01); high dose, 3.97 +/- 0.82 mg/l. Osteocalcin in serum: basal, 3.88 +/- 1.27 ng/ml; low dose, 7.01 +/- 2.20 ng/ml (p < or = 0.01); no further increase. Procollagen-I peptide in serum: basal, 113.6 +/- 36.7 microgram/l; low dose, 211.6 +/- 90.4 microgram/l (p < or = 0.01); no further increase.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Adult; Alkaline Phosphatase; Bone and Bones; Carrier Proteins; Cross-Over Studies; Dose-Response Relationship, Drug; Edema; Female; Growth Hormone; Humans; Hydroxylysine; Insulin-Like Growth Factor Binding Proteins; Insulin-Like Growth Factor I; Insulin-Like Growth Factor II; Male; Osteocalcin; Procollagen

Urinary galactosyl hydroxylysine/creatinine ratio (GHL) was used to assess rates of bone collagen degradation and the activity of the pagetic lesion as well as for monitoring the rate and degree of suppression of bone resorption over 1 year in patients treated with 30 mg of intravenous pamidronate for 3 consecutive days. The clinical utility of GHL was compared with that of urinary hydroxyproline/creatinine and deoxypyridinoline/creatinine and with bone isoenzyme of serum alkaline phosphatase. The results suggest that GHL is a quantitative marker of the activity of Paget's bone disease. GHL is less sensitive than hydroxyproline, deoxypyridinolone and bone alkaline phosphatase in monitoring treatment of Paget's disease. The assay of GHL is easier, faster and less costly than that of hydroxyproline or deoxypyridinoline and it can be easily standardized.

Topics: Adult; Aged; Aged, 80 and over; Alkaline Phosphatase; Biomarkers; Bone and Bones; Bone Resorption; Chromatography, High Pressure Liquid; Creatinine; Female; Humans; Hydroxylysine; Hydroxyproline; Isoenzymes; Male; Middle Aged; Osteitis Deformans; Spectrometry, Fluorescence

Collagens are the most abundant glycoproteins in the body. One characteristic of this protein family is that the amino acid sequence consists of repeats of three amino acids -(X-Y-Gly)n. Within this motif, the Y residue is often 4-hydroxyproline (HyP) or 5-hydroxylysine (HyK). Glycosylation in collagen occurs at the 5-OH group in HyK in the form of two glycosides, galactosylhydroxylysine (Gal-HyK) and glucosyl galactosylhydroxylysine (GlcGal-HyK). In collision induced dissociation (CID), collagen tryptic glycopeptides exhibit unexpected gas-phase dissociation behavior compared to typical N- and O-linked glycopeptides (i.e., in addition to glycosidic bond cleavages, extensive cleavages of the amide bonds are observed). The Gal- or GlcGal- glycan modifications are largely retained on the fragment ions. These features enable unambiguous determination of the amino acid sequence of collagen glycopeptides and the location of the glycosylation site. This dissociation pattern was consistent for all analyzed collagen glycopeptides, regardless of their length or amino acid composition, collagen type or tissue. The two fragmentation pathways-amide bond and glycosidic bond cleavage-are highly competitive in collagen tryptic glycopeptides. The number of ionizing protons relative to the number of basic sites (i.e., Arg, Lys, HyK, and N-terminus) is a major driving force of the fragmentation. We present here our experimental results and employ quantum mechanics calculations to understand the factors enhancing the labile character of the amide bonds and the stability of hydroxylysine glycosides in gas phase dissociation of collagen glycopeptides.

Topics: Amino Acid Sequence; Animals; Binding Sites; Cattle; Chromatography, High Pressure Liquid; Collagen; Glycopeptides; Glycosylation; Hydroxylysine; Molecular Sequence Data; Molecular Structure; Peptide Fragments; Quantum Theory; Spectrometry, Mass, Electrospray Ionization; Tandem Mass Spectrometry; Trypsin

In recent years, glycopeptide purification by hydrazide chemistry has become popular in structural studies of glycoconjugates; however, applications of this method have been almost completely restricted to analysis of the N-glycoproteome. Here we report a novel method for analyzing O-glycosylations unique to collagen, which are attached to hydroxylysine and include galactosyl-hydroxylysine and glucosyl-galactosyl-hydroxylysine. We established a hydrazide chemistry-based glycopeptide purification method using (1) galactose oxidase to introduce an aldehyde into glycopeptides and (2) formic acid with heating to elute the bound glycopeptides by cleaving the hydrazone bond. This method allows not only identification of O-glycosylation sites in collagen but also concurrent discrimination of two types of carbohydrate substitutions. In bovine type I and type II collagens, galactosyl-hydroxylysine /glucosyl-galactosyl-hydroxylysine -containing peptides were specifically detected on subsequent comprehensive liquid chromatography (LC)/MS analysis, and many O-glycosylation sites, including unreported ones, were identified. The position of glycosylated hydroxylysine, which is determined by our unambiguous and simple method, could provide insight into the physiological role of the modifications.

Topics: Amino Acid Motifs; Amino Acid Sequence; Animals; Cattle; Chromatography, Affinity; Collagen; Collagen Type I; Collagen Type II; Glycoproteins; Glycosylation; Hydrazines; Hydroxylysine; Molecular Sequence Data; Oxidation-Reduction; Peptide Fragments; Peptide Mapping; Protein Processing, Post-Translational; Tandem Mass Spectrometry

Lysyl hydroxylase 3 (LH3), encoded by Plod3, is the multifunctional collagen-modifying enzyme possessing LH, hydroxylysine galactosyltransferase (GT), and galactosylhydroxylysine-glucosyltransferase (GGT) activities. Although an alteration in type I collagen glycosylation has been implicated in several osteogenic disorders, the role of LH3 in bone physiology has never been investigated. To elucidate the function of LH3 in bone type I collagen modifications, we used a short hairpin RNA technology in a mouse osteoblastic cell line, MC3T3-E1; generated single cell-derived clones stably suppressing LH3 (short hairpin (Sh) clones); and characterized the phenotype. Plod3 expression and the LH3 protein levels in the Sh clones were significantly suppressed when compared with the controls, MC3T3-E1, and the clone transfected with an empty vector. In comparison with controls, type I collagen synthesized by Sh clones (Sh collagen) showed a significant decrease in the extent of glucosylgalactosylhydroxylysine with a concomitant increase of galactosylhydroxylysine, whereas the total number of hydroxylysine residues was essentially unchanged. In an in vitro fibrillogenesis assay, Sh collagen showed accelerated fibrillogenesis compared with the controls. In addition, when recombinant LH3-V5/His protein was generated in 293 cells and subjected to GGT/GT activity assay, it showed GGT but not GT activity against denatured type I collagen. The results from this study clearly indicate that the major function of LH3 in osteoblasts is to glucosylate galactosylhydroxylysine residues in type I collagen and that an impairment of this LH3 function significantly affects type I collagen fibrillogenesis.

Topics: Animals; Collagen Type I; Glycosylation; HEK293 Cells; Humans; Hydroxylysine; Mice; Osteoblasts; Procollagen-Lysine, 2-Oxoglutarate 5-Dioxygenase

A stereoselective synthesis of the C-glycoside analogue of beta-D-galactosyl-(5R,2S)-hydroxylysine (1) has been achieved starting from tetra-O-benzyl-D-galactopyranosyl lactone. The synthesis involved establishment of three stereogenic centers in an unambiguous manner. A facially selective Grignard reaction followed by a silane reduction was used for the anomeric position of the C-galactose residue. An Evans allylation established the configuration of the delta-aminomethylene group of the hydroxylysine moiety, whereas an asymmetric hydrogenation utilizing Burk's catalyst was used for the alpha-amino acid moiety itself. The synthesis was completed in 17 steps with an overall yield of 18%, resulting in the most complex and functionalized C-glycoside analogue of a naturally occurring glycosylated amino acid prepared to date. In addition, amino acid 1 was incorporated in a glycopeptide from type II collagen known to be crucial for the response of autoimmune T cells obtained in models of rheumatoid arthritis. A preliminary immunological study revealed that four out of five members in a panel of T cell hybridomas were able to recognize this C-linked glycopeptide when presented by A(q) class II MHC molecules.

Topics: Amino Acids; Animals; Collagen Type II; Glycopeptides; Glycosides; Hybridomas; Hydroxylysine; Mice; Monosaccharides; T-Lymphocytes

Two protected derivatives of beta-D-galactopyranosyl-5-hydroxy-L-lysine, in which HO-4 of galactose has been O-methylated or replaced by fluorine, have been prepared. The building blocks were incorporated at position 264 of the peptide fragment CII259-273 from type II collagen by solid-phase synthesis. The ability of these two glycopeptides, and two CII259-273 glycopeptides in which HO-4 of galactose was either unmodified or deoxygenated, to elicit responses from T-cell hybridomas obtained in a mouse model for rheumatoid arthritis was then determined. The hybridomas were all highly sensitive towards modifications at C-4 of the beta-D-galactosyl residue of CII259-273, highlighting the role of HO-4 as an important contact point for the T-cell receptor. Most likely, this glycopeptide hydroxyl group is involved in hydrogen bonding with the T-cell receptor.

Topics: Animals; Arthritis, Rheumatoid; Collagen Type II; Disease Models, Animal; Dose-Response Relationship, Immunologic; Galactose; Glycopeptides; Hybridomas; Hydroxylysine; Magnetic Resonance Spectroscopy; Mice; Peptide Fragments; Receptors, Antigen, T-Cell; T-Lymphocytes

Five protected analogues of beta-D-galactosyl-(5R)-5-hydroxy-L-lysine were prepared, in which the galactosyl moiety was modified by monodeoxygenation or inversion of stereochemistry at C-4. The building blocks were used in the solid-phase synthesis of a set of glycopeptides related to the peptide fragment CII256-273 from type II collagen. Evaluation of the glycopeptides revealed that T-cell hybridomas obtained in collagen-induced arthritis (CIA), which is a common mouse model for rheumatoid arthritis, recognized the galactosyl moiety with high specificity for individual hydroxy groups. Moreover, T-cell hybridomas obtained in a humanized variant of CIA were also found to recognize the glycopeptides in an equally carbohydrate-specific manner. The results allowed the generation of models of the complexes formed between the appropriate class II major histocompatibility complex (MHC) molecule, glycopeptide, and the T-cell receptor, that is, of an interaction that is critical for the stimulation of T cells in the arthritis models. In the structural models, peptide side chains anchor the glycopeptide in pockets in the class II MHC molecule, whereas the galactosylated hydroxylysine residue forms the key contacts with the T-cell receptor. Importantly, the results also suggest that a T-cell response towards glycopeptide fragments from type II collagen could play an important role in the development of rheumatoid arthritis in humans.

Topics: Animals; Arthritis, Rheumatoid; Binding Sites; Collagen Type II; Disease Models, Animal; Glycopeptides; Histocompatibility Antigens Class II; Humans; Hybridomas; Hydroxylysine; Mice; Mice, Transgenic; Peptide Fragments; Structure-Activity Relationship; T-Lymphocytes, Helper-Inducer

Collagen abnormalities of the spinal cord and the skin have been reported in patients with amyotrophic lateral sclerosis (ALS). The urinary concentrations of the hydroxylysine glycosides, i.e., glucosylgalactosyl hydroxylysine (glu-gal Hyl) and galactosyl hydroxylysine (gal Hyl), indicate the tissue origin of the collagen metabolites and the rate of the degradation of collagen. We measured the urinary levels of glu-gal Hyl and gal Hyl in 12 ALS patients, 10 diseased control subjects with other neurologic or muscular diseases (Control Group A), and 10 healthy control subjects (Control Group B). The urinary level of glu-gal Hyl in ALS patients was significantly lower than in the two control groups. In addition, a significant negative relationship between glu-gal Hyl urinary level and duration of illness was found in ALS patients. There was no marked difference in the urinary level of gal Hyl between ALS patients and the control groups. Our data suggest that the decreased urinary level of glu-gal Hyl may be useful in assessing the alteration in collagen metabolism in ALS and may have a relationship with the progression of ALS.

Topics: Aged; Biomarkers; Collagen; Female; Humans; Hydroxylysine; Male; Middle Aged; Motor Neuron Disease; Reference Values; Regression Analysis; Time Factors

Some glycosides of hydroxylysine, viz., alpha-1, 2-glucosylgalactosyl-O-hydroxylysine and beta-1-galactosyl-O-hydroxylysine, appear to be good indicators of collagen turnover. A simple liquid chromatography/tandem mass spectrometry (LC/MS/MS) method is proposed for measuring these analytes in urine, with no sample preparation except for a dilution step. Quantitation is performed using external calibration with no internal standard. A preliminary survey indicates good intra- and inter-day reproducibility (better than 5 and 8%, respectively). With the present method, the estimated limits of detection (S/N > 3) in urine are 0.8 and 0.5 microM/L for beta-1-galactosyl-O-hydroxylysine and alpha-1,2-glucosylgalactosyl-O-hydroxylysine, respectively. The method is proposed as a robust tool for a large-scale research investigation on collagen turnover.

Topics: Chromatography, Liquid; Collagen; Humans; Hydroxylysine; Mass Spectrometry

Alterations of the collagen matrix, e.g., increased hydroxylation and glycosylation of lysyl residues in collagen I, were found in human osteoporotic bone, and it was suggested that they could alter the mechanical properties of skeleton. To test this hypothesis, we evaluated the content of galactosyl-hydroxylysine (GHYL) in bone collagen, as assessed by its urinary excretion, and related it to the occurrence of fracture. Two hundred and fifteen unselected postmenopausal women with osteoporosis were divided in two subgroups (comparable for age, age of menopause, bone mineral density, and biochemical parameters of bone turnover) on the basis of the history of fragility fracture; 115 patients had suffered no fracture and 100 patients had suffered one or more fractures 3 or more years before. Four urinary markers of bone turnover (hydroxyproline, cross-linked N-telopeptide, free deoxypyridoline, and GHYL) were evaluated in all patients. There was no difference between the two groups with regard to all the parameters studied except for GHYL, which was significantly higher in the group with a history of fracture (1.35 +/- 0.82 mmol/mol of creatinine [Cr] versus 1.03 +/- <0.48 mmol/mol Cr, p < 0.001); this marker did not correlate with other markers of bone remodeling in the fracture group, indicating a possible defect in bone collagen. In conclusion, provided that increased levels of urinary GHYL do reflect overglycosylation of hydroxylysine in bone collagen, the GHYL may be considered a marker of bone collagen quality. Our results, showing higher urinary GHYL in osteoporosis patients with fracture, seem to confirm this suggestion.

Topics: Aged; Biomarkers; Biomechanical Phenomena; Bone Density; Collagen; Female; Humans; Hydroxylysine; Middle Aged; Osteoporosis, Postmenopausal; Retrospective Studies

Diurnal variations in the excretion of bone resorption markers were assessed in order to identify the type of urine collection which provides the most information on bone resorption rate and its relation to measuring bone dynamics in a postmenopausal population. Sixty women, ages 43-67 and without disease or treatment known to affect bone mineral density, were divided into two groups on the basis of femoral mineral density T-score: <1.5 (Group I), >1.5 (Group II). Bone formation was assessed by measuring bone alkaline phosphatase activity and osteocalcin concentration, bone resorption by urinary hydroxyproline, pyridinoline and deoxypiridinoline, N-telopeptide, galactosyl hydroxylysine, and CrossLaps. To identify the more appropriate collection times, urine samples were collected from 7 am to 3 pm; from 3 pm to 11 pm; from 11 pm to 7 am. Twenty-four hour urine collection and first morning void urine samples were also measured. The findings suggest that nocturnal collection and first morning void samples provide the most reliable data on the rate of bone degradation, possibly showing bone loss not only in osteopenic patients but also in women with a low T-score. Nocturnal and first morning samples should therefore be recommended in order to standardize sample collection, as they enable an accurate assessment of bone resorption markers and improved comparability to results from different studies, as well as a less cumbersome collection modality.

Topics: Adult; Aged; Alkaline Phosphatase; Amino Acids; Biomarkers; Bone Density; Bone Resorption; Collagen; Collagen Type I; Female; Humans; Hydroxylysine; Hydroxyproline; Middle Aged; Osteocalcin; Peptide Fragments; Peptides

Galactosylhydroxylysine (GHL) is released during bone resorption and has been shown to be elevated in subjects with metabolic bone loss. GHL is relatively specific for bone, it is not recycled or significantly metabolized during collagen turnover, and the levels are not influenced by diet. Previous measurements of GHL levels in urine have been performed using reverse-phase high performance liquid chromatography following pre-column derivatization. We produced polyclonal antibodies to GHL using GHL purified from sea sponges and developed an immunoassay that can recognize GHL in urine. The antibodies have minimal cross-reactivity with a physiological mixture of amino acids (< 1%), galactose (< 0.2%), lactose (< 0.3%), and glucosylgalactosylhydroxylysine (< 1%). This competitive immunoassay requires no dilution or pretreatment of the samples and provides a rapid and easy method for the evaluation of GHL in urine. Analysis of clinical samples from normal individuals, post-menopausal women, osteoporotic patients and individuals with Paget's disease show that the assay can discriminate between groups with differing levels of bone resorption as well as deoxypyridinoline (Dpd).

Topics: Adult; Aged; Amino Acids; Animals; Biomarkers; Bone Resorption; Chromatography, High Pressure Liquid; Collagen; Female; Humans; Hydroxylysine; Immunoassay; Male; Middle Aged; Osteitis Deformans; Osteoporosis; Osteoporosis, Postmenopausal; Porifera; Postmenopause; Rabbits; Reference Values; Reproducibility of Results; Sensitivity and Specificity

The bone mineral density and the biochemical parameters exploring bone cell activities were analyzed in two cosmonauts who spent 1 and 6 months, respectively, in the Russian MIR station. Measurements were performed before the flight, after the flight, and after a recovery period. At the end of the first month, peripheral QCT measurements indicated a slight decrease of trabecular bone mass in the distal tibial metaphysis. However, after 6 months of spaceflight, a more marked loss of trabecular and cortical bones was observed in the tibia, and was still significant after 6 month recovery in the trabecular compartment, whereas a decrease was no longer observed in the cortical envelope. No change was observed in either compartment of the distal radius at any time. Ultrasound BUA of the calcaneus was greatly reduced by the first month, followed by a more dramatic decrease after month 6. Ultrasound SOS detected no change. Parameters reflecting bone formation activity appeared to be depressed after both missions. In contrast, no dramatic change in resorption parameters was observed, except for a trend toward an increase in pyridinoline. In conclusion, the lower weight-bearing bones appeared more sensitive than the upper ones in terms of spaceflight-induced bone loss. This probably explained the absence of marked systemic biochemical data changes. This study further suggests that recovery in the tibial trabecular compartment 6 months after landing was not completed after a 6 month mission.

Topics: Adult; Alkaline Phosphatase; Amino Acids; Astronauts; Biomarkers; Bone and Bones; Bone Density; Bone Development; Bone Resorption; Humans; Hydroxylysine; Male; Osteocalcin; Peptide Fragments; Procollagen; Space Flight; Tomography, X-Ray Computed; Ultrasonography; Weightlessness

The aim of this study was to compare urinary galactosylhydroxylysine (GHyl) and deoxypyridinoline (d-Pyr) as biochemical markers of bone resorption in post-menopausal women treated and untreated with estrogen and cyclic etidronate. Fasting urinary GHyl, D-Pyr, pyridinoline, serum osteocalcin and total alkaline phosphatase were measured in three subgroups, i.e. post-menopausal women undergoing hormone replacement therapy, untreated post-menopausal women and post-menopausal women with low BMD treated with disodium etidronate. The results indicated that GHyl did not significantly discriminate between untreated post-menopausal women and estrogen replated ones unless an osteoporotic untreated group was selected. d-Pyr and GHyl showed similar performances when their values after bisphosphonate treatment were compared to those found in untreated post-menopausal women, thus suggesting that both markers were equal in their ability to detect the bone response to cyclic etidronate administration. This observation further proves the statement that GHyl is prone to confounding factors under estrogen therapy but it is adequate as is d-Pyr in monitoring the bone response to bisphosphonate treatment.

Topics: Aged; Amino Acids; Biomarkers; Bone Resorption; Collagen; Estrogen Replacement Therapy; Etidronic Acid; Female; Humans; Hydroxylysine; Middle Aged; Postmenopause

The ovariectomized rat is the most commonly used animal model of human postmenopausal osteoporosis, exhibiting a high rate of bone turnover with resorption exceeding formation. At present, bone turnover is quantified directly by dynamic histomorphometry. The aim of the present study was to determine whether the measurement of the urinary output of some specific bone collagen catabolites--pyridinolines and hydroxylysine glycosides--could be used to indirectly monitor the initial phase of bone turnover increase in ovariectomized 90-day-old rats. Ninety-day-old female rats were randomly divided into three groups (n = 6): ovariectomized, sham-operated and non-treated controls. Urine samples (24 h) were collected 6 days before surgery and twice weekly for the 4 weeks following ovariectomy. Urinary excretion of pyridinoline (PYD), deoxypyridinoline (DPD), glucosyl-galactosyl-hydroxylysine (GGHYL) and galactosyl-hydroxylysine (GHYL) were measured. As expected, ovariectomy was associated with a significant decrease in bone mineral density in both the proximal tibial and distal femoral metaphysis. Compared with both sham-operated and control animals, ovariectomized rats showed significant increases in PYD, GGHYL, and GHYL urinary output 8 days after surgery and in DPD output after 15 days. These changes were maintained throughout the study. The results confirm that measurement of the urinary excretion of pyridinolines and hydroxylysine glycosides represents a powerful tool for detecting the onset of bone turnover in ovariectomized 90-day-old rats.

Topics: Amino Acids; Animals; Bone and Bones; Female; Glycosides; Hydroxylysine; Ovariectomy; Rats; Rats, Sprague-Dawley; Time Factors

We have previously shown that galactosyl hydroxylysine (GHYL), pyridinoline (PYD), and deoxypyridinoline (DPD) have a better accuracy and discriminate power than hydroxyproline in distinguishing postmenopausal osteoporotic women from premenopausal controls. In this study, we evaluated the clinical performances of GHYL, PYD, and DPD, alone or in combination, in distinguishing postmenopausal osteoporotic women (OPBD, n = 26) from age-matched controls (CBD, n = 19). The diagnosis of osteoporosis was based upon the bone density (BD) of the lumbar spine measured by quantitative computed tomography (CBD: BD > 108 mg/cm3; OPBD: BD < 70 mg/cm3). Urinary excretion of GHYL, PYD, and DPD were measured by HPLC, and all data were expressed as the molar ratio with the creatinine excretion (GHYL/CR, PYD/CR, and DPD/CR). The clinical performances were tested by: Z score analysis (Z), Receiver Operated Characteristic curve analysis (%Acc) and logistic-regression analysis of the posterior probabilities for prediction from a logistic model (LOGIST). GHYL/CR, PYD/CR, and DPD/CR were significantly increased in OPBD compared with CBD. The clinical performances were similar for the three assays, with slightly better performances for GHYL/CR (GHYL/CR: Z = 3.14, %Acc = 70 +/- 8, LOGIST P = 0.01; PYD/CR: Z = 2.19, %Acc = 67 +/- 8, LOGIST P = 0.051; DPD/CR: Z = 2.13, %Acc = 65 +/- 8, LOGIST P = 0.06). None of the possible combinations of the three assays yielded better clinical performances than GHYL/CR alone. In conclusion, this study further confirms the validity of GHYL, PYD, and DPD as markers of bone resorption.

Topics: Aged; Amino Acids; Bone Density; Creatinine; Female; Humans; Hydroxylysine; Middle Aged; Osteoporosis, Postmenopausal; Regression Analysis; Sensitivity and Specificity

Glucosylgalactosylhydroxylysine (GGHYL), galactosylhydroxylysine (GHYL), pyridinoline (PYD) and deoxypyridinoline (DPD) were measured in the urine (6 h serial specimens over 96 and 24 h urine specimens for 4 days) collected from four adult Sprague Dawley rats and in the femoral and tibia] bone as well as in the dorsal skin of the same rats. No significant daily variations were found in the urine excretion of GGHYL, GHYL, PYD and DPD but significant diurnal variations. The GGHYL/GHYL ratio in rat urine (0.46 +/- 0.1) reflected neither the bone collagen ratio (1.9 to 2.4) nor the skin collagen ratio (1.22 +/- 1.07), a finding that may reflect GGHYL conversion into GHYL. The content of both pyridinolines was very low in the skin and high in the bone collagen and the urinary PYD/DPD ratio (1.46 +/- 0.15) reflected essentially the bone collagen ratio (0.8-3.0). These results suggest the usefulness of measuring GGHYL, GHYL, PYD and DPD in 24 h urine specimen and, based on the inter-animal variations, the necessity to consider each animal as its own control when bone turnover needs to be monitored.

Topics: Amino Acids; Animals; Biomarkers; Bone and Bones; Collagen; Cross-Linking Reagents; Glycosides; Humans; Hydroxylysine; Male; Pyridinium Compounds; Rats; Rats, Sprague-Dawley; Skin; Time Factors

Aim of the study was to compare urinary galactosyl-hydroxylysine (GHyl), deoxypyridinoline (D-Pyr) and pyridinoline (Pyr) before and after 5 to 9 months of hormone replacement therapy (HRT) in postmenopausal women. The urinary markers were measured by HPLC in the second void of fasting samples and were expressed as ratio to creatinine. GHyl was also expressed as a ratio to glucosylgalactolysyl-hydroxylysine (GGHyl). After short-term hormone replacement therapy, urinary D-Pyr fell significantly, but Pyr and GHyl, also when expressed as a ratio to GGHyl, remained unmodified. We conclude that GHyl and Pyr are not useful markers in monitoring the bone response to HRT in postmenopausal women.

Topics: Alkaline Phosphatase; Biomarkers; Bone and Bones; Bone Density; Calcium; Chromatography, High Pressure Liquid; Cross-Linking Reagents; Estrogen Replacement Therapy; Female; Humans; Hydroxylysine; Middle Aged; Osteocalcin; Postmenopause; Pyridinium Compounds

The aims of this study were to determine 1) whether primary hyperparathyroidism (PHPT) is associated with accelerated bone loss in postmenopausal women, 2) whether bone mineral density (BMD) and bone turnover change to a similar extent with surgery and hormone replacement therapy (HRT) in these patients, and 3) whether biochemical markers of bone turnover measured at baseline can be used to predict the change in BMD in these patients after different therapies. We studied 33 postmenopausal women with PHPT; their ages at the time of study ranged from 48-80 yr (mean +/- SD, 63 +/- 10). Total body (TB), lumbar spine (LS), and femoral neck (FN) BMD and biochemical markers of bone turnover were measured at baseline and 10-30 months (19 +/- 5) after parathyroid surgery, HRT, or no treatment. BMD was measured in 33 age-matched healthy controls at baseline and at a mean of 24 months. Baseline biochemical markers of bone turnover were measured in controls. In PHPT at baseline, the mean z-score of BMD was -1.25 at TB (95% confidence interval, -1.64 to -0.86), -0.95 at LS (-1.37 to -0.53), and -1.30 at FN (-1.65 to -0.95), whereas the mean z score was 0.45 for serum carboxy-terminal propeptide of human type I procollagen (0.02-0.89), 1.05 for bone alkaline phosphatase (0.38-1.71), 2.38 for 24-h urinary excretion of cross-linked N-terminal telopeptide of type I collagen (NTx; 1.63-3.13), and 2.36 for 24-h urinary excretion of galactosyl hydroxylysine (1.97-2.74). After surgery and HRT, BMD increased and bone turnover decreased during the follow-up. In the untreated group, BMD decreased at TB and FN, and levels of bone alkaline phosphatase, NTx/creatinine, and galactosyl hydroxylysine/creatinine increased. When the rate of change in BMD (percentage per yr) was compared with that in the control group, bone gain was significant at all three skeletal sites after surgery and HRT, and bone loss was significant at TB and FN, but not at LS, in the untreated group. There was a weak, but significant, correlation between baseline urinary NTx and the change in femoral neck BMD in the untreated group (r = -0.36; P = 0.05). We conclude that untreated postmenopausal women with PHPT have low BMD resulting from accelerated bone loss at the TB and FN. Surgery and HRT both restore BMD and bone turnover toward normal in postmenopausal women with PHPT. A single measurement of bone turnover is insufficient to predict BMD changes in individual patients with PHPT.

Topics: Aged; Aged, 80 and over; Alkaline Phosphatase; Bone Density; Bone Remodeling; Collagen; Collagen Type I; Creatinine; Estrogen Replacement Therapy; Female; Humans; Hydroxylysine; Hyperparathyroidism; Middle Aged; Peptide Fragments; Peptides; Postmenopause; Procollagen

This study was undertaken to assess the sensitivity of hydroxylysylpyridinoline (HP), lysylpyridinoline (LP), galactosylhydroxylysine (GHyl) and glucosylgalactosylhydroxylysine (GGHyl) to monitor bone response to estrogen deficiency and replacement by comparing their excretory patterns in ovariectomized aged (11-14 months old) rats. The ovariectomized (OVX) rats were randomized into two groups: (1) OVX plus vehicle; (2) OVX plus 17 beta-estradiol (17-beta E, 10 micrograms/kg, s.c., 4 days/week). Treatment with 17-beta E started immediately after OVX and continued for 60 days. The collagen catabolites were measured in urine for 1 month before OVX and thereafter for 60 days. In temporal coincidence with urine collection, bone area and bone mineral density (BMD) of lumbar vertebrae, femoral diaphysis and distal metaphysis were measured by dual-energy X-ray absorptiometry. In the untreated rats, BMD of the femoral metaphysis and lumbar vertebrae decreased significantly and the urinary excretion of LP, HP, GHyl and GGHyl increased with different patterns. In the treated rats, 17-beta E replacement prevented the increment in LP excretion, partially prevented the increase in HP excretion, but had no effect on the excretion of GHyl and GGHyl. In conclusion pyridinolines and glycosides have different sensitivities to the bone response to OVX. Glycoside excretion after OVX also reflects metabolic processes not strictly related to bone loss and, in contrast with LP, is not sensitive to estrogen replacement.

Topics: Absorptiometry, Photon; Amino Acids; Animals; Biomarkers; Bone and Bones; Estradiol; Estrogen Replacement Therapy; Female; Hydroxylysine; Ovariectomy; Pyridinium Compounds; Random Allocation; Rats; Rats, Sprague-Dawley

Aim of this study was to investigate whether osteoclast activity changes as a consequence of even mild physiological perturbation of plasma calcium as such induced by an oral calcium load. Osteoclast activity was determined indirectly by measuring, in spot urines at two and four hours after oral calcium load, the urinary excretion of hydroxylysylpyridinoline (Pyr), deoxylysylpyridinoline (D-Pyr), hydroxyproline (Hyp) and galactosyl-hydroxylysine (GHyl). The occurrence of the metabolic perturbation of plasma calcium homeostasis was assessed by measuring three indexes: i.e. calcemic response, PTH reduction and calciuric response at times following oral calcium loading. A significant fall of urinary D-Pyr and Pyr followed the perturbation of calcium homeostasis induced by the oral calcium load in two groups of healthy young adult and postmenopausal women. The highest mean percent reduction was observed for D-Pyr and was quantitatively similar in the two groups. Since urinary D-Pyr is the most specific bone resorption marker, it may be inferred that the perturbation of plasma calcium homeostasis induced by an oral calcium load is able to acutely inhibit osteoclast activity. This supports the view that osteoclasts are involved in the short-term error correction of plasma calcium.

Topics: Adult; Aged; Amino Acids; Calcium; Collagen; Creatinine; Female; Homeostasis; Humans; Hydroxylysine; Hydroxyproline; Middle Aged; Osteoclasts; Postmenopause

The composition of collagen was analyzed and the degree of lysyl hydroxylation of individual collagen chains was determined in four osteosarcomas and two osteofibrous dysplasias. In addition, the tumor proliferation (number of mitoses, proliferating-nuclear-antigen-positive cells, MIB) as well as the response to chemotherapy (morphological regression grade) were checked. All tumors contained a high proportion of collagen III and, in all but one osteosarcoma, pepsin-extracted collagens I and III were overmodified. Furthermore, the proportion of diglycosides in collagen I was about four times higher than in controls. The collagen composition and modification resembled those of bones at early stages of human development. One osteosarcoma and both osteofibrous dysplasias were in the normal range of lysyl hydroxylation. There was no correlation between the collagen properties and the histopathological marker of tumor proliferation.

Topics: Adolescent; Adult; Bone Neoplasms; Cell Division; Child; Collagen; Electrophoresis, Polyacrylamide Gel; Fibrous Dysplasia of Bone; Glycosylation; Humans; Hydroxylation; Hydroxylysine; Lysine; Osteosarcoma; Proline; Protein Processing, Post-Translational; Sodium Dodecyl Sulfate

Galactosyl-hydroxylysine (Gal-Hyl) is the predominant product of the posttranslational glycosylation of skeletal collagen. Urinary Gal-Hyl excretion is regarded as a marker of bone resorption in adults, but little information is available on the validity of this parameter in pediatric age groups. Using 24-h urine samples from 88 healthy children and adolescents ages 4-18 yr, reference ranges were established for this age group, and values were compared with measurements in children with overt GH deficiency (n = 14) or Ullrich-Turner syndrome (n = 21). When expressed relative to body weight (Gal-Hyl/wt), urinary Gal-Hyl excretion was 3.2 to 4.7 times higher in subjects 4-16 yr of age than in adults. Highest values were observed in very young children and during the pubertal growth spurt. In the total population, urinary Gal-Hyl/wt was closely related to growth velocity (r = 0.72) and significantly correlated with the urinary excretion of both hydroxyproline (r = 0.74) and deoxypyridinoline (r = 0.88; P < 0.001 each). Urinary Gal-Hyl/wt was significantly lower in children with GH deficiency or Ullrich-Turner syndrome than in healthy children (P < 0.001 each). The urinary excretion of Gal-Hyl was significantly correlated with growth velocity in GH-deficient children (r = 0.69; P = 0.004) but not in patients with Ullrich-Turner syndrome. In the latter, the increase in urinary Gal-Hyl excretion after 3 months of treatment with recombinant human GH correlated significantly with the increase in growth velocity after 12 months of treatment (r = 0.76; P = 0.002). We conclude that the urinary excretion of Gal-Hyl is a valid and potentially useful index of skeletal growth in children.

Topics: Adolescent; Adult; Aging; Amino Acids; Biomarkers; Bone Resorption; Child; Child Development; Child, Preschool; Female; Growth Disorders; Growth Hormone; Humans; Hydroxylysine; Hydroxyproline; Male; Recombinant Proteins; Reference Values

This study evaluated whether pyridinium cross-links, which are positively charged, besides renal clearance are also cleared by the liver into bile. In 13 human bile samples tested, we were able to detect both pyridinoline (PYD) and deoxypyridinoline (DPD) in small amounts which were estimated to be about 1-2% of the amount usually found in urine. To further evaluate the amount of pyridinium cross-links excreted through bile, we studied the stability of these compounds at the alkaline pH of bile. No effect on their stability was detected over a 6-hour incubation. The origin of these molecules in bile and the significance of this finding in the use of PYD and DPD as bone resorption markers are discussed.

Topics: Adult; Aged; Amino Acids; Bile; Cross-Linking Reagents; Female; Humans; Hydroxylysine; Male; Middle Aged; Pyridinium Compounds

Topics: Animals; Bone Remodeling; Chromatography, High Pressure Liquid; Female; Humans; Hydroxylysine; Ovariectomy; Rats

Despite these potential problems, biochemical bone markers are the single most sensitive method for monitoring acute changes in bone metabolism. For example, subcutaneous injections of recombinant human insulin-like growth factor I cause a measurable increase in both procollagen and urinary DPD in as little as 1 day. Similarly, it is possible to measure a significant decrease in bone formation as determined by decreases in serum levels of ALP, OC, and C-PCP within 12 hours after the beginning of a PTH infusion study. Additionally, an increase in DPD/cr was determined within 24 hours of the start of bed rest. These changes, seen within 24 hours, are far earlier than could be detected by any other method of monitoring bone metabolism. Thus, biochemical assays have opened a new era where changes in bone metabolism can be detected in hours to days. This acute detectability should be especially helpful to the development of new drugs and the optimization of the use of approved drugs. Accordingly, definite dose-response studies can now be done in a reasonable time. For osteoporosis therapy there are reasons to consider cyclic drug administration, such as avoiding drug resistance (PTH or calcitonin), avoiding overtreatment (bisphosphonates), or avoiding a possible mineralization defect (fluoride). By using biochemical assays, we can determine the optimum amount of "on time" and "off time" in cyclic therapy. Of the bone formation assays, ALP, OC, and PCP, we recommend for routine use the OC assay because of its high discriminant power and because it has been better characterized, in terms of clinical application, than the PCP assays and the ALP IRMA. If, however, the serum cannot be drawn at a specific time in all patients to be studied, we recommend the ALP assay because, unlike the OC assay, it shows no diurnal variation. Of the bone resorption assays, HYP, TRAP, GHYL, and PYD/DPD, we recommend the urine PYD/DPD assay (adjusted for creatinine) because it is commercially available and because, along with the urine GHYL assay, it is the most sensitive bone resorption assay. Established guidelines for the use of assays in patient care is not yet available, largely because of the large intrapatient variation seen with most assays. Once this problem is resolved, it should be possible to apply biochemical assays to routine clinical practice. For example, if the patient has a urine DPD/cr (indicating a high bone resorption rate), the patient would be selected for anti

Topics: Acid Phosphatase; Alkaline Phosphatase; Amino Acids; Biomarkers; Bone Development; Bone Resorption; Humans; Hydroxylysine; Hydroxyproline; Osteocalcin; Procollagen; Reproducibility of Results

In patients with mild or asymptomatic primary hyperparathyroidism a reliable index of bone resorption might be useful for appropriate management. Hydroxyproline is the most commonly used marker of bone resorption but its low specificity and sensitivity are known. Galactosylhydroxylysine, an amino acid mainly represented in bone collagen, has been proposed as a more suitable index of bone resorption. In this study we evaluated the sensitivity of galactosylhydroxylysine and hydroxyproline assays in following the changes of their urinary levels in 12 patients with mild primary hyperparathyroidism before and after treatment with bisphosphonate and surgery.. Serum and fasting urine specimens were obtained from 12 women with mild primary hyperparathyroidism before and after bisphosphonate treatment (2.5 mg daily for 5 days, intravenously) and after a further 25 days; in 7 patients biochemical tests were also performed 1 and 6 days after parathyroidectomy. Galactosylhydroxylysine was assayed by an HPLC method and hydroxyproline by a RIA commercial kit.. Baseline galactosylhydroxylysine urinary levels were far above the normal range in all the patients whilst in 8 of them baseline hydroxyproline levels were normal. Bisphosphonate treatment significantly decreased bone turnover as shown by a significant fall in serum calcium (from 2.9 to 2.6 mmol/l; P < 0.001) and in galactosylhydroxylysine and hydroxyproline (-55 and -31% respectively). Twenty-five days after the end of treatment, resorption increased again and serum calcium and galactosylhydroxylysine, but not hydroxyproline, rose significantly towards basal levels. One day after parathyroidectomy serum calcium, galactosylhydroxylysine and PTH showed reduction below normal ranges. PTH and galactosylhydroxylysine returned to normal values at day 6 after parathyroidectomy. No changes in hydroxyproline levels were seen. Galactosylhydroxylysine, but not hydroxyproline, correlated significantly with serum calcium and PTH.. Galactosylhydroxylysine appears to be a sensitive index of bone resorption, useful in the clinical assessment of bone involvement and in the management of patients with mild primary hyperparathyroidism.

Topics: Aged; Alendronate; Biomarkers; Bone Resorption; Diphosphonates; Female; Humans; Hydroxylysine; Hydroxyproline; Hyperparathyroidism; Middle Aged; Parathyroidectomy

Glucosyl-galactosyl-hydroxylysine (GGHYL) and galactosyl-hydroxylysine (GHYL) are constituents of collagen protein. The ratio of the two hydroxylysine glycosides varies with the collagen type and, moreover, for a given collagen type, it also varies according to the connective tissue. For example, in type I collagen (the most abundant in the body), the GGHYL/GHYL ratio tends to be greater in soft connective tissues and lower in bone. The hydroxylysine glycosides are not recycled during collagen turnover and are excreted in the urine. Therefore, the urinary GGHYL/GHYL ratio, which reflects the proportion of the two metabolites in the various collagens, may indicate the type of connective tissue affected by pathological turnover, and may thus be a promising marker of bone metabolism. In this paper a method is described for the measurement of urinary hydroxylysine glycosides by reversed-phase liquid chromatography after purification of the sample by solid-phase extraction. The method presented is analytically reliable and suitable for routine use in a clinical laboratory.

Topics: Adult; Biomarkers; Bone and Bones; Chromatography, High Pressure Liquid; Collagen; Female; Humans; Hydroxylysine; Indicators and Reagents; Osteoporosis; Reference Standards; Spectrophotometry, Ultraviolet

Galactosyl hydroxylysine and deoxypyridinoline are at present the most promising markers of bone resorption. Various studies have indeed shown that these two markers discriminate with high accuracy subjects with different rates of bone turnover and that their accuracies and discriminate power are very similar. The aim of this paper is to compare the practicality and the reproducibility of the HPLC galactosyl hydroxylysine and deoxypyridinoline assays. In summary, this review shows that the galactosyl hydroxylysine and deoxypyridinoline HPLC assays differ mainly in the need, in using deoxypyridinoline, for an acid hydrolysis and a preextraction of the urine samples. This implies two major problems for deoxypyridinoline: 1) more time is required due to the cumbersome preanalytical procedures; and 2) a lower reproducibility. Our data, in fact, show that both the intra-assay and inter-assay coefficient of variation of the deoxypyridinoline assay are almost 100% higher than those of the galactosyl hydroxylysine assay.

Topics: Adult; Amino Acids; Biomarkers; Bone Resorption; Child; Chromatography, High Pressure Liquid; Female; Humans; Hydroxylysine; Molecular Weight; Reproducibility of Results

A study was carried out to assess the best use of biochemical bone markers to exclude metastases in patients with breast cancer. Urinary galactosyl-hydroxylysine and serum alkaline phosphatase were used to monitor bone resorption and deposition, respectively. Hydroxyproline was also measured. In a selected population of patients, possibly affected by metastases on the basis of scintigraphic examination, which is highly sensitive but poorly specific, we assessed the efficiency of the markers by a double statistical analysis. In this group, the only marker able to predict metastases was galactosyl-hydroxylysine.

Topics: Alkaline Phosphatase; Biomarkers, Tumor; Bone and Bones; Bone Neoplasms; Bone Resorption; Breast Neoplasms; Female; Humans; Hydroxylysine; Hydroxyproline

Glucosyl-galactosyl-hydroxylysine (GGHYL) and galactosyl-hydroxylysine (GHYL) are metabolites derived from collagen degradation. They are useful biochemical markers provided they are not further processed. Experiments were run to test the activity of alpha- and beta-glycosidases in human kidney cortex preparations. Results allow to exclude the presence of the specific enzymes, in contrast with what is reported for the rat kidney tissue.

Topics: Animals; Collagen; Glycoside Hydrolases; Humans; Hydroxylysine; In Vitro Techniques; Kidney Cortex; Male; Middle Aged; Rats; Rats, Sprague-Dawley

Among some patients, regardless of age, the jaw loses bone mass, leading to loosening and falling out of otherwise healthy teeth. This study seeks to establish whether this bone loss is associated with the metabolic manifestations of other forms of localized decalcifications, such as in Paget's disease, or with generalized osteoporosis. Sixteen women being fitted with dental implants to compensate for bone losses provided 24-hour urine samples for the quantitative determination of calcium and galactosyl hydroxylysine, a bone collagen metabolite. These patients provided demographic information, relevant medical, dental, and dietary history, a profile of their current medications, and the status of their smoking and exercise habits. Urinary excretion of galactosyl hydroxylysine, which is increased in the presence of progressive increased bone resorption, remained within normal values in the patients of this study. These results suggest that the thinning of the jaw bones and subsequent tooth loss of these subjects were osteoporotic processes too limited and too localized to produce measurable increases in urinary bone metabolites.

Topics: Adult; Aged; Alveolar Bone Loss; Calcium; Chi-Square Distribution; Creatinine; Estrogens; Female; Humans; Hydroxylysine; Middle Aged; Mouth, Edentulous; Osteoporosis; Regression Analysis; Tooth Loss

Topics: Adolescent; Alkaline Phosphatase; Bone Resorption; Case-Control Studies; Child; Child, Preschool; Female; Humans; Hydroxylysine; Noonan Syndrome

We compared the clinical performances of four bone-resorption (BR) assays (hydroxyproline, HYP; galactosyl hydroxylysine, GHYL; deoxypyridinoline, DPD; and pyridinoline, PYD) in subjects with different BR rates: normal (adult men and premenopausal women), mildly increased (postmenopausal osteoporotic women), high (Paget disease patients), and very high (children). The discrimination power (Z score) and the accuracy (estimated by receiver-operating characteristic analysis) for GHYL, DPD, and PYD were compared with those for HYP. Discrimination power and accuracy were similar for high- and very-high-BR groups for all four assays. However in the mildly increased-BR group, DPD, GHYL, and PYD showed a higher discrimination power and accuracy than did HYP. The clinical performances of HYP, DPD, GHYL, and PYD are comparable for large changes in BR. For modest changes, DPD, GHYL, and PYD are more accurate and have a higher discrimination power than does HYP.

Topics: Adult; Aged; Amino Acids; Biomarkers; Bone Resorption; Child; Chromatography, High Pressure Liquid; Female; Humans; Hydroxylysine; Hydroxyproline; Male; Middle Aged; Osteitis Deformans; Osteoporosis, Postmenopausal; Pyridinium Compounds

A new mathematical model for the study of bone turnover in growing rats was developed. The model predicts a linear relationship between bone mineral content (BMC) and biochemical markers (BMK) of bone turnover assuming that rats are growing, bone turnover is profoundly affected by skeletal maturation, and resorption and formation are physiologically balanced. The model validation was performed by measuring galactosyl-hydroxylysine (GHYL) and hydroxyproline (HYP) in urines. This mathematical evidence supports our proposed use of the specific bone resorption marker GHYL to predict bone mineral content. Further studies on bone turnover will be possible by the application of the same approach.

Topics: Absorptiometry, Photon; Aging; Animals; Biomarkers; Bone and Bones; Bone Density; Bone Development; Bone Resorption; Female; Hydroxylysine; Hydroxyproline; Male; Mathematics; Models, Biological; Rats; Rats, Inbred Strains; Sex Characteristics

The effect of an organophosphorus pesticide (dimethoate) on the urinary excretion of hydroxyproline (total, nondialysable, dialysable and free fractions) and hydroxylysylglycosides, glucosylgalactosyl hydroxylysine and galactosehydroxylysine was investigated in two groups of female albino rats fed with normal and high protein diets. In comparison to controls, dimethoate treated animals were found to excrete significantly decreased amounts of urinary hydroxyproline fractions from 7th day onwards. The excretion of total hydroxylysylglycoside in urine parallels the excretion of hydroxyproline. The urinary output of both glu-gal-hyl and gal-hyl was also appreciably lower from dimethoate treated animals. The normal ratio of glu-gal-hyl and gal-hyl found in the urine of dimethoate treated animals was discussed in light of decreased turn over of collagen in both bone and skin. The effect of dimethoate in rats fed with high protein diet was comparatively less than those fed with normal diet.

Topics: Animals; Bone and Bones; Carbohydrate Sequence; Collagen; Dietary Proteins; Dimethoate; Female; Hydroxylysine; Hydroxyproline; Molecular Sequence Data; Rats; Skin

We measured the urinary excretion of galactosyl-hydroxylysine (GH) and hydroxyproline in two groups of women with breast cancer, with (M+, n = 24) and without (Mo, n = 30) clinical, scintigraphic, or radiological evidence of bone metastases. Both these compounds are excreted in larger amounts in the M+ group than in the Mo patients. However, GH, which is a specific marker for bone collagen, provides better predictivity for bone metastases than does hydroxyproline: 92% sensitivity and 90% specificity vs 74% and 79%, respectively, for hydroxyproline.

Topics: Adult; Bone Neoplasms; Breast Neoplasms; Diagnostic Errors; Female; Humans; Hydroxylysine; Monitoring, Physiologic; Neoplasm Metastasis; Risk

Immediately after the trauma, spinal cord injury patients have an increased rate of collagen synthesis and an even greater increase of collagen degradation. Collagen lost from bone is implicated in the etiology of osteoporosis and heterotopic ossification, and collagen lost from skin might lead to a propensity to develop pressure ulcers. The urinary excretion of two collagen metabolites has been monitored and their fluctuation related to the onset of skin or bone complications in these patients. One metabolite, glucosyl-galactosyl hydroxylysine, is more abundant in skin collagen; the other, galactosyl hydroxylysine, is more abundant in bone collagen. The excretion of both metabolites increases after injury, reaching a peak between three and six months after injury, and declines gradually, reaching control values about a year after injury. If a skin pressure ulcer develops, the urinary excretion of the diglycoside remains elevated instead of gradually decreasing. Similarly, if osteoporosis or heterotopic ossification is diagnosed, the monoglycoside excretion does not return to control values until the bone turnover stabilizes. Monitoring of the urinary excretion of both glycosides might prove helpful in prompting early examination to establish the presence of emerging skin and bone complications. Thus, aggressive preventive therapy could be given sooner.

Topics: Adolescent; Adult; Bone Diseases, Metabolic; Collagen; Humans; Hydroxylysine; Male; Middle Aged; Pressure Ulcer; Spinal Cord Injuries

Galactosyl-hydroxylysine, a specific bone collagen marker, has been prepared directly from human urine samples by high-performance liquid chromatography (HPLC) on a preparative column. The compound is the didansyl derivative, as proved by HPLC and mass spectrometry under fast atom bombardment conditions. Since this compound is not commercially available, the procedure reported appears to be the simplest way to prepare it, which is necessary to measure the urinary excretion of this collagen metabolite by HPLC.

Topics: Biomarkers; Bone and Bones; Chemical Phenomena; Chemistry; Chromatography, High Pressure Liquid; Collagen; Humans; Hydroxylysine; Mass Spectrometry; Spectrometry, Fluorescence

beta-1-galactosyl-0-hydroxylysine (GH) was measured in the urine of 59 women and 48 men, aged 30-79 years, by High Performance Liquid Chromatography (HPLC) of the dansylated derivative. Vertebral mineral density, measured by quantitative computed tomography (QCT), and urinary GH were inversely correlated (r = -0.74; P less than 0.001). High rate of bone mineral loss is associated with a high urinary GH excretion. Measurement of GH in urine provides a simple and noninvasive method for the evaluation of the extent of bone resorption in large groups of subjects and appears to be more specific than urinary hydroxyproline excretion.

Topics: Aging; Bone and Bones; Circadian Rhythm; Collagen; Creatinine; Female; Humans; Hydroxylysine; Male; Menopause; Minerals; Reference Values; Sex Characteristics

A sensitive and specific method is proposed to follow bone collagen degradation. The procedure consists of the measurement of galactosyl hydroxylysine (GH) in urine by HPLC. The aim of the work is to assess the predictive values of the method for the identification of post-menopausal osteoporotic women. By assuming the value of 12 mumol/g creatinine as the threshold value, the sensitivity of the test is 87% and the specificity 60%. Individuals with a GH/creatinine ratio of 12 or below are not likely to be at risk of bone fractures: an equivalent predictive value is provided by the measurement of bone density by quantitative computed tomography. This biochemical method is however simple and not invasive and may be frequently repeated.

Topics: Adult; Aged; Aged, 80 and over; Aging; Biomarkers; Female; Humans; Hydroxylysine; Middle Aged; Osteoporosis; Predictive Value of Tests

As Alport's syndrome is probably a molecular disorder of basement membrane composition, investigation of the urine on basement membrane components might be a diagnostic aid. It was shown previously that measurement of the excretion of free hydroxylysine and its glycosides, glucosylgalactosylhydroxylysine and galactosylhydroxylysine, was not useful for this purpose. Because the urinary peptide-bound fraction may be more basement membrane specific, a subsequent study was performed with respect to the total (= free and peptide-bound) concentrations and fractions. To establish reference values, urinary specimens of 38 normal subjects of different ages were investigated. These values were used for comparison with data on 30 Alport patients, and 10 Alport siblings. 10 patients with benign recurrent hematuria were also investigated. No marked differences were observed in the excretory rates between normals and patients.

Topics: Adolescent; Adult; Age Factors; Child; Humans; Hydroxylysine; Nephritis, Hereditary; Protein Binding; Sex Factors

Alport's syndrome probably is a molecular disorder of basement membrane composition. Investigation of urine on basement membrane components such as hydroxylysine and its glycosides, glucosylgalactosylhydroxylysine and galactosylhydroxylysine, may be helpful for diagnosis of the disease. Urinary specimens of 33 patients and 12 siblings were investigated, and the results were compared with those of 14 healthy adults and of 29 healthy children. The urine of patients with glomerulopathies, occurring during childhood (IgA nephropathy, benign recurrent hematuria, poststreptococcal glomerulonephritis, Henoch-Schönlein nephropathy, membranoproliferative glomerulonephritis, and nephrotic syndrome due to minimal lesions), was also investigated. No marked differences between normal and diseased subjects could be demonstrated, with respect to excretion of hydroxylysine and its glycosides, in contrast to data reported in the literature.

Topics: Adolescent; Adult; Child; Child, Preschool; Female; Glomerulonephritis; Glomerulonephritis, IGA; Hematuria; Humans; Hydroxylysine; Kidney Diseases; Male; Nephritis, Hereditary; Nephrotic Syndrome

The excretion of the two hydroxylysine glycosides hydroxylysine-galactose-glucose and hydroxylysine-galactose was studied in the urine of normal healthy and experimental rats chronically treated with collagen-like syndrome inductors, hydralazine or binazine. The elevated urinary hydroxylysine-galactose-glucose and hydroxylysine-galactose strongly suggests an altered rate of collagen degradation during induced collagen-like syndrome.

Topics: Animals; Collagen; Collagen Diseases; Glycosides; Hydralazine; Hydroxylysine; Male; Neutrophils; Rats; Rats, Inbred Strains; Syndrome; Todralazine

Topics: Chromatography, High Pressure Liquid; Collagen; Humans; Hydroxylysine

A new technique to evaluate the degradation of skin or bone collagen by measuring glucosylgalactosyl hydroxylysine and galactosyl hydroxylysine is presented. The method utilizes an automated amino acid analyzer. Eluents used are lithium buffers, and the color reagent is ninhydrin. Both glycosides elute in 3.5 h. Samples require minimum preparation. Urinary concentrations of both glycosides in ten patients with cervical spinal cord injuries of less than six months duration were higher than in five healthy controls. Proportional increases were different for each of the two glycosides. Variations in the proportional increase of each glycoside indicate different rates of degradation of skin and bone collagen. Repeated evaluations of the two urinary glycosides may help to predict whether patients are likely to develop skin- or bone-related clinical complications.

Topics: Adult; Amino Acids; Chromatography; Hexoses; Humans; Hydroxylysine; Male; Spinal Cord Injuries

Topics: Adult; Bone and Bones; Child; Collagen; Humans; Hydroxylysine; Hydroxyproline

Topics: Animals; Cadmium; Collagen; Female; Hydroxylysine; Hydroxyproline; Kidney; Rats; Rats, Inbred Strains

Members of a family with dominant epidermolysis bullosa simplex were found to have a deficiency of galactosylhydroxylysyl glucosyltransferase (GGT), an enzyme catalyzing the glucosylation of galactosylhydroxylysyl residues in the biosynthesis of collagen. The enzyme's activity was low in serum, skin tissue, and cultured skin fibroblasts, although no abnormality was found in three other intracellular enzymes of collagen biosynthesis. Mixtures of serum samples from patients and healthy controls gave the expected GGT activity, indicating that the low values were not due to inhibitors. GGT deficiency was accompanied by decreased product formation in vivo, as shown by a markedly decreased urinary excretion of glucosylgalactosylhydroxylysine. Six of 12 affected members had definite GGT deficiency, and five had some evidence suggestive of this abnormality; 13 of 15 unaffected members had no such manifestations. No similar GGT deficiency was found in three other families with the same disease. We conclude that GGT deficiency may be etiologically related to this disease in some families, but that different defects must be the cause in other cases.

Topics: Adolescent; Adult; Child; Collagen; Epidermolysis Bullosa; Female; Fibroblasts; Galactosides; Glucosyltransferases; Glycosides; Humans; Hydroxylysine; Male; Middle Aged; Skin

Topics: Adolescent; Basement Membrane; Child; Child, Preschool; Connective Tissue; Female; Glycosaminoglycans; Humans; Hydroxylysine; Hydroxyproline; Infant; Kidney Glomerulus; Male; Nephritis, Hereditary

A modified purification procedure, consisting of affinity chromatographies on concanavalin A-agarose, collagen-agarose and UDP-glucose-derivative-agarose and one gel filtration, is reported for galactosylhydroxylysyl glucosyltransferase. The enzyme obtained is entirely pure when studied by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. The enzyme protein was rich in glutamic acid + glutamine, aspartic acid + asparagine, glycine and alanine. The enzyme catalysed no significant glucose transfer to any of the glycoproteins tested, except for collagens. This included all the glycoproteins that have previously served as glucosyl acceptors for impure enzyme preparations, thus indicating a high degree of specificity of the enzyme for galactosylhydroxylysine. Galactosylsphingosine would act as a glucosyl acceptor, however. This compound has a close structural similarity to galactosylhydroxylysine in that they both have an unsubstituted amino group next to the hydroxy group to which the galactose is attached.

Topics: Amino Acids; Animals; Chick Embryo; Chromatography, Paper; Collagen; Galactosides; Glucosyltransferases; Glycoproteins; Hydroxylysine; Sphingosine; Substrate Specificity; Uridine Diphosphate Glucose

A rapid and specific assay has been developed for UDPglucose-collagen glucosyltransferase (UDPglucose: 5-hydroxylysine-collagen glucosyltransferase, EC 2.4.1.66) using galactosylhydroxylysine (Gal-Hyl) as acceptor. Studies with intact human platelets and isolated plasma membranes indicated that about 5--10% of the total activity was surface bound and the rest was of cytoplasmic origin. The two forms of the enzyme had similar broad pH optima (6.5--8.0), Km values for UDPglucose (5 muM) and Gal-Hyl (approx. 4 mM) and for optimal manganese concentrations (25 mM). The soluble form of the enzyme was purified 80-fold. The reaction mechanism was determined as being rapid equilibrium random BiBi + dead end complex or ordered BiBi with UDPglucose being the first substrate to bind. Using Gal-Hyl bound in purified alpha 1 chain of chick skin collagen, a Km value three orders of magnitude less (2 muM) was found than for free Gal-Hyl and the manganese requirement decreased to 2 mM. These results suggest that the binding to the enzyme of Gal-Hyl in the collagen molecule is enhanced by the presence of the protein portion so that the enzyme may be capable of recognizing not only the carbohydrate side chains but also the primary structure of collagen.

Topics: Animals; Blood Platelets; Cell Membrane; Chickens; Collagen; Cytoplasm; Glucosyltransferases; Glycosides; Humans; Hydrogen-Ion Concentration; Hydroxylysine; Kinetics; Manganese; Skin; Uridine Diphosphate Glucose

Topics: Animals; Chromatography, Gel; Chromatography, Ion Exchange; Galactose; Glucosyltransferases; Glycosides; Hydrolysis; Hydroxylysine; Porifera