Mass isotopomer study of anaplerosis from propionate in the perfused rat heart

Anaplerosis from propionate was investigated in rat hearts perfused with 0-2mM [(13)C(3)]propionate and physiological concentrations of glucose, lactate, and pyruvate. The data show that when the concentration of [(13)C(3)]propionate was raised from 0 to 2mM, total anaplerosis increased from 5% to 16% of the turnover of citric acid cycle intermediates. Then, [(13)C(3)]propionate abolished anaplerosis from endogenous substrates, glucose, lactate, and pyruvate. Also, while the contents of propionyl-CoA and methylmalonyl-CoA increased with [(13)C(3)]propionate concentration, the content of succinyl-CoA decreased, presumably via activation of succinyl-CoA hydrolysis by a decrease in free CoA. Under our conditions, [(13)C(3)]propionate was a purely anaplerotic substrate since there was no labeling of mitochondrial acetyl-CoA, reflected by the labeling of the acetyl moiety of citrate.     

Assay of the concentration and (13)C isotopic enrichment of propionyl-CoA,methylmalonyl-CoA,and succinyl-CoA by gas chromatography-mass spectrometry

We developed gas chromatography-mass spectrometry assays for the concentration and mass isotopomer distribution of propionyl-CoA, methylmalonyl-CoA, and succinyl-CoA in tissues. The assays involves perchloric acid extraction of the tissue, spiking the extract with [(2)H(5)]propionyl-CoA and [(2)H(4)]succinyl-CoA internal standards, and isolation of short-chain acyl-CoA fraction on an oligonucleotide purification cartridge. Propionyl-CoA is reacted with sarcosine and the formed N-propionylsarcosine is assayed as its pentafluorobenzyl derivative. Methylmalonyl-CoA and succinyl-CoA are hydrolyzed and the corresponding acids assayed as tert-butyl dimethylsilyl derivatives. The assay was applied to a study of [U-(13)C(3)]propionate metabolism in perfused rat livers. While propionyl-CoA is only M3 labeled, succinyl-CoA is M3, M2, and M1 labeled because of isotopic exchanges in the citric acid cycle. Methylmalonyl-CoA is M3 and M2 labeled, reflecting reversal of S-methylmalonyl-CoA mutase. Thus, our assays allow measuring the turnover of the coenzyme A derivatives involved in anaplerosis of the citric acid cycle via precursors of propionyl-CoA, i.e., propionate, odd-chain fatty acids, isoleucine, threonine, and valine.     

Differential effects of heptanoate and hexanoate on myocardial citric acid cycle intermediates following ischemia-reperfusion.

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.     

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

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.     

Isotopomer analysis of myocardial substrate metabolism: a systems biology approach

The increasing accessibility of mass isotopomer data via GC-MS and NMR technology has necessitated the use of a systematic and reliable method to take advantage of such data for flux analysis. Here we applied a nonlinear, optimization-based method to study substrate metabolism in cardiomyocytes using (13)C data from perfused mouse hearts. The myocardial metabolic network used in this study accounts for 257 reactions and 240 metabolites, which are further compartmentalized into extracellular space, cytosol, and mitochondrial matrix. Analysis of the perfused mouse heart showed that the steady-state ATP production rate was 16.6 +/- 2.3 micromol/min . gww, with 30% of the ATP coming from glycolysis. Of the four substrates available in the perfusate (glucose, pyruvate, lactate, and oleate), exogenous glucose forms the majority of cytosolic pyruvate. Pyruvate decaboxylation is significantly higher than carboxylation, suggesting that anaplerosis is low in the perfused heart. Exchange fluxes were predicted to be high for reversible enzymes in the citric acid cycle (CAC), but low in the glycolytic pathway. Pseudoketogenesis amounted to approximately 50% of the net ketone body uptake. Sensitivity analysis showed that the estimated flux distributions were relatively insensitive to experimental errors. The application of isotopomer data drastically improved the estimation of reaction fluxes compared to results computed with respect to reaction stoichiometry alone. Further study of 12 commonly used (13)C glucose mixtures showed that the mixtures of 20% [U-(13)C(6)] glucose, 80% [3 (13)C] glucose and 20% [U-(13)C(6)] glucose, 80% [4 (13)C] were best for resolving fluxes in the current network.     

Metabolomic and mass isotopomer analysis of liver gluconeogenesis and citric acid cycle: II. Heterogeneity of metabolite labeling pattern

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.     

Compartmentation of Metabolism of the C12-, C9-, and C5-n-dicarboxylates in Rat Liver,Investigated by Mass Isotopomer Analysis: ANAPLEROSIS FROM DODECANEDIOATE*

We investigated the compartmentation of the catabolism of dodecanedioate (DODA), azelate, and glutarate in perfused rat livers, using a combination of metabolomics and mass isotopomer analyses. Livers were perfused with recirculating or nonrecirculating buffer containing one fully ¹³C-labeled dicarboxylate. Information on the peroxisomal versus mitochondrial catabolism was gathered from the labeling patterns of acetyl-CoA proxies, i.e. total acetyl-CoA, the acetyl moiety of citrate, C-1 + 2 of β-hydroxybutyrate, malonyl-CoA, and acetylcarnitine. Additional information was obtained from the labeling patterns of citric acid cycle intermediates and related compounds. The data characterize the partial oxidation of DODA and azelate in peroxisomes, with terminal oxidation in mitochondria. We did not find evidence of peroxisomal oxidation of glutarate. Unexpectedly, DODA contributes a substantial fraction to anaplerosis of the citric acid cycle. This opens the possibility to use water-soluble DODA in nutritional or pharmacological anaplerotic therapy when other anaplerotic substrates are impractical or contraindicated, e.g. in propionic acidemia and methylmalonic acidemia.     

Accumulation and disposal of tricarboxylic acid cycle intermediates during propionate oxidation in the isolated perfused rat heart

The role of the metabolite disposal mechanisms in the regulation of the tricarboxylic acid cycle pool size was studied in isolated perfused rat hearts oxidizing 2 mM propionate. Malate and succinate accumulated during the propionate metabolism. A further 118% increase in the malate concentration and 600% increase in the succinate concentration and a slight inhibition of the propionate uptake were observed during a subsequent KCl-induced arrest of the heart metabolizing propionate. When the mechanical activity of the heart was restored, the malate and succinate concentrations returned to the same levels as before the arrest of the heart, but the propionate uptake did not rise significantly. The mean disposal rates of the tricarboxylic acid cycle metabolites during the cardiac arrest and subsequent restoration of the activity were 1.4 and 2.4 μmol/min per g dry weight, respectively. During cardiac arrest the malate carbon disposed was almost totally recovered as C₃ compounds, whereas after the increase in the ATP-consumption most of it was oxidized. The results show that propionate is oxidized by heart muscle at an appreciable rate but the disposal rate of the tricarboxylic acid cycle intermediates is not tightly regulated by the cellular energy state. Although the metabolite pool size of the tricarboxylic acid cycle responds to change in the ATP consumption, the energy state appears to have a greater effect on the fate of the C₃ compounds formed than on the actual rate of C₄ compound disposition.     

Identification and isolation of the phosphorylated intermediate of the calcium pump in rat intestinal basolateral membranes.

High-resolution 13C n.m.r. spectroscopy has been used to examine propionate metabolism in the perfused rat heart. A number of tricarboxylic acid (TCA) cycle intermediates are observable by 13C n.m.r. in hearts perfused with mixtures of pyruvate and propionate. When the enriched 13C-labelled nucleus originates with pyruvate, the resonances of the intermediates appear as multiplets due to formation of multiply-enriched 13C-labelled isotopomers, whereas when the 13C-labelled nucleus originates with propionate, these same intermediates appear as singlets in the 13C spectrum since entry of propionate into the TCA cycle occurs via succinyl-CoA. An analysis of the isotopomer populations in hearts perfused with [3-13C]pyruvate plus unlabelled propionate indicates that about 27% of the total pyruvate pool available to the heart is derived directly from unlabelled propionate. This was substantiated by perfusing a heart for 2 h with [3-13C]propionate as the only available exogenous substrate. Under these conditions, all of the propionate consumed by the heart, as measured by conventional chemical analysis, ultimately entered the oxidative pathway as [2-13C] or [3-13C]pyruvate. This is consistent with entry of propionate into the TCA cycle intermediate pools as succinyl-CoA and concomitant disposal of malate to pyruvate via the malic enzyme. 13C resonances arising from enriched methylmalonate and propionylcarnitine are also detected in hearts perfused with [3-13C] or [1-13C]propionate which suggests that 13C n.m.r. may be useful as a non-invasive probe in vivo of metabolic abnormalities involving the propionate pathway, such as methylmalonic aciduria or propionic acidaemia.     

Triiodothyronine increases myocardial function and pyruvate entry into the citric acid cycle after reperfusion in a model of infant cardiopulmonary bypass

Triiodothyronine (T3) supplementation improves clinical outcomes in infants after cardiac surgery using cardiopulmonary bypass by unknown mechanisms. We utilized a translational model of infant cardiopulmonary bypass to test the hypothesis that T3 modulates pyruvate entry into the citric acid cycle (CAC), thereby providing the energy support for improved cardiac function after ischemia-reperfusion (I/R). Neonatal piglets received intracoronary [2-(13)Carbon((13)C)]pyruvate for 40 min (8 mM) during control aerobic conditions (control) or immediately after reperfusion (I/R) from global hypothermic ischemia. A third group (I/R-Tr) received T3 (1.2 μg/kg) during reperfusion. We assessed absolute CAC intermediate levels and flux parameters into the CAC through oxidative pyruvate decarboxylation (PDC) and anaplerotic carboxylation (PC) using [2-(13)C]pyruvate and isotopomer analysis by gas and liquid chromatography-mass spectrometry and (13)C-nuclear magnetic resonance spectroscopy. When compared with I/R, T3 (group I/R-Tr) increased cardiac power and oxygen consumption after I/R while elevating flux of both PDC and PC (～4-fold). Although neither I/R nor I/R-Tr modified absolute CAC levels, T3 inhibited I/R-induced reductions in their molar percent enrichment. Furthermore, (13)C-labeling of CAC intermediates suggests that T3 may decrease entry of unlabeled carbons at the level of oxaloacetate through anaplerosis or exchange reaction with asparate. T3 markedly enhances PC and PDC fluxes, thereby providing potential substrate for elevated cardiac function after reperfusion. This T3-induced increase in pyruvate fluxes occurs with preservation of the CAC intermediate pool. Our labeling data raise the possibility that T3 reduces reliance on amino acids for anaplerosis after reperfusion.     

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.     

Perspective: emerging evidence for signaling roles of mitochondrial anaplerotic products in insulin secretion

The importance of mitochondrial biosynthesis in stimulus secretion coupling in the insulin-producing beta-cell probably equals that of ATP production. In glucose-induced insulin secretion, the rate of pyruvate carboxylation is very high and correlates more strongly with the glucose concentration the beta-cell is exposed to (and thus with insulin release) than does pyruvate decarboxylation, which produces acetyl-CoA for metabolism in the citric acid cycle to produce ATP. The carboxylation pathway can increase the levels of citric acid cycle intermediates, and this indicates that anaplerosis, the net synthesis of cycle intermediates, is important for insulin secretion. Increased cycle intermediates will alter mitochondrial processes, and, therefore, the synthesized intermediates must be exported from mitochondria to the cytosol (cataplerosis). This further suggests that these intermediates have roles in signaling insulin secretion. Although evidence is quite good that all physiological fuel secretagogues stimulate insulin secretion via anaplerosis, evidence is just emerging about the possible extramitochondrial roles of exported citric acid cycle intermediates. This article speculates on their potential roles as signaling molecules themselves and as exporters of equivalents of NADPH, acetyl-CoA and malonyl-CoA, as well as alpha-ketoglutarate as a substrate for hydroxylases. We also discuss the "succinate mechanism," which hypothesizes that insulin secretagogues produce both NADPH and mevalonate. Finally, we discuss the role of mitochondria in causing oscillations in beta-cell citrate levels. These parallel oscillations in ATP and NAD(P)H. Oscillations in beta-cell plasma membrane electrical potential, ATP/ADP and NAD(P)/NAD(P)H ratios, and glycolytic flux are known to correlate with pulsatile insulin release. Citrate oscillations might synchronize oscillations of individual mitochondria with one another and mitochondrial oscillations with oscillations in glycolysis and, therefore, with flux of pyruvate into mitochondria. Thus citrate oscillations may synchronize mitochondrial ATP production and anaplerosis with other cellular oscillations.     

Differences between mouse and rat pancreatic islets: succinate responsiveness,malic enzyme,and anaplerosis

Succinic acid methyl esters are potent insulin secretagogues in rat pancreatic islets, but they do not stimulate insulin release in mouse islets. Unlike rat and human islets, mouse islets lack malic enzyme and, therefore, are unable to form pyruvate from succinate-derived malate for net synthesis of acetyl-CoA. Dimethyl-[2,3-(14)C]succinate is metabolized in the citric acid cycle in mouse islets to the same extent as in rat islets, indicating that endogenous acetyl-CoA condenses with oxaloacetate derived from succinate. However, without malic enzyme, the net synthesis from succinate of the citric acid cycle intermediates citrate, isocitrate, and alpha-ketoglutarate cannot occur. Glucose and other nutrients that augment alpha-ketoglutarate formation are secretagogues in mouse islets with potencies similar to those in rat islets. All cycle intermediates can be net-synthesized from alpha-ketoglutarate. Rotenone, an inhibitor of site I of the electron transport chain, inhibits methyl succinate-induced insulin release in rat islets even though succinate oxidation forms ATP at sites II and III of the respiratory chain. Thus generating ATP, NADH, and anaplerosis of succinyl-CoA plus the four-carbon dicarboxylic acids of the cycle and its metabolism in the citric acid cycle is insufficient for a fuel to be insulinotropic; it must additionally promote anaplerosis of alpha-ketoglutarate or two intermediates interconvertible with alpha-ketoglutarate, citrate, and isocitrate.     

Quantifying reductive carboxylation flux of glutamine to lipid in a brown adipocyte cell line

We previously reported that glutamine was a major source of carbon for de novo fatty acid synthesis in a brown adipocyte cell line. The pathway for fatty acid synthesis from glutamine may follow either of two distinct pathways after it enters the citric acid cycle. The glutaminolysis pathway follows the citric acid cycle, whereas the reductive carboxylation pathway travels in reverse of the citric acid cycle from alpha-ketoglutarate to citrate. To quantify fluxes in these pathways we incubated brown adipocyte cells in [U-(13)C]glutamine or [5-(13)C]glutamine and analyzed the mass isotopomer distribution of key metabolites using models that fit the isotopomer distribution to fluxes. We also investigated inhibitors of NADP-dependent isocitrate dehydrogenase and mitochondrial citrate export. The results indicated that one third of glutamine entering the citric acid cycle travels to citrate via reductive carboxylation while the remainder is oxidized through succinate. The reductive carboxylation flux accounted for 90% of all flux of glutamine to lipid. The inhibitor studies were compatible with reductive carboxylation flux through mitochondrial isocitrate dehydrogenase. Total cell citrate and alpha-ketoglutarate were near isotopic equilibrium as expected if rapid cycling exists between these compounds involving the mitochondrial membrane NAD/NADP transhydrogenase. Taken together, these studies demonstrate a new role for glutamine as a lipogenic precursor and propose an alternative to the glutaminolysis pathway where flux of glutamine to lipogenic acetyl-CoA occurs via reductive carboxylation. These findings were enabled by a new modeling tool and software implementation (Metran) for global flux estimation.     

Assessing the reversibility of the anaplerotic reactions of the propionyl-CoA pathway in heart and liver

While a number of studies underline the importance of anaplerotic pathways for hepatic biosynthetic functions and cardiac contractile activity, much remains to be learned about the sites and regulation of anaplerosis in these tissues. As part of a study on the regulation of anaplerosis from propionyl-CoA precursors in rat livers and hearts, we investigated the degree of reversibility of the reactions of the propionyl-CoA pathway. Label was introduced into the pathway via NaH13CO3, [U-13C3]propionate, or [U-13C3]lactate + [U-13C3]pyruvate, under various concentrations of propionate. The mass isotopomer distributions of propionyl-CoA, methylmalonyl-CoA, and succinyl-CoA revealed that, in intact livers and hearts, (i) the propionyl-CoA carboxylase reaction is slightly reversible only at low propionyl-CoA flux, (ii) the methylmalonyl-CoA racemase reaction keeps the methylmalonyl-CoA enantiomers in isotopic equilibrium under all conditions tested, and (iii) the methylmalonyl-CoA mutase reaction is reversible, but its reversibility decreases as the flow of propionyl-CoA increases. The thermodynamic dis-equilibrium of the combined reactions of the propionyl-CoA pathway explains the effectiveness of anaplerosis from propionyl-CoA precursors such as heptanoate.     

TCA cycle kinetics in the rat heart by analysis of (13)C isotopomers using indirect (1)H

This study was designed to test the hypothesis that indirect (1)H[(13)C] detection of tricarboxylic acid (TCA) cycle intermediates using heteronuclear multiple quantum correlation-total correlation spectroscopy (HMQC-TOCSY) nuclear magnetic resonance (NMR) spectroscopy provides additional (13)C isotopomer information that better describes the kinetic exchanges that occur between intracellular compartments than direct (13)C NMR detection. NMR data were collected on extracts of rat hearts perfused at various times with combinations of [2-(13)C]acetate, propionate, the transaminase inhibitor aminooxyacetate, and (13)C multiplet areas derived from spectra of tissue glutamate were fit to a standard kinetic model of the TCA cycle. Although the two NMR methods detect different populations of (13)C isotopomers, similar values were found for TCA cycle and exchange fluxes by analyzing the two data sets. Perfusion of hearts with unlabeled propionate in addition to [2-(13)C]acetate resulted in an increase in the pool size of all four-carbon TCA cycle intermediates. This allowed the addition of isotopomer data from aspartate and malate in addition to the more abundant glutamate. This study illustrates that metabolic inhibitors can provide new insights into metabolic transport processes in intact tissues.     

Glycogen turnover and anaplerosis in preconditioned rat hearts

Using (13)C NMR, we tested the hypothesis that protection by preconditioning is associated with reduced glycogenolysis during ischemia. Preconditioned rat hearts showed improved postischemic function and reduced ischemic damage relative to ischemic controls after 30 min stop-flow ischemia and 30 min reperfusion (contractility: 30+/-10 vs. 2+/-2%; creatine kinase release: 41+/-4 vs. 83+/-15 U/g; both P<0.05). Preconditioning decreased preischemic [(13)C]glycogen by 24% (a 10% decrease in total glycogen), and delayed ischemic [(13)C]glycogen consumption by 5-10 min, reducing ischemic glycogenolysis without changing acidosis relative to controls. Upon reperfusion, glycogen synthesis resumed only after preconditioning. Glutamate (13)C-isotopomer analysis showed recovery of Krebs cycle activity with higher anaplerosis than before ischemia (23+/-4 vs. 11+/-3%, P<0.05), but in controls reperfusion failed to restore flux. Compared to control, preconditioning before 20 min ischemia increased contractility (86+/-10 vs. 29+/-14%, P<0.05) and restored preischemic anaplerosis (13+/-3 vs. 39+/-9%, P<0.05). Preconditioning is associated with reduced glycogenolysis early during ischemia. However, protection does not rely on major variations in intracellular pH, as proposed earlier. Our isotopomer data suggest that preconditioning accelerates metabolic and functional recovery during reperfusion by more efficient/active replenishment of the depleted Krebs cycle.     

Stimulatory effect of alcohols (methanol and ethanol) on citric acid productivity by a 2-deoxy D-glucose resistant culture of aspergillus niger GCB-47

The present study describes citric acid fermentation by Aspergillus niger GCB-47 in a 15-1 stainless steel stirred fermentor. Among the alcohols tested as stimulating agents, 1.0% (v/v) methanol was found to give maximum amount of anhydrous citric acid (90.02 +/- 2.2 g/l), 24 h after inoculation. This yield of citric acid was 1.96 fold higher than the control. Methanol has a direct effect on mycelial morphology and it promotes pellet formation. It also increases the cell membrane permeability to provoke more citric acid excretion from the mycelial cells. The sugar consumed and % citric acid was 108 +/- 3.8 g/l and 80.39 +/- 4.5%, respectively. The desirable mycelial morphology was in the form of small round pellets having dry cell mass 14.5 +/- 0.8 g/l. Addition of ethanol, however, did not found to enhance citric acid production, significantly. The maximum value of Yp/x (i.e., 5.825 +/- 0.25 g/g) was observed when methanol was used as a stimulating agent. The best results of anhydrous citric acid were observed, 6 days after inoculation when the initial pH of fermentation medium was kept at 6.0.     

Reverse flux through cardiac NADP(+)-isocitrate dehydrogenase under normoxia and ischemia

Little is known about the role of mitochondrial NADP(+)-isocitrate dehydrogenase (NADP(+)-ICDH) in the heart, where this enzyme shows its highest expression and activity. We tested the hypothesis that in the heart, NADP(+)-ICDH operates in the reverse direction of the citric acid cycle (CAC) and thereby may contribute to the fine regulation of CAC activity (Sazanov and Jackson, FEBS Lett 344: 109-116, 1994). We documented a reverse flux through this enzyme in rat hearts perfused with the medium-chain fatty acid octanoate using [U-(13)C(5)]glutamate and mass isotopomer analysis of tissue citrate (Comte et al., J Biol Chem 272: 26117-26124, 1997). In this study, we assessed the significance of our previous finding by perfusing hearts with long-chain fatty acids and tested the effects of changes in O(2) supply. We showed that under all of these conditions citrate was enriched in an isotopomer containing five (13)C atoms. This isotopomer can only be explained by substrate flux through reversal of the NADP(+)-ICDH reaction, which is evaluated at 3-7% of flux through citrate synthase. Small variations in reversal fluxes induced by low-flow ischemia that mimicked hibernation occurred despite major changes in contractile function and O(2) consumption of the heart as well as citrate and succinate release rates and tissue levels. Our data show a reverse flux through NADP(+)-ICDH and support its hypothesized role in the fine regulation of CAC activity in the normoxic and O(2)-deprived heart.     

Quantification and identification of isotopomer distributions of metabolites in crude cell extracts using 1H TOCSY

Isotopomer analysis is a very powerful technique for determining site enrichment with stable isotopes. Such information helps determine the relative flux through metabolic pathways. We have developed ¹H NMR detection methods to isotopomer analysis of human rhabdomyosarcoma cells grown in the presence of uniformly ¹³C-labeled glucose. We show that TOCSY can be used both to identify the isotopomer distributions in a substantial number of key compounds and to determine the site-specific enrichment with good precision. Effects of differential relaxation have been specifically addressed. We have identified and quantified isotopomer distributions in Ala, Lactate, (glycolysis markers), nucleotide riboses (pentose phosphate markers), Asp, Glu and Gln (citric acid cycle and anaplerosis markers) as well as in nucleotide pyrimidine rings. Due to the high sensitivity of proton experiments, a reasonable throughput was achieved using a cold probe on only 3–5 mg dry cell weight. This methodology can be applied to biological system using different labeled precursors to examine their metabolic phenotypes and their response to external perturbations. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.