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Objectives: Patients with Ehlers-Danlos syndrome were described to contain reduced activities of beta4-galactosyltransferase-7 (beta4Gal-T7). Therefore, measurement of beta4Gal-T7 activity can help to characterize defects in proteoglycan biosynthesis in patients with connective tissue diseases., Design and Methods: We developed a sensitive and specific method to assay beta4Gal-T7 which is based on the transfer of galactose from UDP-galactose to the synthetic peptide Bio-BIK-F-Xyl., Results: Calibration curves exhibited consistent linearity in the range of 10-2000 microg/L Bio-BIK-F-Xyl-Gal, corresponding to a beta4Gal-T7 activity of 3.5-659 microU/L. The limit of detection and the lower limit of quantification were 3.70 microg/L (1.22 microU/L) and 4.50 microg/L Bio-BIK-F-Xyl-Gal (1.48 microU/L beta4Gal-T7 activity), respectively. Interassay imprecision (CV) was 8.1-13.1% in the range from 15.9 to 659 microU/L, and mean recovery was 85.3% (range 61.7-106.3%)., Conclusions: This sensitive, robust and interference-free LC-MS/MS assay allows an accurate determination of beta4Gal-T7 activity in human body fluids.

Objectives: Mycophenolic acid (MPA), the active metabolite of the ester prodrug mycophenolate mofetil is an immunosuppressant which selectively inhibits inosine-monophosphate dehydrogenase. The requirement for therapeutic drug monitoring shown in previous studies raises the necessity of acquiring accurate and sensitive methods to measure MPA and also its metabolite mycophenolic acid glucuronide (MPAG)., Design and Methods: We developed a robust, rapid, sensitive and highly specific HPLC-electrospray ionization mass spectrometry method to assay MPA and its metabolite MPAG in human plasma and serum. Ion suppression was investigated by a post column infusion experiment., Results: Determination of MPA and MPAG were performed during a 3.0-min run time. Multiple calibration curves for the analysis of MPA and MPAG exhibited consistent linearity and reproducibility in the range of 0.05 to 100 mg/L (r>0.999) and 6 to 400 mg/L r>0.998, respectively. Limits of detection were 0.009 mg/L for MPA and 4.5 mg/L for MPAG and lower limits of quantification were 0.011 mg/L for MPA and 4.9 mg/L for MPAG. Interassay imprecision was <6.0% for both substances. Mean recovery was 48.9% (range 43.3-60.0%) for MPA and 112.2% (range 95.0-127.0%) for MPAG. Agreement was relatively good for MPA (n=122) between the presented method and a validated ELISA method (Viva analyzer, Siemens Medicals Solutions Diagnostics, NY). The Passing-Bablok regression line was: EMIT=0.91 (LC-MS/MS)+0.17 [mg/L]; r=0.97., Conclusions: This simple, robust and interference-free LC-MS/MS assay allows the rapid and accurate determination of MPA and MPAG in human plasma and other body fluids.

Objectives: The xylosyltransferases I and II (XT-I, XT-II, EC 2.4.2.26) are the chain-initiating enzymes in the biosynthesis of glycosaminoglycans (GAGs). This is the first investigation of changes in the serum GAG amount and composition in association with polymorphisms in XYLT1 and XYLT2., Design and Methods: Genotyping of three genetic variations in the genes XYLT1 and XYLT2 was performed in 223 healthy blood donor samples. Serum samples were analyzed for their GAG Delta-disaccharide content by reversed-phase high-performance liquid chromatography (RP-HPLC). Furthermore serum XT activity was determined by a radiochemical assay., Results: The single nucleotide polymorphism (SNP) c.343G>T in XYLT1 exon 1 correlated with a significantly decreased GAG content in the serum (p<0.01). For the other two investigated XYLT2 variations (c.166G>A in exon 2 and c.1253C>T in exon 6) no changes in the serum GAG amount were detected. No investigated SNPs were associated with changes to serum XT activities., Conclusions: The XYLT1 SNP c.343G>T is associated with a decreased GAG amount in the serum of healthy blood donors.

Objectives: Pseudoxanthoma elasticum (PXE) is a hereditary disorder predominantly affecting the skin, retina and vascular system. The aim of this study was to measure cell adhesion molecules in PXE patients., Design and Methods: Soluble P-, E- and L-selectins were measured in 61 non-consanguineous PXE patients. The distribution of the variants E-selectin S128R and P-selectin T715R were determined., Results: P-selectin concentrations were significantly increased in male and female PXE patients. Furthermore, P-selectin levels correlated with the ABCC6 gene status of the PXE patients. Patients harboring two mutant ABCC6 alleles had 1.5-fold increased P-selectin concentrations in comparison to patients with at least one wild-type allele. E- and L-selectin levels were within normal range and the allelic frequencies of the investigated polymorphisms did not differ between patients and age- and sex-matched controls., Conclusions: Our data show elevated P-selectin levels in PXE patients potentially due to oxidative stress and elevated protease activity in PXE.

Objectives: Recent studies have revealed the involvement of calcification inhibitory proteins in the pathogenesis of pseudoxanthoma elasticum (PXE)., Design and Methods: We analyzed serum concentrations of the calcification inhibitor matrix Gla protein (MGP) in a large cohort of patients suffering from PXE (n=101), 34 first-degree relatives and 67 healthy controls. Moreover, we determined the distribution of the two MGP promoter polymorphisms c.-7G>A and c.-138T>C in the three cohorts., Results: We found significantly lower total MGP concentrations in the sera of PXE patients compared to healthy controls (p=0.0002). Furthermore, higher serum MGP concentrations could be correlated with a later PXE onset. Analysis of MGP promoter polymorphism frequencies revealed one MGP haplotype to be a potential protective co-factor in PXE., Conclusions: Our findings point to a role of the local calcification inhibitor MGP in PXE manifestation.

Objectives: Increased total homocysteine (tHcy) level is an independent risk factor for atherosclerosis and cardiovascular disease. Here, we describe a rapid tHcy micro-scale assay., Methods: We developed an easy sample preparation and rapid liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the measurement of tHcy in human serum and plasma, respectively., Results: The tHcy assay was linear up to 61.6 micromol/L, the detection limit was 1.0 micromol/L, the lower limit of quantification was 1.8 micromol/L, the imprecision in the range of 9.30-25.9 micromol/L was less than 5.9% and the analytical recovery was 94.7 +/- 6.3%., Conclusions: Our novel tHcy LC-MS/MS assay is a quick, precise and robust method for tHcy determination in routine diagnostics.

Objectives: Hyperhomocysteinemia is a risk factor for cardiovascular diseases, while tissue factor pathway inhibitor (TFPI) regulates the extrinsic pathway of blood coagulation. We investigated the in vivo influence of elevated homocysteine concentrations on TFPI levels., Methods: Total TFPI levels of 82 hyperhomocysteinemic patients were determined., Results: The patients' TFPI levels were significantly elevated compared to blood donors, with a more pronounced effect in females than in males., Conclusions: We have shown a gender-dependent TFPI elevation in hyperhomocysteinemic patients.

Objectives: Serum xylosyltransferase I (XT-I) is a marker for the determination of tissue remodeling in systemic sclerosis. Here, we investigated whether renal insufficiency affects XT-I levels in blood., Methods: We measured serum XT-I activity in 236 patients with different serum creatinine levels., Results: XT-I activities in cohorts with increased creatinine levels were not significantly altered compared to controls., Conclusions: Serum XT-I activity is applicable as a fibrosis marker independent from renal function.