Pharmacokinetics and bioavailability of oral single-dose maleic acid in biofluids of Sprague-Dawley rats.

Maleic acid (MA) was purposefully adulterated in an array of starch-based foods in Taiwan, inciting a food safety incident. Due to limited data on the pharmacokinetics and bioavailability of ingested MA, we studied pharmacokinetic (PK) parameters in serum and urine of Sprague Dawley rats. Three groups of male and female rats were given three doses of MA by oral gavage; biofluid samples were collected accordingly. Data demonstrated that a non-compartment model best described MA's linear kinetic behavior upon ingestion. The mean residence life of maleic acid in serum was 17.58 h and 9.84 h for low-dosed male and female rats, whereas 8.24 h and 4.17 h for high-dosed male and female rats, respectively. Our results revealed oral bioavailability ranged from 30.8 to 41.0% for males and 32.2-39.1% for females. The data confirmed that ingested MA is absorbed and metabolized rapidly, along with low bioavailability. Future pathological studies may determine whether prolonged and low-level exposures of MA produce nephrotoxicity. These data provide additional contribution to current understanding of the kinetics of MA in a rat model and enable the development of a physiologically based model, which is essential to form the basis of evidenced-based food safety guidelines.

Determination of the maleic acid in rat urine and serum samples by isotope dilution-liquid chromatography-tandem mass spectrometry with on-line solid phase extraction.

A rapid and simple on-line solid-phase extraction coupled with isotope dilution-liquid chromatography-tandem mass spectrometry (SPE-ID-LC-MS/MS) method was developed to quantitate maleic acid in serum and urine of SpragueDawley (SD) rats. The aforementioned biological samples were spiked with (13)C2-maleic acid, vigorously vortexed, added with acetonitrile to precipitate proteins, and then injected into the on-line SPE-LC-MS/MS system for quantification. Upon validation, this method demonstrated excellent feasibility and sensitivity: calibration curves for maleic acid in serum and urine display excellent linearity with the coefficient of determination (R(2)) greater than 0.999; the limits of detection and quantitation (LOD and LOQ) for maleic acid were determined at 0.2 and 0.5µg L(-1), respectively. Additionally, intra-day accuracy for maleic acid in serum and urine samples ranged from 94.0% to 100.2% and 101.3% to 104.4%, respectively. Furthermore, inter-day accuracy ranged from 93.6% to 101.0% and from 102.3% to 111.4% in serum and urine samples, respectively. Intra-day precision %RSD of maleic acid in serum and urine samples was 13.8% or less, whereas the inter-day precision was 6.1% or less. The matrix effects were not found to be statistically significant (p=0.9145 and p=0.5378, correspondingly) based on the calculations of recovery functions. The collected serum and urine samples were analyzed using SPE-ID-LC-MS/MS. Our results reveal trace levels of maleic acid in the control rats, demonstrating that this method is capable of analyzing background levels of contaminants in biofluids with excellent sensitivity and specificity at part-per-billion levels concentrations in complex matrices.

Chloral hydrate, through biotransformation to dichloroacetate, inhibits maleylacetoacetate isomerase and tyrosine catabolism in humans.

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 naïve 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.

Haplotype variations in glutathione transferase zeta 1 influence the kinetics and dynamics of chronic dichloroacetate in children.

Dichloroacetate (DCA) is biotransformed by glutathione transferase zeta 1 (GSTZ1), a bifunctional enzyme that, as maleylacetoacetate isomerase (MAAI), catalyzes the penultimate step in tyrosine catabolism. DCA inhibits GSTZ1/MAAI, leading to delayed plasma drug clearance and to accumulation of potentially toxic tyrosine intermediates. Haplotype variability in GSTZ1 influences short-term DCA kinetics in healthy adults, but the impact of genotype in children treated chronically with DCA is unknown. Drug kinetics was studied in 17 children and adolescents with congenital mitochondrial diseases administered 1,2-(13) C-DCA. Plasma drug half-life and trough levels varied 3-6-fold, depending on GSTZ1/MAAI haplotype and correlated directly with urinary maleylacetone, a substrate for MAAI. However, chronic DCA exposure did not lead to progressive accumulation of plasma drug concentration; instead, kinetics parameters plateaued, consistent with the hypothesis that equipoise is established between the inhibitory effect of DCA on GSTZ1/MAAI and new enzyme synthesis. GSTZ1/MAAI haplotype variability affects DCA kinetics and biotransformation. However, these differences appear to be stable in most individuals and are not associated with DCA plasma accumulation or drug-associated toxicity in young children.

Phase 1 trial of dichloroacetate (DCA) in adults with recurrent malignant brain tumors.

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.

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.

Pharmacokinetic comparison of the maleate and free base formulations of LB80380, a novel nucleotide analog, in healthy male volunteers.

OBJECTIVE: LB80380, a dipivoxil ester prodrug of LB80331, is a novel antiviral agent for the treatment of chronic hepatitis B. The aim of this study was to compare the pharmacokinetic differences after single oral administrations of the free base and maleate formulations of LB80380 in healthy male subjects. METHODS: An open-label, single- dose, randomized-sequence, 2-treatment crossover study was conducted in 32 Korean male volunteers. Subjects received either a combination of 60 and 90 mg tablets of the free base LB80380 formulation or a 183 mg (150 mg as a free base) tablet of the maleate LB80380 formulation. Then, after a 14- day washout period, each subject received the other formulation. Plasma and urine concentrations of LB80331 and LB80317 (active metabolites of LB80380) were measured by validated liquid chromatographytandem mass spectrometry assays. A safety assessment, which included vital signs, adverse events, electrocardiograms and clinical laboratory tests, was performed for each subject. RESULTS: A total of 32 healthy subjects was enrolled, and 26 subjects completed the study. Single oral administrations of LB80380 maleate tablets did not result in clinically significant differences in the safety profile compared to the LB80380 free base tablets. The 90% confidence intervals (CIs) for the geometric mean ratios of Cmax and AUClast for LB80331 of the two treatments (maleate versus free base formulation) were 0.986 - 1.1240 and 0.9848 - 1.0533, respectively. The 90% CIs for the geometric mean ratios of Cmax and AUClast for LB80317 were 0.8379 - 0.9696 and 0.7224 - 0.9196. CONCLUSIONS: In healthy male subjects, the 183 mg LB80380 maleate tablet was pharmacokinetically equivalent to the 60 and 90 mg LB80380 free base tablets.

Human polymorphisms in the glutathione transferase zeta 1/maleylacetoacetate isomerase gene influence the toxicokinetics of dichloroacetate.

Dichloroacetate (DCA), a chemical relevant to environmental science and allopathic medicine, is dehalogenated by the bifunctional enzyme glutathione transferase zeta (GSTz1)/maleylacetoacetate isomerase (MAAI), the penultimate enzyme in the phenylalanine/tyrosine catabolic pathway. The authors postulated that polymorphisms in GSTz1/MAAI modify the toxicokinetics of DCA. GSTz1/MAAI haplotype significantly affected the kinetics and biotransformation of 1,2-¹³C-DCA when it was administered at either environmentally (µg/kg/d) or clinically (mg/kg/d) relevant doses. GSTz1/MAAI haplotype also influenced the urinary accumulation of potentially toxic tyrosine metabolites. Atomic modeling revealed that GSTz1/MAAI variants associated with the slowest rates of DCA metabolism induced structural changes in the enzyme homodimer, predicting protein instability or abnormal protein-protein interactions. Knowledge of the GSTz1/MAAI haplotype can be used prospectively to identify individuals at potential risk of DCA's adverse side effects from environmental or clinical exposure or who may exhibit aberrant amino acid metabolism in response to dietary protein.

Clinical characteristics and gene mutation analysis of methylmalonic aciduria.

Methylmalonic aciduria (MMA) is a common inherited autosomal recessive disorder resulting from defects in the enzyme methylmalonyl CoA mutase (MCM, mut complementation group) or in the synthesis of the MCM cofactor adenosylcobalamin (cbl complementation groups). The defects in the mut complementation group accounts for the largest number of patients with isolated MMA. At least 200 mutations in the MUT gene on chromosome 6p12 have been identified in MMA patients until now. This study aimed to investigate the clinical characteristics of MMA and genomic variations in the MUT gene of Chinese patients. Genomic DNA was extracted from 18 patients who were diagnosed as having isolated MMA by gas chromatography/mass spectrometry (GC-MS), and from some of their parents as well. Amplification and direct sequencing of the MUT coding regions (exon 2-13) and their adjacent intronic consensus splice sites were performed in order to identify the disease causing mutations. In this group, six novel mutations in the MUT gene, c.424A>G (p.T142A), c.786T>G (p.S262R), c.808G>C (p.G270R), c.1323_1324insA, c.1445-1G>A and c.1676+77A>C were identified. p.T142A and p.G270R were respectively detected at a heterozygous level in one patient. Two previously reported mutations, c.682C>T (p.R228X) and c.323G>A (p.R108H) were also found in this study. In addition, six previously described single nucleotide polymorphism (SNP), c.636A>G (p.K212K), c.1495G>A (p.A499T), c.1595A>G (p.H532R), c.1992G>A (p.A664A), c.2011G>A (p.V671I) and c.1677-53A>G were identified. In this study, we updated the spectrum of MUT mutations and identified the main MMA-causing mutations in Chinese MMA patients.

Urinary methylmalonic acid in patients with acute myocardial infarction.

OBJECTIVE: To investigate urinary methylmalonic acid (uMMA) levels and their relationship with markers of myocyte necrosis and inflammation in patients with acute myocardial infarction (AMI). SUBJECTS AND METHODS: The study participants consisted of 80 consecutive patients with AMI and 72 age- and sex-matched consecutive controls. Of the patients, 38 had ST segment elevation myocardial infarction (STEMI) and 42 had non-ST segment elevation. All patients with STEMI underwent fibrinolytic therapy. Routine laboratory tests included troponin-I, creatinine phosphokinase MB (CK-MB), high-sensitivity C-reactive protein (hs-CRP), vitamin B(12), folate, homocysteine and methylmalonic acid analyses. uMMA measurements were made by a spectrophotometric method. RESULTS: uMMA levels were significantly higher in patients with AMI than in controls (10.1 vs. 5.2 mmol/mol creatinine, p < 0.001) and higher in patients with anterior MI compared to those with non-anterior MI (18.9 vs. 8.7 mmol/mol creatinine, p < 0.001). In addition, uMMA levels were significantly higher in patients without successful reperfusion compared to those with successful reperfusion. In patients with STEMI, a strong positive association was found between urinary MMA and plasma hs-CRP levels (r = 0.81, p < 0.001), symptom duration (r = 0.91, p < 0.001) and wall motion score (r = 0.60, p = 0.006). More importantly, a strong positive association was observed between uMMA and the size of myocardial infarction in patients without successful reperfusion (for CK-MB r = 0.81, p = 0.013; for wall motion score r = 0.82, p = 0.012). CONCLUSION: uMMA levels were elevated in patients with AMI and, as such, may be a candidate biochemical indicator of larger infarct size and enhanced inflammation in patients with AMI.

A GC-MS/MS method for the quantitative analysis of low levels of the tyrosine metabolites maleylacetone, succinylacetone, and the tyrosine metabolism inhibitor dichloroacetate in biological fluids and tissues.

We developed a sensitive method to quantitate the tyrosine metabolites maleylacetone (MA) and succinylacetone (SA) and the tyrosine metabolism inhibitor dichloroacetate (DCA) in biological specimens. Accumulation of these metabolites may be responsible for the toxicity observed when exposed to DCA. Detection limits of previous methods are 200 ng/mL (1.2 pmol/microL) (MA) and 2.6 microg/mL (16.5 pmol/microL) (SA) but the metabolites are likely present in lower levels in biological specimens. To increase sensitivity, analytes were extracted from liver, urine, plasma and cultured nerve cells before and after dosing with DCA, derivatized to their pentafluorobenzyl esters, and analyzed via GC-MS/MS.

Inhibition and recovery of rat hepatic glutathione S-transferase zeta and alteration of tyrosine metabolism following dichloroacetate exposure and withdrawal.

Dichloroacetate (DCA) is an investigational drug for certain metabolic disorders, a by-product of water chlorination and a metabolite of certain industrial solvents and drugs. DCA is biotransformed to glyoxylate by glutathione S-transferase zeta (GSTz1-1), which is identical to maleylacetoacetate isomerase, an enzyme of tyrosine catabolism. Clinically relevant doses of DCA (mg/kg/day) decrease the activity and expression of GSTz1-1, which alters tyrosine metabolism and may cause hepatic and neurological toxicity. The effect of environmental DCA doses (microg/kg/day) on tyrosine metabolism and GSTz1-1 is unknown, as is the time course of recovery from perturbation following subchronic DCA administration. Male Sprague-Dawley rats (200 g) were exposed to 0 microg, 2.5 microg, 250 microg, or 50 mg DCA/kg/day in drinking water for up to 12 weeks. Recovery was followed after the 8-week exposure. GSTz specific activity and protein expression (Western immunoblotting) were decreased in a dose-dependent manner by 12 weeks of exposure. Enzyme activity and expression decreased 95% after a 1-week administration of high-dose DCA. Eight weeks after cessation of high-dose DCA, GSTz activity had returned to control levels. At the 2.5 or 250 microg/kg/day doses, enzyme activity also decreased after 8 weeks' exposure and returned to control levels 1 week after DCA was withdrawn. Urinary excretion of the tyrosine catabolite maleylacetone increased from undetectable amounts in control rats to 60 to 75 microg/kg/24 h in animals exposed to 50 mg/kg/day DCA. The liver/body weight ratio increased in the high-dose group after 8 weeks of DCA. These studies demonstrate that short-term administration of DCA inhibits rat liver GSTz across the wide concentration range to which humans are exposed.

Perturbation of maleylacetoacetic acid metabolism in rats with dichloroacetic Acid-induced glutathione transferase zeta deficiency.

Glutathione transferase zeta (GSTZ1-1) catalyzes the isomerization of maleylacetoacetate (MAA) to fumarylacetoacetate, the penultimate step in the tyrosine degradation pathway. GSTZ1-1 is inactivated by dichloroacetic acid (DCA), which is used for the clinical management of congenital lactic acidosis and is a drinking-water contaminant. Metabolic changes associated with chemically induced GSTZ1-1 deficiency are poorly understood. The objective of this study was to investigate the biochemical and toxicological effects of giving 0.3-1.2 mmol DCA/kg/day for 5 days on MAA-metabolism in male Fischer rats. Urine from DCA-treated rats inhibited delta-aminolevulinic acid dehydratase (delta-ALAD) activity, which is used for the diagnosis of hereditary tyrosinemia type I. Mass spectrometric analyses of urine from rats given DCA demonstrated elevated excretion of MAA and its decarboxylation product, maleylacetone (MA); succinylacetone (SA), the reduced analogue of MA, was not detected. DCA-induced changes in MA excretion were dose-dependent and were significantly elevated after day 2 of treatment. MA excretion was reversible after discontinuation of DCA treatment and was enhanced 10-fold by the coadministration of homogentisic acid (HGA). MA was cytotoxic to hepatocytes in vitro (EC50 ~ 350 microM) but morphological changes were not observed in liver, kidney, and brain of rats given both DCA and HGA. These data indicate that DCA-induced inactivation of GSTZ1-1 leads to formation of an MAA-derived intermediate, MA, that may be a mediator and biomarker for DCA-associated toxicities.

In vivo percutaneous absorption of fragrance ingredients in rhesus monkeys and humans.

The percutaneous absorption of the fragrance diethyl maleate was measured in vivo in human and monkey studies. With the application sites occluded, 54% of the applied dose of the volatile fragrance penetrated human skin in 24 hr compared with 69% absorption in the monkey skin. It was concluded that the monkey is a good model for human skin with regard to the penetration of this fragrance material since no significant difference in the absorption of diethyl maleate was observed. The percutaneous absorption of the fragrances benzyl acetate and five other benzyl derivatives (benzyl alcohol, benzyl benzoate, benzamide, benzoin and benzophenone) was determined in vivo in monkeys. Absorption through occluded skin was high for all compounds (approximately 70% of the applied dose in 24 hr) and no significant differences between the values for the different compounds were observed. No correlations were seen between skin penetration of these compounds and their octanol-water partition coefficients. Under unoccluded conditions skin penetration of the fragrances was reduced and there was great variability between compounds, presumably because of variations in the rates of evaporation from the site of application. The data suggest that humans may have significant systemic exposure to these fragrance materials.

Methylsuccinate and mesaconate in urine of patients treated with sodium benzoate.

Two unusual peaks were found on gas chromatography of urine from four hyperammonemic patients treated with sodium benzoate. These peaks were identified by gas chromatography/mass spectrometric analyses as methylsuccinate and mesaconate. Of the two unusual substances, methylsuccinate was found to be a contaminant of sodium benzoate administered for the treatment of hyperammonemia. However, mesaconate was not a contaminant of sodium benzoate, though it could be detected in all urine samples from hyperammonemic patients treated with sodium benzoate. Mesaconate can be produced from methylsuccinate in vivo. Considering that mesaconate is an inhibitor of fumarase, the toxic effects of sodium benzoate may be attributable to the mesaconate. It is recommended that methylsuccinate-free sodium benzoate should be used for the treatment of hyperammonemia.

Propionyl-CoA carboxylase deficiency: case report, effect of low-protein diet and identification of 3-oxo-2-methylvaleric acid 3-hydroxy-2-methylvaleric acid, and maleic acid in urine.

Vomiting, lethargy and metabolic acidosis were the main initial symptoms of metabolic disease in a 1 month old girl. Her older sister had died from a similar disease, considered to be Reye's syndrome, at an age of 15 months. The urine of the present case contained 2-methylcitric acid, 3-hydroxypropionic acid, N-propionylglycine, 2-hydroxy-3-methylbutyric acid, N-tiglylglycine, 3-hydroxyvaleric acid and glutaric acid. These metabolites are all known to be associated with propionyl-CoA accumulation. Free propionic acid was not detected in the urine. In addition, the urine contained 3-oxo-2-methylvaleric acid and 3-hydroxy-2-methylvaleric acid, probably formed by condensation of two molecules of propionyl-CoA. The identity of these metabolites was confirmed by synthesis. An elevated urinary concentration of maleic acid and fumaric acid was another constant abnormality. The activity of propionyl-CoA carboxylase in leucocytes was about 20% of the normal activity. The girl was teated with a low-protein diet since the diagnosis was made at an age of 1 month, and her psychomotor development was satisfactory at an age of 2 1/2 years. She had a few episodes of acidosis during infections.

New metabolites in isovaleric acidemia.

Two metabolites, 4-hydroxyisovaleric acid and mesaconic acid, have been identified and quantified in the urine of a patient with isovaleric acidemia. These compounds do not appear to have been reported previously as being components of human metabolism. In addition, large quantities of 3-methylbutyrolactone, the lactone of 4-hydroxyisovaleric acid, were observed in the volatile profile obtained by headspace chromatography. The demonstration of 4-hydroxyisovaleric acid supports the contention that urinary methylsuccinic acid seen in patients with isovaleric acidemia has arisen by omega-oxidation of isovaleric acid. The identification of mesaconic acid may indicate that the methylsuccinic acid formed in these patients is subject to further metabolism.

Organic acids in urine from preterm infants.

The organic acid excretion was studied in urine samples from 26 preterm infants on the 1st and 5th days of life and the results compared to those obtained in 5 samples from full-term neonates. Gas-chromatography-mass spectrometry with a computer system was the method used in this work. The acids tabulated were those more closely related to lactic acidosis and the Krebs cycle. Great variations were found in the excretion of these acids in preterm infants in contrast to the very homogeneous pattern obtained in full-term neonates.