Identification and characterization of N-acetylcysteine conjugates of valproic acid in humans and animals.

Reactive and hepatotoxic metabolites formed from the biotransformation of valproic acid (VPA) are normally detoxified by conjugating with GSH and followed by mercapturic acid metabolism to produce their respective N-acetylcysteine (NAC) conjugates. Hence, the levels of NAC conjugates of VPA in human urine are an indirect measure of exposure of the liver toward reactive metabolites of the anticonvulsant drug. We report here the synthesis, identification, and characterization of a second NAC conjugate of (E)-2-propyl-2, 4-pentadienoic acid in the urine samples (n = 39) of humans on VPA therapy, namely, (E)-5-(N-acetylcystein-S-yl)-2-ene VPA by gas chromatography/mass spectrometry and liquid chromatography with tandem mass spectrometry. In this study, we were able to separate the diastereomers of (E)-5-(N-acetylcystein-S-yl)-3-ene VPA by HPLC. The NAC conjugate of 4,5-epoxy VPA, namely, 5-NAC-4-OH-VPA gamma-lactone, previously identified in rats treated with 2-propyl-4-pentenoic acid (4-ene VPA), was not detected in any of the human urine samples studied. This suggests that in humans, the P-450 metabolism of 4-ene VPA to the reactive epoxide is not a significant pathway. The excretion of the NAC conjugate of (E)-2, 4-diene VPA glucuronide in the urine of seven patients on VPA was also examined and was not detected. The limit of detection of 5-NAC-3-keto VPA and its decarboxylated product, 1-NAC-3-heptanone, was estimated at 25 ng (signal to noise ratio > 3). Neither 5-NAC-3-keto VPA nor 1-NAC-3-heptanone was detected in the urine of patients on VPA therapy or 4-ene VPA-treated guinea pigs, but 1-NAC-3-heptanone was detected in the urine of 4-ene VPA-treated rats.     

The reaction of (E)-2,4-pentadienoic acid with aqueous bromine re-evaluation of the product

The reaction of (E)-2,4-pentadienoic acid with aqueous bromine was reinvestigated to affirm the formation of (E)-5-bromo-4-hydroxy-2-pentenoic acid, whose structure was confirmed by the spectroscopic methods as well as the chemical modification.     

Metabolic fate of valproic acid in the rhesus monkey. Formation of a toxic metabolite, 2-n-propyl-4-pentenoic acid

The metabolic fate of an iv bolus dose (13.5 mg kg-1) of valproic acid (VPA) was studied in adult male rhesus monkeys. Renal excretion proved to be the major route of elimination of the drug and a total of 17 metabolites, accounting collectively for some 82% of the administered dose, were identified in urine by GC-MS techniques. Many of these metabolites were present largely in the form of glucuronide conjugates, as was VPA itself. The principal pathways of VPA biotransformation were, in order of decreasing quantitative importance, ester glucuronide formation, omega-oxidation, beta-oxidation and (omega-1)-hydroxylation. In addition, three mono-unsaturated metabolites, identified as (E)-delta 2-, (E)-delta 3-, and delta 4-VPA, were detected in both plasma and urine. Quantitative analysis of these unsaturated VPA metabolites indicated that the delta 4 olefin, which is known to be a potent hepatotoxic agent, was the predominant isomer of the group.     

Biotransformation and pharmacokinetics in the rhesus monkey of 2-n-propyl-4-pentenoic acid, a toxic metabolite of valproic acid

2-n-Propyl-4-pentenoic acid (delta 4-VPA), a hepatotoxic metabolite of valproic acid (VPA), was administered by iv bolus injection (14 mg kg-1) to two adult male rhesus monkeys. The plasma concentration vs. time curve for delta 4-VPA in these animals was biexponential and the effective half-life values were 0.53 and 0.67 hr. The pharmacokinetic profile of delta 4-VPA was similar to that of VPA in the monkey, although the unbound fraction of delta 4-VPA in plasma was approximately 2.5-fold greater than the value for the parent drug. The major route of elimination of delta 4-VPA was excretion into urine, and studies with a group of eight animals indicated that delta 4-VPA undergoes extensive biotransformation in this species. A total of 20 metabolites was detected in urine by GC-MS techniques, and 19 of these were identified positively by comparison of their gas-liquid chromatographic and mass spectrometric properties with those of the authentic compounds prepared by synthesis. Many of these metabolites were present largely in the form of glucuronide conjugates, as was delta 4-VPA itself. The major pathways of metabolism of delta 4-VPA were found to be ester glucuronide formation and beta-oxidation, whereas omega- and (omega-1)-oxidation processes were of minor quantitative importance. Excretion of unchanged drug and its metabolites into urine over 24 hr accounted collectively for some 59% of the administered dose, a figure which was appreciably less than the corresponding recovery of metabolites of VPA in the same monkeys. The possibility is raised that beta-oxidation of delta 4-VPA leads to the generation of a chemically reactive intermediate(s) which alkylate(s) cellular macromolecules and thereby forms tissue-bound residues. The significance of such a phenomenon is discussed in relation to the etiology of VPA-induced liver injury.     

Formation, characterization, and toxicity of the glutathione and cysteine conjugates of toxic heptapeptide microcystins.

Microcystins LR, YR, and RR, cyclic heptapeptide hepatotoxins produced by cyanobacteria, were synthetically converted into glutathione (GSH) and cysteine (Cys) conjugates. Fast atom bombardment mass spectra showed [M + H]+ ions corresponding to GSH and Cys conjugates of microcystins LR, YR, and RR for the obtained compounds. 1H NMR spectral analyses revealed that two singlet signals of olefinic protons of N-methyldehydroalanine (Mdha) in microcystins disappeared in the conjugates, confirming that thiols of GSH and Cys added nucleophilically to the alpha, beta-unsaturated carbonyl of the Mdha moiety. On examination of the 50% lethal dose (LD50) with intravenous injection using mice, both GSH and Cys conjugates showed reduction in toxicity compared with microcystins, but their toxicity still remained. Microcystin LR and its GSH conjugate were separated and identified in a standard mixture by using a frit-fast atom bombardment liquid chromatography/mass spectrometry (Frit-FAB LC/MS) method. Obtained conjugates in the present study would be important compounds as the standard samples for study of metabolism of microcystins, and the Frit-FAB LC/MS method would be applicable to mass spectrometric identification of metabolites of microcystins.     

Amino acid conjugates: metabolites of 2-propylpentanoic acid (valproic acid) in epileptic patients.

In this study, spectroscopic and chromatographic evidence is presented for the identification and characterization of the metabolites, valproyl glutamate (2-propylpentanoyl glutamate, VPA-GLU) and valproyl glutamine (2-propylpentanoyl glutamine, VPA-GLN) in the urine, serum, and cerebrospinal fluid (CSF) of patients on valproic acid (VPA) therapy. Moreover, the identification of valproyl glycine (2-propylpentanoyl glycine, VPA-GLY) in the serum and urine of patients on VPA, albeit in trace concentrations, is also reported here. The three amino acid conjugates excreted in urine accounted for about 1% of the VPA dose in four patients who were on VPA therapy chronically and had reached steady state. VPA-GLU was quantitatively the most prominent metabolite (0.66-13.1 microg/mg creatinine) compared with VPA-GLN (0.78-9.93 microg/mg creatinine) and VPA-GLY (trace-1.0 microg/mg creatinine) in overnight urine samples of all patients studied (n = 29). The relatively low serum concentrations of the three amino acid conjugates of VPA in six patients suggest that the metabolites are readily excreted once formed. In contrast, whereas VPA GLY was absent in the CSF of one patient on VPA, the concentrations of VPA-GLU and VPA-GLN in this CSF sample were 9 and 5 times, respectively, their corresponding serum concentrations.     

液相色谱-质谱联用法测定癫痫患者血浆中丙戊酸及其代谢产物2-丙基-4-戊烯酸浓度

目的建立液相色谱-质谱联用法同时测定丙戊酸(VPA)及其毒性代谢产物2-丙基-4-戊烯酸(4-ene-VPA)浓度。方法人血浆样品用乙腈直接沉淀蛋白后,色谱柱:Agilent Eclipse Plus-C₁₈(2. 1 mm×10. 0 mm,3. 5μm),流动相:乙腈-10 mmol·L^-1醋酸铵水溶液(均含0. 1%甲酸),梯度洗脱,用电喷雾离子化源,负离子方式,扫描方式为多反应监测(MRM),用于监测的离子反应分别为m/z 143. 2→143. 2(丙戊酸)、m/z 140. 8→140. 8(2-丙基-4-戊烯酸)和m/z 373. 0→329. 1(内标银杏酸(C17:1))。考察该方法的专属性、标准曲线与定量下限、精密度与回收率、基质效应和稳定性。结果 VPA和4-eneVPA分别在0. 50～200. 00μg·m L^-1和0. 05～12. 50μg·m L^-1内线性关系良好(r分别为0. 998 4和0. 997 5),定量下限分别为0. 50和0. 05μg·m L^-1批内、批间RSD均小于8. 59%,提取回收率分别为76. 45%～86. 78%和78. 44%～83. 46%,内标归一化基质效应因子分别为93. 67%～97. 82%和92. 04%～104. 35%。结论本方法简洁、灵敏、专属性好,适用于人血浆中同时测定VPA和4-eneVPA浓度,可用于丙戊酸钠的血药浓度监测。     

Determination of urinary 2-mercaptobenzothiazole (2-MBT), the main metabolite of 2-(thiocyanomethylthio)benzothiazole (TCMTB) in humans and rats

 A method for biological monitoring of urinary 2-(thiocyanomethylthio)benzothiazole (TCMTB), a wood preservative and an industrial chemical, was developed. Three different doses of TCMTB in olive oil were given to male rats by gavage for 3 weeks. Urine was collected daily and the metabolites were analysed as thioethers by derivatization with pentafluorobenzylbromide by gas chromatography-mass spectrometry. The parent chemical was not detected in urine samples, but two metabolites of TCMTB were identified. 2-Mercaptobenzothiazole (2-MBT) was the main metabolite, and its excretion varied according to the dose. The second metabolite was 2-(mercaptomethylthio)benzothiazole. The amount of 2-MBT excreted in rat urine was 66±12% (SD), 51±20% and 44±9% for TCMTB doses of 15, 75 and 150 mg/kg, respectively. Two doses, 75 and 150 mg/kg, caused diuresis in rats during the 1 week of dosing. During the 3-week TCMTB treatment, rat liver microsomal CYP enzyme profile was not significantly changed. Urine samples of sawmill workers exposed to TCMTB were collected after their work shifts for exposure assessment. TCM-TB could not be detected in the urine samples of exposed workers. Most concentrations of 2-MBT were below the limit of the detection, 0.12 μmol/l, the concentrations were 0.12–0.15 μmol/l only in few cases. The determination of 2-MBT in urine, when a sample is taken immediately after a work shift, is a suitable indicator of workers’ exposure to TCMTB. Received: 15 September 1995/Accepted: 30 January 1996     

Identification of a rare sulfonic acid metabolite of andrographolide in rats.

Andrographolide is widely used in clinic as an anti-inflammatory and antibiotic drug. In this paper, the metabolites of andrographolide in rats after single oral doses of 120 mg/kg were investigated. The structures of the metabolites were elucidated by high-resolution mass spectra, NMR spectroscopy including 1H NMR, 13C NMR, and two-dimensional NMR, through comparison to a synthetic standard. The main metabolite of andrographolide in rats was 14-deoxy-12(R)-sulfo andrographolide. In the proposed mechanism, the beta-carbon of alpha, beta-unsaturated carbonyl was attacked by sulfonic acid, to form the sulfonate compound. This was a rare metabolic reaction. It may be the main metabolic pathway of andrographolide in rats. The polarity of the sulfonate metabolite increased greatly and could be easily eliminated from body.     

Valproic acid I: time course of lipid peroxidation biomarkers, liver toxicity, and valproic acid metabolite levels in rats.

A single dose of valproic acid (VPA), which is a widely used antiepileptic drug, is associated with oxidative stress in rats, as recently demonstrated by elevated levels of 15-F(2t)-isoprostane (15-F(2t)-IsoP). To determine whether there was a temporal relationship between VPA-associated oxidative stress and hepatotoxicity, adult male Sprague-Dawley rats were treated ip with VPA (500 mg/kg) or 0.9% saline (vehicle) once daily for 2, 4, 7, 10, or 14 days. Oxidative stress was assessed by determining plasma and liver levels of 15-F(2t)-IsoP, lipid hydroperoxides (LPO), and thiobarbituric acid reactive substances (TBARs). Plasma and liver 15-F(2t)-IsoP were elevated and reached a plateau after day 2 of VPA treatment compared to control. Liver LPO levels were not elevated until day 7 of treatment (1.8-fold versus control, p < 0.05). Liver and plasma TBARs were not increased until 14 days (2-fold vs. control, p < 0.05). Liver toxicity was evaluated based on serum levels of alpha-glutathione S-transferase (alpha-GST) and by histology. Serum alpha-GST levels were significantly elevated by day 4, which corresponded to hepatotoxicity as shown by the increasing incidence of inflammation of the liver capsule, necrosis, and steatosis throughout the study. The liver levels of beta-oxidation metabolites of VPA were decreased by day 14, while the levels of 4-ene-VPA and (E)-2,4-diene-VPA were not elevated throughout the study. Overall, these findings indicate that VPA treatment results in oxidative stress, as measured by levels of 15-F(2t)-IsoP, which precedes the onset of necrosis, steatosis, and elevated levels of serum alpha-GST.     

Structural characterization of in vivo rat glutathione adducts and a hydroxylated metabolite of simvastatin hydroxy acid.

Simvastatin hydroxy acid (SVA), the pharmacologically active form of simvastatin (SV), is a potent inhibitor of 3-hydroxy-3-methylglutaryl (HMG)-coenzyme A reductase and is formed on hydrolysis of the orally administered SV. In this article, we report the structural characterization of two new dihydroxy glutathione adducts and a trihydroxy derivative of SVA, all found in rat bile. Metabolite I is 5'beta,6'beta-dihydroxy-4'a(alpha)-glutathione-SVA, and metabolite II is a pentanoic acid derivative of metabolite I. The two identified GSH conjugates accounted for 16 and 9% in males and 11 and 5% in females of the total radioactivity (metabolites I and II, respectively). Metabolite III is 3',5'beta,6'beta-dihydrotriol-SVA and accounts for 2% (male) and 4% (female) of the total dose in rats. Of these three newly identified metabolites, only metabolite III was also observed in dog bile.     

Identification of glutathione conjugates of the dimethyl ester of bilirubin in the bile of Gunn rats.

The Gunn rat, which is deficient in the UDP-glucuronosyltransferase for bilirubin, promptly excreted polar conjugates of the dimethyl ester of bilirubin in bile after intravenous infusion of this ester. The conjugates proved to be monoglutathione thioether adducts of the vinyl groups of the parent tetrapyrrole. High performance liquid chromatographic analysis of the conjugates as their dipyrrolic azosulfanilates demonstrated that only one of the dipyrroles of each tetrapyrrole was conjugated. The nonconjugated dipyrrole eluted as either the methyl endo- or exovinyl azodipyrrole. The amino acid composition of the pigments was consistent with that of a monoglutathione conjugate. NMR spectroscopy of the two major pigments demonstrated the loss of the proton signals of the C-18 vinyl group, indicating it to be the site of conjugation. Cation fast atomic bombardment tandem mass spectrometry demonstrated a molecular ion, [M + H]+, of m/z 937, which fragmented with a loss of 307 atomic mass units, consistent with glutathione. A molecular ion of m/z 807 was observed for the conjugate treated with gamma-glutamyltranspeptidase, consistent with the loss of glutamate. The mass spectrometry data indicated that the conjugates also contained a functional group whose mass was equivalent to hydroxyl, suggesting initial formation of an epoxide, which then reacts with glutathione. Pretreatment of the rat with 2,3,7,8-tetrachlorodibenzo-p-dioxin to induce cytochrome P-450 resulted in a 6-fold increase of the biliary excretion of the glutathione conjugates. Such induction also resulted in the excretion of a glutathione conjugate of bilirubin itself.     

Inactivation of gamma-aminobutyric acid aminotransferase by (S,E)-4-amino-5-fluoropent-2-enoic acid and effect on the enzyme of (E)-3-(1-aminocyclopropyl)-2-propenoic acid

(S,E)-4-Amino-5-fluoropent-2-enoic acid (6) is synthesized in six steps starting from the known gamma-aminobutyric acid aminotransferase (gamma-Abu-T) inactivator, (S)-4-amino-5-fluoropentanoic acid (1). Compound 6 is a mechanism-based inactivator of gamma-Abu-T: time-dependent inactivation is saturatable and protected by substrate; thiols do not protect the enzyme from inactivation; no enzyme activity returns upon dialysis. This compound (6) binds 50 times more tightly to gamma-Abu-T than does the saturated analogue (1). No transamination of 6 occurs prior to inactivation. However, five molecules of 6 are required to inactivate the enzyme with concomitant release of five fluoride ions. Therefore, four molecules are being converted to product for each inactivation event. (E)-3-(1-Aminocyclopropyl)-2-propenoic acid is synthesized in seven steps from 1-aminocyclopropanecarboxylic acid. It is prepared as a cyclopropyl derivative of the proposed intermediate in the inactivation of gamma-Abu-T by 6. The cyclopropyl derivative, however, is a noncompetitive inhibitor and does not inactivate the enzyme. This study shows the usefulness and hazards of incorporation of a trans double bond into potential gamma-Abu-T inactivators.     

The metabolic disposition of (S)-2-(3-tert-butylamino-2-hydroxypropoxy)-3-cyanopyridine in rats, dogs, and humans

Studies of the metabolic disposition of (S)-2-(3-tert-butylamino-2-hydroxypropoxy)-3-[14C]cyanopyridine (I) have been performed in humans, dogs, and spontaneously hypertensive rats. After an iv injection of I (5 mg/kg), a substantial fraction of the radioactivity was excreted in the feces of rats (32%) and dogs (31%). After oral administration of I (5 mg/kg) the urinary recoveries of radioactivity for rat and dog were 19% and 53%, respectively, and represented a minimum value for absorption because of biliary excretion of radioactivity. In man, bililary excretion of I appeared to be of minor significance because four male subjects, after receiving 6 mg of I p.o., excreted 76% and 9% of the dose of radioactivity in the urine and feces, respectively. Unchanged I represented 58% of the radioactivity excreted in human urine. The half-life for renal elimination of I was determined to be 4.0 +/- 0.9 /hr. In contrast, unchanged I represented 7% and 1% of excreted radioactivity in rat and dog urine, respectively. A metabolite of I common to man, dog, and rat was identified as 5-hydroxy-I, which represented approximately 5% of the excreted radioactivity in all species. Minor metabolites of I in which the pyridine nucleus had undergone additional hydroxylation were present in dog urine along with an oxyacetic acid metabolite, also bearing a hydroxylated pyridine nucleus.