Identification of keto acids in arctic bramble,Rubus arcticus L. as methyl esters of their 2,4-dinitrophenylhydrazones

The major keto acids in arctic bramble,Rubus arcticus L. were investigated. The acids were isolated with anionic and cationic ion-exchange resins, converted to 2,4-dinitrophenylhydrazones, and purified with an Al₂O₃ column. The derivatives were separated on a silica gel G thin-layer plate and esterified with methanol-HCl and the methyl esters of the keto acid 2,4-dinitrophenylhydrazones formed were analyzed on an OV-1-glass capillary gas-liquid chromatography column and with mass spectrometry. 2-Oxoglutaric, pyruvic, oxaloacetic, and glyoxylic acids were identified. The mass spectra of the derivatives are presented.     

Separation and determination of 14C-labelled intermediates of the citric acid cycle and related compounds

A new systematic procedure is presented which permits a complete chromatographic resolution of the intermediates of the citric acid cycle as well as the related amino and keto acids. The adsorption onto ion-exchange columns followed by the subsequent elution therefrom forms the first step of the present procedure. Meanwhile, unstable keto acids are enzymatically converted into the respective amino acids. This preliminary process is very effective in desalting the biological specimen and also results in a broad division of a large number of intermediates into three subgroups, i.e., amino, keto and other organic acid fractions. Each of these subgroups is then readily resolved into individual acids by means of one-dimensional thinlayer chromatography. ¹⁴C-Compounds added to the reaction mixture of rat liver mitochondria were recovered with a good reproducibility throughout the entire course of the present procedure.     

O-trimethylsilylquinoxalinol derivatives of aromatic α-keto acids : Mass spectra and quantitative gas chromatography

As an extension of earlier work on aliphatic α-keto acids, a method is described for the quantitative gas chromatographic determination of urinary aromatic α-keto acids. The keto acids are derivatized with o-phenylenediamine to yield the quinoxalinols. These compounds are chromatographed after trimethylsilyation.The aromatic keto acids are stabilized by sodium dithionite (4 mg/ml urine) and storage below 0°. The final derivatives are stable for weeks at room temperature.Low resolution mass spectra are reported. The fragmentation mechanims are elucidated by analysis of O-trimethylsilyl-(TMS)-quinoxalinols, O-(TMS-d₉)-quinoxalinols and O-TMS-6(7)-chloroquinoxalinols.     

Diet treatment of branched chain ketoaciduria studied by proton magnetic resonance spectroscopy

Summary A novel nuclear magnetic resonance method is proposed for the diagnosis and follow-up of patients affected by branched chain ketoaciduria. The method allows quantitation of the branched chain amino acids (BCAA's) such as leucine, isoleucine and valine and of related keto- and hydroxy acids by means of a single spectrum. The method implies short time of analysis, as opposed to the very long time required by the techniques currently in use (amino acid analyzer combined with gaschromatography/mass spectrometry of keto- and hydroxyacids), it is easy and suitable for adjustements of the dietary treatment even on a daily basis. The case of a 15 days old newborn child, presenting muscular hypertonicity was unambiguously diagnosed in few minutes by means of one single NMR spectrum of urine. More interestingly, NMR spectra of serum in the following days were suitable for quantitating amino-, and keto acids as well as other metabolites of relevance in the follow up of the dietary treatment of the disease. After a diet lacking of BCAA's, to eliminate keto acids, a low BCAA diet was introduced, that succeeded in keeping the serum levels of the three amino acids within the normal range, while dropping the related keto acids.     

Synthesis of branched-chain sugar derivatives from 1,2;5,6-di-O-isopropylidene-alpha-D-ribo-hexofuranos-3-ulose and 1,2;4,5-di-O-isopropylidene-beta-D-erythro-2-hexulopyranos-3-ulose

A facile method for the formation of branched-chain sugar derivatives is described involving the reaction of lithium dianions and carboxylic acids with keto-sugar derivatives. Acetic, propanoic, phenylacetic, 3,3-dimethylacrylic, crotonic and sorbic acids were the acids used for the preparation of the lithium dianions, and glucose and fructose were used for preparation of the keto derivatives.     

Cysteine and keto acids modulate mosquito kynurenine aminotransferase catalyzed kynurenic acid production

Kynurenine aminotransferase (KAT) catalyzes the formation of kynurenic acid (KYNA), the natural antagonist of ionotropic glutamate receptors. This study tests potential substrates and assesses the effects of amino acids and keto acids on the activity of mosquito KAT. Various keto acids, when simultaneously present in the same reaction mixture, display a combined effect on KAT catalyzed KYNA production. Moreover, methionine and glutamine show inhibitory effects on KAT activity, while cysteine functions as either an antagonist or an inhibitor depending on the concentration. Therefore, the overall level of keto acids and cysteine might modulate the KYNA synthesis. Results from this study will be useful in the study of KAT regulation in other animals.     

Formation of keto acids by barotolerant marine bacteria under atmospheric pressure]

Production of keto acids by washed cells of the barotolerant strains was studied under atmospheric pressure on a glucose-mineral medium in balloons made of the copolymer of ethylene with propylene. Production of keto acids in these conditions requires elevated concentrations of glucose and minimum concentrations of ammonium nitrogen. Active aeration inhibits the accumulation of keto acids by the barotolerant bacteria. The majority of the barotolerant bacteria did not form free extracellular keto acids, other bacteria liberated comparatively small amounts of these acids. The ability to accumulate keto acids was different in particular strains.     

Keto acids in tissues and biological fluids: O-t-butyldimethylsilyl quinoxalinols as derivatives for sensitive gas chromatographic/mass spectrometric determination

Quinoxalinol t-butyldimethylsilyl ethers were prepared from three branched-chain and from two aliphatic unbranched 2-keto acids. The electron impact (EI) mass spectra display pronounced [M-57]+ ions. With 39-51% of total ion current contained within them, sensitivity is greater than on chemical ionization (CI) mass spectrometry of O-trimethylsilyl derivatives. Mass spectra and chromatographic behaviour of these novel keto acid derivatives are discussed and preliminary quantitative data from rat muscle are given.     

Common Enzymes of Branched-Chain Amino Acid Catabolism in Pseudomonas putida

Two types of Pseudomonas putida PpG2 mutants which were unable to degrade branched-chain amino acids were isolated after mutagenesis and selection for ability to grow on succinate, but not valine, as a sole source of carbon. These isolates were characterized by growth on the three branched-chain amino acids (valine, isoleucine, and leucine), on the corresponding branched-chain keto acids (2-ketoisovalerate, 2-keto-3-methylvalerate, and 2-ketoisocaproate), and on other selected intermediates as carbon sources, and by their enzymatic composition. One group of mutants lost 2-ketoisovalerate-inducible branched-chain keto acid dehydrogenase that was active on all three keto acids. There was also a concomitant loss of ability to grow on all three branched-chain amino acids as well as on all three corresponding keto acids, but there was retention of ability to use subsequent intermediates in the catabolism of branched-chain amino acids. Another type of mutant showed a marked reduction in branched-chain amino acid transaminase activity and grew poorly at the expense of all three amino acids, but it utilized subsequent intermediates as carbon sources. Both the transaminase and branched-chain keto acid dehydrogenase mutants retained the ability to degrade camphor. These findings are consistent with the view that branched-chain amino acid transaminase and branched-chain keto acid dehydrogenase are common enzymes in the catabolism of valine, isoleucine, and leucine.     

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 K_m 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.     

Why Threonine Is an Essential Amino Acid in Mammals and Birds: Studies at the Enzyme Level

The only pathway for the synthesis of essential amino acids in vertebrates is reversible transamination of their keto analogs with glutamic acid. At the same time, it is commonly accepted that such essential amino acids as lysine and threonine are not involved in transamination and, therefore, cannot be synthesized from their keto analogs. However, using radio-labeled isotopes, synthesis of threonine was demonstrated in rat liver and in a reaction mixture containing chicken liver threonine dehydrogenase. In the review, we discuss why threonine is an essential amino acid in mammals and birds based on the pathways of threonine biosynthesis in these two classes of vertebrates.     

氨基酸脱氢酶的催化机理、分子改造及合成应用

氨基酸脱氢酶催化可逆的氨基酸氧化脱氨和酮酸的不对称还原胺化反应,热力学上反应平衡倾向于生成氨基酸方向,从原子经济学和对环境影响的角度来看,是具有极大优势的氨基酸合成方法之一。本文将主要阐述近年来在?-氨基酸脱氢酶催化机理、分子改造和合成应用方面的研究进展。     

The placenta releases branched-chain keto acids into the umbilical and uterine circulations in the pregnant sheep

There was net uptake of branched-chain keto acids by the fetus from the umbilical circulation. Mean fetal uptake of the 3 keto acids 2-keto isovalerate, 2-keto isocaproate and 2-keto methylvalerate was 1.8 mumol/min per kg of fetus. The concentrations in the umbilical vein for these keto acids were 10.9 +/- 3.8 microM (mean +/- SD: 2-keto isovalerate), 19.7 +/- 6.1 microM (2-keto isocaproate) and 14.8 +/- 5.3 microM (2-keto methylvalerate) respectively. The coefficients of extraction for the same keto acids were 17.2%, 16.8% and 11.9% respectively. Fetal uptakes (both mumol/min and mumol/min per kg fetus) were positively correlated with umbilical supply. There were concentration gradients across the placenta, with fetal concentration: maternal concentration ratios of 3.3 +/- 1.5 for 2-keto isovalerate, 2.1 +/- 0.8 for 2-keto isocaproate and 1.3 +/- 0.6 for 2-keto methylvalerate. The net release of 2-keto acids into the umbilical circulation may conserve the carbon skeleton of branched-chain amino acids for fetal metabolism and growth. In the uterine circulation there was not a consistent pattern of release from or uptake by the uteroplacental tissues. It is suggested that branched-chain keto acids may contribute to fetal growth or energy metabolism.     

Keto Acids in Pollen and Pollen Tubes of Sesbania aegyptica

Pollen and pollen tubes of Sesbania aegyptica Pers. contain α-ketoglutaric acid, oxaloacetic acid and pyruvic acid. Changes in the keto acids have been correlated with their corresponding amino acids during different phases of germination. It is suggested that keto acids were readily turned over during the elongation of pollen tubes.     

Dimeric epoxy fatty acid methyl esters: formation,chromatography and mass spectrometry

The formation of dimers is reported from the thermal treatment of a series of epoxy fatty acid methyl esters. These compounds were isolated from the reaction mixture by steric exclusion chromatography and were subsequently characterised by their high resolution electron impact and ammonia chemical ionisation mass spectra. The spectra were consistent in each case with the presence of a mixture of four possible positional isomers each containing an ether bridge linking a pair of fatty acid methyl esters across the carbon chains, with a keto group on a carbon adjacent to the bridge on one of the esters.     

Easy access to various natural keto polyunsaturated fatty acids and their corresponding racemic alcohols

Various optically active hydroxy derivatives of polyunsaturated fatty acids were easily oxidised to their corresponding keto derivatives using Dess-Martin periodinane. The reaction was run on the millimolar scale with good yields and without appreciable isomerisation of the surrounding double bonds. Reduction of these keto compounds to yield back the starting alcohols, but now as racemic mixtures, was also conducted using CeCl(3)-NaBH(4), once again without noticeable modification of the stereochemistry of the double bonds. These reactions proved the usefulness of the chemoenzymatic access to oxylipins through the use of lipoxygenases with various regiospecificity, combined with chemical transformations of the formed hydro(pero)xides.     

Measurement of alpha-keto acids in plasma using an amino acid analyzer

A method for measuring keto acid concentrations in physiological fluids using an amino acid analyzer was developed. After preliminary deproteinization and removal of amino acids, reduction with sodium cyanoborohydride at 105 degrees C resulted in efficient conversion of the keto acids to their corresponding amino acids. In applying the technique to plasma samples, the use of MeOH for deproteinization was necessary to avoid the large losses of keto acids that occurred during precipitation of proteins with perchloric acid. The method was used to follow plasma ketoisocaproate concentrations in rat plasma after administration of leucine, and was sufficiently sensitive to detect concomitant changes in other branched-chain keto acid concentrations.     

Increased microsomal oxidation of hydroxyl radical scavenging agents and ethanol after chronic consumption of ethanol

The ability of the neonatal rat to oxidize the branched-chain amino acids leucine and valine and their corresponding keto acids was evaluated. In vivo, about 20% of orally administered labeled amino or keto acids were oxidized in 6 h, after which time little further oxidation occurred. In perfused neonatal liver the amino acids were oxidized at only 5-10% the rate of the keto acids. The oxidation of the keto acids showed a saturable dependence on concentration. The decarboxylation of ketoisocaproate (KIC) had a maximal rate of 40.1 +/- 1.6 mumol/h/g liver with an apparent Km of 0.27 +/- 0.03 mM, and decarboxylation of ketoisovalerate (KIV) had a maximal rate of 37.9 +/- 1.9 mumol/h/g liver and an apparent Km of 0.28 +/- 0.04 mM. KIC was ketogenic, producing mainly acetoacetate at a maximal rate of 44.5 +/- 1.6 mumol/h/g liver with an apparent Km of 0.27 +/- 0.03 mM. On the other hand, KIV was not gluconeogenic, although the perfused neonatal liver was able to produce glucose from lactate. During liver perfusion, KIV did not produce measurable quantities of either propionic or beta-aminoisobutyric acids, which are possible end products of KIV metabolism. Decanoic acid inhibited the decarboxylation of both keto acids to the same extent with a maximal effect at 0.4 mM fatty acid. At saturating levels, KIC was less ketogenic than decanoate. Inhibition of endogenous fatty acid oxidation by 2-tetradecylglycidic acid had no effect on keto acid oxidation. These data suggest that branched-chain amino acids derived from milk proteins are probably not quantitatively significant sources of either ketone bodies or glucose in the neonatal rat.     

An aminotransferase specific for the d-enantiomorph of methionine

An enzyme was isolated from germinating peanut seed and shown to be an aminotransferase specific for the d-enantiomorph of methionine. The keto acid of methionine, α-keto-γ-methylthiobutyrate, was isolated from the reaction mixture and identified. Of the keto acids tested pyruvic acid was the most effective acceptor for the amino group of methionine. A small amount of enzyme was isolated which gave only one band on disc gel electrophoresis.     

Effects of abnormal metabolites of maple syrup urine disease on neurotransmitter receptor binding

The deficiency of keto acid decarboxylase in maple syrup urine disease results in the accumulation of branched chain amino acids and their corresponding keto acids in tissues and body fluids. The effects of abnormal metabolites were investigated on neurotransmitter receptor binding in rat brain. alpha-Keto acids caused selective in vitro decrease in alpha-adrenergic, beta-adrenergic receptor binding in synaptosomal preparations from rat brain. No significant changes were observed in binding of cholinergic, GABA, and dopamine receptors binding in appropriate rat brain preparations. These results indicate that selective inhibition of adrenergic receptor binding by branched chain keto acids may presumably account for neural abnormality in maple syrup urine disease.