In-vivo magnetic resonance spectroscopy of lactate as a non-invasive biomarker of dichloroacetate activity in cancer and non-cancer central nervous system disorders.

The adverse effects of lactic acidosis in the cancer microenvironment have been increasingly recognized. Dichloroacetate (DCA) is an orally bioavailable, blood brain barrier penetrable drug that has been extensively studied in the treatment of mitochondrial neurologic conditions to reduce lactate production. Due to its effect reversing aerobic glycolysis (i.e., Warburg-effect) and thus lactic acidosis, DCA became a drug of interest in cancer as well. Magnetic resonance spectroscopy (MRS) is a well-established, non-invasive technique that allows detection of prominent metabolic changes, such as shifts in lactate or glutamate levels. Thus, MRS is a potential radiographic biomarker to allow spatial and temporal mapping of DCA treatment. In this systematic literature review, we gathered the available evidence on the use of various MRS techniques to track metabolic changes after DCA administration in neurologic and oncologic disorders. We included in vitro, animal, and human studies. Evidence confirms that DCA has substantial effects on lactate and glutamate levels in neurologic and oncologic disease, which are detectable by both experimental and routine clinical MRS approaches. Data from mitochondrial diseases show slower lactate changes in the central nervous system (CNS) that correlate better with clinical function compared to blood. This difference is most striking in focal impairments of lactate metabolism suggesting that MRS might provide data not captured by solely monitoring blood. In summary, our findings corroborate the feasibility of MRS as a pharmacokinetic/pharmacodynamic biomarker of DCA delivery in the CNS, that is ready to be integrated into currently ongoing and future human clinical trials using DCA.

The pyruvate dehydrogenase complex: Life's essential, vulnerable and druggable energy homeostat.

Found in all organisms, pyruvate dehydrogenase complexes (PDC) are the keystones of prokaryotic and eukaryotic energy metabolism. In eukaryotic organisms these multi-component megacomplexes provide a crucial mechanistic link between cytoplasmic glycolysis and the mitochondrial tricarboxylic acid (TCA) cycle. As a consequence, PDCs also influence the metabolism of branched chain amino acids, lipids and, ultimately, oxidative phosphorylation (OXPHOS). PDC activity is an essential determinant of the metabolic and bioenergetic flexibility of metazoan organisms in adapting to changes in development, nutrient availability and various stresses that challenge maintenance of homeostasis. This canonical role of the PDC has been extensively probed over the past decades by multidisciplinary investigations into its causal association with diverse physiological and pathological conditions, the latter making the PDC an increasingly viable therapeutic target. Here we review the biology of the remarkable PDC and its emerging importance in the pathobiology and treatment of diverse congenital and acquired disorders of metabolic integration.

Exposure of Rats to Multiple Oral Doses of Dichloroacetate Results in Upregulation of Hepatic Glutathione Transferases and NAD(P)H Dehydrogenase [Quinone] 1.

Dichloroacetate (DCA) is an investigational drug that is used in the treatment of various congenital and acquired disorders of energy metabolism. Although DCA is generally well tolerated, some patients experience peripheral neuropathy, a side effect more common in adults than children. Repetitive DCA dosing causes downregulation of its metabolizing enzyme, glutathione transferase zeta 1 (GSTZ1), which is also critical in the detoxification of maleylacetoacetate and maleylacetone. GSTZ1 (-/-) knockout mice show upregulation of glutathione transferases (GSTs) and antioxidant enzymes as well as an increase in the ratio of oxidized glutathione (GSSG) to reduced glutathione (GSH), suggesting GSTZ1 deficiency causes oxidative stress. We hypothesized that DCA-mediated depletion of GSTZ1 causes oxidative stress and used the rat to examine induction of GSTs and antioxidant enzymes after repeated DCA exposure. We determined the expression of alpha, mu, pi, and omega class GSTs, NAD(P)H dehydrogenase [quinone] 1 (NQO1), gamma-glutamylcysteine ligase complex (GCLC), and glutathione synthetase (GSS). GSH and GSSG levels were measured by liquid chromatography-tandem mass spectrometry. Enzyme activity was measured in hepatic cytosol using 1-chloro-2,4-dinitrobenzene, 1,2-dichloro-4-nitrobenzene, and 2,6-dichloroindophenol as substrates. In comparison with acetate-treated controls, DCA dosing increased the relative expression of GSTA1/A2 irrespective of rodent age, whereas only adults displayed higher levels of GSTM1 and GSTO1. NQO1 expression and activity were higher in juveniles after DCA dosing. GSH concentrations were increased by DCA in adults, but the GSH:GSSG ratio was not changed. Levels of GCLC and GSS were higher and lower, respectively, in adults treated with DCA. We conclude that DCA-mediated depletion of GSTZ1 causes oxidative stress and promotes the induction of antioxidant enzymes that may vary between age groups. SIGNIFICANCE STATEMENT: Treatment with the investigational drug, dichloroacetate (DCA), results in loss of glutathione transferase zeta 1 (GSTZ1) and subsequent increases in body burden of the electrophilic tyrosine metabolites, maleylacetoacetate and maleylacetone. Loss of GSTZ1 in genetically modified mice is associated with induction of glutathione transferases and alteration of the ratio of oxidized to reduced glutathione. Therefore, we determined whether pharmacological depletion of GSTZ1 through repeat administration of DCA produced similar changes in the liver, which could affect responses to other drugs and toxicants.

Effects of Multiple Doses of Dichloroacetate on GSTZ1 Expression and Activity in Liver and Extrahepatic Tissues of Young and Adult Rats.

Glutathione transferase zeta 1 (GSTZ1), expressed in liver and several extrahepatic tissues, catalyzes dechlorination of dichloroacetate (DCA) to glyoxylate. DCA inactivates GSTZ1, leading to autoinhibition of its metabolism. DCA is an investigational drug for treating several congenital and acquired disorders of mitochondrial energy metabolism, including cancer. The main adverse effect of DCA, reversible peripheral neuropathy, is more common in adults treated long-term than in children, who metabolize DCA more quickly after multiple doses. One dose of DCA to Sprague Dawley rats reduced GSTZ1 expression and activity more in liver than in extrahepatic tissues; however, the effects of multiple doses of DCA that mimic its therapeutic use have not been studied. Here, we examined the expression and activity of GSTZ1 in cytosol and mitochondria of liver, kidney, heart, and brain 24 hours after completion of 8-day oral dosing of 100 mg/kg per day sodium DCA to juvenile and adult Sprague Dawley rats. Activity was measured with DCA and with 1,2-epoxy-3-(4-nitrophenoxy)propane (EPNPP), reported to be a GSTZ1-selective substrate. In DCA-treated rats, liver retained higher expression and activity of GSTZ1 with DCA than other tissues, irrespective of rodent age. DCA-treated juvenile rats retained more GSTZ1 activity with DCA than adults. Consistent with this finding, there was less measurable DCA in tissues of juvenile than adult rats. DCA-treated rats retained activity with EPNPP, despite losing over 98% of GSTZ1 protein. These data provide insight into the differences between children and adults in DCA elimination under a therapeutic regimen and confirm that the liver contributes more to DCA metabolism than other tissues. SIGNIFICANCE STATEMENT: Dichloroacetate (DCA) is one of few drugs exhibiting higher clearance from children than adults, after repeated doses, for reasons that are unclear. We hypothesized that juveniles retain more glutathione transferase zeta 1 (GSTZ1) than adults in tissues after multiple DCA doses and found this was the case for liver and kidney, with rat as a model to assess GSTZ1 protein expression and activity with DCA. Although 1,2-epoxy-3-(4-nitrophenoxy)propane was reported to be a selective GSTZ1 substrate, its activity was not reduced in concert with GSTZ1 protein.

Age-Related Changes in miRNA Expression Influence GSTZ1 and Other Drug Metabolizing Enzymes.

Previous work has shown that hepatic levels of human glutathione transferase zeta 1 (GSTZ1) protein, involved in tyrosine catabolism and responsible for metabolism of the investigational drug dichloroacetate, increase in cytosol after birth before reaching a plateau around age 7. However, the mechanism regulating this change of expression is still unknown, and previous studies showed that GSTZ1 mRNA levels did not correlate with GSTZ1 protein expression. In this study, we addressed the hypothesis that microRNAs (miRNAs) could regulate expression of GSTZ1. We obtained liver samples from donors aged less than 1 year or older than 13 years and isolated total RNA for use in a microarray to identify miRNAs that were downregulated in the livers of adults compared with children. From a total of 2578 human miRNAs tested, 63 miRNAs were more than 2-fold down-regulated in adults, of which miR-376c-3p was predicted to bind to the 3' untranslated region of GSTZ1 mRNA. There was an inverse correlation of miR-376c-3p and GSTZ1 protein expression in the liver samples. Using cell culture, we confirmed that miR-376c-3p could downregulate GSTZ1 protein expression. Our findings suggest that miR-376c-3p prevents production of GSTZ1 through inhibition of translation. These experiments further our understanding of GSTZ1 regulation. Furthermore, our array results provide a database resource for future studies on mechanisms regulating human hepatic developmental expression. SIGNIFICANCE STATEMENT: Hepatic glutathione transferase zeta 1 (GSTZ1) is responsible for metabolism of the tyrosine catabolite maleylacetoacetate as well as the investigational drug dichloroacetate. Through examination of microRNA (miRNA) expression in liver from infants and adults and studies in cells, we showed that expression of GSTZ1 is controlled by miRNA. This finding has application to the dosing regimen of the drug dichloroacetate. The miRNA expression profiles are provided and will prove useful for future studies of drug-metabolizing enzymes in infants and adults.

Mitochondrial Glutathione Transferase Zeta 1 Is Inactivated More Rapidly by Dichloroacetate than the Cytosolic Enzyme in Adult and Juvenile Rat Liver.

Dichloroacetate (DCA) has potential for treating mitochondrial disorders and cancer by activating the mitochondrial pyruvate dehydrogenase complex. Repeated dosing of DCA results in reduced drug clearance due to inactivation of glutathione transferase ζ1 (GSTZ1), its metabolizing enzyme. We investigated the time-course of inactivation of GSTZ1 in hepatic cytosol and mitochondria after one oral dose of 100 mg/kg DCA to female Sprague-Dawley rats aged 4 weeks (young) and 52 weeks (adult) as models for children and adults, respectively. GSTZ1 activity with both DCA and an endogenous substrate, maleylacetone (MA), as well as GSTZ1 protein expression were rapidly reduced in cytosol from both ages following DCA treatment. In mitochondria, loss of GSTZ1 protein and activity with DCA were even more rapid. The cytosolic in vivo half-lives of the loss of GSTZ1 activity with DCA were 1.05 ± 0.03 and 0.82 ± 0.02 h (mean ± S.D., n = 6) for young and adult rats, respectively, with inactivation significantly more rapid in adult rats, p < 0.001. The mitochondrial inactivation half-lives were similar in young (0.57 ± 0.02 h) and adult rats (0.54 ± 0.02 h) and were significantly (p < 0.0001) shorter than cytosolic inactivation half-lives. By 24 h after DCA administration, activity and expression remained at 10% or less than control values. The in vitro GSTZ1 inactivation half-lives following incubation with 2 mM DCA in the presence of physiological chloride (Cl-) concentrations (cytosol = 44 mM, mitochondria = 1-2 mM) exhibited marked differences between subcellular fractions, being 3 times longer in the cytosol than in the mitochondria, regardless of age, suggesting that the lower Cl- concentration in mitochondria explained the faster degradation of GSTZ1. These results demonstrate for the first time that rat mitochondrial GSTZ1 is more readily inactivated by DCA than cytosolic GSTZ1, and cytosolic GSTZ1 is inactivated more rapidly in adult than young rats.

Age-Related Changes in Expression and Activity of Human Hepatic Mitochondrial Glutathione Transferase Zeta1.

Glutathione transferase zeta1 (GSTZ1) catalyzes glutathione (GSH)-dependent dechlorination of dichloroacetate (DCA), an investigational drug with therapeutic potential in metabolic disorders and cancer. GSTZ1 is expressed in both hepatic cytosol and mitochondria. Here, we examined the ontogeny and characterized the properties of human mitochondrial GSTZ1. GSTZ1 expression and activity with DCA were determined in 103 human hepatic mitochondrial samples prepared from livers of donors aged 1 day to 84 years. DNA from each sample was genotyped for three common GSTZ1 functional single nucleotide polymorphisms. Expression of mitochondrial GSTZ1 protein increased in an age-dependent manner to a plateau after age 21 years. Activity with DCA correlated with expression, after taking into account the somewhat higher activity of samples that were homo- or heterozygous for GSTZ1A. In samples from livers with the GSTZ1C variant, apparent enzyme kinetic constants for DCA and GSH were similar for mitochondria and cytosol after correcting for the loss of GSH observed in mitochondrial incubations. In the presence of 38 mM chloride, mitochondrial GSTZ1 exhibited shorter half-lives of inactivation compared with the cytosolic enzyme (P = 0.017). GSTZ1 protein isolated from mitochondria was shown by mass spectrometry to be identical to cytosolic GSTZ1 protein in the covered primary protein sequence. In summary, we report age-related development in the expression and activity of human hepatic mitochondrial GSTZ1 does not have the same pattern as that reported for cytosolic GSTZ1. Some properties of cytosolic and mitochondrial GSTZ1 differed, but these were not related to differences in amino acid sequence or post-translationally modified residues.

Regulation of dichloroacetate biotransformation in rat liver and extrahepatic tissues by GSTZ1 expression and chloride concentration.

Biotransformation of dichloroacetate (DCA) to glyoxylate by hepatic glutathione transferase zeta 1 (GSTZ1) is considered the principal determinant of the rate of plasma clearance of the drug. However, several other organismal and subcellular factors are also known to influence DCA metabolism. We utilized a female rat model to study these poorly understood processes. Rats aged 4 weeks (young) and 42-52 weeks (adult) were used to model children and adults, respectively. Hepatic chloride concentrations, which influence the rate of GSTZ1 inactivation by DCA, were lower in rat than in human tissues and rats did not show the age dependence previously seen in humans. We found GSTZ1 expression and activity in rat brain, heart, and kidney cell-free homogenates that were age-dependent. GSTZ1 expression in brain was higher in young rats than adult rats, whereas cardiac and renal GSTZ1 expression levels were higher in adult than young rats. GSTZ1 activity with DCA could not be measured accurately in kidney cell-free homogenates due to rapid depletion of glutathione by γ-glutamyl transpeptidase. Following oral administration of DCA, 100 mg/kg, to rats, GSTZ1 expression and activity were reduced in all rat tissues, but chloride concentrations were not affected. Together, these data extend our understanding of factors that determine the in vivo kinetics of DCA.

Therapeutic Targeting of the Pyruvate Dehydrogenase Complex/Pyruvate Dehydrogenase Kinase (PDC/PDK) Axis in Cancer.

The mitochondrial pyruvate dehydrogenase complex (PDC) irreversibly decarboxylates pyruvate to acetyl coenzyme A, thereby linking glycolysis to the tricarboxylic acid cycle and defining a critical step in cellular bioenergetics. Inhibition of PDC activity by pyruvate dehydrogenase kinase (PDK)-mediated phosphorylation has been associated with the pathobiology of many disorders of metabolic integration, including cancer. Consequently, the PDC/PDK axis has long been a therapeutic target. The most common underlying mechanism accounting for PDC inhibition in these conditions is post-transcriptional upregulation of one or more PDK isoforms, leading to phosphorylation of the E1α subunit of PDC. Such perturbations of the PDC/PDK axis induce a "glycolytic shift," whereby affected cells favor adenosine triphosphate production by glycolysis over mitochondrial oxidative phosphorylation and cellular proliferation over cellular quiescence. Dichloroacetate is the prototypic xenobiotic inhibitor of PDK, thereby maintaining PDC in its unphosphorylated, catalytically active form. However, recent interest in the therapeutic targeting of the PDC/PDK axis for the treatment of cancer has yielded a new generation of small molecule PDK inhibitors. Ongoing investigations of the central role of PDC in cellular energy metabolism and its regulation by pharmacological effectors of PDKs promise to open multiple exciting vistas into the biochemical understanding and treatment of cancer and other diseases.

Therapeutic applications of dichloroacetate and the role of glutathione transferase zeta-1.

Dichloroacetate (DCA) has several therapeutic applications based on its pharmacological property of inhibiting pyruvate dehydrogenase kinase. DCA has been used to treat inherited mitochondrial disorders that result in lactic acidosis, as well as pulmonary hypertension and several different solid tumors, the latter through its ability to reverse the Warburg effect in cancer cells and restore aerobic glycolysis. The main clinically limiting toxicity is reversible peripheral neuropathy. Although administration of high doses to rodents can result in liver cancer, there is no evidence that DCA is a human carcinogen. In all studied species, including humans, DCA has the interesting property of inhibiting its own metabolism upon repeat dosing, resulting in alteration of its pharmacokinetics. The first step in DCA metabolism is conversion to glyoxylate catalyzed by glutathione transferase zeta 1 (GSTZ1), for which DCA is a mechanism-based inactivator. The rate of GSTZ1 inactivation by DCA is influenced by age, GSTZ1 haplotype and cellular concentrations of chloride. The effect of DCA on its own metabolism complicates the selection of an effective dose with minimal side effects.

Pharmacogenetic considerations with dichloroacetate dosing.

The investigational drug dichloroacetate (DCA) is a metabolic regulator that has been successfully used to treat acquired and congenital metabolic diseases and, recently, solid tumors. Its clinical use has revealed challenges in selecting appropriate doses. Chronic administration of DCA leads to inhibition of DCA metabolism and potential accumulation to levels that result in side effects. This is because conversion of DCA to glyoxylate is catalyzed by one enzyme, glutathione transferase zeta 1 (GSTZ1-1), which is inactivated by DCA. SNPs in the GSTZ1 gene result in expression of polymorphic variants of the enzyme that differ in activity and rates of inactivation by DCA under physiological conditions: these properties lead to considerable variation between people in the pharmacokinetics of DCA.

GSTZ1 expression and chloride concentrations modulate sensitivity of cancer cells to dichloroacetate.

Dichloroacetate (DCA), commonly used to treat metabolic disorders, is under investigation as an anti-cancer therapy due to its ability to reverse the Warburg effect and induce apoptosis in tumor cells. While DCA's mechanism of action is well-studied, other factors that influence its potential as a cancer treatment have not been thoroughly investigated. Here we show that expression of glutathione transferase zeta 1 (GSTZ1), the enzyme responsible for conversion of DCA to its inactive metabolite, glyoxylate, is downregulated in liver cancer and upregulated in some breast cancers, leading to abnormal expression of the protein. The cellular concentration of chloride, an ion that influences the stability of GSTZ1 in the presence of DCA, was also found to be abnormal in tumors, with consistently higher concentrations in hepatocellular carcinoma than in surrounding non-tumor tissue. Finally, results from experiments employing two- and three-dimensional cultures of HepG2 cells, parental and transduced to express GSTZ1, demonstrate that high levels of GSTZ1 expression confers resistance to the effect of high concentrations of DCA on cell viability. These results may have important clinical implications in determining intratumoral metabolism of DCA and, consequently, appropriate oral dosing.

Vitamin B6 nutritional status and cellular availability of pyridoxal 5'-phosphate govern the function of the transsulfuration pathway's canonical reactions and hydrogen sulfide production via side reactions.

The transsulfuration pathway (TS) acts in sulfur amino acid metabolism by contributing to the regulation of cellular homocysteine, cysteine production, and the generation of H2S for signaling functions. Regulation of TS pathway kinetics involves stimulation of cystathionine ß-synthase (CBS) by S-adenosylmethionine (SAM) and oxidants such as H2O2, and by Michaelis-Menten principles whereby substrate concentrations affect reaction rates. Although pyridoxal phosphate (PLP) serves as coenzyme for both CBS and cystathionine γ-lyase (CSE), CSE exhibits much greater loss of activity than CBS during PLP insufficiency. Thus, cellular and plasma cystathionine concentrations increase in vitamin B6 deficiency mainly due to the bottleneck caused by reduced CSE activity. Because of the increase in cystathionine, the canonical production of cysteine (homocysteine â†’ cystathionine â†’ cysteine) is largely maintained even during vitamin B6 deficiency. Typical whole body transsulfuration flux in humans is 3-7 µmol/h per kg body weight. The in vivo kinetics of H2S production via side reactions of CBS and CSE in humans are unknown but they have been reported for cultured HepG2 cells. In these studies, cells exhibit a pronounced reduction in H2S production capacity and rates of lanthionine and homolanthionine synthesis in deficiency. In humans, plasma concentrations of lanthionine and homolanthionine exhibit little or no mean change due to 4-wk vitamin B6 restriction, nor do they respond to pyridoxine supplementation of subjects in chronically low-vitamin B6 status. Wide individual variation in responses of the H2S biomarkers to such perturbations of human vitamin B6 status suggests that the resulting modulation of H2S production may have physiological consequences in a subset of people. Supported by NIH grant DK072398. This paper refers to data from studies registered at clinicaltrials.gov as NCT01128244 and NCT00877812.

Pyridoxine supplementation does not alter in vivo kinetics of one-carbon metabolism but modifies patterns of one-carbon and tryptophan metabolites in vitamin B-6-insufficient oral contraceptive users.

BACKGROUND: Low chronic vitamin B-6 status can occur in a subset of women who use oral contraceptives (OCs) with uncertain metabolic consequences. An insufficiency of cellular pyridoxal 5'-phosphate (PLP), which is the coenzyme form of vitamin B-6, may impair many metabolic processes including one-carbon and tryptophan metabolism. OBJECTIVE: We investigated the effects of vitamin B-6 supplementation on the in vivo kinetics of one-carbon metabolism and the concentration of one-carbon and tryptophan metabolites in vitamin B-6-deficient OC users. DESIGN: A primed, constant infusion of [(13)C5]methionine, [3-(13)C]serine, and [(2)H3]leucine was performed on 10 OC users (20-40 y old; plasma PLP concentrations <30 nmol/L) before and after 28 d of supplementation with 10 mg pyridoxine hydrochloric acid/d. In vivo fluxes of total homocysteine remethylation, the remethylation of homocysteine from serine, and rates of homocysteine and cystathionine production were assessed. Targeted metabolite profiling was performed, and data were analyzed by using orthogonal partial least-squares-discriminant analysis and paired t tests adjusted for multiple testing. RESULTS: Pyridoxine supplementation increased the mean ± SD plasma PLP concentration from 25.8 ± 3.6 to 143 ± 58 nmol/L (P < 0.001) and decreased the leucine concentration from 103 ± 17 to 90 ± 20 nmol/L (P = 0.007) and glycine concentration from 317 ± 63 to 267 ± 58 nmol/L (P = 0.03). Supplementation did not affect in vivo rates of homocysteine remethylation or the appearance of homocysteine and cystathionine. A multivariate analysis showed a clear overall effect on metabolite profiles resulting from supplementation. Leucine, glycine, choline, cysteine, glutathione, trimethylamine N-oxide, and the ratios glycine:serine, 3-hydroxykynurenine:kynurenine, 3-hydroxykynurenine:3-hydroxyanthranilic acid, and 3-hydroxykynurenine:anthranilic acid were significant discriminating variables. CONCLUSIONS: Consistent with previous vitamin B-6-restriction studies, fluxes of one-carbon metabolic processes exhibited little or no change after supplementation in low-vitamin B-6 subjects. In contrast, changes in the metabolic profiles after supplementation indicated perturbations in metabolism, suggesting functional vitamin B-6 deficiency. This study was registered at clinicaltrials.gov as NCT01128244.

Dichloroacetate and cancer: new home for an orphan drug?

We reviewed the anti-cancer effects of DCA, an orphan drug long used as an investigational treatment for various acquired and congenital disorders of mitochondrial intermediary metabolism. Inhibition by DCA of mitochondrial pyruvate dehydrogenase kinases and subsequent reactivation of the pyruvate dehydrogenase complex and oxidative phosphorylation is the common mechanism accounting for the drug's anti-neoplastic effects. At least two fundamental changes in tumor metabolism are induced by DCA that antagonize tumor growth, metastases and survival: the first is the redirection of glucose metabolism from glycolysis to oxidation (reversal of the Warburg effect), leading to inhibition of proliferation and induction of caspase-mediated apoptosis. These effects have been replicated in both human cancer cell lines and in tumor implants of diverse germ line origin. The second fundamental change is the oxidative removal of lactate, via pyruvate, and the co-incident buffering of hydrogen ions by dehydrogenases located in the mitochondrial matrix. Preclinical studies demonstrate that DCA has additive or synergistic effects when used in combination with standard agents designed to modify tumor oxidative stress, vascular remodeling, DNA integrity or immunity. These findings and limited clinical results suggest that potentially fruitful areas for additional clinical trials include 1) adult and pediatric high grade astrocytomas; 2) BRAF-mutant cancers, such as melanoma, perhaps combined with other pro-oxidants; 3) tumors in which resistance to standard platinum-class drugs alone may be overcome with combination therapy; and 4) tumors of endodermal origin, in which extensive experimental research has demonstrated significant anti-proliferative, pro-apoptotic effects of DCA, leading to improved host survival.

Pharmacokinetics of oral dichloroacetate in dogs.

We characterized the pharmacokinetics and dynamics of dichloroacetate (DCA), an investigational drug for mitochondrial diseases, pulmonary arterial hypertension, and cancer. Adult Beagle dogs were orally administered 6.25 mg/kg q12h DCA for 4 weeks. Plasma kinetics was determined after 1, 14, and 28 days. The activity and expression of glutathione transferase zeta 1 (GSTZ1), which biotransforms DCA to glyoxylate, were determined from liver biopsies at baseline and after 27 days. Dogs demonstrate much slower clearance and greater inhibition of DCA metabolism and GSTZ1 activity and expression than rodents and most humans. Indeed, the plasma kinetics of DCA in dogs is similar to humans with GSTZ1 polymorphisms that confer exceptionally slow plasma clearance. Dogs may be a useful model to further investigate the toxicokinetics and therapeutic potential of DCA.

Metabolite profile analysis reveals functional effects of 28-day vitamin B-6 restriction on one-carbon metabolism and tryptophan catabolic pathways in healthy men and women.

Suboptimal vitamin B-6 status, as reflected by low plasma pyridoxal 5'-phosphate (PLP) concentration, is associated with increased risk of vascular disease. PLP plays many roles, including in one-carbon metabolism for the acquisition and transfer of carbon units and in the transsulfuration pathway. PLP also serves as a coenzyme in the catabolism of tryptophan. We hypothesize that the pattern of these metabolites can provide information reflecting the functional impact of marginal vitamin B-6 deficiency. We report here the concentration of major constituents of one-carbon metabolic processes and the tryptophan catabolic pathway in plasma from 23 healthy men and women before and after a 28-d controlled dietary vitamin B-6 restriction (<0.35 mg/d). liquid chromatography-tandem mass spectrometry analysis of the compounds relevant to one-carbon metabolism showed that vitamin B-6 restriction yielded increased cystathionine (53% pre- and 76% postprandial; P < 0.0001) and serine (12% preprandial; P < 0.05), and lower creatine (40% pre- and postprandial; P < 0.0001), creatinine (9% postprandial; P < 0.05), and dimethylglycine (16% postprandial; P < 0.05) relative to the vitamin B-6-adequate state. In the tryptophan pathway, vitamin B-6 restriction yielded lower kynurenic acid (22% pre- and 20% postprandial; P < 0.01) and higher 3-hydroxykynurenine (39% pre- and 34% postprandial; P < 0.01). Multivariate ANOVA analysis showed a significant global effect of vitamin B-6 restriction and multilevel partial least squares-discriminant analysis supported this conclusion. Thus, plasma concentrations of creatine, cystathionine, kynurenic acid, and 3-hydroxykynurenine jointly reveal effects of vitamin B-6 restriction on the profiles of one-carbon and tryptophan metabolites and serve as biomarkers of functional effects of marginal vitamin B-6 deficiency.

Metabolomic analysis reveals extended metabolic consequences of marginal vitamin B-6 deficiency in healthy human subjects.

Marginal deficiency of vitamin B-6 is common among segments of the population worldwide. Because pyridoxal 5'-phosphate (PLP) serves as a coenzyme in the metabolism of amino acids, carbohydrates, organic acids, and neurotransmitters, as well as in aspects of one-carbon metabolism, vitamin B-6 deficiency could have many effects. Healthy men and women (age: 20-40 y; n = 23) were fed a 2-day controlled, nutritionally adequate diet followed by a 28-day low-vitamin B-6 diet (<0.5 mg/d) to induce marginal deficiency, as reflected by a decline of plasma PLP from 52.6±14.1 (mean ± SD) to 21.5±4.6 nmol/L (P<0.0001) and increased cystathionine from 131±65 to 199±56 nmol/L (P<0.001). Fasting plasma samples obtained before and after vitamin B6 restriction were analyzed by (1)H-NMR with and without filtration and by targeted quantitative analysis by mass spectrometry (MS). Multilevel partial least squares-discriminant analysis and S-plots of NMR spectra showed that NMR is effective in classifying samples according to vitamin B-6 status and identified discriminating features. NMR spectral features of selected metabolites indicated that vitamin B-6 restriction significantly increased the ratios of glutamine/glutamate and 2-oxoglutarate/glutamate (P<0.001) and tended to increase concentrations of acetate, pyruvate, and trimethylamine-N-oxide (adjusted P<0.05). Tandem MS showed significantly greater plasma proline after vitamin B-6 restriction (adjusted P<0.05), but there were no effects on the profile of 14 other amino acids and 45 acylcarnitines. These findings demonstrate that marginal vitamin B-6 deficiency has widespread metabolic perturbations and illustrate the utility of metabolomics in evaluating complex effects of altered vitamin B-6 intake.

The pyruvate dehydrogenase complex as a therapeutic target for age-related diseases.

Considerable research has been conducted on mitochondrial biology as it pertains to aging. However, relatively little attention has been accorded the pyruvate dehydrogenase complex (PDC) relative to how we grow old and acquire age-related diseases. The purpose of this review is threefold: first, to describe the physiological chemistry of the PDC and define its place in normal cellular bioenergetics; second, to compare and contrast the pathogenesis and clinical features of congenital PDC deficiency with discrete examples of age-associated dysfunction of the complex; and third, to summarize recent findings in Caenorhabditis elegans that shed additional new light on the significance of the PDC to the aging process.

Prenatal and postnatal expression of glutathione transferase ζ 1 in human liver and the roles of haplotype and subject age in determining activity with dichloroacetate.

Glutathione transferase ζ 1 (GSTZ1), also known as maleylacetoacetate isomerase, catalyzes the penultimate step of tyrosine catabolism and metabolizes several α-halocarboxylic acids, including dichloroacetic acid (DCA), an investigational drug used for lactic acidosis and, recently, solid tumors. Age-related differences have been suggested in DCA pharmacotoxicology, but no information is available on GSTZ1 ontogeny in humans. Here, we investigated the cytosolic GSTZ1 developmental expression pattern and the influence of haplotype on GSTZ1 activity with DCA by using human livers from donors between 10 weeks gestation and 74 years. GSTZ1 expression was very low in fetal livers (<2 pmol of GSTZ1/mg cytosol). The expression began to increase after birth in an age-dependent manner until age 7 years. GSTZ1 was then sustained at stable, yet variable, levels (median, 20.0 pmol/mg cytosol; range, 4.8-47.3 pmol/mg cytosol) until age 74 years. GSTZ1 activity with DCA was strongly associated with haplotype and expression level. Samples homozygous or heterozygous for GSTZ1A exhibited ∼3-fold higher DCA dechlorinating activity than samples carrying other alleles at a given level of expression. The correlations (r²) between activity and expression were 0.90 and 0.68, respectively, for GSTZ1A carriers (n = 11) and noncarriers (n = 61). GSTZ1 is expressed in mitochondria in addition to cytosol. The GSTZ1A allele exhibited similar effects in the mitochondrial fraction by conferring a higher activity with DCA. In summary, we report a neonatal onset and an age-related increase in GSTZ1 protein expression during human liver development. Haplotype influenced GSTZ1 activity with DCA but not protein expression.