Validation of fatty acid intakes estimated by a food frequency questionnaire using erythrocyte fatty acid profiling in the Montreal Heart Institute Biobank.

BACKGROUND: To improve the prevention, treatment and risk prediction of cardiovascular diseases, genetic markers and gene-diet interactions are currently being investigated. The Montreal Heart Institute (MHI) Biobank is suitable for such studies because of its large sample size (currently, n = 17 000), the availability of biospecimens, and the collection of data on dietary intakes of saturated (SFAs) and n-3 and n-6 polyunsaturated (PUFAs) fatty acids estimated from a 14-item food frequency questionnaire (FFQ). We tested the validity of the FFQ by correlating dietary intakes of these fatty acids with their red blood cell (RBC) content in MHI Biobank participants. METHODS: Seventy-five men and 75 women were selected from the Biobank. We successfully obtained RBC fatty acids for 142 subjects using gas chromatography coupled to mass spectrometry. Spearman correlation coefficients were used to test whether SFA scores and daily intakes (g day(-1)) of n-3 and n-6 PUFAs correlate with their RBC content. RESULTS: Based on covariate-adjusted analyses, intakes of n-3 PUFAs from vegetable sources were significantly correlated with RBC α-linolenic acid levels (ρ = 0.23, P = 0.007), whereas n-3 PUFA intakes from marine sources correlated significantly with RBC eicosapentaenoic acid (ρ = 0.29, P = 0.0008) and docosahexaenoic acid (ρ = 0.41, P = 9.2 × 10(-7)) levels. Intakes of n-6 PUFAs from vegetable sources correlated with RBC linoleic acid (ρ = 0.18, P = 0.04). SFA scores were not correlated with RBC total SFAs. CONCLUSIONS: The MHI Biobank 14-item FFQ can appropriately estimate daily intakes of n-3 PUFAs from vegetable and marine sources, as well as vegetable n-6 PUFAs, which enables the possibility of using these data in future studies.

Sildenafil and cardiomyocyte-specific cGMP signaling prevent cardiomyopathic changes associated with dystrophin deficiency.

We recently demonstrated early metabolic alterations in the dystrophin-deficient mdx heart that precede overt cardiomyopathy and may represent an early "subclinical" signature of a defective nitric oxide (NO)/cGMP pathway. In this study, we used genetic and pharmacological approaches to test the hypothesis that enhancing cGMP, downstream of NO formation, improves the contractile function, energy metabolism, and sarcolemmal integrity of the mdx heart. We first generated mdx mice overexpressing, in a cardiomyocyte-specific manner, guanylyl cyclase (GC) (mdx/GC(+/0)). When perfused ex vivo in the working mode, 12- and 20-week-old hearts maintained their contractile performance, as opposed to the severe deterioration observed in age-matched mdx hearts, which also displayed two to three times more lactate dehydrogenase release than mdx/GC(+/0). At the metabolic level, mdx/GC(+/0) displayed a pattern of substrate selection for energy production that was similar to that of their mdx counterparts, but levels of citric acid cycle intermediates were significantly higher (36 +/- 8%), suggesting improved mitochondrial function. Finally, the ability of dystrophin-deficient hearts to resist sarcolemmal damage induced in vivo by increasing the cardiac workload acutely with isoproterenol was enhanced by the presence of the transgene and even more so by inhibiting cGMP breakdown using the phosphodiesterase inhibitor sildenafil (44.4 +/- 1.0% reduction in cardiomyocyte damage). Overall, these findings demonstrate that enhancing cGMP signaling, specifically downstream and independent of NO formation, in the dystrophin-deficient heart improves contractile performance, myocardial metabolic status, and sarcolemmal integrity and thus constitutes a potential clinical avenue for the treatment of the dystrophin-related cardiomyopathies.

Higher circulating 4-hydroxynonenal-protein thioether adducts correlate with more severe diastolic dysfunction in spontaneously hypertensive rats.

OBJECTIVE: Accumulating evidence supports a role of 4-hydroxynonenal (4HNE) in oxidative-stress related diseases, but its specific contribution to disease development remains to be clarified. Further to our finding of high circulating 4HNE-protein thioether adducts (4HNE-P) in spontaneously hypertensive rats (SHRs), we aimed at correlating 4HNE-P with cardiac function and testing the impact of antioxidant therapy. MATERIALS AND METHODS: The lipoperoxidation inhibitor probucol (10 mg/kg/day) or vehicle (corn oil) were administered daily (i.p.) for 4 weeks in 18-week-old SHRs (9 rats/group). Cardiac functions were assessed by echocardiography and 4HNE-P by gas chromatography/mass spectrometry. RESULTS: Diastolic dysfunction worsened in SHRs receiving vehicle as reflected by changes (P < 0.05) in indexes of left ventricular relaxation (increased isovolumic relaxation time) and compliance (increased E-wave deceleration rate; EDR). Higher circulating 4HNE-P correlated with diastolic dysfunction (EDR: R(2) = 0.518; P < 0.001) and heart rate (R(2) = 0.225; P < 0.05). Probucol prevented the deterioration of diastolic function, while lowering the mean and median of circulating 4HNE-P by 21% and 35%, respectively. CONCLUSION: Collectively, these results support a role for 4HNE in the pathophysiological events linked to disease progression in SHRs.

Myocardial phenotyping using isotopomer analysis of metabolic fluxes.

Over the past 20 years, stable isotopes combined with isotopomer analysis have proven to be a powerful approach to probe the dynamics of metabolism in various biological systems, including the heart. The aim of this paper is to demonstrate how isotopomer analysis of metabolic fluxes can provide novel insights into the myocardial phenotype. Specifically, building on our past experience using NMR spectroscopy and GC-MS as applied to investigations of cardiac energy metabolism, we highlight specific complex metabolic networks that would not be predicted by classical biochemistry or by static measurements of metabolite, protein and mRNA levels.

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.

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.

Evidence of separate pathways for lactate uptake and release by the perfused rat heart.

The simultaneous release and uptake of lactate by the heart has been observed both in vivo and ex vivo; however, the pathways underlying these observations have not been satisfactorily explained. Consequently, the purpose of this study was to test the hypothesis that hearts release lactate from glycolysis while simultaneously taking up exogenous lactate. Therefore, we determined the effects of fatty acids and diabetes on the regulation of lactate uptake and release. Hearts from control and 1-wk diabetic animals were perfused with 5 mM glucose, 0.5 mM [3-(13)C]lactate, and 0, 0.1, 0.32, or 1.0 mM palmitate. Parameters measured include perfusate lactate concentrations, fractional enrichment, and coronary flow rates, which enabled the simultaneous, but independent, measurements of the rates of 1) uptake of exogenous [(13)C]lactate and 2) efflux of unlabeled lactate from metabolism of glucose. Although the rates of lactate uptake and efflux were both similarly inhibited by the addition of palmitate, (i.e., the ratio of lactate uptake to efflux remained constant), the ratio of lactate uptake to efflux was significantly higher in the controls compared with the diabetic group (1.00 +/- 0.14 vs. 0.50 +/- 0.07, P < 0.002). These data, combined with heterogeneous (13)C enrichment of tissue lactate, pyruvate, and alanine, suggest that glycolytically derived lactate production and oxidation of exogenous lactate operate as functionally separate metabolic pathways. These results are consistent with the concept of an intracellular lactate shuttle.

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.

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.

Dosimetric analysis and clinical implementation of 6 MV X-ray radiosurgery beam.

The dosimetric data on tissue maximum ratios (TMR), output factors, off axis ratios and beam profiles are presented for small circular fields of diameters ranging from 12.5 to 40 mm for 6 MV radiosurgery beam. It is noticed that dmax increases as the collimator field size increases. Comparison of our data with the published TMR and output factors of similar small circular fields shows that our values are higher than those data. Similarities in trend are noticed with the published isodose volumes for 1-5 and 10 arcs. Not much variation is seen beyond two arcs for 80% isodose volumes for all the field sizes. The variation is small in 20% isodose volumes beyond three arcs. Variations are noticed in 5% isodose volumes for 12.5 mm diameter collimated beam. Our experience has been exclusively with malignant neoplasms. An ideal target volume is covered by 80% isodose volume with 3-4 arcs and a single isocenter. Sixteen patients have been treated to date at our institution, including one patient with brain metastases, two patients with meningiomas, one patient with lymphoma and 12 patients with astrocytomas. The majority of tumors have been treated with single isocenter but some as large as 7 cm have been treated safely with two isocenters.

Variable glycation of serum proteins in patients with diabetes mellitus.

OBJECTIVE: To determine whether there were variations in vivo and in vitro in the glycation process among patients with diabetes mellitus and to assess the characteristics of patients with high and low glycation, if this was observed. PATIENTS: Patients (n = 185) attending a Diabetes Day Care Centre or Notre-Dame Hospital in Montreal participated in the in vivo study. Patients found to have high and low glycation were asked to allow the use of their serum for the in vitro part of the study. INTERVENTION: Capillary blood glucose levels were determined by nursing staff 4 times a day over 7.3 (standard deviation [SD]5.3) consecutive days with commercially available glucose oxydase reagent strips and meters. The ratio of the fructosamine concentration to the protein concentration (the F/P ratio) and the glycated hemoglobin were also determined at the same time as the capillary blood glucose level. Glycation was defined as the mean capillary blood glucose/F/P ratio. Patients with high and low glycation (higher or lower than [SD], of the mean) were compared. For the in vitro study, incorporation of carbon-14 glucose in serum proteins incubated with a 30-mmol/L glucose concentration was studied in some of the patients with low and high glycation. RESULTS: The mean capillary blood glucose/F/P ratio was a mean of 2.30 (SD 0.29) g/mL. Of the 185 subjects 31 had high glycation (1.46 [SD 0.19] g/mL) and 27 had low glycation (2.97 [SD 0.035] g/mL, p < 0.001). There was no significant difference in age, sex, diabetic treatment and glycated hemoglobin levels between the 2 groups. However, patients with low glycation had a greater body mass index (29.4 [SD 5.7] kg/m2 v. 26.4 [SD 4.3] kg/m2, p < 0.05). In vitro, incorporation of 14C glucose in serum proteins incubated with a 30mmol/L glucose concentration was higher in the 9 patients with high glycation than in that of the 7 with low glycation (0.031% [SD 0.03%] per gram of proteins v. 0.028% [SD 0.03%] per gram of proteins, p < 0.02). CONCLUSIONS: Glycation may vary among patients with diabetes mellitus who have similar capillary blood glucose concentrations. Glycation appears to be lower in patients with a greater body mass index. Furthermore, alternation in the glycation process itself may explain, in addition to the mean blood glucose level, the difference in fructosamine levels.

Applications of mass isotopomer analysis to nutrition research.

Investigations into regulating metabolic pathways with stable isotopes have, over the past decade, undergone major development with the use of nuclear magnetic resonance and mass spectrometry in studying labeling patterns of newly synthesized biomolecules. In this review, we concentrate on investigations of mass isotopomer distribution (MID) measured by mass spectrometry. We review the applications of MID to analytical problems, in particular the possibility of amplifying the measurement of low isotopic enrichments by incorporating multiple molecules or atoms of a primary analyte into the molecule of a secondary analyte, the MID of which is assayed. We also review new information on the regulation of intermediary metabolism gathered from the analysis of MID patterns of synthesized compounds. Lastly, we review the applications of MID to the synthesis of polymeric molecules, with emphasis on the validity of these techniques. A number of these techniques are applicable to investigations of nutrient metabolism in health and disease.

Instantaneous analysis of aldehydes in biological fluids using a spray interface coupled to a mass spectrometer.

A new interface coupled to a mass spectrometer was developed for the direct analysis of volatile organic compounds from small volumes of aqueous samples, including blood or tissue homogenates (St-Germain et al. 1995, Anal. Chem. 67:4536-4541). The greatest advantages of our system are minimal sample treatment, an instantaneous response time coupled with detection limits in the range of < 1 ppb for most compounds. For the analysis of low-molecular weight aldehydes, such as formaldehyde, acetaldehyde, propanal, and hexanal, lower detection limits were obtained when samples were converted to methoxime derivatives prior to injection. The detection limit for hexanal in water or Krebs-Ringer solution was 0.01 microM (10 pmol injected). The reproducibility of replicate injections was 4.4%. The usefulness of our system was illustrated by measuring aldehyde accumulation in peroxidized solutions of polyunsaturated fatty acids and rat tissue homogenates. Data confirmed that peroxidation of omega-3 fatty acids produces propanal, whereas omega-6 fatty acids form hexanal. Peroxidation of heart and brain homogenates formed predominantly propanal. However, the recovery of hexanal after sample treatment with methoxylamine depended on the derivatization time and temperature, suggesting that this aldehyde may form Schiff base linkages. These results show that spray extraction coupled to mass spectrometry provides a quick (< 1 min), clean and reproducible way to detect aldehydes produced from lipid peroxidation in aqueous samples.

Effects and metabolism of fumarate in the perfused rat heart. A 13C mass isotopomer study.

The cardioprotective effects of fumarate have been linked to its metabolism to succinate through both oxidative and reductive pathways. To date, the relative contribution of these pathways is a subject of controversy. To address this question, we designed a protocol with 13C substrates and took advantage of 13C isotopomer analysis by gas chromatography-mass spectrometry. Rat hearts were perfused with 11 mM glucose, 1 mM lactate, 0.2 mM pyruvate, 0.2 mM [1-13C]octanoate, and 0.04 or 0.4 mM [U-13C4]fumarate. On reoxygenation after 40 min of severe hypoxia, hearts perfused with 0.4 mM fumarate showed a better recovery of contractile function and released less lactate dehydrogenase (an index of cellular necrosis) than those perfused with 0.04 mM fumarate. The 13C data showed that, in hypoxic hearts, fumarate conversion to succinate occurred only through reduction, although it accounted for only 16% of total succinate release. Most of the succinate was formed through the oxidation of alpha-ketoglutarate or its precursors (50 +/- 5%) and by another yet-unidentified pathway (34 +/- 4%). These data show that, in a model of hypoxia-reoxygenation, the cardioprotective effects of fumarate were associated with its predominant metabolism to succinate through the reductive pathway.

Assay of the 13C and 2H mass isotopomer distribution of phosphoenolpyruvate by gas chromatography/mass spectrometry.

The 13C mass isotopomer distribution of liver phosphoenolpyruvate (PEP) yields important information on the regulation of gluconeogenesis and the citric acid cycle. A convenient technique is presented for measuring the mass isotopomer distribution of PEP in tissue extracts. The procedure involves reduction of extant pyruvate to lactate with NaBH4, enzymatic conversion of PEP to pyruvate, extraction of pyruvate hydroxamate and gas chromatographic/mass spectrometric determination of pyruvate hydroxamate di-tert-butyldimethylsilyl derivative. When PEP is labeled with 2H, the enzymatic conversion of PEP to pyruvate results in the loss of 2H. Therefore, to assay the enrichment of [2H]PEP, the tissue extract is chromatographed on an anion-exchange column. The fraction containing PEP is treated to form PEP tri(trimethylsilyl) derivative. The procedures were applied to liver PEP labeled using [U-13C3]lactate, [U-13C3]glycerol or 2H2O. The results show the compatibility between the mass isotopomer distributions of PEP and glucose in rat livers perfused with [U-13C3]lactate or [U-13C3]glycerol. There is a 78% isotopic equilibration of 2H enrichment between the hydrogens on C-3 of liver PEP and the hydrogens of water in 2 day fasted rats.

Correction of 13C mass isotopomer distributions for natural stable isotope abundance.

Metabolism of singly or multiply 13C-labeled substrates leads to the production of molecules that contain 13C atoms at various positions. Molecules differing only in the number of isotopic atoms incorporated are referred to as mass isotopomers. The distribution of mass isotopomers of many molecules can be measured by gas chromatography/ mass spectrometry after chemical derivatization. Quantification of metabolite mass isotopomer abundance resulting from biological processes necessitates correction of the measured mass isotopomer distribution of the derivatized metabolite for contributions due to naturally occurring isotopes of its elements. This correction must take into account differences in the relative natural abundance distribution of each mass isotopomer (skewing). An IBM-compatible computer program was developed which (i) calculates the natural abundance mass isotopomer distribution of unlabeled and labeled standards given the molecular formula of the derivatized molecule or fragment ion, and (ii) calculates the natural abundance mass isotopomer distribution of the singly and multiply labeled molecule or fragment via non-linear fitting to the measured mass isotopomer distribution of the unlabeled molecule or fragment. The output of this program is used to correct measured mass isotopomer distributions for contributions from natural isotope abundances and to verify measured values for theoretical consistency. Differences between predicted and measured unlabeled and 13C-labeled isotopomer distributions for hydroxamate di-t-butyl-dimethylsilyl (di-TBDMS) derivatized pyruvate were measured. The program was applied to the mass isotopomer distribution of glucose labeled from [U-13C3]glycerol and of fatty acids labeled from [U-13C6]glucose and either [2-13C2] acetate or [U-13C2]acetate. In some of these cases, the measured mass isotopomer distributions corrected by the program were different from those corrected by the classical technique. Implications of these differences including those on the calculation of glucose production due to gluconeogenesis in isolated perfused rat liver are discussed.

Effects of N-acetylcysteine in the rat heart reperfused after low-flow ischemia: evidence for a direct scavenging of hydroxyl radicals and a nitric oxide-dependent increase in coronary flow.

The capacity of N-acetylcysteine to directly scavenge hydroxyl radical produced by rat hearts reperfused after 90 min of low-flow ischemia was assessed by the hydroxylation of 4-hydroxybenzoate into 3,4-dihydroxybenzoate using a gas chromatography-mass spectrometric assay. Reperfused hearts showed a massive release of 3,4-dihydroxybenzoate, lactate dehydrogenase, and total glutathione, contained less reduced and oxidized glutathione, but maintained spontaneous beating and coronary flow rates close to preischemic values. Compared to untreated hearts: reperfused hearts treated with N-acetylcysteine from the start of ischemia (i) released four times less 3,4-dihydroxybenzoate, but similar amounts of lactate dehydrogenase or glutathione, (ii) showed a nitric oxide-dependent increase in coronary flow rate, and (iii) contained less oxidized glutathione, but similar amounts of reduced glutathione. Reperfused hearts receiving N-acetylcysteine since the last 5 min of ischemia had also a four-times lower 3,4-dihydroxybenzoate release, but their coronary flow rate response was similar to that of untreated hearts. These results indicate that N-acetylcysteine can directly scavenge hydroxyl radicals produced by reperfused ischemic hearts, although this effect is not associated with any protective effects as indicated by the lactate dehydrogenase and glutathione release and cannot explain the nitric oxide-dependent reperfusion hyperemia.

Isotopomer analysis of citric acid cycle and gluconeogenesis in rat liver. Reversibility of isocitrate dehydrogenase and involvement of ATP-citrate lyase in gluconeogenesis.

We conducted an extensive mass isotopomer analysis of citric acid cycle and gluconeogenic metabolites isolated from livers of overnight fasted rats perfused with 4 mM glucose, 0.2 mM octanoate, 1 mM [U-13C3]lactate, and 0.2 mM [U-13C3]pyruvate, in the anterograde or retrograde mode. In both perfusion modes, two distinct isotopomer patterns were observed: (i) those of phosphoenolpyruvate, glucose, malate, and aspartate and (ii) those of citrate, alpha-ketoglutarate, glutamate, and glutamine. Key citric acid cycle parameters and, hence, rates of gluconeogenesis, calculated (Lee, W.-N.P. (1989) J. Biol. Chem. 264, 13002-13004 and Lee, W.-N.P. (1993) J. Biol. Chem. 268, 25522-25526) from our mass isotopomer data did not only vary, but lead to conclusions inconsistent with Lee's citric acid cycle model. Compared to lactate and pyruvate uptake, which sets an upper limit to glucose production, rates of gluconeogenesis calculated (i) with the phosphoenolpyruvate and citrate data were similar, but those calculated (ii) with the glutamate data amounted to only 60%, which is unlikely. All these conclusions are independent of the perfusion modes. We provide evidence that the following processes contribute to the observed labeling discrepancy: (i) the reversibility of the isocitrate dehydrogenase reaction and (ii) an active citrate cleavage pathway for the transfer of the oxaloacetate carbon skeleton from mitochondria to the cytosol. Also, a good fit of our labeling data was obtained with a model of citric acid cycle and gluconeogenesis which we developed to incorporate the above reactions (Fernandez, C.A., and Des Rosiers, C. (1995) J. Biol. Chem. 270, 10037-10042). The following conclusions can be drawn from the calculated reaction rates: (i) about half of the lactate conversion to glucose occurs via the citrate cleavage pathway, (ii) the flux through the reversal of the isocitrate dehydrogenase reaction is almost as fast as that through the citrate synthase reaction, and (iii) the flux through citrate synthase and alpha-ketoglutarate dehydrogenase is 1.6- and 3.2-fold that through pyruvate carboxylase, respectively.

Modeling of liver citric acid cycle and gluconeogenesis based on 13C mass isotopomer distribution analysis of intermediates.

We have developed and implemented a model that can predict the positional isotopomer distribution of various hepatic metabolites labeled with [U-13C3]lactate and/or [U-13C3]pyruvate for given relative flux rates through the citric acid cycle and gluconeogenesis reactions. Our model includes (i) isotopic exchange between alpha-ketoglutarate and glutamate, (ii) a reversible isocitrate dehydrogenase reaction, (iii) an active ATP-citrate lyase, and (iv) aspartate and malate shuttles with separate cytosolic and mitochondrial pools for oxaloacetate, malate, and fumarate. A parameter estimation routine fit the mass isotopomer distribution of selected metabolites measured by gas chromatography-mass spectrometry to the model predicted distributions. We fit measured mass isotopomer distributions of phosphoenolpyruvate, citrate, alpha-ketoglutarate, glutamate, and pyruvate isolated from fasted rat livers perfused with [U-13C3]lactate + [U-13C3]pyruvate. This fitting yielded rates which we express relative to that of pyruvate carboxylase: citric acid cycle represented by the irreversible alpha-ketoglutarate dehydrogenase = 0.32; citrate synthase = 0.64; reversal of isocitrate dehydrogenase = 0.52; citrate lyase = 0.33, aspartate shuttle = 0.24, and malate shuttle = 0.44. Rates calculated for the cytosolic and mitochondrial fumarate and malate dehydrogenase reactions are subject to uncertainties as indicated by identifiability analyses. Previous forms of our model that did not include pyruvate kinase, exchange of alpha-ketoglutarate with glutamate, reversibility of isocitrate dehydrogenase, and/or ATP-citrate lyase activity were not as successful at predicting our measured values. This model offers a general tool for studying the regulation of the citric acid cycle and gluconeogenesis and can be readily modified for any 13C-labeled lactate or pyruvate substrate.