Simultaneous free and glycosylated pyridinium crosslink determination in urine: validation of an HPLC-fluorescence method using a deoxypyridinoline homologue as internal standard.

Pyridinoline (Pyr), deoxypyridinoline (D-Pyr), galactosyl-pyridinoline (Gal-Pyr) and glucosyl-galactosyl pyridinoline (GluGal-Pyr) are enzymatic mature pyridinium crosslinks. Generally, only total Pyr and D-Pyr urinary amounts (free+bound forms) are evaluated by HPLC as indices of bone resorption. This report describes the validation of an HPLC-fluorescence method for the simultaneous evaluation of free Pyr and D-Pyr, together with GluGal-Pyr and Gal-Pyr, in urine of healthy women (n=20, aged 27-41) and girls (n=20, aged 5-10). The use of an unnatural D-Pyr homologue, here proposed for the first time as internal standard, and of pure Pyr, D-Pyr, GluGal-Pyr and Gal-Pyr synthesized to be used as primary calibrators, guarantees method specificity and correct crosslink quantification. Urine, spiked with IS, was solid-phase extracted prior to HPLC analysis. Total Pyr and D-Pyr amounts were also evaluated after urine hydrolysis. The HPLC method was validated for selectivity, sensitivity, linearity, precision, accuracy, recovery and stability for all measured crosslinks. Both free and total Pyr and D-Pyr as well as GluGal-Pyr and Gal-Pyr amounts were significantly higher in girls than in women (p<0.0001), indicating an increased collagen turnover rather than only bone turnover. Gal-Pyr, for the first time evaluated in girls, was under its lower quantification limit (

Novel biomarkers of 3-chloro-1,2-propanediol exposure by ultra performance liquid chromatography/mass spectrometry based metabonomic analysis of rat urine.

To select early, sensitive biomarkers of 3-chloro-1,2-propanediol (3-MCPD) exposure, a single dose of 30 mg/kg/day 3-MCPD was administered to male Wistar rats for 40 days. Significant elevations of serum creatinine and blood urea nitrogen concentrations were observed on day 40, and urine N-acetyl-beta-D-glucosaminidase and beta-galactosidase (beta-Gal) activities were observed on day 20. Slight renal tubule hydropic degeneration and spermatozoa decreases were observed on day 10. The endogenous metabolite profile of rat urine was investigated by ultra performance liquid chromatography/mass spectrometry with electrospray ionization (ESI). Principal component analysis and partial least-squares enabled clusters to be visualized, with a trend of clustering on day 10 in ESI- and the greatest differences on days 30 and 40. Galactosylglycerol, a marker contributing to the clusters, which had earlier variations than conventional biomarkers and the most significant elevations as compared to other novel biomarkers, was first considered to be an early, sensitive biomarker in evaluating the effect of 3-MCPD exposure. The identification of galactosylglycerol was carried out by beta-Gal catalysis, and the possible mechanism of urine galactosylglycerol variation was elucidated.

Urinary excretion of the main anthocyanin in lingonberry (Vaccinium vitis-idaea), cyanidin 3-O-galactoside, and its metabolites.

In vitro trials have indicated various potential health effects of lingonberries ( Vaccinium vitis-idaea L.). Most of these studies have been performed with berry extract or juice, and the detailed chemical structures of active compounds in these products have not been elucidated. Lingonberry contains cyanidin-3-galactoside as its main anthocyanin. Absorption and metabolism of the compound is not fully understood, and no studies of anthocyanin metabolism have been performed with lingonberries. The aim of this study was to investigate the urinary excretion of cyanidin-3-galactoside and its metabolites in young and healthy subjects receiving a breakfast containing 300 g of lingonberries. A fast, selective, and sensitive ultra-high-performance liquid chromatography-tandem mass spectrometric (uHPLC-MS/MS) method was optimized for the analysis of the anthocyanin metabolites in urine. Both intact cyanidin-3-galactoside and its methylated and glucuronidated metabolites were identified from urine samples. The two metabolites represented >50% of cyanidin excreted in urine. Maximal excretion appeared between 4 and 8 h after the meal. Also, the compounds were absorbed more slowly than reported previously in several studies.

Identification of a novel conjugate in human urine: bile acid acyl galactosides.

We report a novel conjugate, bile acid acyl galactosides, which exist in the urine of healthy volunteers. To identify the two unknown peaks obtained in urine specimens from healthy subjects, the specimens were subjected to solid phase extraction and then to liquid chromatographic separation. The eluate corresponding to the unknown peaks on the chromatogram was collected. Following alkaline hydrolysis and liquid chromatography (LC)/electrospray ionization (ESI)-mass spectrometric (MS) analysis, cholic acid (CA) and deoxycholic acid (DCA) were identified as liberated bile acids. When a portion of the alkaline hydrolyzate was subjected to a derivatization reaction with 1-phenyl-3-methyl-5-pyrazolone, a derivative of galactose was detected by LC/ESI-MS. Finally, the liquid chromatographic and mass spectrometric properties of these unknown compounds in urine specimens were compared to those of authentic specimens and the structures were confirmed as CA 24-galactoside and DCA 24-galactoside. These results strongly imply that bile acid 24-galactosides, a novel conjugate, were synthesized in the human body.

Effects of ibuprofen on molecular markers of cartilage and synovium turnover in patients with knee osteoarthritis.

OBJECTIVE: The aim of this study was to evaluate the effect of ibuprofen on the urinary excretion of C-terminal crosslinking telopeptide of type II collagen (CTX-II) and urinary glucosyl galactosyl pyridinoline (Glc-Gal-PYD), two new molecular markers of cartilage and synovial tissue metabolism, respectively, in patients with knee osteoarthritis (OA). METHODS: We studied 201 patients with knee pain and radiographic evidence of knee OA who were on treatment with non-steroidal anti-inflammatory drugs (NSAIDs) prior to study initiation. After an initial screening visit, patients were withdrawn from their pre-study NSAID and, following a flare of their OA symptoms, were randomised to ibuprofen (2400 mg/day) or placebo. Urinary CTX-II and Glc-Gal-PYD levels were measured at time of randomisation (baseline) and after 4-6 weeks of treatment. RESULTS: After 4 to 6 weeks, urinary CTX-II (+17%, p = 0.023) and Glc-Gal-PYD (+10%, p = 0.020) increased significantly from baseline in the placebo group whereas marginal or no increase was observed in the ibuprofen group (CTX-II +2%, NS and Glc-Gal-PYD +4%, p = 0.045). For urinary CTX-II, the difference in the change from baseline between placebo and ibuprofen treated groups was significant (13%, p = 0.017). At baseline, urinary levels of CTX-II and Glc-Gal-PYD were higher in patients with knee swelling (n = 127) than in those without (n = 74) (p<0.02 for both markers). When patients were stratified according to presence or absence of knee swelling at baseline, the increases over 4-6 weeks of urinary CTX-II and Glc-Gal-PYD in the placebo group were restricted to patients with knee swelling (+22% from baseline, p = 0.001 and +12%, p = 0.011, for urinary CTX-II and Glc-Gal-PYD respectively). In patients with knee swelling who were treated with ibuprofen this increase was not observed and the difference from placebo was significant for urinary CTX-II (p = 0.014). CONCLUSION: In patients with a flare of knee OA, specifically in patients with evidence of joint inflammation documented by knee swelling, there was a significant increase in markers reflecting cartilage and synovium metabolism that could partly be prevented by high doses of ibuprofen. These data suggest that patients with a flare of knee OA are characterised by increased cartilage and synovial tissue degradation, which may be partly prevented by high doses of NSAIDs.

Association of baseline levels of urinary glucosyl-galactosyl-pyridinoline and type II collagen C-telopeptide with progression of joint destruction in patients with early rheumatoid arthritis.

OBJECTIVE: To evaluate whether measurements of urinary glucosyl-galactosyl-pyridinoline (Glc-Gal-PYD) and urinary C-terminal crosslinking telopeptide of type II collagen (CTX-II), 2 new markers of destruction of the synovium and cartilage collagen breakdown, respectively, are associated with the progression of joint damage in patients with early rheumatoid arthritis (RA), and to compare this association with that with serum matrix metalloproteinase 3 (MMP-3), a proteinase expressed by synovial tissue and chondrocytes, and that with serum C-reactive protein (CRP), an index of systemic inflammation. METHODS: The prospective study cohort comprised 116 patients with early RA who were part of a large, double-blind, randomized study comparing the efficacy of etanercept and methotrexate. The relationship between baseline levels of urinary Glc-Gal-PYD, urinary CTX-II, and serum MMP-3 and the progression of joint destruction, as measured by changes in the modified Sharp score (average findings of 2 independent readers) over 1 year, was investigated. RESULTS: Levels of urinary Glc-Gal-PYD (+70%), urinary CTX-II (+104%), and serum MMP-3 (+219%) were elevated compared with the levels in 76 healthy controls. The baseline levels of Glc-Gal-PYD (r = 0.30), CTX-II (r = 0.25), and MMP-3 (r = 0.29) correlated with the changes over 1 year in the total Sharp score (joint space narrowing and bone erosion). Patients with baseline levels of Glc-Gal-PYD, CTX-II, and MMP-3 that were higher than the mean + 2 SD in healthy controls had a significantly greater progression of joint damage, with an increase in the total Sharp score over 1 year that was from 3- to 8-fold higher than that in patients with low baseline levels of these markers. Moreover, patients with these higher levels of Glc-Gal-PYD, CTX-II, and MMP-3 had a higher risk of progression of the disease (increase in total Sharp score > or =0.5 units) than did the other patients (relative risks and 95% confidence intervals [95% CI] 3.3 [95% CI 1.5-7.4], 2.5 [95% CI 1.1-5.7], and 2.5 [95% CI 1.1-5.6], respectively). The baseline serum level of CRP was not significantly associated with the progression of joint damage. Adjustment of the levels of Glc-Gal-PYD, CTX-II, and MMP-3 according to radiologic damage at baseline did not alter their association with progression. After adjustment for serum CRP, the relative risk slightly decreased, but remained significant, for Glc-Gal-PYD (2.6 [95% CI 1.1-6.3]). Patients with both increased levels of the molecular markers and radiologic damage at baseline had a higher risk of progression of joint damage than did those with either high molecular marker levels or radiologic damage. CONCLUSION: High baseline levels of Glc-Gal-PYD, CTX-II, and MMP-3 are associated with increased risk of progression of joint destruction over 1 year in early RA. The association between baseline levels of urinary Glc-Gal-PYD and progression of joint erosion was independent of the severity of radiologic damage and inflammation at baseline. Combining the measurements of these molecular markers with radiologic assessment of joint damage may be useful for identifying patients with RA who are at high risk of rapid progression and for whom early aggressive treatment would be beneficial.

Cross sectional evaluation of biochemical markers of bone, cartilage, and synovial tissue metabolism in patients with knee osteoarthritis: relations with disease activity and joint damage.

OBJECTIVE: To analyse the relations between the urinary levels of type II collagen C-telopeptide (CTX-II) and glucosyl-galactosyl pyridinoline (Glc-Gal-PYD)-two newly developed biochemical markers of type II collagen and synovial tissue destruction respectively-disease activity and the severity of joint destruction in patients with knee osteoarthritis (OA). The clinical performance of these two new markers was compared with that of a panel of other established biochemical markers of connective tissue metabolism. METHODS: The following biochemical markers were measured in a group of 67 patients with knee OA (mean age 64 years, median disease duration eight years ) and in 67 healthy controls: for bone, serum osteocalcin, serum and urinary C-telopeptide of type I collagen (CTX-I); for cartilage, urinary CTX-II, serum cartilage oligomeric matrix protein (COMP), and serum human cartilage glycoprotein 39 (YKL-40); for synovium, urinary Glc-Gal-PYD, serum type III collagen N-propeptide (PIIINP), serum hyaluronic acid (HA); and for inflammation, serum C reactive protein. Biochemical markers were correlated with pain and physical function (WOMAC index) and with quantitative radiographic evaluation of the joint space using the posteroanterior view of the knees flexed at 30 degrees. RESULTS: All bone turnover markers were decreased in patients with knee OA compared with controls (-36%, -38%, and -52%, p<0.0001 for serum osteocalcin, serum CTX-I and urinary CTX-I, respectively). Serum COMP (+16%, p=0.0004), urinary CTX-II (+25%, p=0.0009), urinary Glc-Gal-PYD (+18%, p=0.028), serum PIIINP (+33%, p<0.0001), and serum HA (+ 233%, p<0.0001) were increased. By univariate analyses, increased urinary Glc-Gal-PYD (r=0.41, p=0.002) and decreased serum osteocalcin (r=-0.30, p=0.025) were associated with a higher total WOMAC index. Increased urinary CTX-II (r=-0.40, p=0.0002) and Glc-Gal-PYD (r=-0.30, p=0.0046) and serum PIIINP (r=-0.29, p=0.0034) were the only markers which correlated with joint surface area. By multivariate analyses, urinary Glc-Gal-PYD and CTX-II were the most important predictors of the WOMAC index and joint damage, respectively. CONCLUSION: Knee OA appears to be characterised by a systemic decrease of bone turnover and increased cartilage and synovial tissue turnover. CTX-II, Glc-Gal-PYD, and PIIINP may be useful markers of disease severity in patients with knee OA.

Urinary excretion of glucosyl-galactosyl pyridinoline: a specific biochemical marker of synovium degradation.

OBJECTIVE: Glucosyl-galactosyl pyridinoline (Glc-Gal-PYD), which has been identified in urine, is a glycosylated analogue of pyridinoline. The tissue distribution of this molecule has not been yet determined and its utility as a potential biochemical marker of joint degradation in patients with joint diseases has not been investigated. METHODS AND RESULTS: In this study, we demonstrate that Glc-Gal-PYD is abundant in human synovium tissue, absent from bone and present in minute amounts in cartilage and other soft tissues, such as muscle and liver. Using an ex vivo model of human joint tissue degradation, we found that Glc-Gal-PYD is released from synovium tissue, but not from bone and cartilage. The urinary level of Glc-Gal-PYD was increased by 109% in patients with rheumatoid arthritis (RA) compared with healthy adults, but was normal in patients with Paget's disease of bone. In addition, Glc-Gal-PYD was higher in those patients with destructive disease, as assessed by X-rays of the joints, than in those with non-destructive RA. CONCLUSION: Glc-Gal-PYD may be useful for the clinical investigation of patients with joint disease.

Abnormal galactoside excretion in urine of a patient with early myoclonic epileptic encephalopathy.

An abnormal carbohydrate pattern was found in urine of a patient with early myoclonic epileptic encephalopathy. Three major oligosaccharides have been isolated from the urine; structural studies including sugar analyses, methylation procedure and enzymatic hydrolysis allow us to propose the following structures: beta-Gal-(1 leads to 3)-Gal beta-Gal-(1 leads to 3)-beta-Gal-(1 leads to 3)-Glc beta-Gal-(1 leads to 3)-beta-Gal-(1 leads to 3)-Gal Such oligosaccharide structures have not previously been described in any biological fluid. The origin of these compounds, and the possibility of a specific metabolic defect are discussed.

Deficiency of galactosylhydroxylysyl glucosyltransferase, an enzyme of collagen synthesis, in a family with dominant epidermolysis bullosa simplex.

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.

Urinary excretion of hydroxylysyl glycosides during acute inflammation in the rat.

1. Subcutaneous inflammatory granuloma were induced in young rats and the urinary excretion of hydroxyproline and hydroxylysyl glycosides was observed during the period of acute inflammation. 2. All collagen metabolites were increased in the urine and excretion of glucosyl-galactosyl-hydroxylysine was much greater than excretion of galactosyl-hydroxylysine in the first days. 3. It is argued that urinary glucosyl-galactosyl-hydroxylysine is probably derived from hydroxylysyl residues of soluble collagen. 4. This study affords new arguments in favour of the dermal origin of urinary glucosyl-galactosyl-hydroxylysine, at least in skin inflammation.

Contribution of kidney basement membranes and collagen to the urinary excretion of hydroxylysyl glycosides.

Glucosyl-galactosyl-hydroxylysine and galactosyl-hydroxylysine are two substances present only in collagen and in basement membranes. The purpose of this study is to estimate to what extent the kidney contributes to urinary hydroxylysyl glycoside excretion. "In vitro" perfusion of isolated kidneys allows the measurement of hydroxylysyl glycosides independently of the important amount of interstitial collagen present in the whole body. It is shown that the kidney parenchyma contributes no more than 10 percent of the glycosylated hydroxylysine excreted in urine. GBM is not the only renal source of glucosyl-galactosyl-hydroxylysine in urine. Tubular basement membrane and intersitial collagen of kidney may contribute to the excretion by the isolated organ.