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Henri Unengrabe - One of the best experts on this subject based on the ideXlab platform.
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analysis of the Citric Acid Cycle intermediates using gas chromatography mass spectrometry
Methods of Molecular Biology, 2011Co-Authors: Raja S Kombu, Henri Unengrabe, Michelle PuchowiczAbstract:Researchers view analysis of the Citric Acid Cycle (CAC) intermediates as a metabolomic approach to identifying unexpected correlations between apparently related and unrelated pathways of metabolism. Relationships of the CAC intermediates, as measured by their concentrations and relative ratios, offer useful information to understanding interrelationships between the CAC and metabolic pathways under various physiological and pathological conditions. This chapter presents a relatively simple method that is sensitive for simultaneously measuring concentrations of CAC intermediates (relative and absolute) and other related intermediates of energy metabolism using gas chromatography-mass spectrometry.
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dipropionylcysteine ethyl ester compensates for loss of Citric Acid Cycle intermediates during post ischemia reperfusion in the pig heart
Cardiovascular Drugs and Therapy, 2009Co-Authors: Takha Kasumov, Raja S Kombu, Andrea V Cendrowski, Navee Sharma, Hazel Huang, William C Stanley, Henri UnengrabeAbstract:Purpose During reperfusion, following myocardial ischemia, uncompensated loss of Citric Acid Cycle (CAC) intermediates may impair CAC flux and energy transduction. Propionate has an anaplerotic effect when converted to the CAC intermediate succinyl-CoA, and may improve contractile recovery during reperfusion. Antioxidant therapy with N-acetylcysteine decreases reperfusion injury. To synergize the antioxidant effects of cysteine with the anaplerotic effects of propionate, we synthesized a novel bi-functional compound, N,S-dipropionyl cysteine ethyl ester (DPNCE) and tested its anaplerotic and anti-oxidative capacity in anesthetized pigs.
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metabolomic and mass isotopomer analysis of liver gluconeogenesis and Citric Acid Cycle ii heterogeneity of metabolite labeling pattern
Journal of Biological Chemistry, 2008Co-Authors: Lili Yang, Takha Kasumov, Raja S Kombu, Shu Ha Zhu, Andrea V Cendrowski, Verno E Anderso, Joanne K Kellehe, Henri UnengrabeAbstract:In this second of two companion articles, we compare the mass isotopomer distribution of metabolites of liver gluconeogenesis and Citric Acid Cycle labeled from NaH(13)CO(3) or dimethyl [1,4-(13)C(2)]succinate. The mass isotopomer distribution of intermediates reveals the reversibility of the isocitrate dehydrogenase + aconitase reactions, even in the absence of a source of alpha-ketoglutarate. In addition, in many cases, a number of labeling incompatibilities were found as follows: (i) glucose versus triose phosphates and phosphoenolpyruvate; (ii) differences in the labeling ratios C-4/C-3 of glucose versus (glyceraldehyde 3-phosphate)/(dihydroxyacetone phosphate); and (iii) labeling of Citric Acid Cycle intermediates in tissue versus effluent perfusate. Overall, our data show that gluconeogenic and Citric Acid Cycle intermediates cannot be considered as sets of homogeneously labeled pools. This probably results from the zonation of hepatic metabolism and, in some cases, from differences in the labeling pattern of mitochondrial versus extramitochondrial metabolites. Our data have implications for the use of labeling patterns for the calculation of metabolic rates or fractional syntheses in liver, as well as for modeling liver intermediary metabolism.
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metabolomic and mass isotopomer analysis of liver gluconeogenesis and Citric Acid Cycle i interrelation between gluconeogenesis and cataplerosis formation of methoxamates from aminooxyacetate and ketoAcids
Journal of Biological Chemistry, 2008Co-Authors: Lili Yang, Takha Kasumov, Raja S Kombu, Shu Ha Zhu, Andrea V Cendrowski, Verno E Anderso, Joanne K Kellehe, Henri UnengrabeAbstract:We conducted a study coupling metabolomics and mass isotopomer analysis of liver gluconeogenesis and Citric Acid Cycle. Rat livers were perfused with lactate or pyruvate +/- aminooxyacetate or mercaptopicolinate in the presence of 40% enriched NaH(13)CO(3). Other livers were perfused with dimethyl [1,4-(13)C(2)]succinate +/- mercaptopicolinate. In this first of two companion articles, we show that a substantial fraction of gluconeogenic carbon leaves the liver as Citric Acid Cycle intermediates, mostly alpha-ketoglutarate. The efflux of gluconeogenic carbon ranges from 10 to 200% of the rate of liver gluconeogenesis. This cataplerotic efflux of gluconeogenic carbon may contribute to renal gluconeogenesis in vivo. Multiple crossover analyses of concentrations of gluconeogenic intermediates and redox measurements expand previous reports on the regulation of gluconeogenesis and the effects of inhibitors. We also demonstrate the formation of adducts from the condensation, in the liver, of (i) aminooxyacetate with pyruvate, alpha-ketoglutarate, and oxaloacetate and (ii) mercaptopicolinate and pyruvate. These adducts may exert metabolic effects unrelated to their effect on gluconeogenesis.
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metabolomic assays of the concentration and mass isotopomer distribution of gluconeogenic and Citric Acid Cycle intermediates
Metabolomics, 2006Co-Authors: Lili Yang, Takha Kasumov, Joanne K Kellehe, Kathry A Jobbins, Stephe F Previs, Henri UnengrabeAbstract:We developed gas chromatography-mass spectrometry assays for the relative concentration and for the mass isotopomer distribution of gluconeogenic and Citric Acid Cycle intermediates in tissues. The assay involves (i) spiking the sample with one or more internal standards, (ii) chloroform–methanol extraction at −25 °C, (iii) Folch wash of the extract, (iv) treatment of the water-methanol phase with methoxylamine, (v) evaporation and trimethylsilyl derivatization, and (vi) ammonia positive chemical ionization gas chromatography-mass spectrometry. For metabolomic computations, indices of concentrations for all compounds assayed are calculated as (Area of analyte)/(Area of reference compound). The assay was applied to a study of the effect of mercaptopicolinate, an inhibitor of phosphoenolpyruvate carboxykinase, on the profile of gluconeogenic intermediates in rat livers perfused with pyruvate. Crossover analysis of concentrations indices, compared to a control group, yielded very similar profiles as previous enzymatic assays, and correctly identified the site of action of mercaptopicolinate. Principal component analysis distinguished between control and drug treated samples. A loadings plot was used to identify the site of action of the drug in the metabolic pathway. Since metabolite concentrations do not address the flux through a pathway, perfusions with [1,4-13C2] succinate dimethylester were conducted to assess fluxes around PEPCK. This allowed a dynamic metabolomics analysis which indicated that considerable flux through the pathway remained in the presence of mercaptopicolinate. This study illustrates the power of dynamic metabolomics to complement concentration based metabolomic studies.
Christos Chinopoulos - One of the best experts on this subject based on the ideXlab platform.
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mitochondrial diaphorases as nad donors to segments of the Citric Acid Cycle that support substrate level phosphorylation yielding atp during respiratory inhibition
The FASEB Journal, 2014Co-Authors: Gergely Kiss, Csaba Konrad, Issa Pourghaz, Josef Mansou, Eata Nemeth, Anatoly A Starkov, Vera Adamvizi, Christos ChinopoulosAbstract:Substrate-level phosphorylation mediated by succinyl-CoA ligase in the mitochondrial matrix produces high-energy phosphates in the absence of oxidative phosphorylation. Furthermore, when the electron transport chain is dysfunctional, provision of succinyl-CoA by the α-ketoglutarate dehydrogenase complex (KGDHC) is crucial for maintaining the function of succinyl-CoA ligase yielding ATP, preventing the adenine nucleotide translocase from reversing. We addressed the source of the NAD+ supply for KGDHC under anoxic conditions and inhibition of complex I. Using pharmacologic tools and specific substrates and by examining tissues from pigeon liver exhibiting no diaphorase activity, we showed that mitochondrial diaphorases in the mouse liver contribute up to 81% to the NAD+ pool during respiratory inhibition. Under these conditions, KGDHC's function, essential for the provision of succinyl-CoA to succinyl-CoA ligase, is supported by NAD+ derived from diaphorases. Through this process, diaphorases contribute to the maintenance of substrate-level phosphorylation during respiratory inhibition, which is manifested in the forward operation of adenine nucleotide translocase. Finally, we show that reoxidation of the reducible substrates for the diaphorases is mediated by complex III of the respiratory chain.—Kiss, G., Konrad, C., Pour-Ghaz, I., Mansour, J. J., Nemeth, B., Starkov, A. A., Adam-Vizi, V., Chinopoulos, C. Mitochondrial diaphorases as NAD+ donors to segments of the Citric Acid Cycle that support substrate-level phosphorylation yielding ATP during respiratory inhibition.
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which way does the Citric Acid Cycle turn during hypoxia the critical role of α ketoglutarate dehydrogenase complex
Journal of Neuroscience Research, 2013Co-Authors: Christos ChinopoulosAbstract:The Citric Acid Cycle forms a major metabolic hub and as such it is involved in many disease states involving energetic imbalance. In spite of the fact that it is being branded as a "Cycle", during hypoxia, when the electron transport chain does not oxidize reducing equivalents, segments of this metabolic pathway remain operational but exhibit opposing directionalities. This serves the purpose of harnessing high-energy phosphates through matrix substrate-level phosphorylation in the absence of oxidative phosphorylation. In this Mini-Review, these segments are appraised, pointing to the critical importance of the alpha-ketoglutarate dehydrogenase complex dictating their directionalities. (c) 2013 Wiley Periodicals, Inc.
William C Stanley - One of the best experts on this subject based on the ideXlab platform.
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dipropionylcysteine ethyl ester compensates for loss of Citric Acid Cycle intermediates during post ischemia reperfusion in the pig heart
Cardiovascular Drugs and Therapy, 2009Co-Authors: Takha Kasumov, Raja S Kombu, Andrea V Cendrowski, Navee Sharma, Hazel Huang, William C Stanley, Henri UnengrabeAbstract:Purpose During reperfusion, following myocardial ischemia, uncompensated loss of Citric Acid Cycle (CAC) intermediates may impair CAC flux and energy transduction. Propionate has an anaplerotic effect when converted to the CAC intermediate succinyl-CoA, and may improve contractile recovery during reperfusion. Antioxidant therapy with N-acetylcysteine decreases reperfusion injury. To synergize the antioxidant effects of cysteine with the anaplerotic effects of propionate, we synthesized a novel bi-functional compound, N,S-dipropionyl cysteine ethyl ester (DPNCE) and tested its anaplerotic and anti-oxidative capacity in anesthetized pigs.
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differential effects of heptanoate and hexanoate on myocardial Citric Acid Cycle intermediates following ischemia reperfusion
Journal of Applied Physiology, 2006Co-Authors: Isidore C Okere, Henri Unengrabe, Hazel Huang, Tracy A Mcelfresh, Daniel Z Unengrabe, Wenju Z Martini, Joseph P Sterk, Margare P Chandle, William C StanleyAbstract:In the normal heart, there is loss of Citric Acid Cycle (CAC) intermediates that is matched by the entry of intermediates from outside the Cycle, a process termed anaplerosis. Previous in vitro studies suggest that supplementation with anaplerotic substrates improves cardiac function during myocardial ischemia and/or reperfusion. The present investigation assessed whether treatment with the anaplerotic medium-chain fatty Acid heptanoate improves contractile function during ischemia and reperfusion. The left anterior descending coronary artery of anesthetized pigs was subjected to 60 min of 60% flow reduction and 30 min of reperfusion. Three treatment groups were studied: saline control, heptanoate (0.4 mM), or hexanoate as a negative control (0.4 mM). Treatment was initiated after 30 min of ischemia and continued through reperfusion. Myocardial CAC intermediate content was not affected by ischemia-reperfusion; however, treatment with heptanoate resulted in a more than twofold increase in fumarate and malate, with no change in citrate and succinate, while treatment with hexanoate did not increase fumarate or malate but increased succinate by 1.8-fold. There were no differences among groups in lactate exchange, glucose oxidation, oxygen consumption, and contractile power. In conclusion, despite a significant increase in the content of carbon-4 CAC intermediates, treatment with heptanoate did not result in improved mechanical function of the heart in this model of reversible ischemia-reperfusion. This suggests that reduced anaplerosis and CAC dysfunction do not play a major role in contractile and metabolic derangements observed with a 60% decrease in coronary flow followed by reperfusion.
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regulation of pyruvate dehydrogenase activity and Citric Acid Cycle intermediates during high cardiac power generation
The Journal of Physiology, 2005Co-Authors: Navee Sharma, Hazel Huang, Isidore C Okere, Tracy A Mcelfresh, Daniel Z Unengrabe, Joseph P Sterk, Margare P Chandle, Kriste L King, William C StanleyAbstract:A high rate of cardiac work increases Citric Acid Cycle (CAC) turnover and flux through pyruvate dehydrogenase (PDH); however, the mechanisms for these effects are poorly understood. We tested the hypotheses that an increase in cardiac energy expenditure: (1) activates PDH and reduces the product/substrate ratios ([NADH]/[NAD+] and [acetyl-CoA]/[CoA-SH]); and (2) increases the content of CAC intermediates. Measurements were made in anaesthetized pigs under control conditions and during 15 min of a high cardiac workload induced by dobutamine (Dob). A third group was made hyperglycaemic (14 mm) to stimulate flux through PDH during the high work state (Dob + Glu). Glucose and fatty Acid oxidation were measured with 14C-glucose and 3H-oleate. Compared with control, the high workload groups had a similar increase in myocardial oxygen consumption ( and cardiac power. Dob increased PDH activity and glucose oxidation above control, but did not reduce the [NADH]/[NAD+] and [acetyl-CoA]/[CoA-SH] ratios, and there were no differences between the Dob and Dob + Glu groups. An additional group was treated with Dob + Glu and oxfenicine (Oxf) to inhibit fatty Acid oxidation: this increased [CoA-SH] and glucose oxidation compared with Dob; however, there was no further activation of PDH or decrease in the [NADH]/[NAD+] ratio. Content of the 4-carbon CAC intermediates succinate, fumarate and malate increased 3-fold with Dob, but there was no change in citrate content, and the Dob + Glu and Dob + Glu + Oxf groups were not different from Dob. In conclusion, compared with normal conditions, at high myocardial energy expenditure (1) the increase in flux through PDH is regulated by activation of the enzyme complex and continues to be partially controlled through inhibition by fatty Acid oxidation, and (2) there is expansion of the CAC pool size at the level of 4-carbon intermediates that is largely independent of myocardial fatty Acid oxidation.
Douwe Molenaa - One of the best experts on this subject based on the ideXlab platform.
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functions of the membrane associated and cytoplasmic malate dehydrogenases in the Citric Acid Cycle of escherichia coli
Journal of Bacteriology, 2000Co-Authors: Michel Eduard Van Der Res, Christia Frank, Douwe MolenaaAbstract:ABSTRACT Oxidation of malate to oxaloacetate in Escherichia colican be catalyzed by two enzymes: the well-known NAD-dependent malate dehydrogenase (MDH; EC 1.1.1.37 ) and the membrane-associated malate:quinone-oxidoreductase (MQO; EC 1.1.99.16 ), encoded by the genemqo (previously called yojH). Expression of themqo gene and, consequently, MQO activity are regulated by carbon and energy source for growth. In batch cultures, MQO activity was highest during exponential growth and decreased sharply after onset of the stationary phase. Experiments with the β-galactosidase reporter fused to the promoter of the mqo gene indicate that its transcription is regulated by the ArcA-ArcB two-component system. In contrast to earlier reports, MDH did not repressmqo expression. On the contrary, MQO and MDH are active at the same time in E. coli. For Corynebacterium glutamicum, it was found that MQO is the principal enzyme catalyzing the oxidation of malate to oxaloacetate. These observations justified a reinvestigation of the roles of MDH and MQO in the Citric Acid Cycle of E. coli. In this organism, a defined deletion of the mdh gene led to severely decreased rates of growth on several substrates. Deletion of the mqo gene did not produce a distinguishable effect on the growth rate, nor did it affect the fitness of the organism in competition with the wild type. To investigate whether in an mqo mutant the conversion of malate to oxaloacetate could have been taken over by a bypass route via malic enzyme, phosphoenolpyruvate synthase, and phosphenolpyruvate carboxylase, deletion mutants of the malic enzyme genessfcA and b2463 (coding for EC 1.1.1.38 and EC1.1.1.40 , respectively) and of the phosphoenolpyruvate synthase (EC2.7.9.2 ) gene pps were created. They were introduced separately or together with the deletion of mqo. These studies did not reveal a significant role for MQO in malate oxidation in wild-type E. coli. However, comparing growth of themdh single mutant to that of the double mutant containingmdh and mqo deletions did indicate that MQO partly takes over the function of MDH in an mdh mutant.
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functions of the membrane associated and cytoplasmic malate dehydrogenases in the Citric Acid Cycle of corynebacterium glutamicum
Journal of Bacteriology, 2000Co-Authors: Douwe Molenaa, M E Van Der Res, Andre Drysch, R YucelAbstract:Like many other bacteria, Corynebacterium glutamicum possesses two types of L-malate dehydrogenase, a membrane-associated malate:quinone oxidoreductase (MQO; EC 1.1.99.16) and a cytoplasmic malate dehydrogenase (MDH; EC 1.1.1.37) The regulation of MDH and of the three membrane-associated dehydrogenases MQO, succinate dehydrogenase (SDH), and NADH dehydrogenase was investigated. MQO, MDH, and SDH activities are regulated coordinately in response to the carbon and energy source for growth. Compared to growth on glucose, these activities are increased during growth on lactate, pyruvate, or acetate, substrates which require high Citric Acid Cycle activity to sustain growth. The simultaneous presence of high activities of both malate dehydrogenases is puzzling. MQO is the most important malate dehydrogenase in the physiology of C. glutamicum. A mutant with a site-directed deletion in the mqo gene does not grow on minimal medium. Growth can be partially restored in this mutant by addition of the vitamin nicotinamide. In contrast, a double mutant lacking MQO and MDH does not grow even in the presence of nicotinamide. Apparently, MDH is able to take over the function of MQO in an mqo mutant, but this requires the presence of nicotinamide in the growth medium. It is shown that addition of nicotinamide leads to a higher intracellular pyridine nucleotide concentration, which probably enables MDH to catalyze malate oxidation. Purified MDH from C. glutamicum catalyzes oxaloacetate reduction much more readily than malate oxidation at physiological pH. In a reconstituted system with isolated membranes and purified MDH, MQO and MDH catalyze the cyclic conversion of malate and oxaloacetate, leading to a net oxidation of NADH. Evidence is presented that this cyclic reaction also takes place in vivo. As yet, no phenotype of an mdh deletion alone was observed, which leaves a physiological function for MDH in C. glutamicum obscure.
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another unusual type of Citric Acid Cycle enzyme in helicobacter pylori the malate quinone oxidoreductase
Journal of Bacteriology, 2000Co-Authors: K Stingl, M E Van Der Res, K Altendorf, Douwe MolenaaAbstract:The only enzyme of the Citric Acid Cycle for which no open reading frame (ORF) was found in the Helicobacter pylori genome is the NAD-dependent malate dehydrogenase. Here, it is shown that in this organism the oxidation of malate to oxaloacetate is catalyzed by a malate:quinone oxidoreductase (MQO). This flavin adenine dinucleotide-dependent membrane-associated enzyme donates electrons to quinones of the electron transfer chain. Similar to succinate dehydrogenase, it is part of both the electron transfer chain and the Citric Acid Cycle. MQO activity was demonstrated in isolated membranes of H. pylori. The enzyme is encoded by the ORF HP0086, which is shown by the fact that expression of the HP0086 sequence from a plasmid induces high MQO activity in mqo deletion mutants of Escherichia coli or Corynebacterium glutamicum. Furthermore, this plasmid was able to complement the phenotype of the C. glutamicum mqo deletion mutant. Interestingly, the protein predicted to be encoded by this ORF is only distantly related to known or postulated MQO sequences from other bacteria. The presence of an MQO shown here and the previously demonstrated presence of a 2-ketoglutarate:ferredoxin oxidoreductase and a succinyl-coenzyme A (CoA):acetoacetyl-CoA transferase indicate that H. pylori possesses a complete Citric Acid Cycle, but one which deviates from the standard textbook example in three steps.
M Eylo - One of the best experts on this subject based on the ideXlab platform.
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modifications of Citric Acid Cycle activity and gluconeogenesis in streptozotocin induced diabetes and effects of metformin
Diabetes, 1999Co-Authors: V Large, M EyloAbstract:To better define the modifications of liver gluconeogenesis and Citric Acid Cycle, or Krebs' Cycle, activity induced by insulin deficiency and the effects of metformin on these abnormalities, we infused livers isolated from postabsorptive or starved normal and streptozotocin-induced diabetic rats with pyruvate and lactate (labeled with [3-13C]lactate) with or without the simultaneous infusion of metformin. Lactate and pyruvate uptake and glucose production were calculated. The 13C-labeling pattern of liver glutamate was used to calculate, according to Magnusson's model, the relative fluxes through Krebs' Cycle and gluconeogenesis. These relative fluxes were converted into absolute values using substrate balances. In normal rats, starvation increased gluconeogenesis, the flux through pyruvate carboxylase-phosphoenolpyruvate carboxykinase (PC-PEPCK), and the ratio of PC to pyruvate dehydrogenase (PDH) flux (P < 0.05); metformin induced only a moderate decrease in the PC:PDH ratio. Livers from postabsorptive diabetic rats had increased lactate and pyruvate uptakes (P < 0.05); their metabolic fluxes resembled those of starved control livers, with increased gluconeogenesis and flux through PC-PEPCK. Starvation induced no further modifications in the diabetic group. Metformin decreased glucose output from the liver of starved diabetic rats (P < 0.05). The flux through PC-PEPCK and also pyruvate kinase were decreased (P < 0.05) by metformin in both groups of diabetic rats. In conclusion, insulin deficiency increased in this model of diabetes gluconeogenesis through enhanced uptake of substrate and increased flux through PC-PEPCK; metformin decreased glucose production by reducing the flux through PC-PEPCK.
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use of labeling pattern of liver glutamate to calculate rates of Citric Acid Cycle and gluconeogenesis
American Journal of Physiology-endocrinology and Metabolism, 1997Co-Authors: V Large, Henri Unengrabe, Michele Odeo, M EyloAbstract:The use of the labeling pattern of hepatic glutamate during infusion of L-[3-13C]- or [3-14C]lactate to calculate rates of Citric Acid Cycle activity and gluconeogenesis has been proposed. We teste...
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tracing hepatic gluconeogenesis relative to Citric Acid Cycle activity in vitro and in vivo comparisons in the use of 3 c lactate 2 c acetate and α keto 3 c isocaproate
Journal of Biological Chemistry, 1995Co-Authors: M Eylo, M V Soloviev, Ernard R Landau, Henri UnengrabeAbstract:Abstract The validity of the use of a carbon tracer for investigating liver intermediary metabolism in vivo requires that the labeling pattern of liver metabolites not be influenced by metabolism of the tracer in other tissues. To identify such specific tracer, livers from 48-h starved rats were perfused with recirculating buffer containing [3-C]lactate, [2-C]acetate, or α-keto[3-C]isocaproate. Conscious 48-h starved rats were infused with the same tracers for 5 h. The labeling patterns of liver glutamate and extracellular glucose were assayed by gas chromatography-mass spectrometry. In vivo data were corrected for CO2 reincorporation into C-1 of glutamate and C-3 and C-4 of glucose, using data from control rats infused with NaHCO3. With [3-C]lactate the labeling pattern of liver glutamate was the same in perfused organs and in vivo. In contrast, with [2-C]acetate and α-keto[3-C]isocaproate the labeling pattern of liver glutamate in vivo was clearly influenced by the expected labeling pattern of Citric Acid Cycle intermediates formed in non-gluconeogenic organs, presumably glutamine made in muscle. Indeed, the labeling pattern of plasma glutamine and liver glutamate were similar in experiments with [3-C]lactate but different in experiments with [2-C]acetate and α-keto[3-C]isocaproate. Similar conclusions were drawn from the labeling patterns of glucose. Therefore, labeled lactate appears as the best tracer for studies of liver intermediary metabolism in vivo. Our data also show that a substantial fraction of α-ketoisocaproate metabolism occurs in peripheral tissues.
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assay of the human liver Citric Acid Cycle probe phenylacetylglutamine and of phenylacetate in plasma by gas chromatography mass spectrometry
Analytical Biochemistry, 1993Co-Authors: Dawei Yang, M Eylo, K C Agarwal, M V Soloviev, Henri UnengrabeAbstract:Phenylacetate, derived from phenylalanine, is converted in human and primate liver to phenylacetylglutamine. The latter has been used to assess the labeling pattern of liver Citric Acid Cycle intermediates. We present gas chromatographic-mass spectrometric assays of phenylacetylglutamine, phenylacetate, and phenylalanine in biological fluids. The compounds are derivatized with dimethylformamide dimethyl acetal. Limits of detection are 0.1 nmol for phenylacetylglutamine and phenylacetate and 2 nmol for phenylalanine. Baseline plasma concentrations of phenylacetate and phenylacetylglutamine and 1 and 3 microM, respectively. The 24-h urinary excretions of phenylacetate and phenylacetylglutamine are about 4 mumol and 1 mmol, respectively. Ingestion of phenylalanine (in the form of aspartame) by a human is followed by sequential increases in phenylacetate and phenylacetylglutamine concentrations in plasma and urine. This assay opens the way to noninvasive probing of the 13C-labeling pattern of liver Citric Acid Cycle intermediates in humans.