Kazuo Igarashi

Participation of a medium chain acyl-CoA synthetase in glycine conjugation of the benzoic acid derivatives with the electron-donating groups

Glycine conjugation of a series of benzoic acid derivatives was investigated in bovine liver mitochondria. Benzoic acids with chlorine, methyl, methoxy or ethoxy substituents in the para-or meta-positions of the benzene ring showed a high degree of glycine conjugation. In contrast, the acids with cyano, nitro, amino, or acetylamino groups were conjugated to a small extent with glycine. A medium chain acyl-CoA synthetase that activates carboxylic acids was purified from bovine liver mitochondria. The purified medium chain acyl-CoA synthetase accepted not only medium chain fatty acids but also aromatic and arylacetic acids as substrates. There was a good correlation between the activity of the purified medium chain acyl-CoA synthetase and glycine conjugation of ten benzoic acids with electron-donating substituents. These findings indicate that the purified medium chain acyl-CoA synthetase is a major enzyme for glycine conjugation of benzoic acids with electron-donating groups in bovine live mitochondria.

The analysis of cocaine and its metabolites by liquid chromatography/atmospheric pressure chemical ionization-mass spectrometry (LC/APCI-MS)

The method for simultaneous determination of cocaine and its four metabolites (benzoylecgonine, ecgonine methyl ester, ecgonine and norcocaine) in urine by liquid chromatography/atmospheric pressure chemical ionization-mass spectrometry (LC/APCI-MS) was studied. The mass spectra showed the quasi-molecular ions, [M+H]+ as the base peak. LC/APCI-MS analysis was performed by focusing the characteristic ions at m/ = 186, 290, 200, 304 and 290 for ecgonine, benzoylecgonine, ecgonine methyl ester, cocaine and norcocaine, respectively. Cocaine and its four metabolites were well separated by high performance liquid chromatography (HPLC). The recoveries of cocaine and its metabolites from the spiked urine were 40.3-94.7% by solid-phase extraction with two type cartridges (Bond Elut Certify and Bond Elut SCX).

Determination of cabergoline and l-dopa in human plasma using liquid chromatography–tandem mass spectrometry

We determined cabergoline and L-dopa in human plasma using liquid chromatography-mass spectrometry with tandem mass spectrometry (LC-MS-MS). The deproteinized plasma samples with organic solvent or acid were analyzed directly by reversed-phase liquid chromatography. Using multiple reaction monitoring (MRM, product ions m/z 381 of m/z 452 for cabergoline and m/z 152 of m/z 198 for L-dopa) on LC-MS-MS with electrospray ionization (ESI), cabergoline and L-dopa in human plasma were determined. Calibration curves of the method showed a good linearity in the range 5-250 pg/ml for cabergoline and 1-200 ng/ml for L-dopa, respectively. The limit of determination was estimated to be approximately 2 pg/ml for cabergoline and approximately 0.1 ng/ml for L-dopa, respectively. The method was applied to the analysis of cabergoline and L-dopa in plasma samples from patients treated with these drugs. The precision of analysis showed coefficients of variation ranging from 3.8% to 10.5% at cabergoline concentration of 13.8-26.2 pg/ml and from 2.9% to 8.9% at an L-dopa concentration of 302.5-522.1 ng/ml in patient plasma. As a result, the procedure proved to be very suitable for routine analysis.

Difference of the liver and kidney in glycine conjugation of ortho-substituted benzoic acids

The relative importance of the liver and kidney for glycine conjugation of ortho-substituted benzoic acids was investigated. Glycine conjugation of ortho-substituted benzoic acids was investigated in mouse liver and kidney mitochondria. The extent of glycine conjugation of benzoic acids with the halogen group decreased in the order F > Cl > Br > I. The conjugation of salicylic acid with glycine took place in only the kidney. 2-Methoxybenzoic acid exhibited no activity in the liver and kidney. The difference in glycine conjugation of ortho-substituted benzoic acids was observed between liver and kidney. The kidney was more active in glycine conjugation of ortho-substituted acids than the liver. In addition, the relationship between glycine conjugation and the chemical structure of ortho-substituted acids was examined in the liver and kidney. The size of the substituent had a far greater influence over glycine conjugation in the liver and kidney. Glycine conjugation was also dependent on the substituent electronegativity. It may be important that the substrates undergoing glycine conjugation contain a flat region coplanar to the carboxylate group.

Inhibition of a medium chain acyl-CoA synthetase involved in glycine conjugation by carboxylic acids.

Molecular characteristics of carboxylic acids were investigated for the ability to inhibit a purified medium chain acyl-CoA synthetase, using hexanoic acid as a substrate. Salicylic acid, 4-methylsalicylic acid, 2-hydroxynaphtoic acid, and 2-hydroxyoctanoic acid, which do not act as substrates for the medium chain acyl-CoA synthetase, were potent as inhibitors. Valproic acid was not an inhibitor. Salicylic acid, 2-hydroxynaphthoic acid, and 2-hydroxyoctanoic acid inhibited the medium chain acyl-CoA synthetase with Ki values of 37, 5.2, and 500 microM, respectively. 4-Methylsalicylic acid was more potent than salicylic acid. The inhibitory carboxylic acids were competitive with respect to hexanoic acid. The distance of the hydroxyl group from the carboxylic acid group of the benzene ring influenced the inhibitory activity. The hydroxyl group on the carbon adjacent to the carboxylic acid group was required for inhibitory activity. In addition, there was a good correlation between the lipophilicity of the carboxylic acids and the Ki values, suggesting that the lipophilicity of the carboxylic acids is a major determinant for inhibition of the medium chain acyl-CoA synthetase.

Molecular specificity of a medium chain acyl-CoA synthetase for substrates and inhibitors: conformational analysis.

Amino acid conjugation is an important route of detoxification of xenobiotic and endogenous carboxylic acids. The specificity of the purified medium chain acyl-CoA synthetase catalyzing the first reaction of amino acid conjugation was investigated further for substrates and inhibitors. Molecular modeling techniques were applied to derive the molecular characteristics of substrates and inhibitors for the medium chain acyl-CoA synthetase. The purified enzyme accepted not only straight medium chain fatty acids but also aromatic acids. Of the arylacetic acids, activity was obtained with naphthylacetic acids, whereas introduction of a methyl group at the alpha-position caused loss of activity. High activity was also observed with cyclohexanoic acid. Diflunisal, 2-hydroxydodecanoic acid, and nalidixic acid inhibited the medium chain acyl-CoA synthetase activity for hexanoic acid, with Ki values of 0.8, 4.4, and 12.3 microM, respectively. The inhibitory carboxylic acids were competitive with respect to hexanoic acid. The hydroxyl or ketone (oxo) groups at the beta-position of carboxylic acids were an important determinant for inhibitory activity. All substrates and inhibitors contained a flat hydrophobic region coplanar to the carboxylate group. In addition, the substrates had negative values for charge on the carbon in the beta-position of carboxylic acids.

Characterization of a renal medium chain acyl-CoA synthetase responsible for glycine conjugation in mouse kidney mitochondria.

Glycine conjugation of a series of benzoic acid derivatives was investigated in mouse kidney mitochondria. The chlorine and methyl substitutions in the para- and meta-positions of the benzene ring yielded an increase in glycine conjugation. The acids with a methoxy group showed a low degree of glycine conjugation. In addition, the acids with nitro or amino groups were conjugated to a slight extent with glycine. The in vitro conjugation of salicylic acid with glycine occurred not in liver but in kidney. The specificity of the renal medium chain acyl-CoA synthetase catalyzing the first reaction of glycine conjugation was also examined. The enzyme accepted not only medium chain fatty acids but also aromatic and arylacetic acids. The highest activity was shown with hexanoic acid. High activities were observed for benzoic acid derivatives with alkyl and alkoxyl groups in the para- and meta-positions of the benzene ring. An ortho-substituted acid exhibited no activity. In addition, the enzyme was less active with valproic acid, tranexamic acid, indomethacin and ketoprofen. The enzyme was inhibited by diflunisal, 2-hydroxydodecanoic acid and salicylic acid, which did not act as substrates. There was a poor correlation between the activity of the medium chain acyl-CoA synthetase and glycine conjugation of eleven substituted benzoic acids. These findings suggest that the present medium chain acyl-CoA synthetase is involved in glycine conjugation of the substituted acids in mouse kidney mitochondria, but there may be a larger contribution of another isoenzyme.

CYP2D6 HhaI Genotype and the Neuroleptic Malignant Syndrome

To investigate the relationship between CYP2D6 genotypes (reported to be associated with the susceptibilities to Parkinson’s disease and multisystem atrophy) and the possible susceptibility to neuroleptic malignant syndrome (NMS) and subacute myelo-optico-neuropathy (SMON), we analyzed the CYP2D6 gene by polymerase chain reaction and restriction fragment length polymorphism in Japanese schizophrenia patients with a history of NMS. There was no significant difference in the frequency of the poor metabolizer genotype of CYP2D6 between the cases with a history of NMS and controls (p > 0.05). The frequency of the mutation located at the HhaI site in exon 6 of CYP2D6 in the cases was higher, but not significantly (p > 0.05; the mutated allele frequency was 0.25), than that in the controls, schizophrenia patients without NMS (0.11) and healthy controls (0.09). The frequency (0.10) of the HhaI mutation type in patients with a diagnosis of SMON was also not significantly higher than in healthy controls. These results suggest that the poor metabolizer and HhaI polymorphism of CYP2D6 may not be a useful molecular marker for predicting the onset of NMS and SMON.

Analysis of the Metabolism of Haloperidol and Its Neurotoxic Pyridinium Metabolite in Patients with Drug-Induced Parkinsonism

The blood levels of the neurotrophic drug haloperidol (HP) and its pyridinium metabolite, HPP+, have been analyzed by liquid chromatography/electrospray ionization-mass spectrometry in 10 schizophrenic patients treated with HP, without carbamazepine (HP, oral daily dose of 0.3–0.5 mg/kg body weight for more than 1 year, females, aged 41 ± 8.5 years). There was a significant difference (t-test, d.f. = 8, p (t₀ = 7.2) <0.005) in the blood HPP+ level between the 5 patients with (18.5 ± 6.4 ng/ml) and the 5 without (6.3 ± 2.4 ng/ml) severe side effects such as drug-induced parkinsonism (Extrapyramidal Symptom Rating Scale (ESRS) parkinsonism severity scores 2.8 ± 1.5 and 1.8 ± 1.1, respectively). Moreover, it is suggested that vitamin E may be effective for drug-induced parkinsonism through a change in the blood HPP+ level. It is necessary to investigate the HPP+ metabolism in psychiatric patients to avoid severe side effects such as drug-induced parkinsonism and cardiac functional disorders.

Inhibitory effect of quinolone antimicrobial and nonsteroidal anti-inflammatory drugs on a medium chain acyl-CoA synthetase

The inhibitory effects of quinolone antimicrobial agents and nonsteroidal anti-inflammatory drugs on purified mouse liver mitochondrial medium chain acyl-CoA synthetase catalyzing the first reaction of glycine conjugation were examined, using hexanoic acid as a substrate. Enoxacin, ofloxacin, nalidixic acid, diflunisal, salicylic acid, 2-hydroxynaphthoic acid, and 2-hydroxydodecanoic acid, which do not act as substrates, were potent inhibitors. Diflunisal, nalidixic acid, salicylic acid, 2-hydroxynaphthoic acid, and 2-hydroxydodecanoic acid inhibited competitively this medium chain acyl-CoA synthetase with K(i) values of 0.6, 12.4, 19.6, 13.4, and 15.0 microM, respectively. Enoxacin and ofloxacin inhibited this medium chain acyl-CoA synthetase in a mixed-type manner with K(i) values of 23.7 and 38.2 microM, respectively. Felbinac, which is a substrate, inhibited the activity of this medium chain acyl-CoA synthetase for hexanoic acid (IC50 = 25 microM). The concomitant presence of enoxacin and felbinac strongly inhibited this medium chain acyl-CoA synthetase. These findings indicate that medium chain acyl-CoA synthetases may be influenced by quinolone antimicrobial and nonsteroidal anti-inflammatory drugs.