Structural, functional and metabolic remodeling of rat left ventricular myocytes in normal and in sodium-supplemented pregnancy.

OBJECTIVES: Pregnancy is an important physiological condition associated with hemodynamic and endocrine changes that affect the heart. Nevertheless, very little is known about cardiomyocyte remodeling in this condition. Here, we studied the morphological, functional and metabolic remodeling of rat left ventricular myocytes that occurs in late stages of normal pregnancy (P) and in experimental preeclampsia induced by elevated (0.9%) sodium intake (P0.9). METHODS: We applied confocal microscopy to examine the morphology and the contractility of single cells, while the patch clamp technique was used to assay ionic currents. RESULTS: Our results revealed a significant increase in the volume of single left ventricular cardiac myocytes in P, mainly resulting from cell elongation. In P0.9, further increase in the cell length led to a significant rise in the length/width ratio. Cell contractility was significantly decreased in glucose-based solutions in response to stimulation at 0.5 Hz and 6 Hz in P as well as in P0.9. The density of L-type calcium current (I(Ca)L) was not significantly altered in P or in P0.9. Metabolic substrates lactate and pyruvate, increased in the blood of P and P0.9 rats, enhanced contractility in P, without affecting I(Ca)L. The same effect, present but blunted in P0.9, was associated with a significant increase in I(Ca)L. CONCLUSION: Our results demonstrate that processes of adaptive remodeling take place in normal pregnancy, while maladaptive components are identified in experimental preeclampsia; they also reveal an adaptation in the use of energy substrates in pregnancy and its impairment by sodium supplementation.

A conformational model for the human liver microsomal glucose-6-phosphatase system: evidence from rapid kinetics and defects in glycogen storage disease type 1.

Rapid kinetics of glucose-6-phosphate (G6P) uptake and hydrolysis as well as of orthophosphate uptake were investigated in microsomes prepared from normal and glycogen storage disease type 1a (GSD 1a) human livers using a fast sampling, rapid filtration apparatus and were compared to those of rat liver microsomes. As shown before with rat microsomes, the production of [U-14C]glucose from 0.2 mmol/L [U-14C]G6P by untreated normal human microsomes was characterized by a burst in activity during the first seconds of incubation, followed by a slower linear rate. The initial velocity of the burst was equal to the rate of glucose production in detergent-treated microsomes. In untreated and detergent-treated GSD 1a microsomes, no glucose-6-phosphatase activity was observed. When untreated normal human or rat microsomes were incubated in the presence of 0.2 mmol/L [U-14C]G6P, an accumulation of [U-14C]glucose was observed, whereas no radioactive compound (G6P and/or glucose) was taken up by GSD 1a microsomes. Orthophosphate uptake was, however, detectable in both GSD 1a and normal untreated vesicles. These results do not support a rate-limiting transport of G6P in untreated normal human microsomes and further show that in this case of GSD 1a, no distinct G6P transport activity is present.

Hormonal control of glucose production and pyruvate kinase activity in isolated rat liver cells: influence of hypothyroidism.

Hormonal control of glucose production and of L-pyruvate kinase activity has been measured in isolated liver cells from fed control and thyroidectomized rats. In hypothyroid rats, sensitivity to isoproterenol as measured by these parameters was increased: the apparent K0.5 for isoproterenol-induced stimulation of glucose production decreased from 8.0 +/- 3 X 10(-6) M in control rats to 2.0 +/- 0.2 X 10(-8) M in hypothyroid rats (P less than 0.001) and the apparent K0.5 for inhibition of L-pyruvate kinase was 5 +/- 2 X 10(-7) M vs. 7 +/- 2 X 10(-9) M (P less than 0.001) in control and thyroidectomized rats, respectively. Utilisation of specific adrenergic antagonists confirmed increased beta-adrenergic responsiveness in hypothyroid rats. This phenomenon was not reversed by 3 days of T3 treatment (10 micrograms/100 g body weight). Sensitivity to the alpha-agonist was unchanged by thyroid status. Stimulation of glucose production and inhibition of L-pyruvate kinase activity by glucagon and their reversal by insulin were not affected by hypothyroidism. The dose-response curve to vasopressin and its maximal effect measured on stimulation of glucose production were unchanged in thyroidectomized rats. Thus, hypothyroidism produces a specific enhancement of liver beta-adrenergic responsiveness without affecting sensitivity to glucagon, insulin and vasopressin.

Influence of thyroid hormones on gluconeogenesis from glycerol in rat hepatocytes: a dose-response study.

The role of L-3,3'-5 triiodothyronine (T3), in a pathophysiological range, on gluconeogenesis from low concentration of glycerol (2 mmol/L), was investigated in isolated liver cells from 24-hour fasted rats either thyroidectomized, normal, or treated by a T3 dose ranging from 1, 5, or 10 micrograms/d/100 g body weight (BW) during 3 days to 50 micrograms during 7 days. Gluconeogenesis from glycerol was decreased by 63% in hypothyroid rats and increased by 35% in severely hyperthyroid rats. However, in cells from mild hyperthyroid rats no increase of gluconeogenesis was observed. Nevertheless, in mild hyperthyroidism, alpha-glycerophosphate (G3P) was significantly decreased and gluconeogenesis from glycerol was not inhibited by the addition of ethanol (10 mmol/L), both of which have a drastic effect in cells from thyroidectomized rats. The decrease of gluconeogenesis observed in cells from thyroidectomized rats was reversed by the addition of pyruvate (10 mmol/L). Thus, when the cells were in a "reduced state" (addition of ethanol) the differences between the group were magnified, and when the cells were in an "oxidized state" (addition of pyruvate) the differences were suppressed. These findings suggest that alteration of the capacity of reducing equivalents transfer from the cytoplasmic compartment to the mitochondria is the main mechanism by which mild hyperthyroidism can stimulate gluconeogenesis.

Routes of transmission during a nosocomial influenza A(H3N2) outbreak among geriatric patients and healthcare workers.

BACKGROUND: Influenza presents a life-threatening infection for hospitalized geriatric patients, who might be nosocomially infected via healthcare workers (HCWs), other patients or visitors. In the 2011/2012 influenza season an influenza A(H3N2) outbreak occurred in the geriatric department at the Hôpital Edouard Herriot, Lyon. AIM: To clarify the transmission chain for this influenza A(H3N2) outbreak by sequence analysis and to identify preventive measures. METHODS: Laboratory testing of patients with influenza-like illness in the acute care geriatric department revealed 22 cases of influenza between 19th February and 15th March 2012. Incidences for patients and HCWs were calculated and possible epidemiological links were analysed using a questionnaire. Neuraminidase and haemagglutinin genes of culture-positive samples and community influenza samples were sequenced and clustered to detect patients with identical viral strains. FINDINGS: Sixteen patients and six HCWs were affected, resulting in an attack rate of 24% and 11% respectively. Six nosocomial infections were recorded. The sequence analysis confirmed three independent influenza clusters on three different sections of the geriatric ward. For at least two clusters, an HCW source was determined. CONCLUSION: Epidemiological and microbiological results confirm influenza transmission from HCWs to patients. A higher vaccination rate, isolation measures and better hand hygiene are recommended in order to prevent outbreaks in future influenza seasons.

Detection of bile salt-dependent lipase, a 110 kDa pancreatic protein, in urines of healthy subjects.

Bile salt-dependent lipase (BSDL), a 110 kDa glycoprotein secreted by the pancreatic acinar cells, participates in the duodenal hydrolysis of dietary lipid esters. Recent in vitro and in vivo studies demonstrated that the BSDL reaches the blood via a transcytosis motion through enterocytes, suggesting that this enzyme may play a role in vascular biology. Once in the blood, BSDL should be eliminated. We address the hypothesis that BSDL may be filtered by the glomerulus and eliminated in urines. Immunological methods and proteomic were used to detect and to characterize BSDL in urine. The immunoreactive form of BSDL was detected in urines of 36 male subjects devoid of renal failure. Proteomic demonstrated that the immunoreactive protein is BSDL. Experiments using a monoclonal antibody to the oncofetal glycoform of pancreatic BSDL suggested that the protein is not expressed by renal cells but originates from the pancreas via circulation. We demonstrate that under normal physiological conditions, BSDL, a high-molecular weight blood glycoprotein, can be filtered by the renal glomerulus to be eliminated in urines.

Inhibition of glucose oxidation by vasoactive intestinal peptide in isolated rat enterocytes.

Glucose metabolism and its hormonal regulation have been investigated in isolated enterocytes from rat small intestine. About 70% of the glucose consumed by the cells was transformed into lactate, 5% into pyruvate, and 4% into alanine. The remaining 20% was oxidized. Among several tested gastrointestinal peptides and hormones, only vasoactive intestinal peptide (VIP) was found to affect the metabolic fate of glucose. VIP (10(-7) M) induced a 40% inhibition of glucose oxidation without significant modification of either glucose uptake or production of lactate, pyruvate, and alanine. This acute inhibition was dose-dependent (Ki = 3.10(-11) M) and appeared to be dependent on the stimulation of cAMP production (K0.5 = 3.10(-9) M) since dibutyryl-cAMP and forskolin reproduced all the effects of VIP. Similar inhibition of cell respiration by VIP was observed when pyruvate, fructose, and dihydroxyacetone were used as substrates, while the oxidation of glutamine, ketone bodies, and octanoate was unaffected, suggesting that the peptide acts on pyruvate metabolism. The suppression of VIP effects by dichloroacetate (5 mM) and pyruvate (10 mM) and the significant decrease (18%) of the activity of the pyruvate dehydrogenase complex after incubation of the cells with the neuropeptide, support the hypothesis that the effects of VIP on glucose oxidation may occur through an inhibition of the pyruvate dehydrogenase complex. The total suppression of the inhibitory effects of VIP by sodium 2-[6-(4-chlorophenoxy)hexyl]oxirane-2-carboxylate, a potent inhibitor of long-chain fatty acid oxidation, suggests that VIP did not affect the pyruvate dehydrogenase directly, but more probably acted through modifications of fatty acid oxidation.

Probing the origin of acetyl-CoA and oxaloacetate entering the citric acid cycle from the 13C labeling of citrate released by perfused rat hearts.

We present a strategy for simultaneous assessment of the relative contributions of anaplerotic pyruvate carboxylation, pyruvate decarboxylation, and fatty acid oxidation to citrate formation in the perfused rat heart. This requires perfusing with a mix of 13C-substrates and determining the 13C labeling pattern of a single metabolite, citrate, by gas chromatography-mass spectrometry. The mass isotopomer distributions of the oxaloacetate and acetyl moieties of citrate allow calculation of the flux ratios: (pyruvate carboxylation)/(pyruvate decarboxylation), (pyruvate carboxylation)/(citrate synthesis), (pyruvate decarboxylation)/(citrate synthesis) (pyruvate carboxylation)/(fatty acid oxidation), and (pyruvate decarboxylation)/(fatty acid oxidation). Calculations, based on precursor-product relationship, are independent of pool size. The utility of our method was demonstrated for hearts perfused under normoxia with [U-13C3](lactate + pyruvate) and [1-13C]octanoate under steady-state conditions. Under these conditions, effluent and tissue citrate were similarly enriched in all 13C mass isotopomers. The use of effluent citrate instead of tissue citrate allows probing substrate fluxes through the various reactions non-invasively in the intact heart. The methodology should also be applicable to hearts perfused with other 13C-substrates, such as 1-13C-labeled long chain fatty acid, and under various conditions, provided that assumptions on which equations are developed are valid.

Citrate release by perfused rat hearts: a window on mitochondrial cataplerosis.

Cytosolic citrate is proposed to play a crucial role in substrate fuel selection in the heart. However, little is known about factors regulating the transfer of citrate from the mitochondria, where it is synthesized, to the cytosol. Further to our observation that rat hearts perfused under normoxia release citrate whose (13)C labeling pattern reflects that of mitochondrial citrate (B. Comte, G. Vincent, B. Bouchard, and C. Des Rosiers. J. Biol. Chem. 272: 26117-26124, 1997), we report here data indicating that this citrate release is a specific process reflecting the mitochondrial efflux of citrate, a process referred to as cataplerosis. Indeed, measured rates of citrate release, which vary between 2 and 21 nmol/min, are modulated by the nature and concentration of exogenous substrates feeding acetyl-CoA (fatty acid) and oxaloacetate (lactate plus pyruvate) for the mitochondrial citrate synthase reaction. Such release rates that represent at most 2% of the citric acid cycle flux are in agreement with the activity of the mitochondrial tricarboxylate transporter whose participation is also substantiated by 1) parallel variations in citrate release rates and tissue levels of citrate plus malate, the antiporter, and 2) a lowering of the citrate release rate by 1,2, 3-benzenetricarboxylic acid, a specific inhibitor of the transporter. Taken together, the results from the present study indicate that citrate cataplerosis is modulated by substrate supply, in agreement with the role of cytosolic citrate in fuel partitioning, and occurs, at least in part, through the mitochondrial tricarboxylate transporter.

Assay of blood and tissue oxaloacetate and alpha-ketoglutarate by isotope dilution gas chromatography-mass spectrometry.

The assay of oxaloacetate and alpha-ketoglutarate in biological samples is complicated by their chemical instability and low concentrations. We present a quantitative assay for physiological concentrations of these metabolites by isotope dilution gas chromatography-mass spectrometry. Samples are spiked with the corresponding internal standards of [U-13C4]oxaloacetate and [U-13C5] alpha-ketoglutarate prior to their treatment with hydroxylamine. After ethyl acetate extraction and evaporation of the organic phases, the oximes are converted to t-butyldimethylsilyl ethers and analyzed by selected ion monitoring gas chromatography-mass spectrometry of the [M-57]+ ion in electron impact. Although the internal standards of [U-13C4]oxaloacetate and [U-13C5] alpha-ketoglutarate are not commercially available, they can easily be synthesized in 30 min by reacting [1,2,3,6-13C4]citrate with citrate lyase, and L-[U-13C5]glutamate with pyruvate and glutamate-pyruvate transaminase, respectively. Because of their chemical instability, the internal standards are prepared on the day of the analysis. A stock solution of [1,2,3,6-13C4]citrate is prepared from L-[U-13C4]aspartate using citrate synthase and glutamate-oxaloacetate transaminase and then purified and kept frozen until required. The detection limit of the method is 0.05 nmol in a given sample. The method was applied to measurements of oxaloacetate and alpha-ketoglutarate in human blood and rat liver.

Vasoactive intestinal peptide stimulates long-chain fatty acid oxidation and inhibits acetyl-coenzyme A carboxylase activity in isolated rat enterocytes.

The effects of vasoactive intestinal peptide (VIP) on fatty acid oxidation in isolated rat enterocytes were investigated. VIP (10(-7) M) increased more than 2-fold the production of 14CO2 from [U-14C]palmitate. This effect was dose-dependent (K0.5 = 5.10(-11) M) and appeared to be related to the stimulation of cAMP production since it was mimicked by forskolin (10(-4) M). VIP also stimulated oxygen consumption of the cells, an effect accounted for by the stimulation of the oxidation of both exogenous added palmitate (0.12 mM) and endogenous fatty acids produced by lipolysis. VIP appeared to specifically enhance the oxidation of long-chain fatty acids since its effects were counteracted by 5.10(-5) M sodium 2-[6-(chlorophenoxy)hexyl]oxirane-2-carboxylate, a potent inhibitor of carnitine palmitoyltransferase 1, and since VIP did not affect cell respiration in the presence of octanoate. These results suggested that VIP stimulated long-chain fatty acid oxidation by increasing their translocation into the mitochondria. Therefore, we examined the effect of VIP on the activity of acetyl-coenzyme A carboxylase, the enzyme responsible for the biosynthesis of malonyl-CoA, a physiological inhibitor of carnitine acyltransferase 1. VIP produced an acute, dose-dependent (Ki = 3.10(-11) M), 90% inhibition of acetyl-coenzyme A carboxylase activity. These results allow us to elucidate the mechanism of the recently reported inhibitory effect of VIP on glucose oxidation (Vidal, H., Comte, B., Beylot, M., and Riou, J. P. (1988) J. Biol. Chem. 263, 9206-9211) and demonstrate for the first time that balance between fatty acids and glucose as energetic fuels is under neurohormonal control in isolated rat enterocytes.

Dexfenfluramine modulates hepatic glycogen metabolism by a calcium-dependent pathway.

In this study, the mechanism of action of dexfenfluramine (DEXF) at the hepatic level was investigated. The drug is shown to bind to the alpha 1-adrenergic receptor and to increase intracellular calcium in isolated rat hepatocytes, thereby activating phosphorylase via a calcium-dependent mechanism. Moreover, phosphorylase activation by DEXF was inhibited by different agents that interfere with the alpha 1-adrenergic signalling system: prazosin, phorbol 12 alpha-myristate 13 beta-acetate (PMA), and DEXF itself. We also show that phosphorylase activation induced by catecholamines and analogues (epinephrine, phenylephrine), whose actions are mediated by a calcium-dependent mechanism, was counteracted by the drug in the submillimolar range (0.1-1 mM). The activation of glycogenolysis by the drug is accompanied by a stimulation of the glycolytic flux (54% increase in lactate plus pyruvate accumulation), consistent with an increase in fructose-2,6-bisphosphate (F-2,6-BP) levels (36%). These results indicate that the interaction of DEXF with the alpha 1-adrenergic receptor channels glucose 6-phosphate derived from glycogen away from glucose production into the glycolytic pathway.

A 13C mass isotopomer study of anaplerotic pyruvate carboxylation in perfused rat hearts.

Anaplerotic pyruvate carboxylation was examined in hearts perfused with physiological concentrations of glucose, [U-13C3]lactate, and [U-13C3]pyruvate. Also, a fatty acid, [1-13C]octanoate, or ketone bodies were added at concentrations providing acetyl-CoA at a rate resulting in either low or substantial pyruvate decarboxylation. Relative contributions of pyruvate and fatty acids to citrate synthesis were determined from the 13C labeling pattern of effluent citrate by gas chromatography-mass spectrometry (see companion article, Comte, B., Vincent, G., Bouchard, B., and Des Rosiers, C. (1997) J. Biol. Chem. 272, 26117-26124). Precision on flux measurements of anaplerotic pyruvate carboxylation depended on the mix of substrates supplied to the heart. Anaplerotic fluxes were precisely determined under conditions where acetyl-CoA was predominantly supplied by beta-oxidation, as it occurred with 0.2 or 1 mM octanoate. Then, anaplerotic pyruvate carboxylation provided 3-8% of the OAA moiety of citrate and was modulated by concentrations of lactate and pyruvate in the physiological range. Also, the contribution of pyruvate to citrate formation through carboxylation was equal to or greater than through decarboxylation. Furthermore, 13C labeling data on tissue citric acid cycle intermediates and pyruvate suggest that (i) anaplerosis occurs also at succinate and (ii) cataplerotic malate decarboxylation is low. Rather, the presence of citrate in the effluent perfusate of hearts perfused with physiological concentrations of glucose, lactate, and pyruvate and concentrations of octanoate leading to maximal oxidative rates suggests a cataplerotic citrate efflux from mitochondria to cytosol. Taken altogether, our data raise the possibility of a link between pyruvate carboxylation and mitochondrial citrate efflux. In view of the proposed feedback regulation of glycolysis by cytosolic citrate, such a link would support a role of anaplerosis and cataplerosis in metabolic signal transmission between mitochondria and cytosol in the normoxic heart.

Modulation of glucagon-induced glucose production by dexfenfluramine in rat hepatocytes.

The mechanism of the antihyperglycaemic action of dexfenfluramine (DEXF) was investigated in isolated rat hepatocytes exposed to glucagon. Preincubation of hepatocytes with DEXF caused a dose-dependent inhibition of cyclic AMP formation by 100 nM glucagon (Ki = 0.29 mM) that was almost complete at 1 mM DEXF. Surprisingly, glucagon-induced phosphorylase activation was not affected by DEXF despite the significant drop in cyclic AMP levels. Glucose production stimulated by glucagon was inhibited by up to 48% by 1 mM DEXF, and the rate of glucose production correlated positively with the steady-state concentration of glucose 6-phosphate. DEXF also partially restored lactate + pyruvate production which was abolished by an optimal concentration of glucagon. Although DEXF was not able to prevent the inactivation of pyruvate kinase by glucagon, the lack of further accumulation of phosphoenolpyruvate in DEXF-treated cells supports the conclusion that the flux through pyruvate kinase is stimulated, probably via the increase in fructose 2,6-bisphosphate, thereby increasing glycolysis. Our results thus indicate that DEXF counteracts the inhibition of glycolysis by glucagon and that this property might contribute to the antihyperglycaemic effect of this drug. Furthermore, this study shows that, in the presence of the drug, glucagon caused phosphorylase activation and pyruvate kinase inactivation without a significant increase in cyclic AMP levels.

Effect of hepatic portal infusion of pyruvate on pancreatic hormone response during exercise.

The purpose of the present investigation was to evaluate the effects of a small infusion of pyruvate into the hepatic portal vein on the pancreatic hormone response during exercise (30-min treadmill run; 26 m/min, 0% grade) in adrenodemedullated rats. Resting and exercising rats were infused with either pyruvate (5% solution; 0.016 ml/min) into the portal vein, pyruvate into the jugular vein, or saline into the portal vein. Peripheral and portal blood glucose concentrations were decreased (P < 0.01) similarly in all groups after the exercise period. Peripheral insulin, glucagon, and norepinephrine levels, either at rest or after exercise, were not significantly affected by the infusions. The response of portal pancreatic hormone concentrations to exercise was, however, reduced by the pyruvate infused into the portal and jugular veins for insulin and into the portal vein only for glucagon. The normal increase in peripheral glucagon-insulin molar ratio during exercise was shut down by the infusion of pyruvate into the portal vein but not by the infusion of pyruvate into the jugular vein or by the infusion of saline. These results indicate that a small blood infusion of pyruvate, even in the presence of a decreasing blood glucose level, can attenuate substantially the pancreatic hormone response during exercise in adrenodemedullated rats.

Acute hibernation decreases myocardial pyruvate carboxylation and citrate release.

In the well-perfused heart, pyruvate carboxylation accounts for 3-6% of the citric acid cycle (CAC) flux, and CAC carbon is lost via citrate release. We investigated the effects of an acute reduction in coronary flow on these processes and on the tissue content of CAC intermediates. Measurements were made in an open-chest anesthetized swine model. Left anterior descending coronary artery blood flow was controlled by a extracorporeal perfusion circuit, and flow was decreased by 40% for 80 min to induce myocardial hibernation (n = 8). An intracoronary infusion of [U-(13)C(3)]lactate and [U-(13)C(3)]pyruvate was given to measure the entry of pyruvate into the CAC through pyruvate carboxylation from the (13)C-labeled isotopomers of CAC intermediates. Compared with normal coronary flow, myocardial hibernation resulted in parallel decreases of 65% and 79% in pyruvate carboxylation and net citrate release by the myocardium, respectively, and maintenance of the CAC intermediate content. Elevation of the arterial pyruvate concentration by 1 mM had no effect. Thus a 40% decrease in coronary blood flow resulted in a concomitant decrease in pyruvate carboxylation and citrate release as well as maintenance of the CAC intermediates.

Partitioning of pyruvate between oxidation and anaplerosis in swine hearts.

The goal of this study was to measure flux through pyruvate carboxylation and decarboxylation in the heart in vivo. These rates were measured in the anterior wall of normal anesthetized swine hearts by infusing [U-(13)C(3)]lactate and/or [U-(13)C(3)] pyruvate into the left anterior descending (LAD) coronary artery. After 1 h, the tissue was freeze-clamped and analyzed by gas chromatography-mass spectrometry for the mass isotopomer distribution of citrate and its oxaloacetate moiety. LAD blood pyruvate and lactate enrichments and concentrations were constant after 15 min of infusion. Under near-normal physiological concentrations of lactate and pyruvate, pyruvate carboxylation and decarboxylation accounted for 4.7 +/- 0.3 and 41.5 +/- 2.0% of citrate formation, respectively. Similar relative fluxes were found when arterial pyruvate was raised from 0.2 to 1.1 mM. Addition of 1 mM octanoate to 1 mM pyruvate inhibited pyruvate decarboxylation by 93% without affecting carboxylation. The absence of M1 and M2 pyruvate demonstrated net irreversible pyruvate carboxylation. Under our experimental conditions we found that pyruvate carboxylation in the in vivo heart accounts for at least 3-6% of the citric acid cycle flux despite considerable variation in the flux through pyruvate decarboxylation.

Assay of the concentration and 13C-isotopic enrichment of malonyl-coenzyme A by gas chromatography-mass spectrometry.

We developed gas chromatography-mass spectrometry assays for the concentration and mass isotopomer distribution of malonyl-CoA in tissues. The assay involves perchloric acid extraction of the tissue, spiking the extract with [U-13C3]malonyl-CoA or dimethylmalonyl-CoA internal standard, isolation of short-chain acyl-CoA fraction on an oligonucleotide purification cartridge, alkaline hydrolysis to malonate, trimethylsilyl derivatization, and analysis of the mass isotopomer distribution of malonate. The assay was applied to labeling of malonyl-CoA from various [13C]substrates in perfused rat livers and hearts. In livers perfused with [1,2-13C2]acetate, malonyl-CoA is doubly labeled from [1,2-13C2]acetate and singly labeled from 13CO2. In livers perfused with either NaH13CO3 or [3-13C]lactate + [3-13C]pyruvate, the half-lives of singly labeled malonyl-CoA were less than 20 s and 6.95 min, respectively. In rat heart, the half-life of malonyl-CoA, traced with NaH13CO3, was about 1.25 min. Thus, our assay allows us to measure the turnover of tissue malonyl-CoA, the contribution of various [13C]substrates to its production in lipogenic and nonlipogenic organs, and the cycling between acetyl-CoA and malonyl-CoA in nonlipogenic organs.