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In the mammalian brain, d -serine acts as a co-agonist at the glycine-binding site on the N-methyl- d -aspartate receptor. Because plasma d -serine levels are significantly lower in patients with schizophrenia than in healthy subjects, d -serine has been proposed as a potential therapeutic agent for schizophrenia treatment. However, d -serine has a nephrotoxic effect in rats at high doses. The purpose of this study was to investigate the relationship between the plasma kinetics of d -serine and nephrotoxicity in rats. We administered d -serine intravenously (iv), orally (po), or intraperitoneally (ip) to male Wistar rats, and performed gas chromatography-mass spectrometry to measure the plasma concentrations of d - and l -serine. After iv administration (0.1 mmol/kg body weight (bw)), plasma d -serine declined multiexponentially with an elimination t1/2 of 108 ± 16 min, and the total clearance was 7.9 ± 0.9 ml/min/kg bw. The oral bioavailability of d -serine was estimated to be 94 ± 27%. To evaluate the dose–response relationship of d -serine-induced kidney injury and the plasma kinetics of d -serine, we injected d -serine into rats ip in doses ranging from 0.6 to 4.8 mmol/kg bw. Twenty-four hours after d -serine administration, histological changes indicating renal damage were observed in the kidneys of rats who received d -serine at doses of 1.8–4.8 mmol/kg bw; the severity of the tubular injury increased with increasing d -serine dose. When the Cmax value of d -serine was approximately >2 μmol/ml, the plasma creatinine increased remarkably 24 h after d -serine administration. This suggests that the Cmax of d -serine could be a good predictor of d -serine-induced nephrotoxicity.

Mutations in the gene encoding hypoxanthine-guanine phosphoribosyltransferase (HPRT) cause Lesch-Nyhan disease (LND) and its variants (LNV). Due to the technical problems for measuring the HPRT activity in vitro, discordances between the residual HPRT activity and the clinical severity were found. 5-Phosphoribosyl 1-pyrophosphate (PRPP) is a substrate for HPRT. Since increased PRPP concentrations were observed in erythrocytes from patients with LND and LNV, we have turned our attention to erythrocyte PRPP as a biomarker for the phenotype classification. In the present work, a method for determination of PRPP concentration in erythrocyte was developed using liquid chromatography-tandem mass spectrometry (LC-MS/MS) with multiple reaction monitoring (MRM). Packed erythrocyte samples were deproteinized by heating and the supernatants were injected into the LC-MS/MS system. All measurement results showed good precision with RSD6%. PRPP concentrations of nine normal male subjects, four male patents with LND and six male patients with LNV were compared. The PRPP concentrations in erythrocyte from patients with LND were markedly increased compared with those from normal subjects, and those from patients with LNV were also increased but the degree was smaller than those with LND. The increase pattern of PRPP concentration in erythrocyte from patients with HPRT deficiency was consistent with the respective phenotypes and was correlated with the disease severity. PRPP concentration was suggested to give us supportive information for the diagnosis and the phenotype classification of LND and LNV.

Hereditary xanthinuria is an extremely rare purine metabolism disorder caused by a genetic abnormality in xanthine dehydrogenase. A new case of hereditary xanthinuria type 1 accompanied by bilateral renal calculi was encountered. We performed an allopurinol loading test and diagnosed classical type 1 xanthinuria. Through genetic diagnosis, we identified a mutation site in the xanthine dehydrogenase gene. Genetic analysis revealed a homozygous deletion of cytosine 2,567 in the xanthine dehydrogenase gene, and as a result, a stop codon was formed at position 928. Renal failure caused by the deposition of xanthine crystals is a known complication because xanthine is poorly soluble in water. With high fluid intake and low purine diet, no significant increase in calculi has been observed in this patient for 2 years.

5-Phosphoribosyl-1-pyrophosphate (PRPP) is an important regulator of de novo purine synthesis. A method for the measurement of PRPP in erythrocytes was designed, which is based on the determination of [(13)C(5)]glutamate derived from [(13)C(5)]glutamine following the utilization of PRPP by the action of amidophosphoribosyltransferase. The present study describes a gas chromatographic-mass spectrometric method for determination of [(13)C(5)]glutamate using [(13)C(2)]glutamate as an internal standard. The methods involved purification by anion-exchange chromatography using a BondElut SAX and derivatization with isobutyl chlorocarbonate in water-methanol-pyridine. Quantitation was performed by selected ion monitoring of the protonated molecular ions in the chemical ionization mode. The intra-day reproducibility in the amounts of [(13)C(5)]glutamate determined was in good agreement with the actual amounts added in erythrocytes. A linear relationship was found between the amount of PRPP added and the amount of [(13)C(5)]glutamate formed from [(13)C(5)]glutamine using amidophosphoribosyltransferase.

D-Serine is a co-agonist of the N-methyl-D-aspartate receptor in glutamate neurotransmission and has been proposed as a potential therapeutic agent for schizophrenia. However, D-serine also acts as a nephrotoxic substance in rats at high doses. To investigate the pharmacokinetics and toxicokinetics of D-serine, a method for the stereoselective determination of serine enantiomers in rat plasma was developed using GC-MS with selected ion monitoring (GC-MS-SIM). DL-[2H3]Serine was used as an internal standard to account for losses associated with the extraction, derivatization and chromatography. Serine enantiomers were purified by cation-exchange chromatography using BondElut SCX cartridge and derivatized with HCl in methanol to form methyl ester followed by subsequent N,O-diacylation with optically active (+)-α-methoxy-α-trifluoromethylphenylacetyl chloride to form epimeric amide. Quantitation was performed by SIM of the molecular-related ions of the epimers in the chemical ionization mode. The intra- and inter-day reproducibility of the assay was less than 5% for D-serine and 3% for L-serine. The method was successively applied to study the pharmacokinetics of D-serine in rats. Copyright © 2011 John Wiley & Sons, Ltd.

D: -Amino acids are now recognized to be widely present in mammals. In rats, exogenously administered D: -methionine is almost converted into the L: -enantiomer via 2-oxo-4-methylthiobutylic acid as an intermediate. D: -Amino acid oxidase is associated with conversion of D: -methionine into the 2-oxo acid. Since D: -amino acid oxidase is present at the highest activity in the kidney compared to other organ, kidney injury is suggested to cause accumulation of D: -methionine. The purpose of the present study is to assess the role of kidney in the elimination of D: -methionine and metabolic conversion into L: -methionine in rats using a stable isotope methodology. After a bolus i.v. administration of D: -[²H₃)]methionine to 5/6-nephrectomized rats, plasma concentrations of D: -[²H₃]methionine, L: -[²H₃]methionine, and endogenous L: -methionine were determined by a stereoselective GC-MS method. Renal mass reduction slowed down the elimination of D: -[²H₃]methionine. The clearance values of conversion of D: -[²H₃]methionine into the L: -enantiomer in 5/6-nephrectomized rats were one-sixth of those in sham-operated rats. The elimination behavior of D: -[²H₃]methionine observed in rats demonstrated that kidney was the principal organ responsible for chiral inversion of D: -methionine.

Optically pure (R)- and (S)-3-aminotetradeutero-2-thiophenone (D- and L-[2H4]homocysteine thiolactone) for use as substrates for metabolic and pharmacokinetic studies has been prepared. The racemic [2H4]homocysteine thiolactone hydrochloride was prepared from commercially available and highly enriched DL-[3,3,4,4-2H4]methionine by intramolecular condensation with hydriodic acid. The racemate was derivatized with (S)-(+)-2-methoxyphenylacetic acid to form the diastereomeric amides. The diastereomers were separated by silica gel column chromatography. Hydrolysis of the D- and L-isomer amide in 4 M HCl–ethanol (1:2 v/v) afforded the optically pure D- and L-[2H4]homocysteine thiolactone, respectively. Copyright © 2010 John Wiley & Sons, Ltd.

This study describes a GC–MS method for the simultaneous determination of androstenedione (AD), 11β-hydroxyandrostenedione (11β-OHAD), and testosterone (TS) in human plasma. [19,19,19- 2 H 3 ]Androstenedione (AD- 2 H 3 ), 11β-hydroxy-[1,2,4,19- 13 C 4 ]androstenedione (11β-OHAD- 13 C 4 ), and [1,16,16,17- 2 H 4 ]testosterone (TS- 2 H 4 ) were used as internal standards. Pentafluoropropionic (PFP) derivatization with good GC behavior was employed for the GC–MS analysis of the three steroids. The detection limit of the present GC–MS–SIM method was found to be 1 pg per injection for AD (S/N ratio = 4.5), 5 pg for 11β-OHAD (S/N ratio = 5.0), and 1 pg for TS (S/N ratio = 4.4), respectively. Calibration curves were linear from 0.22 to 2.80 ng/mL ( r = 0.9998) for AD, from 0.56 to 3.19 ng/mL ( r = 0.9996) for 11β-OHAD, and from 2.05 to 10.3 ng/mL ( r = 0.9996) for TS. The intra- and inter-day assay reproducibilities in the amounts of the three androgens determined were in good agreement with the actual amounts added, the relative errors (R.E.) were −3.1 to 2.4%. The inter-assay relative standard deviation (R.S.D.) was less than 5.3%. The present method provides a sensitive and reliable technique for the simultaneous determination of AD, 11β-OHAD, and TS in plasma. The method can be applied to pharmacokinetic and metabolic studies of androgens with a particular interest in evaluating the conversion of AD to 11β-OHAD and the interconversion of AD and TS in humans.

An equation is theoretically derived which describes the relative standard deviation (RSD) of the amount ratios of analyte to its isotope-labeled variant in gas chromatography/mass spectrometry (GC/MS) using the stable isotope dilution method. The determination of methyltestosterone is taken as an example. The uncertainty equation proposed is justified by comparing the theoretical RSD values with the experimental RSD values obtained by replication over a wide range of analyte amount. The detection limit and quantitation limit are estimated from the continuous plot (precision profile) of the theoretical RSD against analyte amount.

A method for the stereoselective determination of D- and L-enantiomers of both methionine and [(2)H3]methionine in rat plasma was developed using gas chromatography-mass spectrometry with selected-ion monitoring (GC-MS-SIM). DL-[(2)H7]Methionine was used as analytical internal standard to account for losses associated with the extraction, derivatization and chromatography. The amino acids were purified by cation-exchange chromatography using BondElut SCX cartridge and derivatized with HCl in methanol to form methyl ester followed by subsequent N-acylation with optically active (+)-alpha-methoxy-alpha-trifluoromethylphenylacetyl chloride to form diastereomeric amide. Quantification was performed by SIM of the molecular-related ions of the diastereomers on the chemical ionization mode. Endogenous L-methionine concentrations in 50 microl of rat plasma were measured with relative intra- and inter-day precision of 4.0 and 6.3%, respectively. The intra- and inter-day reproducibility in the amounts of D- and L-[(2)H3]methionine determined were in good agreement with actual amount added.

Osaterone acetate (17 alpha-acetoxy-6-chloro-2-oxa-4,6-pregnadiene-3,20-dione; OA) is a steroidal antiandrogen. In order to clarify the species differences, metabolites of OA were examined in plasma, urine, and feces of dogs and humans after oral administration of OA. Eleven metabolites in plasma, urine, and feces were identified by their spectral properties and comparison to appropriate standards. The primary routes of OA metabolism involve 11 beta-, 15 beta- and 21-hydroxylation, 17 alpha-deacetylation, and dechlorination. Other metabolites arise from combinations of these pathways to form multiple oxidized metabolites. All metabolites observed in humans occurred in dogs. 11 beta-Hydroxylated metabolites (11 beta-OH OA and 11-oxo OA) were found in the plasma and urine of dogs, but there was no evidence of their presence in humans. 11 beta-Hydroxylation of exogenous steroids represents a distinctive biotransformation pathway.

d-Amino acids are now recognized to be widely present in mammals. Renal d-amino-acid oxidase (DAO) is associated with conversion of d-amino acids to the corresponding α-keto acids, but its contribution in vivo is poorly understood because the α-keto acids and/or l-amino acids formed are indistinguishable from endogenous compounds. First, we examined whether DAO is indispensable for conversion of d-amino acids to their α-keto acids by using the stable isotope tracer technique. After a bolus intravenous administration of d-[2H7]leucine to mutant mice lacking DAO activity (ddY/DAO−) and normal mice (ddY/DAO+), elimination of d-[2H7]leucine and formation of α-[2H7]ketoisocaproic acid ([2H7]KIC) and l-[2H7]leucine in plasma were determined. The ddY/DAO−mice, in contrast to ddY/DAO+mice, failed to convert d-[2H7]leucine to [2H7]KIC and l-[2H7]leucine. This result clearly revealed that DAO was indispensable for the process of chiral inversion of d-leucine. We further investigated the effect of renal mass reduction by partial nephrectomy on elimination of d-[2H7]leucine and formation of [2H7]KIC and l-[2H7]leucine. Renal mass reduction slowed down the elimination of d-[2H7]leucine. The fraction of conversion of d-[2H7]leucine to [2H7]KIC in sham-operated rats was 0.77, whereas that in five-sixths-nephrectomized rats was 0.25. The elimination behavior of d-[2H7]leucine observed in rats suggested that kidney was the principal organ responsible for converting d-leucine to KIC.

The stereoselective pharmacokinetics of selenomethionine enantiomers in rats has been studied to evaluate the chiral inversion of d -selenomethionine to the l -enantiomer. After bolus intravenous administration of d - or l -selenomethionine to rats, the plasma concentrations of these two enantiomers were determined by stereoselective gas chromatography–mass spectrometry with selected ion monitoring. This method involved derivatization of selenomethionine enantiomers with HCl in methanol to form methyl ester followed by N -acylation with (+)-α-methoxy-α-trifluoromethylphenylacetyl chloride to form the diastereomeric amide, and separation of the diastereomer on GC with an achiral column. Plasma concentrations of administered d - and l -selenomethionine declined with terminal half-lives of 96 ± 17 min and 91 ± 6 min, respectively. l -Selenomethionine appeared rapidly in plasma after administration of d -selenomethionine, whereas d -selenomethionine was not detected in plasma after administration of l -selenomethionine. The fraction of conversion of d -selenomethionine to l -selenomethionine was estimated to be 61.3 ± 14.5%. The present method evaluates the stereoselective pharmacokinetics of selenomethionine enantiomers, including the estimation of the metabolic chiral inversion.

D-Leucine is considered to be converted into the L-enantiomer by two steps: oxidative deamination to form alpha-ketoisocaproic acid (KIC) and subsequent stereospecific reamination of KIC. We investigated the pharmacokinetics of leucine enantiomers and KIC in rats to evaluate how deamination of D-leucine, reamination of KIC, and decarboxylation of KIC were affected to the overall extent that converted D-leucine into the L-enantiomer. After intravenous administrations of D-[(2)H(7)]leucine, L-[(2)H(7)]leucine, or [(2)H(7)]KIC, their plasma concentrations together with endogenous L-leucine and KIC were determined by gas chromatography-mass spectrometry. The rapid appearances of [(2)H(7)]KIC and L-[(2)H(7)]leucine were observed after administration of D-[(2)H(7)]leucine, whereas no detectable amount of D-[(2)H(7)]leucine was found after administrations of [(2)H(7)]KIC or L-[(2)H(7)]leucine. The fraction of conversion from D-[(2)H(7)]leucine into [(2)H(7)]KIC (F(D-->KIC)) was estimated by using the area under the curve (AUC) of [(2)H(7)]KIC on the D-[(2)H(7)]leucine administration [AUC(KIC(D))] and that of [(2)H(7)]KIC on the [(2)H(7)]KIC administration (AUC(KIC)) to yield 70.1%. The fraction of conversion from [(2)H(7)]KIC to L-[(2)H(7)]leucine (F(KIC-->L)) was 40.2%. The fraction of conversion from D-leucine to the L-enantiomer (F(D-->L)) was considered to be the product of F(D-->KIC) and F(KIC-->L), indicating that 28.2% of D-[(2)H(7)]leucine was metabolized to L-[(2)H(7)]leucine via [(2)H(7)]KIC. These results suggested that the relatively low conversion of D-leucine into the L-enantiomer might depend on irreversible decarboxylation of KIC. Regardless of [(2)H(7)]KIC, F(D-->L) was also calculated directly using AUC(L(D)) and AUC(L) to yield 27.5%. There were no differences between the two F(D-->L) values, suggesting that almost all of the formation of L-[(2)H(7)]leucine from D-[(2)H(7)]leucine occurred via [(2)H(7)]KIC as an intermediate.

The pharmacokinetics of methionine has been studied in rats by means of stable isotope methodology. After the i.v. bolus injection of [ 2 H 7 ]methionine (5 mg/kg body wt.), the plasma concentrations of [ 2 H 7 ]methionine, demethylated [ 2 H 4 ]homocysteine and remethylated [ 2 H 4 ]methionine were determined simultaneously with endogenous methionine and homocysteine by gas chromatography-mass spectrometry. The half-life for [ 2 H 7 ]methionine were 35.0 ± 6.9 min. The appearance of the metabolites, [ 2 H 4 ]homocysteine and [ 2 H 4 ]methionine, in the plasma was very rapid. The fraction of [ 2 H 7 ]methionine that remethylated to [ 2 H 4 ]methionine through [ 2 H 4 ]homocysteine were 0.185 ± 0.028. The administered [ 2 H 7 ]methionine did not influence the plasma levels of endogenous methionine and homocysteine. The present stable isotope methodology has made it possible to evaluate the pharmacokinetics of methionine, including the estimation of remethylation.

A method for determination of alpha-ketoisocaproic acid (KIC) and [4,5,5,5,6,6,6-2H7]alpha-ketoisocaproic acid ([2H7]KIC) in rat plasma was developed using gas chromatography-mass spectrometry-selected ion monitoring (GC-MS-SIM). [5,5,5-2H3]alpha-Ketoisocaproic acid ([2H3]KIC) was used as an analytical internal standard to account for losses associated with the extraction, derivatization and chromatography. The keto acids were extracted by cation-exchange chromatography using BondElut SCX cartridge and derivatized with N-phenyl-1,2-phenylenediamine to form N-phenylquinoxalinone derivatives. Quantitation was performed by SIM of the respective molecular ions at m/z 278, 281 and 285 for the derivatives of KIC, [2H3]KIC and [2H7]KIC on the electron impact method. The limit of detection was found to be 70 fmol per injection (S/N=3) and the limit of quantitation for [2H7]KIC was around 50 nM in rat plasma. Endogenous KIC concentrations in 50 microl of rat plasma were measured with relative intra- and inter-day precision of 4.0% and 3.3%, respectively. The intra- and inter-day precision for [2H7]KIC spiked to rat plasma in the range of 0.1 to 10 microM gave good reproducibility with relative standard deviation (RSD) of 6.5% and 5.4%, respectively. The intra- and inter-day relative errors (RE) for [2H7]KIC were less than 6.4% and 3.8%, respectively. The method was applied to determine the plasma concentration of [2H7]KIC after an intravenous administration of [2H7]KIC in rat.

Optically pure L-[3,3,4,4,S-methyl- 2 H 7 ]methionine (L-[ 2 H 7 ]-methionine) for use as a substrate for the methionine loading test has been prepared. The racemic [ 2 H 7 ]methionine was prepared from DL-[3,3,4,4- 2 H 4 ]methionine (DL-[ 2 H 4 ]methionine) by conversion of the S-CH 3 group to a C 2 H 3 group. The racemate was resolved by stereospecific hydrolysis of the N-acetylated derivative with acylase. After an intravenous administration of L-[ 2 H 7 ]methionine in a rat, the concentration of L-[ 2 H 7 ]methionine and the metabolites in plasma were determined by GC-MS-SIM. L-[ 2 H 4 ]Homocysteine and L-[ 2 H 4 ]methionine were detected in plasma at 30 min after dosing. These results show that L-[ 2 H 7 ]methionine was converted by de-methylation and subsequent remethylation to form L-[ 2 H 4 ]methionine.

A method based on gas chromatography-mass spectrometry-selected-ion monitoring was developed to measure the main metabolites of 17alpha-methyltestosterone, 17alpha-methyl-5alpha-androstan-3alpha,17beta-di ol and 17alpha-methyl-5beta-androstan-3alpha,17beta-dio l, in human urine. 17alpha-Methyl-[(2)H3]-5alpha-androstan-3alpha,1 7beta-diol and 17alpha-methyl-[(2)H3]-5beta-androstan-3alpha,17 beta-diol were used as internal standards. The methods involved purification using a Sep-Pak C(18) cartridge, hydrolysis by beta-glucuronidase from Ampullaria and derivatization with N-methyl-N-trimethylsilyl-trifluoroacetamide/dithioerythriol/ammon ium iodide. Quantitation was achieved by selected-ion monitoring of the characteristic fragment ions ([(M+H)-2xTMSOH]+) of the di-TMS derivatives on the chemical ionization mode. The method provides a specific, sensitive and reliable technique to determine the urine levels of 17alpha-methyl-5alpha-androstan-3alpha,17beta-di ol and 17alpha-methyl-5beta-androstan-3alpha,17beta-dio l, and can be applied to pharmacokinetic studies of 17alpha-methyltestosterone.

Internal acyl migration reactions of 1beta-O-acyl glucuronides of 2-arylpropionic acids (profens) are of interest because of their possible role in covalent binding to serum proteins and consequent allergic reactions. The stereoselective degradation of 1beta-O-acyl glucuronides of enantiomeric 2-phenylpropionic acids (PAs), the basic structures of profens, in phosphate buffer (pH 7.4) at 37 degrees C, has been investigated using HPLC. Apparent first-order degradation of 1beta-O-acyl glucuronide and the sequential appearance of 2-, 3- and 4-O-acyl isomers were observed for each enantiomer. Acyl migration was observed to predominate over hydrolysis as in the other profen glucuronides. All the positional isomers and anomers were characterized using NMR and HPLC-NMR. The overall degradation half-life of (R)- and (S)-PA glucuronides was 1.8 and 3.3 h, respectively. These results suggest that (R)-PA glucuronide could be more susceptible to covalent binding to proteins via acyl migration than the corresponding antipode. The lability of the (R)-diastereomer over the antipode is consistent with previous reports on other profen glucuronides. Thus, the diastereomeric PA glucuronides are considered to be the best model compounds for the computation of structural physicochemical parameters to control the stereoselective internal acyl migration of profen glucuronides because PA has the simplest chemical structure of all the profens.

A method for the stereoselective assay of d - and l -enantiomers of both leucine and [2H7]leucine in rat plasma was developed using gas chromatography–mass spectrometry–selected-ion monitoring. dl -[2H3]leucine was used as an internal standard. The method involved purification by cation-exchange chromatography using BondElut SCX cartridge and derivatization with hydrochloric acid in methanol to form methyl ester followed by subsequent chiral derivatization with (+)-α-methoxy-α-trifluoromethylphenylacetyl chloride to form diastereomeric amide. The derivatization made the separation of the leucine enantiomers possible with good gas chromatographic behavior. Quantitation was performed by selected-ion monitoring of the quasi-molecular ions of the diastereomers on the chemical ionization method. The sensitivity, specificity, accuracy and reproducibility of the method were demonstrated to be satisfactory for application to pharmacokinetic studies of leucine enantiomers.

(2S)-2-Amino-3-(2′,6′-dibromo-4′-hydroxy)phenylpropionic acid (2,6-dibromo-L-tyrosine) 1a has been synthesized by two methods. The first is by enantioselective hydrolysis of the N-trifluoroacetyl derivative of the racemic 2,6-dibromotyrosine with carboxypeptidase A. The kinetic parameters of activity of carboxypeptidase A against N-trifluoroacetyl-2,6-dibromo-L-tyrosine have also been estimated (Vmax, 0.32 mM min–1 mg–1; Km 16.15 mM). The second is an asymmetric synthesis by Schollkopf’s bis-lactim ether method. Alkylation of the lithiated bis-lactim ether of cyclo(-D-Val-Gly) 6 with 2,6-dibromo-4-methoxybenzyl bromide and subsequent hydrolysis with aqueous trifluoroacetic acid gives 2,6-dibromo-4-methoxy-L-tyrosine methyl ester 8. Demethylation of the ester 8 with boron tribromide affords the amino acid 1a. The enantiomeric purity of 2,6-dibromo-L-tyrosine 1a obtained by both methods has been determined by HPLC with a chiral stationary column and is found to be more than 95% (ee).

1-O-Acyl-beta-D-glucopyranuronates can undergo irreversible binding to proteins mainly through internal acyl migration reactions, which may have toxicological significance. A new method based on the 13C-labeling and nuclear magnetic resonance (NMR) spectroscopy has been developed to study the reactivity of the 1-O-acyl-beta-D-glucopyranuronate of benzoic acid. In phosphate buffer (pH 7.4) solution at 37 degrees C, the glucuronide showed apparent first-order degradation kinetics (T1/2, 125 min), and concurrent and sequential appearance of 2-, 3- and 4-O-acyl isomers as both alpha- and beta-anomers was observed. The isomeric glucuronides were identified by two-dimensional NMR of the reaction mixture. The direct approach using 13C-labeling and NMR could also provide insights into the reactivities of other labile drug acylglucuronides and their isomeric glucuronides.

The preparation of enantiomeric [1- 13 C]ketoprofens (KPs) and their acylglucuronides has been reported for the nuclear magnetic resonance (NMR) spectroscopic studies on the stereoselective pharmacokinetics and reactivities of KP acylglucuronides. The racemic [1- 13 C]KP was prepared by a three-step synthetic scheme from [ 13 C]potassium cyanide in overall yield of 23 %. The racemate was optically resolved by the formation of diastereomeric amides with (R)-(+)-a-phenylethylamine, separation of the amides by column chromatography on silica gel, and nonhydrolytic cleavage of the amide bond using nitrogen tetroxide. The yields of KP enantiomers were 30 % based on the racemate. The acylglucuronides of (R)- and (S)-[1- 13 C]KP were isolated from human urine after dosing of each labelled KP (100 mg) using preparative high-performance liquid chromatography following Sep-Pak C 18 pretreatments. The yields of the conjugates from 0-4 h post-dose urine were roughly 50 mg.

A new method based on the 13 C-labeling and nuclear magnetic resonance (NMR) spectroscopy was developed to study the human urinary excretion of diastereomeric acylglucuronides after the oral administration of 100 mg racemic [3- 13 C]ketoprofen (KP). The urinary excretion of diastereomeric [3- 13 C]KP glucuronides formed from [3- 13 C]KP was followed by proton-decoupled 13 C NMR spectroscopy (9.4 T, 10min accumulation time) without any separation procedures such as extraction and chromatography. The C3 resonances due to the diastereomers were satisfactorily separated from each other by the addition of methyl-β-cyclodextrin as an achiral shift reagent through the formation of inclusion complexes. The C3 resonances were detected with an acceptable signal-to-noise ratio over 8 h at a therapeutic dosage. The concentrations of [ 13 C]KP glucuronides were calculated from the inverse gated decoupling and proton-decoupling experiments using [2- 13 C]glycine as an internal standard. The urinary excretion of diastereomeric [3- 13 C]KP glucuronides over 8 h amounted to 29% (downfield position) and 25% (upfield position) of the administered dose, respectively, and the former was presumed to have S -configuration. The elimination rate constants of both glucuronides were virtually the same ( K = 0.19 h −1 ). In addition, the susceptibility of the glucuronides to acyl migration as well as hydrolysis was suggested under the physiological conditions (37 °C, pH 7.3). The present direct approach is simple and convenient, and has the potential for wide application to the analysis of the labile acylglucuronides of 2-arylpropionic acids in biofluids.

An off-line counting method for the determination of carbon-14 in HPLC eluates was developed using radioluminography (RLG). A succession of aliquots of the eluate were collected in the flat-bottomed wells of a polystyrene microplate, evaporated to dryness and contacted with an imaging plate, and radioactivity was determined with a Bio-image analyser. The limit of detection was 0.35 Bq per injection. The inter- assay relative standard deviation was less than 3% over the range 2–20 Bq. The RLG off-line counting method was utilized to determine [ 14 C]eicosapentaenoic acid metabolites formed by rat hepatic microsomes. The results were compared with those obtained with an off- line liquid scintillation counting method and an on-line counting method.

ABCG2, also known as BCRP, is a high-capacity urate exporter, the dysfunction of which raises gout/hyperuricemia risk. Generally, hyperuricemia has been classified into urate 'overproduction type' and/or 'underexcretion type' based solely on renal urate excretion, without considering an extra-renal pathway. Here we show that decreased extra-renal urate excretion caused by ABCG2 dysfunction is a common mechanism of hyperuricemia. Clinical parameters, including urinary urate excretion, are examined in 644 male outpatients with hyperuricemia. Paradoxically, ABCG2 export dysfunction significantly increases urinary urate excretion and risk ratio of urate overproduction. Abcg2-knockout mice show increased serum uric acid levels and renal urate excretion, and decreased intestinal urate excretion. Together with high ABCG2 expression in extra-renal tissues, our data suggest that the 'overproduction type' in the current concept of hyperuricemia be renamed 'renal overload type', which consists of two subtypes—'extra-renal urate underexcretion' and genuine 'urate overproduction'—providing a new concept valuable for the treatment of hyperuricemia and gout., Hyperuricemia, or gout, is thought to arise either from urate overproduction or from decreased renal excretion of urate. Ichida et al. show that the extra-renal excretion of urate also has a role in the pathogenesis of hyperuricemia, and propose a new classification for patients with this disease.

A method alternative to liquid scintillation counting for detecting 14C was developed. The new method involves putting an aqueous radioactive sample onto the flat-bottomed wells of a polystyrene microplate, preparing a pellicle by lyophilization, and determining the radioactivity using radioluminography. It provides a simple, inexpensive, sensitive and reliable technique for determining the radioactivity of a few hundred samples simultaneously.

A method for profiling arachidonic acid metabolites by radio gas chromatography (GC) is described. The incubation mixture of rabbit platelets with [14C]arachidonic acid was purified on a Sep-Pak C18 cartridge and derivatized with diazomethane, O-methylhydroxylamine and dimethyl-isopropylsilylimidazole. The recovery of total 14C-radioactivity was 93.1 +/- 7.2%. Loss of radioactivity during derivatization was negligible. Baseline separations for [14C]arachidonic acid and its metabolites were obtained in a single run within 45 min by GC using a synchronized accumulating radioisotope detector (GC/SARD). The recovery of radioactivity from the GC column was virtually 100%. The chemical structures of the metabolites were confirmed by GC/mass spectrometry; peaks of arachidonic acid metabolites were assigned by comparison of the methylene unit values with those of radioactive peaks in GC/SARD analyses. The intra-assay coefficients of variation in GC/SARD analyses were less than 10%. The method was used to map the profile of arachidonic acid metabolites formed by rabbit platelets in the presence of indomethacin, baicalein or glutathione.

A method for the stereoselective determination of d - and l -enantiomers of selenomethionine in mouse plasma was developed using gas chromatography–mass spectrometry with selected-ion monitoring (GC–MS-SIM). dl -[ 2 H 3, 82 Se]selenomethionine was used as analytical internal standard to account for losses associated with the extraction, derivatization and chromatography. Selenomethionine enantiomers in mouse plasma were purified by cation-exchange chromatography using BondElut SCX cartridge and derivatized with HCl in methanol to form methyl ester followed by subsequent N -acylation with optically active (+)-α-methoxy-α-trifluoromethylphenylacetyl chloride to form diastereomeric amide. Quantification was performed by SIM of the molecular-related ions of the diastereomers on the chemical ionization mode. The intra- and inter-day precision for d - and l -selenomethionine spiked to mouse plasma gave good reproducibility with relative standard deviation of 3% and 3% for d -selenomethionine and 6% and 3% for l -selenomethionine, respectively. The estimated amounts were in good agreement with the actual amounts spiked, the intra- and inter-day relative error being 5% and 2% for d -selenomethionine and 2% and 1% for l -selenomethionine, respectively. The present method is sensitive enough to determine pharmacokinetics of selenomethionine enantiomers.

The synthesis of D- and L-selenomethionine labeled with ⁸²Se and three deuteriums at Se-methyl group (D- and L-[²H₃, ⁸²Se]selenomethionine) was described. D- And L-[²H₃, ⁸²Se]selenomethionine were prepared by condensation of (R)- and (S)-2-amino-4-bromobutylic acid with lithium [²H₃, ⁸²Se]methaneselenolate, which was prepared from metal (82)Se and [²H₃]methyl iodide. The optical purities of D- and L-[²H₃, ⁸²Se]selenomethionine were determined by HPLC with a chiral stationary phase column and were found more than 99% ee. The chemical ionization mass spectra showed that the molecular related ion for N-isobutyloxycarbonyl ethyl ester derivatives of [²H₃, ⁸²Se]selenomethionine did not overlap with the m/z values known from that of non-labeled selenomethionine.

(2S)-2-Amino-3-(2′,6′-dibromo-4′-hydroxy)phenylpropionic acid (2,6-dibromo-L-tyrosine) 1a has been synthesized by two methods. The first is by enantioselective hydrolysis of the N-trifluoroacetyl derivative of the racemic 2,6-dibromotyrosine with carboxypeptidase A. The kinetic parameters of activity of carboxypeptidase A against N-trifluoroacetyl-2,6-dibromo-L-tyrosine have also been estimated (Vmax, 0.32 mM min–1 mg–1; Km 16.15 mM). The second is an asymmetric synthesis by Schollkopf’s bis-lactim ether method. Alkylation of the lithiated bis-lactim ether of cyclo(-D-Val-Gly) 6 with 2,6-dibromo-4-methoxybenzyl bromide and subsequent hydrolysis with aqueous trifluoroacetic acid gives 2,6-dibromo-4-methoxy-L-tyrosine methyl ester 8. Demethylation of the ester 8 with boron tribromide affords the amino acid 1a. The enantiomeric purity of 2,6-dibromo-L-tyrosine 1a obtained by both methods has been determined by HPLC with a chiral stationary column and is found to be more than 95% (ee).

Homocysteine plays a key role in several pathophysiological conditions. To assess the methionine-homocysteine kinetics by stable isotope methodology, we developed a simultaneous quantification method of [(2)H(7)]methionine, [(2)H(4)]methionine, methionine, [(2)H(4)]homocysteine and homocysteine in rat plasma by gas chromatography-mass spectrometry (GC-MS). [(13)C]Methionine and [(13)C]homocysteine were used as analytical internal standards to account for losses associated with the extraction, derivatization and chromatography. For labeled and non-labeled homocysteine measurements, disulfide bonds between homocysteine and other thiols or proteins were reduced by dithiothreitol. The reduced homocysteine and methionine species were purified by cation-exchange chromatography and derivatized with isobutyl chlorocarbonate in water-ethanol-pyridine. Quantification was carried out by selected ion monitoring of the molecular-related ions of N(O,S)-isobutyloxycarbonyl ethyl ester derivatives on the chemical ionization mode. The intra- and inter-day precision of the assay was less than 6% for all labeled and non-labeled methionine and homocysteine species. The method is sensitive enough to determine pharmacokinetics of labeled methionine and homocysteine.

We examined the relationship between change in the redox state of the plasma albumin molecule and the metabolic disorder of sulfur amino acid observed being accompanied by reduction of renal function. Thirty-seven cases of pre-dialysis renal failure with conservative treatment and thirteen cases of chronic hemodialysis were selected as the subjects of this examination. The fraction of plasma albumin and the concentration of plasma cysteine and homocysteine were respectively measured by the HPLC and GC/MS (gas chromatography/mass spectrometry) methods. In the case of pre-dialysis renal failure with conservative treatment, the reduction rate of plasma albumin significantly decreased in correspondence with reduction of the glomerular filtration rate (GFR). It is well known that the reduction rate of plasma albumin also decreases with the aging process. However, in regard to chronic hemodialysis, a correlation with aging was not found, where the transient reduction rate of plasma albu- min increased after the hemodialysis session. However, in correspondence with the decrease in renal function, the concentration of plasma cysteine and homocysteine increased. This shows that there was a negative correlation with GFR in cases of pre-dialysis renal failure with conservative treatment. In cases of chronic hemodialysis, the concentration of free cysteine and free homocysteine rapidly decreased after a hemodialysis session. Therefore, a negative correlation was recognized between the reduction rate of plasma albumin and the concentration of plasma cysteine and homocysteine. The result of this examination shows the following mechanisms: plasma albumin plays an important role in the reaction of oxidation/reduction in blood plasma, and sulfur amino acid in blood plasma, especially the abnormality of cysteine concentration, plays an important role in changing the redox state of the blood plasma observed in the decrease in renal function.

Homocysteine, a sulfur amino acid, is an intermediate metabolite of methionine. In 1969, McCully reported autopsy evidence of extensive arterial thrombosis and atherosclerosis in children with elevated plasma homocysteine concentrations and homocystinuria. On the basis of this observation, he proposed that elevated plasma homocysteine (hyperhomocysteinemia) can cause atherosclerotic vascular disease. Hyperhomocysteinemia is now well established as an independent risk factor for atherosclerotic vascular disease. Mild hyperhomocysteinemia is quite prevalent in the general population. It can be caused by genetic defects in the enzymes involved in homocysteine metabolism or nutritional deficiencies in vitamin cofactors, certain medications or renal disease. An increase of 5 micromol per liter in the plasma homocysteine concentration raises the risk of coronary artery disease by as much as an increase of 20 mg per deciliter in the cholesterol concentration. In this article, we review the biochemical, experimental and clinical studies on hyperhomocysteinemia, with emphasis on the metabolism and pharmacokinetics of homocysteine.

Both folate and betaine, a choline metabolite, play essential roles in the remethylation of homocysteine to methionine. We have studied the effects of folate and choline deficiency on the plasma kinetics of methionine, especially remethylation of homocysteine to methionine, by means of stable isotope methodology. After a bolus intravenous administration of [(2)H(7)]methionine (5 mg/kg body weight) into the rats fed with folate-, choline-, folate + choline-deficient or control diets, the plasma concentrations of [(2)H(7)]methionine, demethylated [(2)H(4)]homocysteine, and remethylated [(2)H(4)]methionine were determined simultaneously with endogenous methionine and homocysteine by gas chromatography-mass spectrometry-selected ion monitoring. The total plasma clearance of [(2)H(7)]methionine was not significantly different among groups, suggesting that the formation of [(2)H(4)]homocysteine from [(2)H(7)]methionine was not influenced by deficiencies of folate and choline. The area under concentration-time curve of [(2)H(4)]homocysteine significantly increased in the folate- and folate + choline-deficient group as compared with the control, but not in the choline-deficient group. The time profile of plasma concentrations of [(2)H(4)]methionine in the folate-deficient group was the same as the control group, whereas the appearance of [(2)H(4)]methionine in plasma was delayed in the choline- and folate + choline-deficient group. These results suggested plasma levels of remethylated methionine were influenced by choline deficiency rather than folate deficiency.

The stereoselective kinetics of methionine enantiomers in rats was investigated to evaluate the fraction that converted from D-methionine to the L-enantiomer using a stable isotope methodology. After bolus i.v. administration of D -o rL-( 2 H3)methionine, their plasma concentrations and that of endogenous L-methionine were determined by a stereoselective GC-MS method. L-( 2 H3)Methionine appeared rapidly after administration of D-( 2 H3)methionine, whereas D-( 2 H3)methionine was not detected after administration of L-( 2 H3)methionine. The fraction of conversion of D-( 2 H3)methionine into L-( 2 H3)methionine was estimated using the area under the plasma concentration vs. time curve of L-( 2 H3)methionine on D-( 2 H3)methionine administration and total clearance of L-( 2 H3)methionine on L-( 2 H3)methionine administration, and that fraction was 90%. This result demonstrates that almost all i.v. administered D-methionine is converted into the L-enantiomer in vivo. J. Nutr. 135: 2001-2005

Urate, the final oxidation product of purine metabolism, is excreted into urine in humans. Clinically, increased serum urate levels are indicative of pregnancy‐induced hypertension (PIH). However, how urate is handled in the placenta is still largely unknown. In this study, we compared maternal serum urate levels with those of umbilical cord blood and investigated urate transport mechanisms in BeWo cells, a trophoblast‐derived cell line. The maternal and umbilical cord blood samples and placentas were collected from patients undergoing cesarean section at Kyorin University Hospital after obtaining informed consents. There were no significant differences in serum urate levels between maternal blood and umbilical cord blood, and between umbilical cord vein and arterial blood, suggesting that urate is freely movable at the placenta and that fetus is not a major source of urate production. RT‐PCR and immunohistochemistry showed that urate transporters including OAT4, OAT10, GLUT9/URATv1 and ABCG2 were expressed in the syncytiotrophoblast cells in the placenta as well as BeWo cells. Despite expressing aforementioned urate transporters BeWo cells did not take up urate. After confirming the formation of tight junctions of these cells cultured on the transwell, urate transport between upper and lower chambers was measured. Urate moved through BeWo cell monolayers with nonsaturation kinetics and this movement was observed even when the cells were incubated at 4°C, suggesting that urate moves through the paracellular route by simple diffusion., Serum urate concentration was identical between mother and fetus, indicating that urate can pass through blood‐placental barrier. Using trophoblast‐derived BeWo cell monolayer, this study demonstrated that urate moved through BeWo cell monolayer via paracellular route.

A gas chromatographic–mass spectrometric method for the simultaneous determination of methionine and total homocysteine in human plasma is described. dl-[2H4]Methionine and dl-[2H8]homocystine were used as internal standards. The method involved reduction of the disulfide bond with dithiothreitol, purification by cation-exchange chromatography using a BondElut SCX cartridge and derivatization with isobutyl chlorocarbonate in water–ethanol–pyridine. Quantitation was performed by selected-ion monitoring of the quasi-molecular ions of N(O,S)-isobutyloxycarbonyl ethyl ester (IBC-OEt) derivatives for methionine and [2H4]methionine, respectively, and the fragment ions ([M+H–COOisoBu–COOEt]+) for IBC-OEt derivatives for homocysteine and [2H4]homocysteine, respectively. The sensitivity, specificity, accuracy and precision of the method were demonstrated to be satisfactory for measuring concentrations of methionine and total homocysteine in human plasma.

The stereoselective acyl migration of diastereomeric 1beta-O-acyl glucuronides of (R)- and (S)-2-phenylpropionic acid [(R)-1PG and (S)-IPG, respectively] in phosphate buffer (pH 7.4) at 310K was investigated using HPLC. The disappearance of (R)-1PG was faster than that of (S)-1PG according to pseudo first-order kinetics. A kinetic model describing the degradation reactions was constructed. The rate constant for acyl migration from the 1beta-O-isomer to the 2-O-acyl isomer (k12) was about one order magnitude larger than that for hydrolysis from 1beta-O-acyl isomer to aglycone (k10). The k12 of (R)-IPG (0.377 +/- 0.005 h(-1)) was about two times larger than that of (S)-IPG (0.184 +/- 0.003 h(-1)). The results indicated that the stereoselectivity in the degradation of 1PG was apparently governed by the acyl migration from 1-isomer to 2-isomer. The kinetic parameters for acyl migration from 1-isomer to 2-isomer were estimated from temperature-dependent experiments using the transition state theory. The value of the free energy of activation at 310 K for (R)-1PG (99.67 kJ/mol) was smaller than that of (S)-IPG (101.60kJ/mol), suggesting that (R)-IPG showed thermodynamically higher reactivity in acyl migration than (S)-1PG.

A high-performance liquid chromatographic procedure has been developed for the determination of [ d -Ala2, d -Leu5]enkephalin (DADLE) and the fragments containing d -leucine in rat blood. The procedure was applied to the determination of blood levels of [3H- d -Leu5]DADLE and the C-terminal fragments after intravenous administration of [3H- d -Leu5]DADLE to a rat. Unlabelled DADLE and the C-terminal fragments were spiked as carriers to rat blood samples and the blood samples were extracted with 1% trifluoroacetic acid in methanol. The recoveries from rat blood were quantitative for all compounds. DADLE and the C-terminal four fragments were well separated on a reversed-phase column with gradient elution using a mobile phase composed of 0.14% HClO4 and acetonitrile.

The stereoselective pharmacokinetics of suprofen enantiomers has been studied in humans by means of stable isotope-labeled pseudoracemate-diastereomer methodology. After a single oral dose of a near equimolar mixture of unlabeled-(R)-(−)- and [2H3]-(S)-(+)-suprofen [or unlabeled-(S)- and [2H3]-(R)-suprofen] to three healthy male subjects, the plasma concentrations of drug were determined by a stereospecific gas chromatography-mass spectrometry method. Racemic [2H7]suprofen was used as an internal standard. The method involved chiral derivatization with (S)-(−)-1 -(naphthyl)ethylamine to form the diastereomeric amide. The plasma concentrations were consistently higher for the (R)-isomer than the (S)-isomer. No significant difference in the elimination half-life of the enantiomers was observed. An average of 6.8% of an administered dose of the (R)-isomer was stereospecifically inverted to the (S)-isomer. There was no measurable inversion of the (S)-to (R)-isomer. The present stable isotope-labeled pseudoracemate-diastereomer methodology has made it possible to evaluate the pharmacokinetics of each enantiomer, including the estimation of chiral inversion after administration of the racemic mixture.

The application of a stable isotope coadministration technique for estimating the fraction (fm) of imipramine (IP) that is converted to desipramine (DMI) is described. Four healthy male subjects received 25 mg of IP-d4 hydrochloride orally with 25 mg of DMI hydrochloride. The plasma concentrations of IP-d4, DMI-d4, and DMI were determined by capillary gas chromatography-mass spectrometry-selected ion monitoring using d8 analogues as internal standards. The fm values, calculated from the ratio of the area under the plasma concentration–time curve of DMI-d4 to that of DMI, varied from 0.54 to 0.85.

A method for the stereoselective assay of the (+)- and (-)-enantiomers of suprofen and [2H3]suprofen in human plasma was developed using gas chromatography-mass spectrometry-selected-ion monitoring. (+/-)-[2H7]Suprofen was used as an internal standard. The method involved diethyl ether extraction and chiral derivatization with S-(-)-1-(naphthyl)ethylamine to form diastereomeric amide. The diastereoisomers were separated on a capillary gas chromatograph-mass spectrometer. Quantitation was achieved by selected-ion monitoring of the quasi-molecular ions of the diastereoisomers. The sensitivity, specificity, accuracy and reproducibility of the method were demonstrated to be satisfactory for application to pharmacokinetic studies of suprofen enantiomers.