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Background: Glutathione plays various protective roles in the human body. Vitamin B-6 as pyridoxal-5′-phosphate (PLP) is required as the coenzyme in the formation of glutathione precursors. Despite this obligatory role of PLP, previous studies from this laboratory showed that vitamin B-6 deficiency caused elevated glutathione concentrations in rat liver and human plasma. Objective: Our aim was to determine the effect of marginal vitamin B-6 deficiency (plasma PLP 20–30 nmol/L) on the rate of red blood cell (RBC) glutathione synthesis. Design: We measured plasma and RBC glutathione concentrations and the fractional and absolute synthesis rates of RBC glutathione using the stable-isotope-labeled glutathione precursor [1,2-13C2]glycine in 13 healthy volunteers aged 21–39 y. Results: Dietary vitamin B-6 restriction did not significantly affect the glutathione concentration in plasma (6.9 ± 1.9 compared with 6.7 ± 1.1 μmol/L) or RBCs (2068 ± 50 compared with 2117 ± 48 μmol/L). For RBC glutathione, the mean fractional synthesis rates were 54 ± 5%/d and 43 ± 4%/d (P = 0.10), and the absolute synthesis rates were 1116 ± 100 and 916 ± 92 μmol · L−1 · d−1 (P = 0.14) before and after vitamin B-6 restriction, respectively. Conclusions: Marginal vitamin B-6 deficiency tended to decrease mean RBC glutathione synthesis with no effect on RBC glutathione concentration, but the responses varied widely among individuals. Because the cysteine concentration in plasma and RBC did not change during vitamin B-6 restriction, we conclude that the effects of marginal vitamin B-6 deficiency on glutathione synthesis are not caused by altered precursor concentrations.

Glycine undergoes decarboxylation in the glycine cleavage system (GCS) to yield CO(2), NH(3), and a 1-carbon unit. CO(2) also can be generated from the 2-carbon of glycine by 10-formyltetrahydrofolate-dehydrogenase and, after glycine-to-serine conversion by serine hydroxymethyltransferase, from the tricarboxylic acid cycle. To evaluate the relative fates of glycine carbons in CO(2) generation in healthy volunteers (3 male, 3 female, aged 21-26 y), primed, constant infusions were conducted using 9.26 micromol x h(-1) x kg(-1) of [1,2-(13)C]glycine and 1.87 micromol x h(-1) x kg(-1) of [5,5,5-(2)H(3)]leucine, followed by an infusion protocol using [1-(13)C]glycine as the glycine tracer. The time period between the infusion protocols was6 mo. In vivo rates of whole-body glycine and leucine flux were nearly identical in protocols with [1,2-(13)C]glycine and [5,5,5-(2)H(3)]leucine and with [1-(13)C]glycine and [5,5,5-(2)H(3)]leucine tracers, which showed high reproducibility between the tracer protocols. Using the [1-(13)C]glycine tracer, breath CO(2) data showed a total rate of glycine decarboxylation of 96 +/- 8 micromol x h(-1) x kg(-1), which was 22 +/- 3% of whole-body glycine flux. In contrast, infusion of [1,2-(13)C]glycine yielded a glycine-to-CO(2) flux of 146 +/- 37 micromol x h(-1) x kg(-1) (P = 0.026). By difference, this implies a rate of CO(2) formation from the glycine 2-carbon of 51 +/- 40 micromol x h(-1) x kg(-1), which accounts for approximately 35% of the total CO(2) generated in glycine catabolism. These findings also indicate that approximately 65% of the CO(2) generation from glycine occurs by decarboxylation, primarily from the GCS. Further, these results suggest that the GCS is responsible for the entry of 5,10-methylenetetrahydrofolate into 1-carbon metabolism at a very high rate ( approximately 96 micromol x h(-1) x kg(-1)), which is approximately 20 times the demand for methyl groups for homocysteine remethylation.

Dichloroacetate (DCA) is an investigational drug for the treatment of genetic mitochondrial diseases. Its primary site of action is the pyruvate dehydrogenase (PDH) complex, which it stimulates by altering its phosphorylation state and stability. DCA is metabolized by and inhibits the bifunctional zeta-1 family isoform of glutathione transferase/maleylacetoacetate isomerase. Polymorphic variants of this enzyme differ in their kinetic properties toward DCA, thereby influencing its biotransformation and toxicity, both of which are also influenced by subject age. Results from open label studies and controlled clinical trials suggest chronic oral DCA is generally well-tolerated by young children and may be particularly effective in patients with PDH deficiency. Recent in vitro data indicate that a combined DCA and gene therapy approach may also hold promise for the treatment of this devastating condition.

Dichloroacetate has been administered for several years to children and adults with genetic mitochondrial diseases. The primary rationale for its use is its ability to stimulate the activity of the pyruvate dehydrogenase complex, thereby facilitating aerobic glucose and lactate oxidation and energetics.1 Most published information on the safety and efficacy of dichloroacetate in this patient population is derived from open-label studies in 1 or a few individuals. Recently, a randomized, controlled trial evaluated oral dichloroacetate at a dose of 12.5 mg/kg twice daily for the chronic treatment of older adolescents and adults (mean age at entry: 30 years) with the common A3243G mutation in mitochondrial DNA that gives rise to the syndrome of mitochondrial encephalomyopathy, lactic acidosis, and stroke-like episodes.2 This study was terminated prematurely because of an undue incidence of worsening or new onset of peripheral neuropathy associated with dichloroacetate administration compared with placebo administration. Another recently completed randomized, controlled trial investigated the same dichloroacetate dose in 43 young children (mean age at entry: 5.6 years) with congenital lactic acidosis (CLA) because of defects in pyruvate dehydrogenase or in ≥1 complex of the respiratory chain or a mitochondrial DNA mutation.3 In contrast to the findings in older subjects with mitochondrial encephalomyopathy, lactic acidosis, and stroke-like episodes, this study demonstrated good tolerability of dichloroacetate, and there were no significant differences in the severity or frequency of adverse events between the drug and placebo groups after 6 months of treatment. Here we report our experience with long-term administration of dichloroacetate in the 36 children on the trial who received dichloroacetate, from their starting times on the drug. Some of these patients have received treatment continuously for ~10 years.

Dichloroacetate (DCA) is an investigational drug for certain metabolic diseases. It is biotransformed principally by the zeta-1 family isoform of glutathione transferase (GSTz1), also known as maleylacetoacetate isomerase (MAAI), which catalyzes the penultimate step in tyrosine catabolism. DCA causes a reversible peripheral neuropathy in several species, including humans. However, recent clinical trials indicate that adults are considerably more susceptible to this adverse effect than children. We evaluated the kinetics and biotransformation of DCA and its effects on tyrosine metabolism in nine patients treated for 6 months with 25 mg/kg/day and in rats treated for 5 days with 50 mg/kg/day. We also measured the activity and expression of hepatic GSTz1/MAAI. Chronic administration of DCA causes a striking age-dependent decrease in its plasma clearance and an increase in its plasma half-life in patients and rats. Urinary excretion of unchanged DCA in rats increases with age, whereas oxalate, an end product of DCA metabolism, shows the opposite trend. Low concentrations of monochloroacetate (MCA), which is known to be neurotoxic, increase as a function of age in the urine of dosed rats. MCA was detectable in plasma only of older animals. Hepatic GSTz1/MAAI-specific activity was inhibited equally by DCA treatment among all age groups, whereas plasma and urinary levels of maleylacetone, a natural substrate for this enzyme, increased with age. We conclude that age is an important variable in the in vivo metabolism and elimination of DCA and that it may account, in part, for the neurotoxicity of this compound in humans and other species.

Glycine plays several roles in human metabolism, e.g. as a 1-carbon donor, in purine synthesis, and as a component of glutathione. Glycine is decarboxylated via the glycine cleavage system (GCS) that yields concurrent generation of a 1-carbon unit as 5,10-methylenetetrahydrofolate (methyleneTHF). Serine hydroxymethyltransferase (SHMT) catalyzes the interconversion of glycine and serine, another 1-carbon donor. The quantitative role of glycine in human 1-carbon metabolism has received little attention. The aim of this protocol was to quantify whole body glycine flux, glycine to serine flux, and rate of glycine cleavage in humans. A primed, constant infusion with 9.26 micromol x kg(-1) x h(-1) [1,2-(13)C2]glycine and 1.87 micromol x kg(-1) x h(-1) [(2)H3]leucine was used to quantify the kinetic behavior of glycine in young, healthy volunteers (n = 5) in a fed state. The isotopic enrichment of infused tracers and metabolic products in plasma, as well as breath (13)CO2 enrichment, were determined for use in kinetic analysis. Serine synthesis by direct conversion from glycine via SHMT occurred at 193 +/- 28 micromol x kg(-1) x h(-1) (mean +/- SEM), which comprised 41% of the 463 +/- 55 micromol x kg(-1) x h(-1) total glycine flux. Nearly one-half (46%) of the glycine-to-serine conversion occurred using GCS-derived methyleneTHF 1-carbon units. Based on breath (13)CO2 measurement, glycine decarboxylation (190 +/- 41 micromol x kg(-1) x h(-1)) accounted for 39 +/- 6% of whole body glycine flux. This study is the first to our knowledge to quantify human glycine cleavage and glycine-to-serine SHMT kinetics. GCS is responsible for a substantial proportion of whole body glycine flux and constitutes a major route for the generation of 1-carbon units.

The direct analysis of tissue from both the central and peripheral nervous systems of control rats and those administered the potential neurotoxin dichloroacetate (DCA) was investigated using an intermediate-pressure matrix-assisted laser desorption/ionization (IP-MALDI) source coupled to a linear ion trap (LIT) mass spectrometer. The matrix, 2,5-dihydroxybenzoic acid, was applied to the tissue using a novel automated inkjet printer system. The MSn capabilities of the LIT allowed identification of lipids desorbed directly from tissue. A marked decrease is observed in the intensity of lipid ions in spinal cord and sciatic nerve tissues from rats exposed to DCA. The results also demonstrate the rapid, sensitive, and semiquantitative capabilities of this method.

Dichloroacetate (DCA) is an investigational drug for genetic mitochondrial diseases whose use has been mitigated by reversible peripheral neuropathy. We investigated the mechanism of DCA neurotoxicity using cultured rat Schwann cells (SCs) and dorsal root ganglia (DRG) neurons. Myelinating SC-DRG neuron co-cultures, isolated SCs and DRG neurons were exposed to 1-20 mm DCA for up to 12 days. In myelinating co-cultures, DCA caused a dose- and exposure-dependent decrease of myelination, as determined by immunolabeling and immunoblotting for myelin basic protein (MBP), protein zero (P0), myelin-associated glycoprotein (MAG) and peripheral myelin protein 22 (PMP22). Partial recovery of myelination occurred following a 10-day washout of DCA. DCA did not affect the steady-state levels of intermediate filament proteins, but promoted the formation of anti-neurofilament antibody reactive whirls. In isolated SC cultures, DCA decreased the expression of P0 and PMP22, while it increased the levels of p75(NTR) (neurotrophin receptor), as compared with non-DCA-treated samples. DCA had modest adverse effects on neuronal and glial cell vitality, as determined by the release of lactate dehydrogenase. These results demonstrate that DCA induces a reversible inhibition of myelin-related proteins that may account, at least in part, for its clinical peripheral neuropathic effects.

BACKGROUND/OBJECTIVES The zeta-1 family isoform of GST biotransforms the investigational drug dichloroacetate (DCA) and certain other halogenated carboxylic acids. Haplotype variability in GSTZ1 influences the kinetics and, possibly, the toxicity of DCA. DCA metabolism correlates with expression of the GSTZ1 protein, so it is important to document variables that affect expression. Following up on a limited previous study, we tested the hypothesis that a coding single nucleotide polymorphism (SNP), the lysine (K) amino acid (E32>K) in GSTZ1 haplotypes linked to a promoter region SNP results in lower hepatic expression of GSTZ1. MATERIALS AND METHODS The influence of K carrier and non-K carrier haplotypes on GSTZ1 expression was determined by analyzing 78 liver samples from individuals aged 7-84 years of various racial and ethnic backgrounds. GSTZ1 expression data were analyzed on the basis of the presence or absence of lysine 32. RESULTS GSTZ1 protein expression differed significantly between K carrier and non-K carrier haplotypes (P=0.001) in Whites, but not in African-Americans (P=0.277). We attribute this difference in GSTZ1 expression among K carrier haplotypes in Whites to the linkage disequilibrium between the K or A allele from the G>A SNP (rs7975), within the promoter G>A-1002 SNP (rs7160195) A allele. There is no linkage disequilibrium between these two polymorphisms in African-Americans. CONCLUSION We conclude that the lower expression of GSTZ1 in Whites who possess the K carrier haplotype results in lower enzymatic activity and slower metabolism of DCA, compared with those who possess the non-K carrier haplotype. These results further define safe, genetics-based dosing regimens for chronic DCA administration.

Background Chloral hydrate (CH), a sedative and metabolite of the environmental contaminant trichloroethylene, is metabolized to trichloroacetic acid, trichloroethanol, and possibly dichloroacetate (DCA). DCA is further metabolized by glutathione transferase zeta 1 (GSTZ1), which is identical to maleylacetoacetate isomerase (MAAI), the penultimate enzyme in tyrosine catabolism. DCA inhibits its own metabolism through depletion/inactivation of GSTZ1/MAAI with repeated exposure, resulting in lower plasma clearance of the drug and the accumulation of the urinary biomarker maleylacetone (MA), a metabolite of tyrosine. It is unknown if GSTZ1/MAAI may participate in the metabolism of CH or any of its metabolites and, therefore, affect tyrosine catabolism. Stable isotopes were utilized to determine the biotransformation of CH, the kinetics of its major metabolites, and the influence, if any, of GSTZ1/MAAI. Methods Eight healthy volunteers (ages 21-40 years) received a dose of 1 g of CH (clinical dose) or 1.5 μg/kg (environmental) for five consecutive days. Plasma and urinary samples were analyzed by gas chromatography-mass spectrometry. Results Plasma DCA (1.2-2.4 μg/mL), metabolized from CH, was measured on the fifth day of the 1 g/day CH dosage but was undetectable in plasma at environmentally relevant doses. Pharmacokinetic measurements from CH metabolites did not differ between slow and fast GSTZ1 haplotypes. Urinary MA levels increased from undetectable to 0.2-0.7 μg/g creatinine with repeated CH clinical dose exposure. Kinetic modeling of a clinical dose of 25 mg/kg DCA administered after 5 days of 1 g/day CH closely resembled DCA kinetics obtained in previously naive individuals. Conclusions These data indicate that the amount of DCA produced from clinically relevant doses of CH, although insufficient to alter DCA kinetics, is sufficient to inhibit MAAI and tyrosine catabolism, as evidenced by the accumulation of urinary MA.

Cysteine synthesis from homocysteine is catalyzed by two pyridoxal 5'-phosphate (PLP)-dependent enzymes. This suggests that vitamin B-6 status might affect cysteine and glutathione homeostasis, but it is unclear whether this occurs in humans. We assessed the effects of vitamin B-6 status on static and kinetic parameters of cysteine and glutathione metabolism in healthy female (n=5) and male (n=4) volunteers (20-30 y) before and after 4 wk of dietary vitamin B-6 restriction (

Peripheral neuropathy is an underrecognized but common occurrence in genetic mitochondrial disorders. To gain insight into the frequency and clinical presentation of this complication, nerve conduction studies were performed on 43 subjects with congenital lactic acidosis enrolled in a controlled clinical trial of oral dichloroacetate. Median and peroneal motor conduction studies and median and sural sensory conduction studies were performed on each patient. The mean amplitude of the peroneal motor nerve (P < 0.001) and the conduction velocities of the median (P < 0.001) and peroneal (P < 0.001) motor nerves were uniformly lower in our subjects than in healthy literature control subjects. There were no significant differences in sensory nerve conduction studies. A generalized reduction in motor nerve conduction velocity was the dominant electrophysiological abnormality in the patients in this study and was independent of age, sex, or congenital mitochondrial disorder. We postulate that cellular energy failure is the most likely common cause of peripheral neuropathy in patients with genetic mitochondrial diseases, owing to the high demand for adenosine triphosphate via aerobic carbohydrate metabolism by nerve tissue.

Whether folate status and the methylenetetrahydrofolate reductase (MTHFR) 677C-->T polymorphism interact to affect methionine-cycle metabolite concentrations is uncertain. We evaluated the effects of dietary folate restriction on relations among folate status indices and plasma concentrations of methionine cycle metabolites in women with the MTHFR 677 C/C and T/T genotypes. Healthy, normohomocysteinemic women (n = 18; 20-30 y old) of adequate B vitamin status, and equally divided according to MTHFR 677C-->T genotype (9 C/C and 9 T/T) were recruited. Folate status indices and methionine cycle metabolites were measured in blood samples collected at baseline and after 7 wk of dietary folate restriction (115 microg dietary folate equivalents/d). Significant negative correlations between plasma total homocysteine concentrations and total or 5-methyl folate concentrations (P = 0.041 and 0.023, respectively) in RBCs were found only in T/T subjects. Formylated folates were detected in RBCs of T/T subjects only, and their abundance was predictive of plasma total homocysteine concentration despite no significant alteration by folate restriction. Plasma concentrations of S-adenosylmethionine and S-adenosylhomocysteine were not significantly affected by dietary folate restriction and the MTHFR 677 T/T genotype. In conclusion, plasma total homocysteine concentrations in subjects with the MTHFR 677 T/T genotype were inversely related to 5-methyl folate concentrations and directly related to formylated folate concentrations in RBCs, even though the latter were not significantly affected by moderate folate restriction.

Background: The effects of vitamin B-6 status on steady-state kinetics of homocysteine metabolism in humans are unclear. Objective: The objective was to determine the effects of dietary vitamin B-6 restriction on the rates of homocysteine remethylation and synthesis in healthy humans. Design: Primed, constant infusions of [ 13 C5]methionine, [3- 13 C]serine, and [ 2 H3]leucine were conducted in healthy female (n5)andmale(n4)volunteers(20–30y)beforeandafter4wk of dietary vitamin B-6 restriction (0.5 mg vitamin B-6/d) to establish whether vitamin B-6 status affects steady-state kinetics of homocysteine metabolism in the absence of concurrent methionine intake. Effects of dietary vitamin B-6 restriction on vitamin B-6 status, plasma amino acid concentrations, and the rates of reactions of homocysteine metabolism were assessed. Results: Dietary vitamin B-6 restriction significantly reduced plasma pyridoxal 5'-phosphate (PLP) concentrations (55.1 8.3 compared with 22.6 1.3 nmol/L; P 0.004), significantly increased plasma glycine concentrations (230 14 compared with 296 15; P 0.008), and significantly reduced basal (43%; P 0.001) and PLP-stimulated (35%; P 0.004) lymphocyte serine hydroxymethyltransferase activities in vitro. However, the in vivo fluxes of leucine, methionine, and serine; the rates of homocysteine synthesisandremethylation(totalandvitaminB-6–dependent);and the concentrations of homocysteine, methionine, and serine in plasma were not significantly affected by dietary vitamin B-6 restriction. Conclusions: Moderate vitamin B-6 deficiency does not significantly alter the rates of homocysteine remethylation or synthesis in healthy young adults in the absence of dietary methionine intake. Am J Clin Nutr 2005;81:648–55.

Several acquired and congenital human disorders perturb the concentrations of delta-aminolevulinate (delta-ALA), creatinine and tyrosine in biological fluids. There is currently no facile, sensitive and specific method to measure these analytes simultaneously.We developed an LC-MS/MS method to quantify delta-ALA, creatinine and tyrosine in urine that requires minimal sample preparation and no derivatization. The method is also applicable to the analysis of tyrosine in plasma.All calibration plots were linear, with R(2)or=0.996. Intra- and interday CVs were10%. The limit of quantitation for delta-ALA was approximately 0.1 micromol/l, and for creatinine and tyrosine it was well below the lowest measured physiological concentrations. The method was applied to analyze urine from 75 healthy volunteers and 43 patients with hereditary tyrosinemia type I (HT I). The mean urinary concentration of delta-ALA in patients (38+/-35 micromol/l, 53+/-30 mg/g creatinine) was higher than that measured in healthy subjects (5.5+/-2.6 micromol/l, 0.9+/-0.2 mg/g creatinine; p0.001). Treatment with 2-(2-nitro-4-trifluoromethylbenzyl)-1,3-cyclohexanedione (NTBC), an inhibitor of an early step in tyrosine catabolism, decreased urinary delta-ALA (6.4+/-4.8 micromol/l, 13+/-24 mg/g creatinine; p0.001). The average plasma tyrosine concentration in healthy volunteers (56+/-14 micromol/l) was within normal reference interval used in clinical practice.The method is simple, specific and precise and allows simultaneous quantitation of delta-ALA, creatinine and tyrosine at concentrations present under physiological or pathophysiological conditions.

Common cardiovascular disease risk factors (e.g., insulin and aerobic fitness) are improved with exercise; however, few studies have addressed the potential for training to modify emerging cardiovascular disease risk factors such as homocysteine and high-sensitivity C-reactive protein.Sedentary adults (n = 324, 48.9 +/- 8.4 years) were randomized to four groups differing in training intensity (moderate = 45-55% or high = 65-75% of heart rate reserve) and frequency (low = 3-4, 30-min sessions/week or high = 5-7, 30 min-sessions/week).Within-group changes in homocysteine, insulin, and aerobic fitness were significant (all P0.0125). Furthermore, homocysteine increased in the high-intensity-low-frequency (0.98 +/- 2.32 micromol/L) and high-intensity-high-frequency (0.93 +/- 2.56 micromol/L) groups, while aerobic fitness increased in the moderate-intensity-high-frequency (0.99 +/- 2.01 mL min(-1) kg(-1)) and high-intensity-high-frequency (1.77 +/- 2.97 mL min(-1) kg(-1)) groups (all P0.003). The change in aerobic fitness was greater in the high-intensity-high-frequency compared to the moderate-intensity-low-frequency group (1.77 +/- 2.97 vs. 0.36 +/- 2.10 mL min(-1) kg(-1), P = 0.0014) (effect size estimate = 0.60 mL min(-1) kg(-1)). The main effects for intensity, with respect to the change in insulin (effect size estimate = 0.46 microU/mL), and frequency, with respect to the change in aerobic fitness (effect size estimate = 0.38 mL min(-1) kg(-1)), were significant (P0.0125).Although frequent bouts of higher intensity exercise were particularly effective in reducing fasting insulin and improving fitness, they resulted in slightly increased homocysteine levels.

Ketogenic diets (KDs) are used for treatment of refractory epilepsy and metabolic disorders. The classic saturated fatty acid-enriched (SAT) KD has a fat:carbohydrate plus protein ratio of 4:1, in which the predominant fats are saturated. We hypothesized that a polyunsaturated fat-enriched (POLY) KD would induce a similar degree of ketosis with less detrimental effects on carbohydrate and lipid metabolism. Twenty healthy adults were randomized to two different weight-maintaining KDs for 5 d. Diets were 70% fat, 15% carbohydrate, and 15% protein. The fat contents were 60 or 15% saturated, 15 or 60% polyunsaturated, and 25% monounsaturated for SAT and POLY, respectively. Changes in serum beta-hydroxybutyrate, insulin sensitivity (S(I)), and lipid profiles were measured. Mean circulating beta-hydroxybutyrate levels increased 8.4 mg/dl in the POLY group (P = 0.0004), compared with 3.1 mg/dl in the SAT group (P = 0.07). S(I) increased significantly in the POLY group (P = 0.02), whereas total and low-density lipoprotein cholesterol increased significantly in the SAT group (both P = 0.002). These data demonstrate that a short-term POLY KD induces a greater level of ketosis and improves S(I), without adversely affecting total and low-density lipoprotein cholesterol, compared with a traditional SAT KD. Thus, a POLY KD may be superior to a classical SAT KD for chronic administration.

We determined acute and chronic effects of dichloroacetate (DCA) on maximal (MAX) and submaximal (SUB) exercise responses in patients with abnormal mitochondrial energetics. Subjects (n = 9) completed a MAX treadmill bout 1 h after ingesting 25 mg/kg DCA or placebo (PL). A 15-min SUB bout was completed the next day while receiving the same treatment. After a 1-d washout, MAX and SUB were repeated while receiving the alternate treatment (acute). Gas exchange and heart rate were measured throughout all tests. Blood lactate (Bla) was measured 0, 3, and 10 min after MAX, and 5, 10, and 15 min during SUB. MAX and SUB were repeated after 3 months of daily DCA or PL. After a 2-wk washout, a final MAX and SUB were completed after 3 months of alternate treatment (chronic). Average Bla during SUB was lower (P < 0.05) during both acute (1.99 ± 1.10 vs. 2.49 ± 1.52 mmol/liter) and chronic (1.71 ± 1.37 vs. 2.39 ± 1.32 mmol/liter) DCA vs. PL despite similar exercise intensities between conditions (∼75 and 70% maximal exercise capacity during acute and chronic treatment). Thus, although DCA does not alter MAX responses, acute and chronic DCA attenuate the Bla response to moderate exercise in patients with abnormal mitochondrial energetics.

Haloacetic acids (HAAs) are environmentally and medically important chemicals. No analytical method is currently available to analyze EPA-regulated HAAs in biological samples at environmentally relevant low concentrations. Clinical studies of this class of chemicals are also limited by the lack of analytical techniques of high sensitivity and precision. We now report a new analytical method using gas chromatography/ion trap mass spectrometry for quantifying nine HAAs present inplasma, urine, and water at picogram per milliliter levels. The derivatization reactions of HAAs with pentafluorobenzyl bromide were optimized and detection with an electron capture negative ion chemical ionization mode was employed to enhance the sensitivity. Selected ion monitoring and selected reaction monitoring methods were utilized for quantitation. The detection limits of HAAs in plasma, urine, and water were 25-1000 pg/mL. Accuracies varied from 86.6 to 118.1% (intraday) and 81.7 to 119.6% (interday). Precisions (CV) varied from 0.9 to 19.9% (intraday) and 0.8 to 19.8% (interday), and linearities (r2) varied from 0.9732 to 0.9998 (intraday) and 0.9422 to 0.9987 (interday), respectively. Methyl tertbutyl ether and diethyl ether provided the highest extraction recoveries for the HAAs (74.9-107.2%). The method was applied successfully to a kinetic investigation of low levels of HAAs in humans consuming chlorinated drinking water.

Here we review the rationale for considering the pyruvate dehydrogenase multienzyme complex (PDC) as a target for gene therapy for defects in mitochondrial energetics. PDC is entirely nuclear encoded and is situated in the mitochondrial inner membrane. The complex catalyzes the rate-determining step in aerobic carbohydrate metabolism and plays a critical role in the efficient conversion of substrate fuel into energy by cells. PDC activity is regulated in large part by reversible phosphorylation (inactivation) of its E1alpha subunit. Congenital defects in PDC are usually due to mutations in E1alpha and are typified by lactic acidosis, neurodegeneration and early death. Acquired deficiency in PDC has been implicated in the etiopathology of several other metabolic or neurodegenerative disorders. Recently, a vector using recombinant adeno-associated virus (rAAV) that contained a fusion protein of full-length E1alpha and the reporter gene green fluorescent protein was used to deliver wild type E1alpha into mitochondria after injection of the construct in vivo into the central nervous system of rats and in vitro into human cells. Transduction of cultured fibroblasts from a male patient with E1alpha deficiency led to partial restoration of PDC activity, as determined by decarboxylation of 14C-pyruvate. These data indicate that at least partial correction of PDC defects may be feasible by gene transfer. Furthermore, the combination of AAV-mediated delivery of E1alpha with pharmacologic activation (dephosphorylation) of the wild type enzyme subunit may provide an optimal therapeutic strategy for patients with acquired or congenital deficiencies in mitochondrial energy metabolism.

The authors conducted a randomized, double-blind, placebo-controlled trial of intravenous dichloroacetate (DCA) for the purpose of treating lactic acidosis in 124 West African children with severe Plasmodium falciparum malaria. Lactic acidosis independently predicts mortality in severe malaria, and DCA stimulates the oxidative removal of lactate in vivo. A single infusion of 50 mg/kg DCA was well tolerated. When administered at the same time as a dose of intravenous quinine, DCA significantly increased the initial rate and magnitude of fall in blood lactate levels and did not interfere with the plasma kinetics of quinine. The authors developed a novel population pharmacokinetic-pharmacodynamic indirect-response model for DCA that incorporated characteristics associated with disease reversal. The model describes the complex relationships among antimalarial treatment procedures, plasma DCA concentrations, and the drug's lactate-lowering effect. DCA significantly reduces the concentration of blood lactate, an independent predictor of mortality in malaria. Its prospective evaluation in affecting mortality in this disorder appears warranted.

We provide a simple method for fitting a one-compartment, zero-order absorption pharmacokinetics model in the presence of observations below the detection limit. This method may be extended to more complex pharmacokinetics models. We demonstrate, using a small simulation study, that the method provides accurate parameter estimates over a range of detection limits and we compare it to an ad hoc midpoint method. An applied example is provided from a pharmacokinetic investigation of a nicotine nasal spray.

To determine the feasibility of gene transfer to correct defects in the E1alpha subunit of the pyruvate dehydrogenase (PDH) complex (PDC), we constructed rAAV vectors that expressed PDH E1alpha, either alone or with a green fluorescent protein tag, from a hybrid cytomegalovirus (CMV) enhancer/chicken beta-actin (CB) promoter. These vectors were functional in vitro, as judged by increased expression of mRNA in vector-transduced deficient cell lines and correction of the biochemical defect in PDH activity in these cells. Approximately 30% of wild-type levels of PDH activity were restored under conditions with which only about 15% of cells were transduced. These same vectors were then used in vivo to transduce neurons within the rat striatum. Gene transfer, expression, and translocation into mitochondria were observed, without any obvious untoward effects. In vivo vector-mediated PDH expression persisted for at least 1 year after injection, indicating the stability of gene transfer. These studies provide the basis for future efforts to develop a recombinant AAV (rAAV)-based gene therapy approach for the correction of PDC deficiency.

Congenital lactic acidoses (CLAs) constitute a group of rare inborn errors of mitochondrial metabolism in which cellular energy failure is the defining biochemical abnormality. We report the principal manifestations of renal dysfunction in 35 children with CLA caused by defects in either the pyruvate dehydrogenase multienzyme complex or one or more components of the respiratory chain. The most prominent renal abnormalities included bicarbonaturia, phosphaturia, hypercalciuria, complete Fanconi's syndrome, proteinuria, and decreased glomerular filtration rate. These data were compared with those from 79 previously published cases. Clinical manifestations of renal dysfunction in CLA are common and may be the first presenting sign of the disease. The glomerulus and proximal renal tubule appear to be the anatomic sites most vulnerable to abnormal mitochondrial energy transduction. We propose that the primary defect in mitochondrial energy metabolism, together with the consequent intracellular accumulation of lactate and hydrogen ions, precipitates a state of tissue injury that, unless interrupted, becomes self-perpetuating and ultimately leads to renal cell death.

Primary mitochondrial diseases are caused by heritable or spontaneous mutations in nuclear DNA or mitochondrial DNA. Such pathological mutations are relatively common in humans and may lead to neurological and neuromuscular complication that could compromise normal sleep behavior. To gain insight into the potential impact of primary mitochondrial disease and sleep pathology, we reviewed the relevant English language literature in which abnormal sleep was reported in association with a mitochondrial disease.We examined publication reported in Web of Science and PubMed from February 1976 through January 2014, and identified 54 patients with a proven or suspected primary mitochondrial disorder who were evaluated for sleep disturbances.Both nuclear DNA and mitochondrial DNA mutations were associated with abnormal sleep patterns. Most subjects who underwent polysomnography had central sleep apnea, and only 5 patients had obstructive sleep apnea. Twenty-four patients showed decreased ventilatory drive in response to hypoxia and/ or hyperapnea that was not considered due to weakness of the intrinsic muscles of respiration.Sleep pathology may be an underreported complication of primary mitochondrial diseases. The probable underlying mechanism is cellular energy failure causing both central neurological and peripheral neuromuscular degenerative changes that commonly present as central sleep apnea and poor ventilatory response to hyperapnea. Increased recognition of the genetics and clinical manifestations of mitochondrial diseases by sleep researchers and clinicians is important in the evaluation and treatment of all patients with sleep disturbances. Prospective population-based studies are required to determine the true prevalence of mitochondrial energy failure in subjects with sleep disorders, and conversely, of individuals with primary mitochondrial diseases and sleep pathology.

Dichloroacetate (DCA) is an inhibitor of pyruvate dehydrogenase kinase and an investigational drug used to treat mitochondrial disorders, pulmonary arterial hypertension, and cancer. The enzyme that metabolizes it, glutathione transferase Z1-1 (GSTZ1-1), is irreversibly inactivated upon metabolism of DCA. This leads to decreased clearance of subsequent doses and a buildup of the toxic physiological substrates of GSTZ1-1, maleylacetone and maleylacetoacetate, possibly causing the observed side effect of peripheral neuropathy upon chronic administration. We have observed that the extent of this inactivation is dependent on chloride ion concentration, with high concentrations providing a protective effect. Here, we present data measuring intracellular [Cl-] in human tissues and the effects that it has on the mechanism of inactivation, adduct formation. These results have important implications in the use of DCA, including predicting patient response, and may provide information to advance the use of DCA in p...

A novel index of insulin sensitivity, the quick insulin sensitivity check index, termed QUICKI (1/[log (insulin) + log (glucose)]), was recently developed. We examined whether QUICKI accurately reflects changes in insulin sensitivity after exercise training, a perturbation known to improve insulin sensitivity. Sedentary, nondiabetic adults underwent a frequently sampled iv glucose tolerance test before and after 6 months of training. Insulin sensitivity was estimated from the glucose tolerance test using Bergman's minimal model (insulin sensitivity-minimal model), and QUICKI was calculated from basal insulin and glucose. Exercise increased (P = 0.003) insulin sensitivity-minimal model but did not change (P = 0.12) QUICKI. Before and after training, the rank-correlation between QUICKI and insulin sensitivity-minimal model was significant (r = 0.79, P = 0.0005; r = 0.56, P = 0.03, respectively). However, the rank-correlation between fasting insulin alone with insulin sensitivity-minimal model was as good (before training r = -0.77, P = 0.0009; after training r = -0.55, P = 0.03) as that between QUICKI and insulin sensitivity-minimal model. Fasting glucose was not related to insulin sensitivity-minimal model at either time. When difference scores (i.e. after pretraining values) were examined, neither QUICKI nor fasting insulin correlated with insulin sensitivity-minimal model (QUICKI vs. insulin sensitivity-minimal model r = 0.24, P = 0.39; fasting insulin vs. insulin sensitivity-minimal model r = -0.40, P = 0.14). We conclude that fasting insulin is equivalent to fasting insulin plus glucose (i.e. QUICKI) at estimating basal insulin sensitivity in nondiabetic adults. However, QUICKI does not accurately reflect exercise-induced changes in insulin sensitivity within individual subjects.

We present the first population pharmacokinetic analysis of quinine in patients with Plasmodium falciparum malaria. Ghanaian children ( n = 120; aged 12 months to 10 years) with severe malaria received an intramuscular loading dose of quinine dihydrochloride (20 mg/kg of body weight). A two-compartment model with first-order absorption and elimination gave post hoc estimates for pharmacokinetic parameters that were consistent with those derived from non-population pharmacokinetic studies (clearance [CL] = 0.05 liter/h/kg of body weight; volume of distribution in the central compartment [ V 1 ] = 0.65 liter/kg; volume of distribution at steady state = 1.41 liter/kg; half-life at β phase = 19.9 h). There were no covariates (including age, gender, acidemia, anemia, coma, parasitemia, or anticonvulsant use) that explained interpatient variability in weight-normalized CL and V 1 . Intramuscular quinine was associated with minor, local toxicity in some patients (13 of 108; 12%), and 11 patients (10%) experienced one or more episodes of postadmission hypoglycemia. A loading dose of intramuscular quinine results in predictable population pharmacokinetic profiles in children with severe malaria and may be preferred to the intravenous route of administration in some circumstances.

Recent studies show alarming decreases in the proportions of physicians applying for federal resources and of graduating medical students who declare strong interest in pursuing careers as physician—scientists. To expose medical students in their formative years to hypothesis-driven experimental investigations in a clinical setting, the first-year curriculum at the University of Florida has involved students as both investigators and study subjects in patient-oriented research conducted in the General Clinical Research Center (GCRC). Each year a hypothesis-driven experiment is conceived by first-year medical students in the university's MD-PhD program. Later in the year, the protocol is implemented in the GCRC by the entire freshman class, whose members serve as volunteer study subjects or as investigators. The experimental data are analyzed by the MD-PhD students, who report their findings at national biomedical research meetings and submit a manuscript on their project to a peer-reviewed journal. The authors describe students' research projects over the first six years of this GCRC-based program. They also describe the responses of former students to a questionnaire about their perceptions of the value of the research program. Most respondents considered the GCRC research exercise to have been useful and relevant to their overall education, and many more declared a current interest in pursuing research careers compared with the number who had declared such interest as freshmen. The authors conclude that early integration of hands-on, patient-oriented research into the medical school curriculum is a positive educational experience for students, and may contribute to their ultimate pursuit of academic research careers.

Children with severe malaria often present with lactic acidosis and hypoglycemia. Although both complications independently predict mortality, mechanisms underlying their development are poorly understood. To study these metabolic derangements we sequentially allocated 21 children with falciparum malaria and capillary lactate concentrations of 5 mmol/L or more to receive either quinine or artesunate as antimalarial therapy, and dichloroacetate or saline placebo for lactic acidosis. We then administered a primed infusion (90 min) of l-[3-13C1]sodium lactate and d-[6,6-D2]glucose to determine the kinetics of these substrates. The mean (sd) glucose disposal rate in all patients was 56 (16) μmol/kg·min, and the geometric mean (range) lactate disposal rate was 100 (66–177)μ mol/kg·min. Glucose and lactate disposal rates were positively correlated (r = 0.62; P = 0.005). Artesunate was associated with faster parasite clearance, lower insulin/glucose ratios, and higher glucose disposal rates than quinine. Lactate...

We determined the independent effects of hypoxia, glucose deprivation and ischemia (hypoxia plus glucose deprivation) on steady-state levels of mRNA coding for specific nuclear and mitochondrially encoded enzymes of oxidative metabolism in cultured rat neurons and glia. Neither hypoxia nor low glucose alone changed steady-state message levels for any transcript. However, ischemia induced a biphasic effect on mitochondrially encoded transcripts for cytochrome oxidase subunit two (CO2) and the subunits 8 and 6 of ATPase (A 8/6), initially decreasing and then increasing mRNA levels to or above the levels recorded prior to ischemia. In contrast, three nuclear encoded transcripts for mitochondrial proteins were decreased by ischemia. These data demonstrate a lack of coordination between the expression of nuclear and mitochondrial genes in the initial response to ischemia and suggest that a selective, primary reaction to brain cell insults exists within the mitochondrion.

In 27 of 28 children with congenital lactic acidosis, cerebrospinal fluid lactate was higher than venous blood lactate. The mean +/- SEM difference between these variables was 2.4 +/- 0.3 mmol/L (P =.0001). Girls or patients with pyruvate dehydrogenase deficiency had higher cerebrospinal fluid lactate concentrations than boys or patients with respiratory chain defects or mitochondrial DNA mutations.

The effect on apolipoprotein B kinetics of a diet enriched in either fish oil or safflower oil was investigated in five hypertriglyceridemic (HTG), non-insulin-dependent diabetic subjects. The fish oil diet decreased plasma triglycerides and VLDL-apoB but increased LDL-apoB and LDL-cholesterol. Total plasma apoB concentration did not change, nor did the increased VLDL-apoB secretion present in these HTG subjects, which, accompanied by impaired lipolysis, accounted for their elevated VLDL. The fish oil-induced fall in VLDL resulted from a decrease in secretion without a change in residence time. The IDL fraction, which also contained small VLDL, was the primary site for the secretion of apoB particles in the HTG subjects. On the fish oil diet there was a further, compensatory increase in the secretion of these lipoproteins such that the transport of apoB in IDL remained the same, as did its mass. In the HTG subjects the major portion of IDL lipoproteins was catabolized, with LDL-apoB production comprising the lesser quantity. On the fish oil diet, a shift in the channeling of the lipoprotein output from IDL resulted in a decrease in the catabolic pathway and an increase in conversion to LDL. As the residence time of LDL did not change, this increased input gave rise to the larger mass of LDL-apoB seen in these hypertriglyceridemic subjects when receiving a fish oil diet.—Fisher, W. R., L. A. Zech, and P. W. Stacpoole. Apolipoprotein B metabolism in hypertriglyceridemic diabetic patients administered either a fish oil- or vegetable oil-enriched diet.

(1998). Pharmacokinetics, Metabolism, and Toxicology of Dichloroacetate. Drug Metabolism Reviews: Vol. 30, No. 3, pp. 499-539.

The term congenital lactic acidosis (CLA) refers to a group of inborn errors of mitochondrial metabolism variably characterised by progressive neuromuscular deterioration and accumulation of lactate and hydrogen ions in blood, urine and/or cerebrospinal fluid, frequently resulting in early death.1-4 The incidence and prevalence of CLA are unknown, although it has been estimated that there are approximately 250 new cases recognised in the US per year (personal communication). Thus, with an estimated annual mortality attrition rate of 20%, at least 1000 cases exist in the general US population. Recent diagnostic advances have allowed the biochemical or molecular identification of specific enzyme defects in the majority of infants and children with CLA. Most identifiable cases involve inherited or spontaneous mutations in the pyruvate dehydrogenase complex (PDC) or in one or more enzymes of the respiratory chain.2 4 A few cases have been reported that involve deficiencies in enzymes of the tricarboxylic acid cycle, such as fumarase, or of gluconeogenesis, such as pyruvate carboxylase (PC) or phosphoenolpyruvate carboxykinase (PEPCK). In a substantial number of patients, however, the specific biochemical defect fails to be determined by established techniques. Hyperlactataemia is the defining biochemical abnormality in children with CLA and, in the absence of hypoxia, should be considered a surrogate marker for underlying failure of mitochondrial energy metabolism.5 This concept is most readily appreciated by considering mitochondrial enzyme deficiencies. Figure 1 summarises the major steps of carbohydrate oxidation in mammalian cells. Note that the oxidative fate of pyruvate is to be irreversibly decarboxylated to acetyl CoA. This reaction is catalysed by PDC, a series of linked enzymes located in the inner mitochondrial membrane (fig 2). Under aerobic conditions, the activity of PDC determines the rate at which all cells oxidise glucose, pyruvate, and lactate. Figure 1 Pathways of pyruvate metabolism and oxidative phosphorylation. …

This article critically reviews the pharmacologic effects of the investigational drug dichloroacetate (DCA), which activates the mitochondrial pyruvate dehydrogenase enzyme complex in cardiac tissue and thus preferentially facilitates aerobic oxidation of carbohydrate over fatty acids. The pharmacologic effects of DCA are compared with other interventions, such as glucose plus insulin, inhibitors of long chain fatty acid oxidation and adenosine, that are also thought to exert their therapeutic effects by altering myocardial energy metabolism. Short-term clinical and laboratory experiments demonstrate that intravenous DCA rapidly stimulates pyruvate dehydrogenase enzyme complex activity and, therefore, aerobic glucose oxidation in myocardial cells. Typically these effects are associated with suppression of myocardial long chain fatty acid metabolism and increased left ventricular stroke work and cardiac output without changes in coronary blood flow or myocardial oxygen consumption. Although long-term studies are lacking, short-term parenteral administration of DCA appears to be safe and capable of significantly improving myocardial function in conditions of limited oxygen availability by increasing the efficient conversion of myocardial substrate fuels into energy.

The pharmacokinetic properties of the lactate-lowering drug dichloroacetate were investigated in 111 adult patients with lactic acidosis who were randomized to receive dichloroacetate as part of a placebo-controlled clinical trial. The clinical symptoms and etiology of lactic acidosis varied markedly among patients. Dichloroacetate, at a dose of 50 mg per kilogram of body weight, was administered in a 30-minute intravenous infusion into a peripheral vein. A second dose, identical to the first, was administered 2 hours after beginning the first infusion. Plasma levels of dichloroacetate were determined from blood samples collected periodically up to 288 hours after administration and the data were subjected to pharmacokinetic modeling. The pharmacokinetic properties of dichloroacetate in these acutely ill patients were complex and differed markedly from those in healthy volunteers, whose data fitted a one-compartment pharmacokinetic model. In contrast, the data from patients fitted one-, two-, or three-compartment pharmacokinetic models or even none of these, depending on the individual. Drug clearance in plasma tended to decrease as the number of compartments required to fit the data increased or as the number of drug treatments increased.

Background Recurrent malignant brain tumors (RMBTs) carry a poor prognosis. Dichloroacetate (DCA) activates mitochondrial oxidative metabolism and has shown activity against several human cancers. Design We conducted an open-label study of oral DCA in 15 adults with recurrent WHO grade III – IV gliomas or metastases from a primary cancer outside the central nervous system. The primary objective was detection of a dose limiting toxicity for RMBTs at 4 weeks of treatment, defined as any grade 4 or 5 toxicity, or grade 3 toxicity directly attributable to DCA, based on the National Cancer Institute’s Common Toxicity Criteria for Adverse Events, version 4.0. Secondary objectives involved safety, tolerability and hypothesis-generating data on disease status. Dosing was based on haplotype variation in glutathione transferase zeta 1/maleylacetoacetate isomerase (GSTZ1/MAAI), which participates in DCA and tyrosine catabolism. Results Eight patients completed at least 1 four week cycle. During this time, no dose-limiting toxicities occurred. No patient withdrew because of lack of tolerance to DCA, although 2 subjects experienced grade 0–1 distal parasthesias that led to elective withdrawal and/or dose-adjustment. All subjects completing at least 1 four week cycle remained clinically stable during this time and remained on DCA for an average of 75.5 days (range 26–312). Conclusions Chronic, oral DCA is feasible and well-tolerated in patients with recurrent malignant gliomas and other tumors metastatic to the brain using the dose range established for metabolic diseases. The importance of genetic-based dosing is confirmed and should be incorporated into future trials of chronic DCA administration.

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.

Lactic acidosis is a buildup of lactic acid in the blood and tissues, which can be due to several inborn errors of metabolism as well as nongenetic conditions. Deficiency of pyruvate dehydrogenase complex (PDHC) is the most common genetic disorder leading to lactic acidosis. Phosphorylation of specific serine residues of the E1 alpha subunit of PDHC by pyruvate dehydrogenase kinase (PDK) inactivates the enzyme, whereas dephosphorylation restores PDHC activity. We found that phenylbutyrate enhances PDHC enzymatic activity in vitro and in vivo by increasing the proportion of unphosphorylated enzyme through inhibition of PDK. Phenylbutyrate given to C57BL/6 wild-type mice results in a significant increase in PDHC enzyme activity and a reduction of phosphorylated E1 alpha in brain, muscle, and liver compared to saline-treated mice. By means of recombinant enzymes, we showed that phenylbutyrate prevents phosphorylation of E1 alpha through binding and inhibition of PDK, providing a molecular explanation for the effect of phenylbutyrate on PDHC activity. Phenylbutyrate increases PDHC activity in fibroblasts from PDHC-deficient patients harboring various molecular defects and corrects the morphological, locomotor, and biochemical abnormalities in the noa(m631) zebrafish model of PDHC deficiency. In mice, phenylbutyrate prevents systemic lactic acidosis induced by partial hepatectomy. Because phenylbutyrate is already approved for human use in other diseases, the findings of this study have the potential to be rapidly translated for treatment of patients with PDHC deficiency and other forms of primary and secondary lactic acidosis.

The kinetics of apolipoproteins A-I and A-II were examined in human subjects using leucine tracers administered intravenously. High density lipoproteins were separated and apoA-I and A-II were isolated. The specific activity or enrichment data for these apolipoprotein were analyzed by mathematical compartmental modeling. In 11 of 14 subjects studied with a bolus-injected [3H]leucine tracer, in 3 subjects studied similarly with [3H]leucine, and in one subject studied by primed dose, constant infusion of [3H]leucine, a rapidly turning-over apoA-I fraction was resolved. A similar component was observed in 7 of 10 studies of apoA-II. The apoA-I data were analyzed using a compartmental model (Zech, L.A. et al. 1983. J. Lipid Res. 24: 60-71) modified to incorporate plasma leucine as a precursor for apoprotein synthesis. The data permitted resolution of two apoA-I pools, one, C(2), turned-over with a residence time of less than 1 day, the other, C(1), a slowly turning-over pool, appeared in plasma after a delay of less than half a day. C(1) comprised the predominant mass of apoA-I and was also the primary determinant of the residence time of apoA-I. Although the mass of the fast pool, C(2), was considerably less than that of C(1), because of its rapid turnover, the quantities of apoA-I transported through this fast pathway were 2- to 4-fold greater. These kinetic studies indicate that apoA-I is secreted into both fast and slowly turning-over plasma pools. The latter is predominantly measured with radioiodinated apoA-I tracers. The data can be analyzed by postulating either separate input pathways to each of the pools or by assuming the fast pool is the precursor to the slow pool. Thus, apoA-I could be initially secreted as a family of particles that are rapidly cleared from plasma, and a portion of this apoprotein then reappears in a slowly turning-over pool that constitutes the major mass of apoA-I. The physiologic identity of these kinetically distinct apoA-I species is unknown; however, the fast pool of apoA-I demonstrated in these studies is strikingly similar to that seen in subjects with Tangier disease who lack the slow pool.

Dichloroacetate (DCA), a small molecule 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. While DCA's mechanism of action is well-studied, other factors that influence its success as a cancer treatment have not been thoroughly studied. Here we present data showing that expression of glutathione transferase zeta 1 (GSTZ1), the enzyme responsible for the metabolism of DCA, is often misregulated in cancer, resulting in expression in tissues where there generally is none and a lack of expression in sites that have a high level of basal expression. Expression of GSTZ1 in a tumor would be expected to reduce intratumoral DCA concentrations. As we see a disconnect between GSTZ1 mRNA and protein expression in these tumors, control of expression is likely through miRNA. We also report that chloride concentrations, which strongly impact the stability of GSTZ1 in the presence of DCA, may be abnormal in tumors, further influencing DCA metabolism, concentration, and treatment success. Both two- and three-dimensional cell culture of cells expressing or not expressing GSTZ1 provide a proof of principle that GSTZ1 expression confers a level of resistance to DCA. These results have important clinical implications as GSTZ1 expression and chloride concentration could be used as markers to predict outcomes of DCA anticancer therapy. Citation Format: Stephan C. Jahn, Mohamed H. Solaymen, Ryan J. Lorenzo, Taimour Langaee, Peter W. Stacpoole, Margaret O. James. Chemoresistance through the intratumoral metabolism of dichloroacetate. [abstract]. In: Proceedings of the AACR Special Conference: Metabolism and Cancer; Jun 7-10, 2015; Bellevue, WA. Philadelphia (PA): AACR; Mol Cancer Res 2016;14(1_Suppl):Abstract nr A62.

We report the design and implementation of the first phase 3 trial of CoenzymeQ₁₀ (CoQ₁₀) in children with genetic mitochondrial diseases. A novel, rigorous set of eligibility criteria was established. The trial, which remains open to recruitment, continues to address multiple challenges to the recruitment of patients, including widely condoned empiric use of CoQ₁₀ by individuals with proven or suspected mitochondrial disease and skepticism among professional and lay mitochondrial disease communities about participating in placebo-controlled trials. These attitudes represent significant barriers to the ethical and scientific evaluation--and ultimate approval--of nutritional and pharmacological therapies for patients with life-threatening inborn errors of energy metabolism.

Previous animal studies showed that severe vitamin B-6 deficiency altered fatty acid profiles of tissue lipids, often with an increase of linoleic acid and a decrease of arachidonic acid. However, little is known about the extent to which vitamin B-6 deficiency affects human fatty acid profiles. The aim of this study was to determine the effects of marginal vitamin B-6 deficiency on fatty acid profiles in plasma, erythrocytes, and peripheral blood mononuclear cells (PBMC) of healthy adults fed a 28-d, low-vitamin B-6 diet. Healthy participants (n = 23) received a 2-d, controlled, vitamin B-6–adequate diet followed by a 28-d, vitamin B-6–restricted diet to induce a marginal deficiency. Plasma HDL and LDL cholesterol concentrations, FFA concentrations, and erythrocyte and PBMC membrane fatty acid compositions did not significantly change from baseline after the 28-d restriction. Plasma total arachidonic acid, EPA, and DHA concentrations decreased from (mean ± SD) 548 ± 96 to 490 ± 94 μmol/L, 37 ± 13 to 32 ± 13 μmol/L, and 121 ± 28 to 109 ± 28 μmol/L [positive false discovery rate (pFDR) adjusted P < 0.05], respectively. The total (n-6):(n-3) PUFA ratio in plasma exhibited a minor increase from 15.4 ± 2.8 to 16.6 ± 3.1 (pFDR adjusted P < 0.05). These data indicate that short-term vitamin B-6 restriction decreases plasma (n-3) and (n-6) PUFA concentrations and tends to increase the plasma (n-6):(n-3) PUFA ratio. Such changes in blood lipids may be associated with the elevated risk of cardiovascular disease in vitamin B-6 insufficiency.

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 (