hexanoylcarnitine and propionylcarnitine

We applied untargeted mass spectrometry-based metabolomics to the diseases methylmalonic acidemia (MMA) and propionic acidemia (PA).. We used a screening platform that used untargeted, mass-based metabolomics of methanol-extracted plasma to find significantly different molecular features in human plasma samples from MMA and PA patients and from healthy individuals. Capillary reverse phase liquid chromatography (4 microL/min) was interfaced to a TOF mass spectrometer, and data were processed using nonlinear alignment software (XCMS) and an online database (METLIN) to find and identify metabolites differentially regulated in disease.. Of the approximately 3500 features measured, propionyl carnitine was easily identified as the best biomarker of disease (P value 1.3 x 10(-18)), demonstrating the proof-of-concept use of untargeted metabolomics in clinical chemistry discovery. Five additional acylcarnitine metabolites showed significant differentiation between plasma from patients and healthy individuals, and gamma-butyrobetaine was highly increased in a subset of patients. Two acylcarnitine metabolites and numerous unidentified species differentiate MMA and PA. Many metabolites that do not appear in any public database, and that remain unidentified, varied significantly between normal, MMA, and PA, underscoring the complex downstream metabolic effects resulting from the defect in a single enzyme.. This proof-of-concept study demonstrates that metabolomics can expand the range of metabolites associated with human disease and shows that this method may be useful for disease diagnosis and patient clinical evaluation.

Topics: Acetylcarnitine; Adult; Amino Acid Metabolism, Inborn Errors; Betaine; Biomarkers; Carnitine; Child; Chromatography, High Pressure Liquid; Humans; Methylmalonic Acid; Plasma; Propionates; Spectrometry, Mass, Electrospray Ionization

A sensitive method of continuous on-line radio-high performance liquid chromatography (HPLC) was used to detect the specific radio-labelled acyl-carnitine esters derived from the oxidation of [U-14C]3-methyl-2-oxopentanoate by rat liver and muscle mitochondrial fractions. The recoveries of carnitine, acetyl-carnitine, propionyl-carnitine, 2-methylbutyryl-carnitine, and hexanoyl-carnitine were 98.7% (+/- 5.4; SEM, n = 3), 91.4% (+/- 7.6), 89.4% (+/- 5.2), 84.6% (+/- 6.8), and 87.9% (+/- 7.8), respectively, from quenched mitochondrial incubations. This method demonstrated that rat liver and muscle mitochondria generate acetyl-carnitine, propionyl-carnitine and 2-methylbutyryl-carnitine when incubated with [U-14C]3-methyl-2-oxopentanoate in the presence of carnitine. The production of acetyl-carnitine was almost similar in the 2 tissues. Muscle mitochondria produced higher amounts of propionyl-carnitine and 2-methylbutyryl-carnitine than liver mitochondria. These observations suggest a limited utilization of propionyl-CoA by muscle mitochondria which, through a mechanism of feed-back inhibition, may have contributed to the accumulation of 2-methylbutyryl-CoA. This study provides further evidence for the importance of carnitine in the modulation of the mitochondrial [acyl-CoA/[CoA] pool.

Topics: Acetylcarnitine; Amino Acids, Branched-Chain; Animals; Carnitine; Chromatography, High Pressure Liquid; Keto Acids; Kinetics; Male; Mitochondria, Liver; Mitochondria, Muscle; Oxidation-Reduction; Rats; Rats, Wistar

This paper describes a method for the quantitative determination of free carnitine, acetylcarnitine, propionylcarnitine, hexanoylcarnitine, octanoylcarnitine, and total carnitine in plasma. Carnitine and acylcarnitines were extracted from 100 microliters of plasma with acetonitrile/methanol and isolated using 0.5-ml columns of silica gel. Samples were then derivatized with 4'-bromophenacyl trifluoromethanesulfonate and quantified by high-performance liquid chromatography with detection at 260 nm. Carnitine and acylcarnitines were quantified in normal human plasma and the plasma of patients diagnosed with methylmalonic aciduria, propionic acidemia, and medium-chain acyl-CoA dehydrogenase deficiency.

Topics: Acetylcarnitine; Acyl-CoA Dehydrogenase; Acyl-CoA Dehydrogenases; Acylation; Carnitine; Chromatography, High Pressure Liquid; Humans; Methylmalonic Acid; Molecular Weight; Propionates; Reference Standards; Reproducibility of Results