Lactate Removal Research Articles

In‐situ removal of ammonium and lactate through electrical means for hybridoma cultures

Ammonium and lactate are two known toxic products detrimental to mammalian cell growth and productivity. An electrokinetic technique, utilizing an electrophoretic mechanism, was developed to remove these cellular wastes in-situ from suspension hybridoma (ATCC CRL-1606) cultures to enhance cell growth and productivity. This technique applies continuously a dc electric field to selectively remove the electrically charged wastes. The experiments were shown to be successful in the removal of externally added 10 rnM ammonium and 45 mM lactate while maintaining the chemostatic condition of culture medium in a cell-free condition under an electric current density of 50 A/m(2). Toxic levels of ammonium were added, ranging from 7.5 to 12.5 mM, at the start of the hybridoma culture, and the applied dc electric fields were able to completely remove these added materials. This in turn released the inhibition and restored the cell growth. Finally, this electrokinetic technique was applied to the batch and glutamine fed-batch hybridoma cultures. At an applied electric current density of 50 A/m(2), this was able to completely remove cell-produced ammonium and increased the cell growth and antibody titer by 30% to 50%, respectively, compared to the control experiment in the absence of the electric field. Lastly, the applied electric current density of 50 A/m(2) did not affect cellular functionalities such as glucose and glutamine consumption and antibody productivity.(c) 1995 John Wiley & Sons, Inc.

Nutrient enrichment and in‐situ waste removal through electrical means for hybridoma cultures

In-situ dc electric fields were applied to remove ammonium and lactate from suspension hybridoma cultures (ATCC-CRL-1606) which used enriched media. Nutrient concentration was increased fourfold above the normal concentration of DMEM to study enhanced protein product formation in a dc electric field. In the presence of the electric field, hybridoma growth and antibody production were increased 1.5-fold (from 3.7 x 10(6) to 9.1 x 10(6) viable cells/mL) and twofold (from 170 to 505 mg IgG/L), respectively, compared with the control. The effective removal of ammonium and lactate and increased concentrations of the various nutrients accounted for this enhancement. The enriched media caused the overflow metabolism of glucose, glutamine, and various essential amino acids. The overconsumption of glucose also produced substantial amounts of lactate, which in turn greatly increased the medium osmolarity. The increase in medium osmolarity is believed to be one of the causes of cell death in these culture systems.(c) 1995 John Wiley & Sons, Inc.

Muscle and blood metabolic responses to intense exercise during acute hypoxia and hyperoxia

SummaryThree horses performed exercise on a treadmill under normoxic conditions (F1O2–0.21) and during exposure to acute hypoxia (F1O2–0.17) and hyperoxia (F1O2–0.35) achieved by addition of nitrogen and oxygen to inspired air by means by an open flow system. The gases were added after the horses had walked and trotted at 2 submaximal speeds (2 and 4 m/s for 7–15 min). Thereafter, the treadmill speed was increased in 3 steps to attain near maximal oxygen uptake within 5–8 min (8–10.5 m/s for normoxia and hyperoxia and 7–10 m/s for hypoxia). Heart rate and oxygen uptake were recorded continuously. By use of indwelling catheters in the pulmonary and carotid arteries, blood samples were drawn at rest and immediately after exercise. Muscle biopsies (m. gluteus) were taken before and immediately after exercise. In all horses, at the end of exercise, heart rates were lower and oxygen uptake, arterial and venous PO2, haemoglobin saturation and oxygen content were higher following hyperoxia than following hypoxia. At the end of exercise, all horses had lower concentrations of lactate, ammonia and potassium in plasma and lower lactate in muscle after hyperoxia than after hypoxia and normoxia. Concentrations of ATP were higher and of G‐6‐P lower after hyperoxia than after normoxia. Significant correlations were observed between post exercise concentrations of lactate and ammonia (r=0.80) and between lactate and potassium (r=0.96) in plasma. There was also a correlation between muscle and plasma lactate (r=0.92). Post exercise ATP concentrations were correlated with IMP (r=0.85), ammonia (r=0.94) and potassium (r=0.84) concentrations in plasma. The results indicate that acute hypoxia and hyperoxia during intense exercise influence the production and/or removal of lactate.

Crithidia fasciculata as Feeder Cells for Malaria Parasites

Crithidia fasciculata was used to replace murine peritoneal wash cells as feeder cells for the adaptation of Plasmodium falciparum isolates to continuous culture in vitro, thus avoiding the need to sacrifice animals. Fourteen of 17 malaria parasite isolates in one study, and 12 of 12 isolates in a second study, were successfully adapted to continuous culture in the presence of C. fasciculata, while only 5 of 17 parallel control isolates in the first study, and 2 of 12 isolates in the second study, were adapted in the absence of any feeder cells. Biochemical assays were performed to investigate various hypotheses put forward to explain the mode of action of feeder cells. No effect of C. fasciculata feeder cells was observed on lactate removal, osmotic pressure, or glucose or amino acid content of the malaria culture media. This feeder cell system was shown to reduce the pH of the malaria culture medium. Neither this feeder system nor another system, murine peritoneal macrophages, had any effect on the cysteine content of the culture medium. C. fasciculata was shown to reduce the redox potential of the culture medium, as were other malaria growth enhancers including cysteine and glutathione. This effect on the redox potential of the culture medium is proposed to be a possible mode of action for the feeder cell systems studied.

The contribution of glucose cycling to the maintenance of steady-state levels of lactate by hepatocytes during glycolysis and gluconeogenesis.

When hepatocytes from fasted rats were incubated with 10 mM glucose, there was a linear accumulation of lactate and pyruvate for about 80 min after which steady-state concentrations of these metabolites became established. The rate of glycolysis, determined with [6-3H]glucose, was constant over the entire incubation period and was 50% greater than that calculated from carbon balance studies. This suggests that one-third of the glycolytic products formed were recycled to glucose. To enable study of the factors associated with the generation and maintenance of the lactate steady state and to measure accurately the carbon balance, incubations were performed using supraphysiological concentrations of glucose (20-80 mM). Under these conditions the initial rate of lactate accumulation and its concentration at steady state were shown to be dependent on the concentration of extracellular glucose. Rates of glycolysis were also measured using 40 mM [6-3H]glucose and [U-14C]glucose added alone, or in combination with a steady-state lactate concentration (3 mM). There was no effect on the rate of glycolysis determine with [6-3H]glucose, even when lactate was present in the medium. The difference in rates between measurements with the two isotopes reflect the apparent degree of glucose recycling which in the absence and presence of added lactate increased from 0.26 to 0.54 mumol C3 equivalents min-1.g-1 respectively. Identical studies employing [U-14C]lactate showed that glucose and CO2 were the major products of lactate metabolism under steady-state conditions and that the formation of lactate from [U-14C]glucose exactly balanced the rate of lactate removal as a result of oxidation and gluconeogenesis. These studies provide evidence for the concomitant operation of glycolysis and gluconeogenesis, even in the presence of high glucose concentrations. They also demonstrate that lactate steady states are achieved not by the cessation of glycolysis but rather by the removal of lactate and pyruvate at a rate equal to that of their production.

Lactate release and uptake in hepatoma 7288CTC perfused in situ with l-[(U)- 14C] lactate or d-[(U)- 14C] glucose

Arteriovenous differences (AVD) for glucose and lactic acid measured across tissue-isolated rat tumors in vivo have shown that individual tumors with similar rates of glucose consumption may either release or utilize lactic acid. The experiments described here investigated the relationships among arterial blood lactate concentrations and tumor lactate and glucose balances. AVDs for lactate, pyruvate, glucose, 14CO 2, Po 2, Pco 2, pH, and lactate specific activities were measured across 17 tissue-isolated 7288CTC hepatomas perfused in situ with arterial blood containing 2.5 to 14.4 mmol/L lactate and either l-[(U)- 14C]lactic acid or d-[(U)- 14C]glucose. Measurements were made over a range of blood flow rates from 60% to 200% of the mean in vivo rate, 0.11 mL/min. Data collected during steady states were compared by regression analysis. Tumor lactate balance and the arterial blood lactate concentration were directly related ( r = .895, n = 22, P < .01). Net negative and positive balances occurred below and above approximately 6.5 mmol/L arterial blood lactate, respectively. The mean intratumor lactate concentration for all tumors was 6.9 ± 1.0 mmol/L (mean ± SD, n = 13). Rates of 14C-lactate oxidation to 14CO 2 ( r = .716, n = 18, P < .01) and tumor venous / arterial blood 14C-lactate specific activity ratios ( r = .845, n = 19, P < .01) were low during lactate release and were increased during lactate uptake. Total arterial blood lactate removal estimated from chemical and isotopic analyses was 23.1% ± 11% and 43.0% ± 16% ( P < .05), respectively, for six lactate-utilizing tumors. Perfusions performed with 14C-glucose showed that approximately 50% of the glucose consumed during net negative lactate balance was released as 14C-lactate to the tumor venous blood, whereas only 5% was released as 14C-lactate during net positive lactate balance. The data support the following conclusions: Arterial blood lactate controls net lactate balance in solid tumors; high concentrations increase uptake. Lactate uptake inhibits lactate formation from glucose without changing the glucose balance. Lactate is released during net lactate uptake. Since lactate uptake may exceed glucose uptake, arterial blood lactate can be a substrate for tumor energy metabolism and growth.

Facilitated Transport of Lactate by Rabbit Corneal Endothelium

Proton coupled lactate transport across the rabbit corneal endothelium was studied using a pH-sensitive intracellular fluorescent probe. Functional indications that lactate transport is carrier-mediated and coupled to H + at the apical endothelium (aqueous humor facing) that were found were: (1) proton influx was a saturable function of factate concentration; (2) L-lactate produced a faster maximal H + influx and had a higher affinity for the transporter ( V max = 1·6 mM min -1, apparent K1/2 = 31 mM) than its optical isomer, d -Lactate ( V max = 0·9 mM min -1, apparent K 1/2 = 59 mM); (3) the lactate-induced acidification was inhibitable by apical mersalyl acid. These results are consistent with the presence of an apical lac -:H + cotransporter. On basolateral addition of lactate to a de-epithelialized cornea, the endothelial cells transiently acidified by 0·05 units, but then alkalinized by 0·07, 0·02 units over baseline. The basolateral lactate-induced acidification was inhibited by 75% by mersalyl acid, while the net alkalinization observed under control conditions was unaffected. The alkalinization was Na +-dependent yet amiloride-insensitive. Alkalinization on addition of basolateral lactate could also be inhibited by apical mersalyl. These results suggest that the acidification represents lactate influx through basolateral Lac -:H + cotransport while the net alkalinization is due to basolateral Na +-dependent lactate uptake and lactate efflux through apical Lac -:H + cotransport. These facilitated transport mechanisms could provide for the efficient removal of lactate from the highly glycolytic cornea.

Effect of endurance training on oxidation of lactate in mice after supramaximal exercise

The effect of endurance training on lactate metabolism after supramaximal exercise was investigated in male ddY mice. The blood lactate concentration 20 min after supramaximal exercise in the trained mice was significantly lower than that in the control mice. No significant effect of endurance training on the amount of 14CO 2 expired from [ 14C]lactate injected was found during the recovery period from supramaximal exercise. The muscle [ 14C]glycogen concentration after 20 min of recovery in the trained mice was significantly higher than that in the control mice. These results suggest that endurance training does not enhance oxidative removal of lactate, which is the most important pathway of lactate metabolism, and activates muscle glycogen resynthesis from lactate in mice after supramaximal exercise.

Extracellular pH and buffering power determine intracellular pH in cortical brain slices during and following hypoxia.

1H and 31P nuclear magnetic resonance (NMR) spectroscopy were used to study the role of external pH (pHo) in regulation of intracellular pH (pHi) in the cerebral cortex in vitro. Lowering the pH of the oxygenated superfusion medium (pHb) from 7.5 to 7.0-6.8 decreased pHi from 7.33 +/- 0.08 to approximately 7.00 independently of the presence of HCO3- and medium buffering power. Reduction of medium (pHb 6.8) buffering power during hypoxia severely acidified pHi independently of the presence of HCO3-. Following hypoxia, recovery of pHi was not sensitive to pHb provided that medium buffering was maintained. Lactate removal proceeded independently of pHb, HCO3- and external buffering power. These results indicate that decrease in extracellular buffering capacity has a feed-back effect on pHi in the cerebral cortex.

Plasma catecholamine and corticosterone and their in vitro effects on lizard skeletal muscle lactate metabolism

Lizard skeletal muscles utilize primarily lactate as a gluconeogenic substrate for glycogen replenishment following exercise. To understand the influence of selected hormones on this process, we measured changes in plasma catecholamines and corticosterone resulting from exercise in the lizard Dipsosaurus dorsalis and then investigated the physiological effects of those hormones on skeletal muscle lactate and glucose metabolism in vitro. Plasma epinephrine (Epi), norepinephrine, and corticosterone (Cort) increased 5.8, 10.2, and 2.2 times, respectively, after 5 min of exhaustive exercise. Epi and Cort levels remained elevated after 2 h of recovery. Skeletal muscle fiber bundles isolated from the red and white regions of the iliofibularis muscle were incubated 2 h at 40 degrees C in the presence of postexercise concentrations of [14C]lactate (15 mM) and glucose (8.5 mM) in the presence and absence of Epi or Cort. Red muscle oxidized both substrates at 2-3 times the rate of white muscle, and both red and white fibers oxidized lactate at 5-10 times the rate of glucose oxidation. Epi had a stimulatory effect on lactate oxidation by white muscle. Lactate incorporation into glycogen proceeded at 2-3 times the rate of glucose incorporation in both muscle types, with rates in red muscle again 2-3 times that for white muscle. Epi stimulated lactate carbon incorporation into glycogen by 50-140% in both red and white muscle but had no effect on glucose incorporation into glycogen in either tissue. We interpret these data as evidence that epinephrine stimulates lactate removal by skeletal muscle. Cort had no effect on lactate metabolism in either muscle type.(ABSTRACT TRUNCATED AT 250 WORDS)

Blood Lactate Concentrations in Elite Skiers During a Series of On-Snow Downhill Ski Runs

Lactic acid levels were evaluated during the training of 5 female members of the U.S. Ski Team. Fingerstick lactate samples and heart rates (HR) were obtained before and after three consecutive downhill ski runs. Post-ski measurements were taken within 1 min after each run. The lactate analysis demonstrated the high intensity of these 2-min downhill efforts (post-ski La M = 6.0 mM ± 1.7). Statistical analysis of the serial lactates (1–3) revealed that skiing significantly increased blood lactate levels (p=0.01). In addition, the pre and post-ski blood lactate levels measured on the first run were significantly lower than the corresponding lactate levels on Runs 2 and 3. Maximal HR recorded at the end of the final ski run was significantly lower than in the two preceding runs (p=0.016). These data suggest that significant lactate accumulates in training; subsequent runs occur without complete lactate removal and inadequate recovery. Light exercise between each run may aid this process, delay fatigue, and enhance the quality of performance.

Lactic Acidosis

Lactic acidosis is the most common metabolic acidosis. At clinical presentation, several causes usually can be identified. The liver is a major site of removal of lactate and hydrogen ions, and abnormalities in the aerobic metabolism of lactate by mitochondria in hepatocytes and other cells may contribute to many clinical conditions in which overproduction and underuse of lactate occur. To date, no therapy specifically designed to lower arterial blood lactate levels has reduced mortality significantly. Prompt recognition and treatment of the underlying causes of lactic acidosis remain the cornerstone of treatment.

Heat stress does not modify lactate exchange and removal abilities during recovery from short exercise

Arterial and femoral venous lactate concentrations were measured before, during, and after short intermittent exercise (55-118% of maximal O2 consumption) in thermoneutral (N, 25 degrees C, 10.5 Torr) and hot (H, 45 degrees C, 17.5 Torr) conditions. The thermal load induced significantly higher heart rate and rectal temperature in H relative to N. All the arterial lactate (La) recovery curves were fitted to an equation containing two exponential time functions of the form La(t) = La(0) + A1a(1 - e-gamma 1at) + A2a(1 - e-gamma 2at) where the velocity constants gamma 1a and gamma 2a are the body's overall ability to exchange and remove lactate after exercise, respectively, and t is time. There was no significant difference in these constants, regardless of thermal conditions. The arterial lactate concentration at the end of exercise, the peak lactate concentration during recovery, the amplitudes A1a and A2a of the biexponential function, and the arteriofemoral venous lactate concentration difference during recovery were not significantly different in H relative to N. However, measured and computed arterial lactate concentrations during recovery, especially at the end of the tests, were higher in H (P < 0.04). The more elevated lactate concentrations in H at rest at the end of recovery denote a higher basal lactate production, and they were not due to muscle hypoxia.

Lactate kinetics in resting and exercising forearms during moderate-intensity supine leg exercise

Arterial blood lactate was elevated by supine leg exercise (20 min at approximately 65% maximal oxygen uptake) in five untrained male subjects, and the contribution to blood lactate removal from passive uptake vs. metabolic disposal was compared in resting and lightly exercising (15% maximal voluntary contraction static handgrip) forearm skeletal muscle. An integrated form of the Fick equation was used to predict venous lactate levels resulting solely from passive equilibration of lactate between incoming arterial blood and the forearm muscles. In the resting forearm, predicted and measured venous lactate levels were closely correlated during the exercise period (r = 0.995, P < 0.001), indicating that lactate removal could be accounted for in terms of passive uptake alone. In the lightly exercising forearm, measured venous lactate levels were higher than both the arterial and predicted venous levels, indicating net lactate production. It was concluded that most of the blood lactate generated by moderate-intensity supine leg exercise is taken up passively and not metabolized by resting skeletal muscle and that the rate of lactate disposal is unlikely to be enhanced in lightly exercising muscle.

Lactate exchange and removal abilities in sickle cell patients and in untrained and trained healthy humans.

Arterial blood lactate concentrations obtained on seven black males with hemoglobin sickle cell disease (SC) before, during, and after graded bicycle exercise up to exhaustion were compared with those of seven untrained (HU) and seven trained (HT) healthy males of the same ethnic origin. Lactate recovery curves were fitted by a biexponential time function consisting of a rapidly increasing and a slowly decreasing component. Higher work rates were reached by the HU and HT than by the SC group. Blood lactate rose distinctly over the corresponding preexercise resting values after the 25-, 50-, and 100-W exercise steps for the SC, HU, and HT groups, respectively. The arterial oxygen content was significantly lower for the SC than for the HU group at rest and at the end of exercise. The velocity constants of the slowly decreasing component of the lactate recovery curves were similar for the SC, HU, and HT groups despite the fact that they cycled up to different absolute work rates. The velocity constant of the rapidly increasing component was significantly higher for the HT. In terms of the functional meaning given to these constants and in view of their inverse relationship with absolute work rate (Freund et al. J. Appl. Physiol. 61: 932-939, 1986), these results indicate that, relative to the HU, the HT and the SC display improved and impaired abilities, respectively, to exchange and to remove lactate.

Endurance training vs. lactate production and removal

Endurance training vs. lactate production and removal

Is accelerated oxidation of lactate required for dichloroacetate to lower the level of lactate in blood?

We examined mechanisms by which dichloroacetate (DCA), an activator of pyruvate dehydrogenase (PDH), led to a decrease in the concentration of lactate in blood in a unique "metabolic setting," where the concentration of lactate in blood was 5.4 +/- 0.5 mmol/L. Elevated levels of lactate were induced in anaesthetized rabbits by the administration of a large dose of insulin. The rate of consumption of oxygen was 1.2 +/- 0.1 mmol/min, the respiratory quotient was close to unity, and close to half of the PDH was in its active form; therefore, virtually all ATP synthesis should require flux through PDH. Hence, we predicted that DCA should not cause a significant decrease in the concentration of lactate in blood in this model. In contrast, if DCA was effective, new insights could be obtained into its mechanisms of action, at least in this setting. During steady-state hyperlactatemia, DCA was given as its sodium salt, 2 mmol/kg (n = 10); a control group (n = 5) received equimolar NaCl. Forty minutes later, the level of lactate in blood in the DCA group was 1.3 +/- 0.2 mmol/L, significantly lower than in the NaCl group (4.2 +/- 0.6 mmol/L). To determine the organ(s) responsible for removing lactate, arteriovenous differences were measured in organs drained by the jugular, femoral, and hepatic veins. There was no net uptake of lactate in these drainage beds after DCA was administered. From a quantitative analysis of the rate of removal of lactate and the rate of consumption of oxygen, it seems unlikely that the majority of the decrease in lactate could be directly attributed to an increase in its oxidation.

Effect of pedal rate on cardiorespiratory responses during continuous exercise

The role of cycle ergometer pedal rate on the gradual increase in ventilation (VE), heart rate (HR), and oxygen uptake (VO2) accompanying continuous submaximal exercise is unknown. To examine this problem, five trained males (VO2peak = 4.00 +/- 0.27 l.min-1) performed 45 min of moderate intensity (MI, 127 W) and high-moderate intensity (HMI, 166 W) cycle ergometry both at pedal rates of 60 rpm and 90 rpm. Power output and pedal rate had an additive effect on the overall mean responses for VE, HR, and VO2, producing significantly higher values as power output and pedal rate increased. During continuous exercise, VE, HR, and VO2 increased progressively from the 10th to the 45th minute for all tests. However, the rates of increase and factors modifying the VE, HR, and VO2 responses were different. HR increased during all exercise tests an average of 10.8% independent of power output and pedal rate. VE increased 7.4% during MI exercise and 10% during HMI exercise independent of pedal rate. Similar power output dependent responses were observed for rectal temperature (Tr) and blood lactate. VO2 increased 4.4% for MI and HMI exercise at 60 rpm, and 8.2% for the same power outputs at 90 rpm, respectively. Increases in Tr, the oxygen cost of pulmonary ventilation and fat oxidation, and lactate removal were estimated to account for only 31-36% of the slow rise in VO2 for any single test. This suggests that 64-69% of the rise in VO2 was due to factors related to muscle use.(ABSTRACT TRUNCATED AT 250 WORDS)

Metabolic state of the in situ perfused trout heart during severe hypoxia.

An in situ perfused heart preparation was used to study the effects of severe hypoxia in the rainbow trout, Oncorhynchus mykiss. Hypoxic trout hearts were capable of generating similar power outputs and ATP turnovers to normoxic counterparts at subphysiological work regimes. However, lactate efflux was 35-fold higher and glycolytic rate was calculated to be > 10-fold higher in hypoxic than in normoxic hearts. The surprising ability of trout hearts to withstand severe hypoxia appears to be related to the rapid removal of lactate and associated protons from the heart. An increase in power demand to normal in vivo levels caused rapid failure in hypoxic hearts. Failure was caused by a decline in stroke volume (contractility) and was not a consequence of heart rate deterioration. Hypoxia caused marked declines in the concentration of creatine phosphate but not ATP, and we suggest that an increase in intracellular phosphate was the primary cause of failure.

Operation and energy dependence of the reducing-equivalent shuttles during lactate metabolism by isolated hepatocytes

The participation and energy dependence of the malate-aspartate shuttle in transporting reducing equivalents generated from cytoplasmic lactate oxidation was studied in isolated hepatocytes of fasted rats. Both lactate removal and glucose synthesis were inhibited by butylmalonate, aminooxyacetate or cycloserine confirming the involvement of malate and aspartate in the transfer of reducing equivalents from the cytoplasm to mitochondria. In the presence of ammonium ions the inhibition of lactate utilization by butylmalonate was considerably reduced, yet the transfer of reducing equivalents into the mitochondria was unaffected, indicating a substantially lesser role for butylmalonate-sensitive malate transport in reducing-equivalent transfer when ammonium ions were present. Ammonium ions had no stimulatory effect on uptake of sorbitol, a substrate whose oxidation principally involves the α-glycerophosphate shuttle. The role of cellular energy status (reflected in the mitochondrial membrane electrical potential (ΔΨ) and redox state), in lactate oxidation and operation of the malate-aspartate shuttle, was studied using a graded concentration range of valinomycin (0–100 nM). Lactate oxidation was strongly inhibited when ΔΨ fell from 130 to 105 mV whereas O 2 consumption and pyruvate removal were only minimally affected over the valinomycin range, suggesting that the oxidation of lactate to pyruvate is an energy-dependent step of lactate metabolism. Our results confirm that the operation of the malate-aspartate shuttle is energy-dependent, driven by ΔΨ. In the presence of added ammonium ions the removal of lactate was much less impaired by valinomycin, suggesting an energy-independent utilization of lactate under these conditions. The oxidizing effect of ammonium ions on the mitochondrial matrix apparently alleviates the need for energy input for the transfer of reducing equivalents between the cytoplasm and mitochondria. It is concluded that, in the presence of ammonium ions, the transport of lactate hydrogen to the mitochondria is accomplished by malate transfer that is not linked to the electrogenic transport of glutamate across the inner membrane, and, hence, is clearly distinct from the butylmalonate-sensitive, energy-dependent, malate-aspartate shuttle.