Artefactual pyruvate and 2-oxobutyrate produced by trimethylsilylation of methylmalonic and ethylmalonic acids in the presence of oxygen
Trimethylsilylation of methylmalonic and ethylmalonic acids in the presence of headspace atmospheric oxygen is shown to produce the trimethylsilyl derivatives of pyruvic and 2-oxobutyric acids, along with 2-hydroxy-2-methylmalonic and 2-hydroxy-2-ethylmalonic acids, respectively. This may lead to overestimation of these keto acids, if they were not oximated in the original sample, and the mistaken reporting of the 2-hydroxymalonates.
Experiment-originated variations, and multi-peak and multi-origination phenomena in derivatization-based GC-MS metabolomics
Gas chromatography combined with mass spectrometry (GC-MS), in particular derivatization-based GC-MS, is one of the most commonly used analytical methods in metabolomics. However, experiment-derived variations and originations of chromatographic peaks are two fundamental issues that could influence interpretation of metabolic pathways. Here, we focus on some pitfalls and limitations, especially the commonly ignored multi-origination and multi-peak phenomena in derivatization-based GC-MS metabolomics. We use examples to illustrate that ignoring these two factors could lead to wrong interpretations of results and serious metabolic implications. We also discuss strategies to overcome some of these confounding factors.
TRANSAMINATIONS BETWEEN AMINO ACIDS AND KETO ACIDS ELEVATED IN PHENYLKETONURIA AND MAPLE SYRUP URINE DISEASE
Abstract— The transamination between amino acids and aliphatic and aromatic keto acids has been investigated in homogenates of human and rat brain. Tryptophan, phenylalanine and 3,4‐dihydroxyphenylalanine (DOPA) at concentrations of 3.6 min and below trans‐aminated aromatic keto acids more rapidly than α‐ketoglutarate; lower Km values were found for tryptophan and phenylalanine in the presence of the aromatic keto acid. Rat brain and liver arninotransferases exhibited similar affinities for tryptophan in the presence of different keto acids. Branched chain keto acids were also acceptors of the amino groups of tryptophan and DOPA. In brain homogenates α‐ketoglutarate and p‐hydroxyphenyl‐pyruvate were transaminated by tyrosine and 5‐hydroxytryptophan at about equal rates, whereas a‐ketoglutarate was transaminated more rapidly with aliphatic amino acids. At concentrations of 1.6 m DOPA and 0.8 mM p‐hydroxyphenylpyruvate, transamination was 6‐fold greater than the rate of formation of dopamine. The dihydroxyphenylpyruvate formed during arninotransfer from DOPA by brain tissue was not readily decarboxylated, whereas 65–70 per cent of the indolepyruvate formed from tryptophan was decarboxylated. We suggest that an increased rate or degree of transamination between tryptophan and aromatic and branched chain keto acids may explain the increased excretion of non‐hydroxylated indolic acids in phenylketonuria and‘maple syrup urine’disease, respectively. Increased aminotransfers from tryptophan and DOPA may reduce the amounts of precursors available for the synthesis of serotonin and catecholamines, both of which are at low levels in the sera of untreated phenylketonurics.