A novel method for quantitation of acylglycines in human dried blood spots by UPLC-tandem mass spectrometry.

BACKGROUND: Several acylcarnitines used as primary markers on dried blood filter papers (DBS) for newborn screening lack specificity and contribute to a higher false positive rate. The analysis of urine acylglycines is useful in the diagnosis of inborn errors of metabolism (IEM) including medium chain acyl-CoA dehydrogenase deficiency (MCADD), isovaleric acidemia, and beta-ketothiolase deficiency (BKTD). Currently, no method for analyzing acylglycines from DBS has been published. METHODS: Acylglycines were extracted from two 3.2 mm DBS punches and butylated using Butanol-HCl. Ultra Performance Liquid Chromatography (UPLC-MS/MS) with run time of 10 min permits resolution and quantitation of 15 acylglycines; including several isobaric. Method development was completed. Reference intervals (n = 573) were established for four birth weight groups. Furthermore, samples from patients with a confirmed IEM (n = 11), and false positive screens (n = 78) were analyzed to validate the interpretation obtained from the newly established reference intervals. RESULTS: Calibration curves were linear from 0.005 to 25.0 µM. Ion suppression was evaluated as minimal (2 to 10%). Samples from known patients were used to validate the reference intervals. For C5OH-related disorders, tiglylglycine (TG), TG/acetylglycine (AG) ratio, 3methylcrotonylglycine (3MCG) and 3MCG/AG ratio increased specificity. Propionylglycine (PG) and PG/acetylglycine ratio were two discriminatory markers in the investigation of C3-related disorders. Hexanoylglycine (HG), octanoylglycine (OG), suberylglycine (SG), and the ratios HG/AG, OC/AG and SG/AG were excellent markers of MCADD deficiency. CONCLUSION: This method shows potential application as a second tier screen in order to reduce the false positive rate for a number of IEM targeted by newborn screening.

Pyruvate uptake is inhibited by valproic acid and metabolites in mitochondrial membranes.

The pyruvate uptake rate in inverted submitochondrial vesicles prepared from rat liver was optimized and further characterized; the potential inhibitory effects of the anticonvulsive drug valproic acid or 2-n-propyl-pentanoic acid (VPA), Delta4-valproic acid or 2-n-propyl-4-pentenoic acid and the respective coenzyme A (CoA) conjugates were studied in the presence of a proton gradient. All tested VPA metabolites inhibited the pyruvate uptake, but the CoA esters were stronger inhibitors (40% and 60% inhibition, respectively, for valproyl-CoA and Delta4-valproyl-CoA, at 1mM). At the same concentration, the specific inhibitor 2-cyano-4-hydroxycinnamate decreased the pyruvate uptake rate by 70%. The reported inhibition of the mitochondrial pyruvate uptake may explain the significant impairment of the pyruvate-driven oxidative phosphorylation induced by VPA.

Studies on the extra-mitochondrial CoA -ester formation of valproic and Delta4 -valproic acids.

The hypothesis whether valproic acid (VPA) and its main microsomal metabolite, Delta(4)-valproic acid, can be activated to the respective CoA esters in the cell cytosol was investigated. The valproyl-CoA formation was measured in different subcellular fractions obtained by differential centrifugation of liver homogenates of rats treated with VPA (studies ex vivo) and digitonin fractionation of rat hepatocytes incubated with VPA and cofactors (studies in vitro). The results show that VPA activation may occur in the cytosol and is not restricted to the mitochondrial matrix as believed until now. Furthermore, the activation of Delta(4)-VPA is demonstrated in vitro. Valproyl-CoA and Delta(4)-valproyl-CoA were detected after in vitro incubations and the former also in the mitochondrial and cytosolic fractions obtained from liver cells of treated rats. The activation to valproyl-CoA was characterized in cytosolic fractions, optimized with respect to time and protein and the kinetic constants (K(m)(app)) were estimated for the reaction substrates. Other medium-chain fatty acids decreased the formation of valproyl-CoA suggesting a competition for both mitochondrial and extra-mitochondrial VPA activating enzymes. The present findings suggest additional mechanisms of mitochondrial dysfunction associated with VPA, and they may contribute to the further understanding of the toxic effects associated with this drug.

Metabolism of gamma-hydroxybutyrate to d-2-hydroxyglutarate in mammals: further evidence for d-2-hydroxyglutarate transhydrogenase.

gamma-Hydroxybutyratic acid (GHB), and its prodrugs 4-butyrolactone and 1,4-butanediol, represent expanding drugs of abuse, although GHB is also used therapeutically to treat narcolepsy and alcoholism. Thus, the pathway by which GHB is metabolized is of importance. The goal of the current study was to examine GHB metabolism in mice with targeted ablation of the GABA degradative enzyme succinic semialdehyde dehydrogenase (SSADH(-/-) mice), in whom GHB persistently accumulates, and in baboons intragastrically administered with GHB immediately and persistently. Three hypotheses concerning GHB metabolism were tested: (1) degradation via mitochondrial fatty acid beta-oxidation; (2) conversion to 4,5-dihydroxyhexanoic acid (a putative condensation product of the GHB derivative succinic semialdehyde); and (3) conversion to d-2-hydroxyglutaric acid (d-2-HG) catalyzed by d-2-hydroxyglutarate transhydrogenase (a reaction previously documented only in rat). Both d-2-HG and 4,5-dihydroxyhexanoic acid were significantly increased in neural and nonneural tissue extracts derived from SSADH(-/-) mice. In vitro studies demonstrated the ability of 4,5-dihydroxyhexanoic acid to displace the GHB receptor ligand NCS-382 (IC(50) = 38 micromol/L), although not affecting GABA(B) receptor binding. Blood and urine derived from baboons administered with GHB also accumulated d-2-HG, but not 4,5-dihydroxyhexanoic acid. Our results indicate that d-2-HG is a prominent GHB metabolite and provide further evidence for the existence of d-2-hydroxyglutarate transhydrogenase in different mammalian species.

Measurement of Nepsilon-(carboxymethyl)lysine and Nepsilon-(carboxyethyl)lysine in human plasma protein by stable-isotope-dilution tandem mass spectrometry.

BACKGROUND: N(epsilon)-(Carboxymethyl)lysine (CML) and N(epsilon)-(carboxyethyl)lysine (CEL) are two stable, nonenzymatic chemical modifications of protein lysine residues resulting from glycation and oxidation reactions. We developed a tandem mass spectrometric method for their simultaneous measurement in hydrolysates of plasma proteins. METHODS: CML and CEL were liberated from plasma proteins by acid hydrolysis after addition of deuterated CML and CEL as internal standards. Chromatographic separation was performed by gradient-elution reversed-phase chromatography with a mobile phase containing 5 mmol/L nonafluoropentanoic acid as ion-pairing agent. Mass transitions of 205.1-->84.1 and 219.1-->84.1 for CML and CEL, respectively, and 209.1-->88.1 and 223.1-->88.1 for their respective internal standards were monitored in positive-ion mode. RESULTS: CML and CEL were separated with baseline resolution with a total analysis time of 21 min. The lower limit of quantification was 0.02 micromol/L for both compounds. Mean recoveries from plasma samples to which CML and CEL had been added were 92% for CML and 98% for CEL. Within-day CVs were <7.2% for CML and <8.2% for CEL, and between-day CVs were <8.5% for CML and <9.0% for CEL. In healthy individuals (n = 10), mean (SD) plasma concentrations of CML and CEL were 2.80 (0.40) micromol/L (range, 2.1-3.4 micromol/L) and 0.82 (0.21) micromol/L (range, 0.5-1.2 micromol/L), respectively. In hemodialysis (n = 17) and peritoneal dialysis (n = 9) patients, plasma concentrations of CML and CEL were increased two- to threefold compared with controls, without significant differences between dialysis modes [7.26 (1.36) vs 8.01 (3.80) micromol/L (P = 0.89) for CML, and 1.84 (0.39) vs 1.71 (0.42) micromol/L (P = 0.53) for CEL]. CONCLUSIONS: This stable-isotope-dilution tandem mass spectrometry method is suitable for simultaneous analysis of CML and CEL in hydrolysates of plasma proteins. Its robustness makes it suitable for assessing the value of these compounds as biomarkers of oxidative stress resulting from sugar and lipid oxidation.