Search

Cancer cells meet their energy need by predominantly increased uptake of glucose, high rate of glycolysis, and increased production of lactate even in the presence of adequate oxygen.  This process was proposed by Otto Warburg and named after him as the Warburg effect. The development of drugs that target glucose intake and aerobic glycolysis or lactic acid secretion of cancer cells is a newer approach for drug discovery. We have tested five purified plants-derived compounds such as curcumin, quercetin, ellagic acid, resveratrol, and indole-3-carbinol in HeLa cells for cytotoxicity, inhibition of metastasis, and modulation of lactate-pyruvate metabolism. Standard biochemical methods were used for glucose, lactic acid, and pyruvic acid measurement. The cell viability was determined by MTT assay. Cell migration was checked by wound healing assay. A dose-dependent cytotoxic effect and inhibition of cell migration were observed in all the tested compounds. A decrease in the lactate and increase in pyruvate level was observed in all the tested compounds except ellagic acid. Our finding suggests that tested phytocompounds are associated with the metabolic reprogramming of cancer cells and execute the cytotoxic effect. These compounds could be used for cancer prevention and therapy., (© 2020 International Union of Biochemistry and Molecular Biology, Inc.)

Backhousia citriodora (lemon myrtle) extract has been found to inhibit glucansucrase activity, which plays an important role in biofilm formation by Streptococcus mutans. In addition to glucansucrase, various virulence factors in S. mutans are involved in the initiation of caries. Lactate produced by S. mutans demineralizes the tooth enamel. This study investigated whether lemon myrtle extract can inhibit S. mutans lactate production. Lemon myrtle extract reduced the glycolytic pH drop in S. mutans culture and inhibited lactate production by at least 46%. Ellagic acid, quercetin, hesperetin, and myricetin, major polyphenols in lemon myrtle, reduced the glycolytic pH drop and lactate production, but not lactate dehydrogenase activity. Furthermore, these polyphenols reduced the viable S. mutans cell count. Thus, lemon myrtle extracts may inhibit S. mutans-mediated acidification of the oral cavity, thereby preventing dental caries and tooth decay., (© The Author(s) 2021. Published by Oxford University Press on behalf of Japan Society for Bioscience, Biotechnology, and Agrochemistry.)

Following the decline in Physical Activity (PA) due to COVID-19 restrictions in the form of government mandated lockdowns and closures of public spaces, the modulatory effect of physical exercise on immunity is being heavily revisited. In an attempt to comprehend the wide discrepancy in patient response to COVID-19 and the factors that potentially modulate it, we summarize the findings relating PA to inflammation and immunity. A distinction is drawn between moderate intensity and high intensity physical exercise based on the high lactate production observed in the latter. We hypothesize that, the lactate production associated with high intensity anaerobic exercise is implicated in the modulation of several components of the innate and adaptive immunity. In this review, we also summarize these immunomodulatory effects of lactate. These include increasing serum IL-6 levels, the main mediator of cytokine storms, as well as affecting NK cells, Macrophages, Dendritic cells and cytotoxic T-lymphocytes. The implications of high lactate levels in athletic performance are highlighted where athletes should undergo endurance training to increase VO2 max and minimize lactate production. Tumor models of hypoxia were also reported where lactate levels are elevated leading to increased invasiveness and angiogenesis. Accordingly, the novel lactate blocking strategy employed in cancer treatment is evaluated for its potential benefit in COVID-19 in addition to the readily available beta-blockers as an antagonist to lactate. Finally, we suggest the diagnostic/prognostic purpose of the elevated lactate levels that can be determined through sweat lactate testing. It is the detrimental effect of lactate on immunity and its presence in sweat that qualify it to be used as a potential non-invasive marker of poor COVID-19 outcome., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Many global infectious diseases are not well-controlled, underlining a critical need for new, more effective therapies. Pathogens and pathogen-infected host cells, like cancer cells, evade immune surveillance via immune evasion mechanisms. The present study indicates that pathogenic bacteria, endoparasites, and virus-infected host cells can have immune evasion mechanisms in common with cancers. These include entry into dormancy and metabolic reprogramming to aerobic glycolysis leading to excessive secretion of lactic acid and immobilization of local host immunity. The latter evasion tactic provides a therapeutic target for cancer, as shown by our recent finding that patient-derived cancer xenografts can be growth-arrested, without major host toxicity, by inhibiting their lactic acid secretion (as mediated by the MCT4 transporter)-with evidence of host immunity restoration. Accordingly, the multiplication of bacteria, endoparasites, and viruses that primarily depend on metabolic reprogramming to aerobic glycolysis for survival may be arrested using cancer treatment strategies that inhibit their lactic acid secretion. Immune evasion mechanisms shared by pathogens and cancer cells likely represent fundamental, evolutionarily-conserved mechanisms that may be particularly critical to their welfare. As such, their targeting may lead to novel therapies for infectious diseases.

Energy and biomass production in cancer cells are largely supported by aerobic glycolysis in what is called the Warburg effect. The process is regulated by key enzymes, among which phosphofructokinase PFK-2 plays a significant role by producing fructose-2,6-biphosphate; the most potent activator of the glycolysis rate-limiting step performed by phosphofructokinase PFK-1. Herein, the synthesis, biological evaluation and structure-activity relationship of novel inhibitors of 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 3 (PFKFB3), which is the ubiquitous and hypoxia-induced isoform of PFK-2, are reported. X-ray crystallography and docking were instrumental in the design and optimisation of a series of N-aryl 6-aminoquinoxalines. The most potent representative, N-(4-methanesulfonylpyridin-3-yl)-8-(3-methyl-1-benzothiophen-5-yl)quinoxalin-6-amine, displayed an IC 50 of 14 nm for the target and an IC 50 of 0.49 μm for fructose-2,6-biphosphate production in human colon carcinoma HCT116 cells. This work provides a new entry in the field of PFKFB3 inhibitors with potential for development in oncology., (© 2019 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Introduction: We hypothesize that the combination of transarterial embolization (TAE) plus inhibition of lactate export will limit anaerobic metabolism and reduce tumor survival compared to TAE alone. The purpose of this study was to test this hypothesis in a rat model of hepatocellular carcinoma (HCC)., Methods: Rat N1-S1 hepatoma cells were assayed in vitro using the Seahorse XF analyzer to measure extracellular acidification (lactate excretion) comparing effects of the addition of caffeic acid (CA) or ferulic acid (FA) or UK-5099 with control. Monocarboxylate transporter Slc16a3 was knocked down by RNAi. N1S1 tumors were orthotopically implanted in rats and 4 groups evaluated: (1) Control, (2) TAE-only, (3) TAE plus CA, and (4) TAE plus FA. Tumor size was determined by ultrasound and analyzed by repeated measures statistics. Tumors harvested at 4 weeks were examined by microscopy., Results: Seahorse assays showed that CA and FA caused a significant reduction by >90% in lactate efflux by N1S1 tumor cells (p < 0.01). Knockdown of Slc16a3 prevented inhibition by CA. In vivo tumors grew 30-fold in volume over 4 weeks in untreated controls. By comparison, TAE resulted in near cessation of growth (10% in 4-week time period). However, both TAE + CA and TAE + FA caused a significant reduction of tumor volumes (87 and 72%, respectively) compared to control and TAE (p < 0.05). Pathologic evaluation revealed residual tumor in the TAE group but no residual viable tumor cells in the TAE + CA and TAE + FA groups., Conclusion: Addition of CA or FA enhances the effectiveness of TAE therapy for HCC in part by blocking lactate efflux.

Background: Some studies have demonstrated elevated concentrations of lactate both in the cerebrospinal fluid (CSF) and blood samples of multiple sclerosis (MS) patients as a pathological condition. We designed an experimental study first to investigate the serum level of lactate as a biomarker of MS progression and also to investigate the effect of methylprednisolone on serum lactate., Methods: Experimental autoimmune encephalomyelitis (EAE) was inducted in Lewis rats, and then rats were treated intraperitoneally with methylprednisolone (30mg/kg/d), at the disease onset, and the clinical scores were recorded. After seven days of treatment, the serum levels of lactate were determined using high performance liquid chromatography (HPLC). Moreover, lymphocyte infiltration and the demyelinated area was analysed in spinal cord., Results: Compared to the untreated-EAE rats, methylprednisolone remarkably improved the clinical score of EAE and ameliorated the spinal cord inflammation and demyelination. In addition, the marked decline in IFN-γ and the increase in IL-4 confirmed improvement in the rats treated with methylprednisolone. Measurement of lactate using HPLC indicated enhancement in the serum level of lactate in the untreated-EAE rats; the lactate level significantly decreased after methylprednisolone therapy. Moreover, serum lactates and disease severity were correlated positively and significantly., Conclusion: These data confirmed for the first time, that methylprednisolone can decreases the enhanced level of serum lactate in EAE model. In addition, it was shown that measurement of serum lactate could be an inexpensive and accurate laboratory test to determine the response to treatment and to assess disease severity in MS patients., (Copyright © 2016 Elsevier Masson SAS. All rights reserved.)

Astrocyte-derived lactate supports pathologically enhanced neuronal metabolism, but its role under physiological conditions is still a matter of debate. Here, we determined the contribution of astrocytic neuronal lactate shuttle for maintenance of ion homeostasis and energy metabolism. We tested for the effects of α-cyano-4-hydroxycinnamic acid (4-CIN), which could interfere with energy metabolism by blocking monocarboxylate-transporter 2 (MCT2)-mediated neuronal lactate uptake, on evoked potentials, stimulus-induced changes in K + , Na + , Ca 2+ , and oxygen concentrations as well as on changes in flavin adenine dinucleotide (FAD) autofluorescence in the hippocampal area CA3. MCT2 blockade by 4-CIN reduced synaptically evoked but not antidromic population spikes. This effect was dependent on the activation of K ATP channels indicating reduced neuronal ATP synthesis. By contrast, lactate receptor activation by 3,5-dihydroxybenzoic acid (3,5-DHBA) resulted in increased antidromic and orthodromic population spikes suggesting that 4-CIN effects are not mediated by lactate accumulation and subsequent activation of lactate receptors. Recovery kinetics of all ion transients were prolonged and baseline K + concentration became elevated by blockade of lactate uptake. Lactate contributed to oxidative metabolism as both baseline respiration and stimulus-induced changes in Po 2 were decreased, while FAD fluorescence increased likely due to a reduced conversion of FAD into FADH 2 These data suggest that lactate shuttle contributes to regulation of ion homeostatsis and synaptic signaling even in the presence of ample glucose., (Copyright © 2016 the American Physiological Society.)

Tumor cells display a shift in energy metabolism from oxidative phosphorylation to aerobic glycolysis. A subset of papillary thyroid carcinoma (PTC) is refractory to surgery and radioactive iodine ablation. Doxorubicin and sorafenib are the drugs of choice for treating advanced thyroid cancer but both induce adverse effects. In this study, we assessed the anti-cancer activity of 2-deoxy-d-glucose (2-DG) alone and in combination with doxorubicin or sorafenib in PTC cell lines with (BCPAP) and without (CG3) the BRAFV600E mutation. BCPAP cells were more glycolytic than CG3 cells, as evidenced by their higher extracellular l-lactate production, lower intracellular ATP level, lower oxygen consumption rate (OCR), and lower ratio of OCR/extracellular acidification rate. However, dose-dependent reduction in cell viability, intracellular ATP depletion, and extracellular l-lactate production were observed after 2-DG treatment. Regression analysis showed that cell growth in both cell lines was dependent on ATP generation. 2-DG increased the chemosensitivity of BCPAP and CG3 cell lines to doxorubicin and sorafenib. These results demonstrate that the therapeutic effects of low combined doses of 2-DG and doxorubicin or sorafenib are similar to those of high doses of doxorubicin or sorafenib alone in PTC cell lines regardless of the BRAFV600E mutation.

Neuroprotective properties of the new derivative of glutamic and apovincaminic acids, ethyl -(3-alpha,16-alpha)-eburnamenin-14-carbopxylate of 2-aminopentadionic acid (LHT 1-02) were studied on a model of acute brain ischemia in cats. LHT 1-02 has proved to be more effective than the reference drugs vinpocetin and glycine in preventing the reperfusive damage, which was manifested by decreased postischemic hyperglycemia, activated utilization of oxygen in the brain, and suppressed postischemic metabolic lactate acidosis. Thus, the results of this comparative study show expediency of further investigations of LHT 1 - 02 as a potential neuroprotective drug.

A head-to-head study of representative examples of N-hydroxyindole-2-carboxylates (NHI) and malonic derivatives (Mal) as LDH-A inhibitors was conducted, comparing the enzyme inhibition potency, cellular uptake, reduction of lactate production in cancer cells and anti-proliferative activity. Among the compounds tested, methyl 1-hydroxy-6-phenyl-4-(trifluoromethyl)-1H-indole-2-carboxylate (2, NHI-2), a methyl ester belonging to the NHI class, displayed optimal properties in the cell-based assays, proving to be an efficient anti-glycolytic agent against cancer cells.

Studies in animal models of cancer have demonstrated that targeting tumor metabolism can be an effective anticancer strategy. Previously, we showed that inhibition of glucose metabolism by the pyruvate analog, 3-bromopyruvate (3-BrPA), induces anticancer effects both in vitro and in vivo. We have also documented that intratumoral delivery of 3-BrPA affects tumor growth in a subcutaneous tumor model of human liver cancer. However, the efficacy of such an approach in a clinically relevant orthotopic tumor model has not been reported. Here, we investigated the feasibility of ultrasound (US) image-guided delivery of 3-BrPA in an orthotopic mouse model of human pancreatic cancer and evaluated its therapeutic efficacy. In vitro, treatment of Panc-1 cells with 3-BrPA resulted in a dose-dependent decrease in cell viability. The loss of viability correlated with a dose-dependent decrease in the intracellular ATP level and lactate production confirming that disruption of energy metabolism underlies these 3-BrPA-mediated effects. In vivo, US-guided delivery of 3-BrPA was feasible and effective as demonstrated by a marked decrease in tumor size on imaging. Further, the antitumor effect was confirmed by (1) a decrease in the proliferative potential by Ki-67 immunohistochemical staining and (2) the induction of apoptosis by terminal deoxynucleotidyl transferase-mediated deoxyuridine 5-triphospate nick end labeling staining. We therefore demonstrate the technical feasibility of US-guided intratumoral injection of 3-BrPA in a mouse model of human pancreatic cancer as well as its therapeutic efficacy. Our data suggest that this new therapeutic approach consisting of a direct intratumoral injection of antiglycolytic agents may represent an exciting opportunity to treat patients with pancreas cancer.

Most malignant tumors exhibit the Warburg effect, which consists in increased glycolysis rates with production of lactate, even in the presence of oxygen. Monocarboxylate transporters (MCTs), maintain these glycolytic rates, by mediating the influx and/or efflux of lactate and are overexpressed in several cancer cell types. The lactate and pyruvate analogue 3-bromopyruvate (3-BP) is an inhibitor of the energy metabolism, which has been proposed as a specific antitumor agent. In the present study, we aimed at determining the effect of 3-BP in breast cancer cells and evaluated the putative role of MCTs on this effect. Our results showed that the three breast cancer cell lines used presented different sensitivities to 3-BP: ZR-75-1 ER (+)>MCF-7 ER (+)>SK-BR-3 ER (-). We also demonstrated that 3-BP reduced lactate production, induced cell morphological alterations and increased apoptosis. The effect of 3-BP appears to be cytotoxic rather than cytostatic, as a continued decrease in cell viability was observed after removal of 3-BP. We showed that pre-incubation with butyrate enhanced significantly 3-BP cytotoxicity, especially in the most resistant breast cancer cell line, SK-BR-3. We observed that butyrate treatment induced localization of MCT1 in the plasma membrane as well as overexpression of MCT4 and its chaperone CD147. Our results thus indicate that butyrate pre-treatment potentiates the effect of 3-BP, most probably by increasing the rates of 3-BP transport through MCT1/4. This study supports the potential use of butyrate as adjuvant of 3-BP in the treatment of breast cancer resistant cells, namely ER (-).

Oxidative stress-energy depletion therapy using oxidative stress induced by D-amino acid oxidase (DAO) and energy depletion induced by 3-bromopyruvate (3BP) was reported recently (El Sayed et al., Cancer Gene Ther., 19, 1-18, 2012). Even in the presence of oxygen, cancer cells oxidize glucose preferentially to produce lactate (Warburg effect) which seems vital for cancer microenvironment and progression. 3BP is a closely related structure to lactate and pyruvate and may antagonize their effects as a novel mechanism of its action. Pyruvate exerted a potent H(2)O(2) scavenging effect to exogenous H(2)O(2), while lactate had no scavenging effect. 3BP induced H(2)O(2) production. Pyruvate protected against H(2)O(2)-induced C6 glioma cell death, 3BP-induced C6 glioma cell death but not against DAO/D-serine-induced cell death, while lactate had no protecting effect. Lactate and pyruvate protected against 3BP-induced C6 glioma cell death and energy depletion which were overcome with higher doses of 3BP. Lactate and pyruvate enhanced migratory power of C6 glioma which was blocked by 3BP. Pyruvate and lactate did not protect against C6 glioma cell death induced by other glycolytic inhibitors e.g. citrate (inhibitor of phosphofructokinase) and sodium fluoride (inhibitor of enolase). Serial doses of 3BP were synergistic with citrate in decreasing viability of C6 glioma cells and spheroids. Glycolysis subjected to double inhibition using 3BP with citrate depleted ATP, clonogenic power and migratory power of C6 glioma cells. 3BP induced a caspase-dependent cell death in C6 glioma. 3BP was powerful in decreasing viability of human glioblastoma multiforme cells (U373MG) and C6 glioma in a dose- and time-dependent manner.

Glioblastoma multiforme (GBM) are the most malignant among brain tumors. They are frequently refractory to chemotherapy and radiotherapy with mean patient survival of approximately 6 months, despite surgical intervention. The highly glycolytic nature of glioblastomas describes their propensity to metabolize glucose to lactic acid at an elevated rate. To survive, GBMs efflux lactic acid to the tumor microenvironment through transmembrane transporters denoted monocarboxylate transporters (MCTs). We hypothesized that inhibition of MCT function would impair the glycolytic metabolism and affect both glioma invasiveness and survival. We examined the effect on invasiveness with α-cyano-4-hydroxy-cinnamic acid (ACCA, 4CIN, CHCA), a small-molecule inhibitor of lactate transport, through Matrigel-based and organotypic (brain) slice culture invasive assays using U87-MG and U251-MG glioma cells. We then conducted studies in immunodeficient rats by stereotaxic intracranial implantation of the glioma cells followed by programmed orthotopic application of ACCA through osmotic pumps. Effect on the implanted tumor was monitored by small-animal magnetic resonance imaging. Our assays indicated that glioma invasion was markedly impaired when lactate efflux was inhibited. Convection-enhanced delivery of inhibitor to the tumor bed caused tumor necrosis, with 50% of the animals surviving beyond the experimental end points (3 months after inhibitor exhaustion). Most importantly, control animals did not display any adverse neurologic effects during orthotopic administration of ACCA to brain through programmed delivery. These results indicate the clinical potential of targeting lactate efflux in glioma through delivery of small-molecule inhibitors of MCTs either to the tumor bed or to the postsurgical resection cavity.

Objective: Additional treatments for therapeutic hypothermia are required to maximize neuroprotection for perinatal asphyxial encephalopathy. We assessed neuroprotective effects of combining inhaled xenon with therapeutic hypothermia after transient cerebral hypoxia-ischemia in a piglet model of perinatal asphyxia using magnetic resonance spectroscopy (MRS) biomarkers supported by immunohistochemistry., Methods: Thirty-six newborn piglets were randomized (all groups n = 9), with intervention from 2 to 26 hours, to: (1) normothermia; (2) normothermia + 24 hours 50% inhaled xenon; (3) 24 hours hypothermia (33.5°C); or (4) 24 hours hypothermia (33.5°C) + 24 hours 50% inhaled xenon. Serial MRS was acquired before, during, and up to 48 hours after hypoxia-ischemia., Results: Mean arterial blood pressure was lower in all treatment groups compared with normothermia (p < 0.01) (although >40mmHg); the combined therapy group required more fluid boluses (p < 0.05) and inotropes (p < 0.001). Compared with no intervention, both hypothermia and xenon-augmented hypothermia reduced the temporal regression slope magnitudes for phosphorus-MRS inorganic phosphate/exchangeable phosphate pool (EPP) and phosphocreatine/EPP (both p < 0.05); for lactate/N-acetylaspartate (NAA), only xenon-augmented hypothermia reduced the slope (p < 0.01). Xenon-augmented hypothermia also reduced transferase-mediated deoxyuridine triphosphate nick-end labeling (TUNEL)(+) nuclei and caspase 3 immunoreactive cells in parasagittal cortex and putamen and increased microglial ramification in midtemporal cortex compared with the no treatment group (p < 0.05). Compared with hypothermia, however, combination treatment did not reach statistical significance for any measure. Lactate/NAA showed a strong positive correlation with TUNEL; nucleotide triphosphate/EPP showed a strong negative correlation with microglial ramification (both p < 0.01)., Interpretation: Compared with no treatment, xenon-augmented hypothermia reduced cerebral MRS abnormalities and cell death markers in some brain regions. Compared with hypothermia, xenon-augmented hypothermia did not reach statistical significance for any measure. The safety and possible improved efficacy support phase II trials., (Copyright © 2011 American Neurological Association.)

A novel inhibitor of lactate transport, AR-C122982, was used to study the effect of inhibiting the monocarboxylate transporters MCT1 and MCT2 on cortical brain slice metabolism. We studied metabolism of L-[3-13C]lactate, and D-[1-13C]glucose under a range of conditions. Experiments using L-[3-13C]lactate showed that the inhibitor AR-C122982 altered exchange of lactate. Under depolarizing conditions, net flux of label from D-[1-13C]glucose was barely altered by 10 or 100 nM AR-C122982. In the presence of AMPA or glutamate there were increases in net flux of label and metabolic pool sizes. These data suggest lactate may supply compartments in the brain not usually directly accessed by glucose. In general, it would appear that movement of lactate between cell types is not essential for metabolic activity, with the heavy metabolic workloads imposed being unaffected by inhibition of MCT1 and MCT2. Further experiments investigating the mechanism of operation of AR-C122982 are necessary to corroborate this finding.

The central nervous system regulates food intake (FI) and body weight (BW), but the associated mechanisms remain to be elucidated. Here we report that central injections of lactate reduced FI and BW in rodents. Inhibition of central lactate metabolism to pyruvate with the lactate dehydrogenase inhibitor oxamate abolished the central effects of lactate on FI and BW. Conversely, central injections of pyruvate recapitulated the effects of lactate. Finally, inhibition of central lactate metabolism prevented the ability of circulating lactate to lower FI and BW. Together, the data indicate that activation of central lactate metabolism lowers FI and BW.

The reasons for bacterial proliferation in the lungs of mechanically ventilated patients are poorly understood. We hypothesized that prolonged cyclic stretch of lung cells influenced bacterial growth. Human alveolar type II-like A549 cells were submitted in vitro to prolonged cyclic stretch. Bacteria were cultured in conditioned supernatants from cells submitted to stretch and from control static cells. Escherichia coli had a marked growth advantage in conditioned supernatants from stretched A549 cells, but also from stretched human bronchial BEAS-2B cells, human MRC-5 fibroblasts, and murine RAW 264.7 macrophages. Stretched cells compared with control static cells acidified the milieu by producing increased amounts of lactic acid. Alkalinization of supernatants from stretched cells blocked E. coli growth. In contrast, acidification of supernatants from control cells stimulated bacterial growth. The effect of various pharmacological inhibitors of metabolic pathways was tested in this system. Treatment of A549 cells and murine RAW 264.7 macrophages with the Na(+)/K(+)-ATPase pump inhibitor ouabain during cyclic stretch blocked both the acidification of the milieu and bacterial growth. Several pathogenic bacteria originating from lungs of patients with ventilator-associated pneumonia (VAP) also grow better in vitro at slightly acidic pH (pH 6-7.2), pH similar to those measured in the airways from ventilated patients. This novel metabolic pathway stimulated by cyclic stretch may represent an important pathogenic mechanism of VAP. Alkalinization of the airways may represent a promising preventive strategy in ventilated critically ill patients.

Oxygen binding to hemoglobin (Hb) depends on allosteric effectors (CO(2), lactate and protons) that may increase drastically in concentration during exercise. The effectors share common binding sites on the Hb molecules, predicting mutual interaction in their effects on Hb (de)oxygenation. We analysed the effects of lactate and CO(2), separately and in combination, on O(2) binding of purified human Hb at 37 degrees C and physiological pH and chloride values. We demonstrate pH-dependent, inhibitory interactions between lactate binding and CO(2) binding (carbamate formation); at pH 7.4, physiological CO(2) tension ( approximately 43 mm Hg) reduced lactate binding more markedly ( approximately 75%), than lactate (50 mM) inhibited carbamate formation ( approximately 25%). In contrast to previous studies on blood and Hb solutions, we moreover find that added lactate neither 'reverses' oxylabile carbamate formation (resulting in lower carbamate levels in deoxyHb than in oxyHb) nor exerts greater allosteric effects on Hb-O(2) affinity than equal increases in chloride ion concentrations.

While in medium containing glucose, schistosomes exhibit homolactic fermentation. Accumulation of lactate acid in tissue fluid causes lowering of pH and a resultant inhibition of metabolic pathways. This requires lactate transporter protein in homolactic fermentors to facilitate the translocation of lactate(-) and [H(+)] across their plasma membrane. The ex-vivo experiment assessed lactic acid secretion by adult worms in absence and the presence of lactic acid transporter protein inhibitors. Phloretin and alpha-cyano-4-hydroxycinnamate caused a combined 25-35% inhibition of lactic acid secretion and probenecid increased this inhibition to 65% of control values. The removal of inhibitors resulted in 80% recovery of lactic acid secretion. In the in-vitro studies using vesicles isolated from adult worms and from schistosomula, the effects of phloretin and alpha-cyano-4-hydroxycinnamate were greater, each causing approximately 80% inhibition independently. The data obtained in this study demonstrate the presence of lactic acid transporters or similar proteins in Schistosoma mansoni.

Alterations in cellular metabolism are among the most consistent hallmarks of cancer. Herein we have investigated the relationship between increased aerobic lactate production and mitochondrial physiology in tumor cells. To diminish the ability of malignant cells to metabolize pyruvate to lactate, lactate dehydrogenase A (LDH-A) levels were knocked down by means of LDH-A short hairpin RNAs. Reduction in LDH-A activity resulted in stimulation of mitochondrial respiration and decrease of mitochondrial membrane potential. It also compromised the ability of these tumor cells to proliferate under hypoxia. The tumorigenicity of the LDH-A-deficient cells was severely diminished, and this phenotype was reversed by complementation with the human ortholog LDH-A protein. These results demonstrate that LDH-A plays a key role in tumor maintenance.

Prior work in animals suggests that muscle mechanoreceptor control of sympathetic activation (MSNA) during exercise in heart failure (HF) is heightened and that muscle mechanoreceptors are sensitized by metabolic by-products. We sought to determine whether 1) muscle mechanoreceptor control of MSNA is enhanced in HF patients and 2) lactic acid sensitizes muscle mechanoreceptors during rhythmic handgrip (RHG) exercise in healthy humans and patients with HF. Dichloroacetate (DCA), which reduces the production of lactic acid, or saline control was infused in 12 patients with HF and 13 controls during RHG. MSNA was recorded (microneurography). After saline was administered and during exercise thereafter, MSNA increased earlier in HF compared with controls, consistent with baseline-heightened mechanoreceptor sensitivity. In both HF and controls, MSNA increased during the 3-min exercise protocol, consistent with further sensitization of muscle mechanoreceptors by metabolic by-product(s). During posthandgrip circulatory arrest, MSNA returned rapidly to baseline levels, excluding the muscle metaboreceptors as mediators of the sympathetic excitation during RHG. To isolate muscle mechanoreceptors from central command, we utilized passive exercise in 8 HF and 11 controls, and MSNA was recorded. MSNA increased significantly during passive exercise in HF but not in controls. In conclusion, muscle mechanoreceptors mediate the increase in MSNA during low-level RHG exercise in healthy humans, and this muscle mechanoreceptor control is augmented further in HF. Neither lactate generation nor the fall in pH during RHG plays a central role in muscle mechanoreceptor sensitization. Finally, muscle mechanoreceptors in patients with HF have heightened basal sensitivity to mechanical stimuli resulting in exaggerated early increases in MSNA.

The development and characteristics of a bioreactor employing bacteria (Streptococcus thermophilus) encapsulated in Ca-alginate beads coupled with an L-lactate biosensor are reported. The biosensor comprises a carbon paste electrode modified with enzymes HRP (horseradish peroxidase), LOD (lactate oxidase), and FcH (ferrocene) as redox mediator. The measurement of L-lactate is based on the signal produced by H(2)O(2), the product of the enzymatic oxidation of L-lactate by LOD. The detection of H(2)O(2) is performed at the electrode surface via HRP/FcH at low operating potential (-100mV vs Ag/AgCl). Optimization studies were performed using the bioreactor in conjunction with an L-lactate electrode operating in a flow injection system to assess the ability of encapsulated bacteria to ferment carbohydrate solutions. The possibility of using the developed method to assess the fermentation capability of milk samples was evaluated. Bronopol (2-bromo-2-nitro propane-1,3-diol) was chosen to simulate the effect of an inhibitory agent of milk fermentation. The obtained results indicated that the evaluation of the amount of L-lactate amount produced through the bioreactor could be used as a measure of inhibition of lactic acid production in milk samples.

The relationship between the numbers of salivary mutans streptococci and the acid production in dental plaque after a single application of the 40% chlorhexidine varnish EC40 has been studied. Thirteen healthy subjects were treated with EC40 varnish. Saliva samples were taken before and up to 12 weeks after treatment to count mutans streptococci and lactobacilli. At the same time points plaque samples were taken before and after sucrose challenge and analyzed for protein and organic acid. Suppression of salivary mutans streptococci was observed together with a reduced production of lactic acid in sucrose-challenged dental plaque in 9 subjects while inhibition of acid production without significant suppression of mutans streptococci was observed in the other 4 participants. The duration of the effects differed among the individuals but never exceeded 6 weeks. We conclude that a prolonged suppression of mutans streptococci and acid production was not achieved by a single treatment with EC40 varnish in all subjects. Moreover, reduced acidogenicity of dental plaque after chlorhexidine treatment was not necessarily predicted by suppression of mutans streptococci in saliva., (Copyright 2003 S. Karger AG, Basel)

The aim of this study was two-fold: firstly, to study the effect of high fluoride concentrations on carbohydrate metabolism in Streptococcus mutans present in biofilms on hydroxyapatite; and, secondly, to evaluate the effect of fluoride-bound hydroxyapatite on lactic acid formation in growing biofilms of Strep. mutans. Biofilms of a clinical strain of Strep. mutans on saliva-coated hydroxyapatite beads were incubated with sodium fluoride over a wide range of concentrations. At high fluoride concentrations (>10 mM) the incorporation of [14C]-labeled glucose decreased by 80-85%, at both pH 7.0 and 5.6. At lower fluoride concentrations, the effect of fluoride on the incorporation of labeled glucose was pH-dependent in both biofilm cells and in planktonic cells. At pH 7.0, fluoride at concentrations < 10 mM had little or no effect. Pretreatment of hydroxyapatite discs with fluoride varnish (Fluor Protector) or fluoride solutions caused a statistically significant reduction of lactic acid formation in associated, growing biofilms of Strep. mutans. Fluoride varnish and 0.2% (47.6 mM) sodium fluoride solution exhibited a statistically significant inhibitory effect on lactate production.

Lactic acid produced by the malignant tumor is what keeps the malignancy going. Suppression or the removal of lactic acid is the prerequisite for any cancer treatment., (Copyright 2001 Harcourt Publishers Ltd.)

Hyperinsulinemia increases lactate release by various organs and tissues. Whereas it has been shown that aerobic glycolysis is linked to Na+-K+-ATPase activity, we hypothesized that stimulation by insulin of skeletal muscle Na+-K+-ATPase is responsible for increased muscle lactate production. To test this hypothesis, we assessed muscle lactate release in healthy volunteers from the [13C]lactate concentration in the effluent dialysates of microdialysis probes inserted into the tibialis anterior muscles on both sides and infused with solutions containing 5 mmol/l [U-13C]glucose. On one side, the microdialysis probe was intermittently infused with the same solution additioned with 2.10(-5) M ouabain. In the basal state, [13C]lactate concentration in the dialysate was not affected by ouabain. During a euglycemic-hyperinsulinemic clamp, [13C]lactate concentration increased by 135% in the dialysate without ouabain, and this stimulation was nearly entirely reversed by ouabain (56% inhibition compared with values in the dialysate collected from the contralateral probe). These data indicate that insulin stimulates muscle lactate release by activating Na+-K+-ATPase in healthy humans.

Monocarboxylate transporters (MCTs) comprise a group of highly homologous proteins that reside in the plasma membrane of almost all cells and which mediate the 1:1 electroneutral transport of a proton and a lactate ion. The isoform MCT3 is restricted to the basal membrane of the retinal pigment epithelium where it regulates lactate levels in the neural retina. Kinetic analysis of this transporter poses formidable difficulties due to the presence of multiple lactate transporters and their complex interaction with MCTs in adjacent cells. To circumvent these problems, we expressed both the MCT3 gene and a green fluorescent protein-tagged MCT3 construct in Saccharomyces cerevisiae. Since L-lactate metabolism in yeast depends on the CYB2 gene, we disrupted CYB2 to study the MCT3 transporter activity free from the complications of metabolism. Under these conditions L-lactate uptake varied inversely with pH, greater uptake being associated with lower pH. Whereas the V(max) was invariant, the K(m) increased severalfold as the pH rose from 6 to 8. In addition, MCT3 was highly resistant to a number of "classical" inhibitors of lactate transport. Last, studies with diethyl pyrocarbonate and p-chloromercuribenzenesulfonate set limitations on the locus of potential residues involved in the critical site of lactate translocation.

LY354740 is a potent and selective agonist at the group II metabotropic glutamate receptors and is shown to be an effective inhibitor of glutamate release with significant anxiolytic and drug withdrawal alleviating properties in certain animal models. Rats with chronic inhibition of GABA synthesis in the dorsomedial hypothalamus (DMH) are highly anxious and exhibit panic-like responses to peripheral lactate infusions similar to patients with panic disorder. Using these panic-prone rats, we tested the efficacy of LY354740 in preventing the lactate-induced panic-like response, comparing it to alprazolam, a clinically effective anti-panic drug. Rats were fitted with femoral arterial and venous catheters and implanted with Alzet pumps infusing the GABA synthesis inhibitor L-allylglycine into the DMH. After four days of recovery, they were confirmed to be panic-prone to lactate infusions as indicated by increases in heart rate, blood pressure, respiratory rate and "anxiety" measured in the social interaction test. Next, they were pretreated with either vehicle, LY354740 (0.3 and 0.6 mg/kg) or alprazolam (0.5 and 1.0 mg/kg) and re-challenged with lactate infusions. LY354740 treatment was equally efficacious as alprazolam in preventing lactate-induced panic attacks in this model. These data suggest that LY354740 could be a novel anti-panic drug, as effective as alprazolam in acute treatment.

Butyrivibrio fibrisolvens strains D1 and A38 produced little lactate, but strain 49 converted as much as 75% of its glucose to lactate. Strain 49 had tenfold more lactate dehydrogenase activity than strains D1 or A38, this activity was stimulated by fructose 1,6-bisphosphate, and had a pH optimum of 6.25. A role for fructose 1,6-bisphosphate or pH regulation of lactate production in strain 49 was, however, contradicted by the observations that very low concentrations (< 0.2 mM) of fructose 1,6-bisphosphate gave maximal activity, and continuous cultures did not produce additional lactate when the pH was decreased. The lactate production of strain 49 was clearly inhibited by the presence of acetate in the growth medium. When strain 49 was supplemented with as little as 5 mM acetate, lactate production decreased dramatically, and most of the glucose was converted to butyrate. Strain 49 did not possess butyrate kinase activity, but it had a butyryl-CoA/acetate CoA transferase that converted butyryl-CoA directly to butyrate, using acetate as an acceptor. The transferase had a low affinity for acetate (K(m) of 5 mM), and this characteristic explained the acetate stimulation of growth and butyrate formation. Strains D1 and A38 had butyrate kinase but not butyryl-CoA/acetate CoA transferase, and it appeared that this difference could explain the lack of acetate stimulation and lactate production. Based on these results, it is unlikely that B. fibrisolvens would ever contribute significantly to the pool of ruminal lactate. Since relatives of strain 49 (strains Nor37, PI-7, VV1, and OB156, based on 16S rRNA sequence analysis) all had the same method of butyrate production, it appeared that butyryl-CoA/acetate CoA transferase might be a phylogenetic characteristic. We obtained a culture of strain B835 (NCDO 2398) that produced large amounts of lactate and had butyryl-CoA/acetate CoA transferase activity, but this strain had previously been grouped with strains A38 and D1 based on 16S rRNA sequence analysis. Our strain B835 had a 16S rRNA sequence unique from the one currently deposited in GenBank, and had high sequence similarity with strains 49 and Nor37 rather than with strains A38 or D1.

1. Capsaicin activation of the pulmonary C fibre vanilloid receptor (VR1) evokes the pulmonary chemoreflex and reflex bronchoconstriction. Among potential endogenous ligands of C fibre afferents, lactic acid has been suggested as a promising candidate. We tested the hypotheses that (a) lactic acid behaves as a stimulant of C fibre receptors in the newborn dog to cause reflex bronchoconstriction, and (b) lactic acid causes reflex bronchoconstriction via the same pulmonary C fibre receptor mechanism as capsaicin using the competitive capsaicin/VR1 receptor antagonist capsazepine. 2. Right heart injection of lactic acid caused a significant increase (47 +/- 8.0 %) in lung resistance (RL) that was atropine sensitive (reduced by 75 %; P < 0.05), consistent with reflex activation of muscarinic efferents by stimulation of C fibre afferents. 3. Infusion of the competitive capsaicin antagonist capsazepine caused an 80 % reduction (P < 0.01) in the control bronchoconstrictor response (41 +/- 8.5 % increase in RL) to right heart injections of capsaicin. The effects of capsazepine are consistent with reversible blockade of the VR1 receptor to abolish C fibre-mediated reflex bronchoconstriction. 4. Lactic acid-evoked increases in RL were unaffected by VR1 blockade with capsazepine, consistent with a separate lactic acid-induced reflex mechanism. 5. We conclude that (a) putative stimulation of C fibres with lactic acid causes reflex bronchoconstriction in the newborn dog, (b) capsazepine reversibly antagonizes reflex bronchoconstriction elicited by right heart injection of capsaicin, presumably by attenuating capsaicin-induced activation of the C fibre 'capsaicin' receptor (VR1), and (c) capsazepine resistance of lactic acid-induced bronchoconstriction indicates that lactic acid evokes reflex bronchoconstriction by a separate mechanism, possibly via the acid-sensing ionic channel.

We already showed that group IV muscle afferents are activated during electrically-induced fatigue. The purpose of this study is to identify the mechanisms of stimulation of these muscle afferents by electrically-induced fatigue at a high (100 Hz; high frequency fatigue, HFF) or a low rate (10 Hz; low frequency fatigue, LFF) of stimulation. In 23 paralyzed and anaesthetized rabbits, group IV afferent activity from the tibialis anterior muscle was recorded before and after 3-min HFF or 5-min LFF runs eliciting the same force failure. Plasma lactic acid concentration (LA) was also measured in leg venous blood. We tested the effects of dichloroacetate (DCA), which reduces lactic acid production, and of acetylsalicylic acid (ASA), a blocker of cyclooxygenase, on fatigue-induced activation of group IV muscle afferents after HFF and LFF trials. Pretreatment by ASA or DCA did not modify HFF-induced activation of muscle afferents. On the other hand, LFF-induced response was markedly depressed by each pharmacological agent (-44% after ASA and -75% after DCA). We verified that DCA markedly lowered LA production after LFF. The present results show that the activation of group IV muscle afferents by LFF or HFF results from different mechanisms and also demonstrate the major role played by lactic acid production and, to a lesser extent, the release of inflammatory mediators in LFF-induced activation of group IV muscle afferents.

Lactate production, glucose utilization, glucose oxidation, and insulin release were studied in islets from rat and ob/ob mice. Lactate was determined with a highly sensitive method, based on esterification, subsequent separation, and quantitation with high-performance liquid chromatography. There was a significant lactate production in the absence of glucose, which increased with glucose concentrations up to 3 mmol/l, reaching its half-maximal rate in the presence of 0.2-1.0 mmol/l glucose in both species. Glucose utilization displayed a wider glucose concentration dependence, with a K0.5 value between 3 and 10 mmol/l glucose. The rates of glucose utilization and lactate production were similar at 3 mmol/l glucose in rat islets and at about 6 mmol/l glucose in ob/ob mice islets. Saturation of lactate production at low glucose concentrations is probably contributing to the observed preferential stimulation of oxidative metabolism at higher concentrations. D-Mannoheptulose caused a marked inhibition of glucose utilization and glucose oxidation at 20 mmol/l glucose in islets from rat or ob/ob mice, as would be expected from a competitive inhibition of glucokinase. By contrast, D-mannoheptulose reduced only marginally the islet metabolism at 3 mmol/l glucose, which is consistent with an effective mannoheptulose-induced inhibition of the glucokinase-dependent, minor part of glucose phosphorylation at this low glucose concentration.

Selected esters of D-glucose were recently proposed as tools to provide the sugar to cells, whilst bypassing the carrier system for hexose transport across the plasma membrane. In the present study, alpha-D-glucose pentaacetate, beta-D-glucose pentaacetate, alpha-D-mannose pentaacetate and, to a lesser extent, 6-O-acetyl-D-glucose, all tested at a 1.7 mM concentration, were found to increase lactate production above basal value in rat erythrocytes. Over 90 min incubation, the increment in lactate production ranged from about 1.2 (alpha-D-glucose pentaacetate) to 0.6 (6-O-acetyl-D-glucose) micromol/microl of erythrocytes. Little or no change in lactate production was observed in cells exposed to beta-L-glucose pentaacetate, alpha-D-glucose pentaethylsuccinate, alpha-D-galactose pentaacetate or beta-D-galactose pentaacetate. The metabolic response to alpha-D-glucose pentaacetate was resistant to 3-O-methyl-D-glucose (10-80 mM) which suppressed, however, that evoked by D-glucose. D-mannoheptulose (10 mM) virtually failed to affect the response to D-glucose and its pentaacetate ester. On the contrary, 2-deoxy-D-glucose (10.6 mM) inhibited to the same relative extent (55% decrease) lactate production in erythrocytes exposed to either unesterified D-glucose or alpha-D-glucose pentaacetate. The tetraacetic ester of 2-deoxy-D-glucose was more efficient than unesterified 2-deoxy-D-glucose in inhibiting lactate production from alpha-D-glucose pentaacetate. It is proposed that selected esters of saccharides represent useful tools to bypass defects in hexose transport, and to increase their nutritional or therapeutic efficiency.

To study in standardbred horses interindividual variation in the influx of lactate into red blood cells, venous blood samples were collected from 89 horses from 2 wk to 9 yr of age. For 62 horses, the rate of influx was normally distributed with a mean rate of 4.09 nmol.mg protein-1.min-1 at a lactate concentration of 10 mM, and the respective value for the other 27 horses was 0.58 nmol.mg protein-1.min-1. At 30 mM of lactate, the rates were 8.71 and 1.97 nmol.mg protein-1.min-1, respectively. This bimodal distribution was independent of age. In horses with high transport activity, the monocarboxylate transporter (MCT) appears to be the major carrier, whereas, in those with low transport activity, no activity of the MCT could be detected. The band 3 protein may account for 18-39% of transport activity. With all age groups combined, the transport activity tended to be higher in mares than in stallions. Lactate transport into red blood cells seems thus to be an inherent property in which participation of various transporters varies interindividually.

Membrane lactate transport was studied in skeletal muscle cells and membrane vesicles from the L6 line in relation to in vitro myogenesis. In myoblasts, lactate was transported by simple diffusion and insensitive to classical inhibitors: a positive correlation between onset of creatine kinase activity and lactate transport in differentiated myotubes was observed and could be considered to be a functional marker of cell differentiation. In myotubes, complete analysis of the velocity curves (direct coordinates, Eadie-Scatchard plots, Hill plots) gave parameters showing that lactate was carried by an allosteric or multisite system. This was confirmed by using sarcolemmal vesicles and specific inhibitors. In whole cells, alpha-cyano-4-hydroxycinnamic acid (CIN) and parachloromercuribenzylsulphonic acid (pCMBS) inhibited the maximal velocity without modifying the global cooperativity of the system. The weak effect of 4,4'-diisothiocyanostilbene-2,2'-disulphonic acid (DIDS), which has a low affinity constant (Ki = 22.5 microM), implicated the monocarboxylate system rather than the anionic exchanger as a carrier system in muscle cells. CIN and DIDS exhibited one type of interaction with lactate carriers, and the curvilinear shape of the lactate Hill plot with or without inhibitors suggested that inhibitors were active at the same family of interaction sites and had a common range of affinities. The apparent competitive inhibition of pyruvate (Ki = 3.2 mM) did not modify the transport pathway of lactate in L6 myotubes. In conclusion, kinetic analysis of lactate transport in the presence or absence of inhibitors gave evidence for a multisite lactate carrier activity in myotubes composed of two systems at least, related to two or three isoforms of lactate carriers.

The relationship between glial lactate release and glial intracellular pH (pHi) regulation is studied using C6 glioma cells and rat astrocytes in vitro, and the lactate transport inhibitors quercetin and alpha-cyano-4-hydroxycinnamate (CHC). pHi is measured using 2',7'-bis(carboxyethyl)-5,6-carboxyfluorescein (BCECF). The results show that lactate release is mediated partly by a specific lactate transport system inhibitable by quercetin (50 microM), but not by CHC (5 mM). Inhibition by quercetin results in a significant 3-4-fold increase of intracellular lactate and a decrease of intracellular pH to 6.9. A participation of quercetin-inhibitable lactate transport in glial pHi-regulation is suggested by the observation that pHi-homeostasis after acidification by diffusion of undissociated lactic acid into the cell is inhibited by quercetin. The existence of a system controlling lactate release in glial cells may also reflect a function of astrocytes to supply neurons with lactate.

Although a linkage between aerobic glycolysis and sodium-potassium transport has been demonstrated in diaphragm, vascular smooth muscle, and other cells, it is not known whether this linkage occurs in skeletal muscle generally. Metabolism of intact hind-leg muscles from young rats was studied in vitro under aerobic incubation conditions. When sodium influx into rat extensor digitorum longus (EDL) and soleus muscles was facilitated by the sodium ionophore monensin, muscle weight gain and production of lactate and alanine were markedly stimulated in a dose-dependent manner. Although lactate production rose in both muscles, it was more pronounced in EDL than in soleus. Monensin-induced lactate production was inhibited by ouabain or by incubation in sodium-free medium. Preincubation in potassium-free medium followed by potassium re-addition also stimulated ouabain-inhibitable lactate release. Replacement of glucose in the incubation medium with pyruvate abolished monensin-induced lactate production but exacerbated monensin-induced weight gain. Muscles from septic or endotoxin-treated rats exhibited an increased rate of lactate production in vitro that was partially inhibited by ouabain. Increases muscle lactate production in sepsis may reflect linked increases in activity of the Na+, K+-ATPase, consumption of ATP and stimulation of aerobic glycolysis.

We have used the intracellular pH-sensitive fluorescent dye 2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein (BCECF) to characterize the substrate and inhibitor specificity of monocarboxylate transport into isolated rat heart cells. Further evidence was obtained for the presence of two lactate carriers present in heart cells (Wang et al., Biochem. J. 290: 249-258, 1993) both distinct from the recently cloned monocarboxylate transporter isoform 1 (MCT-1) found in many other cell types. Only one isoform was potently inhibited by alpha-cyano-4-hydroxycinnamate [CHC; inhibitor constant (Ki) 190 microM] and the stilbene disulfonates 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (Ki 79 microM) and 4,4'-dinitrostilbene-2,2'-disulfonate (Ki of cis- and trans-isomers 38 and 171 microM, respectively; neither isomer inhibits MCT-1). The second carrier had a Ki of approximately 3 mM for CHC and 0.5-2 mM for the stilbene disulfonates. Thus, unlike in many other tissues, in rat heart cells these inhibitors are not effective at blocking lactate transport totally unless used at very high concentrations. Both carriers were inhibited by 3-isobutyl-1-methylxanthine (Ki 340 microM) and neither by 5-nitro-2-(3-phenylpropylamino)benzoate (a potent inhibitor of MCT-1). The overall Michaelis constant (Km) and maximum reaction rate (Vmax) for transport of a variety of substituted monocarboxylates (C2-C5) were determined, although it was not possible to elucidate the kinetic parameters of the two isoforms. Of physiological interest, the ketone bodies D-beta-hydroxybutyrate and acetoacetate had K(m) values of 10 and 5.4 mM, respectively. Vmax values were similar to those of L-lactate and pyruvate and indicate that transport could limit rates of utilization of ketone bodies. No stereoselectivity for L-over D-isomers of 2-chloro or 2-hydroxy acids was observed.