A mechanism for epoxidation of cholesterol by hepatic microsomal lipid hydroperoxides.

Evidence was obtained, using cis-stilbene as a model substrate, for the participation of peroxy and/or oxy radicals in epoxidation of cholesterol by rat liver microsomal phospholipid hydroperoxides and a ferrous ion-ADP complex. Under the conditions used, cholesterol was epoxidised to the alpha- and beta-epoxides in the ratio 1:2-4, and cis-stilbene to trans-stilbene oxide without concomitant formation of the cis-oxide. Microsomal phospholipid hydroperoxides could be replaced with methyllinoleate monohydroperoxide for the epoxidation of both substrates. The hydroperoxide-mediated epoxidations were completely inhibited by alpha-tocopherol and t-butylhydroxyanisole. A GLC study suggested that highly polyunsaturated fatty acyl constituents of the microsomal phospholipids might play an important role in epoxidation of the olefinic substrates.

Photogeneration of 3beta-hydroxy-5alpha-cholest-6-ene-5-hydroperoxide in rat skin: evidence for occurrence of singlet oxygen in vivo.

We identified singlet oxygen adduct of cholesterol, 3beta-hydroxy-5alpha-cholest-6-ene-5-hydroperoxide (5alpha-OOH), in skin of rats pretreated with oral doses of pheophorbide a and subsequent visible irradiation, that have been known to induce photosensitive diseases in animals and humans. In a living animal body, this is the first demonstration of presence of 5alpha-OOH, that is known to be formed exclusively by reaction in vitro between singlet oxygen and cholesterol. By the quantitative determination with high performance liquid chromatography equipped with a chemiluminescence detector, we observed time-dependent increase in concentrations of 5alpha-OOH in skin of rats pretreated with oral doses of pheophorbide a and subsequent visible irradiation, suggesting the occurrence of a labile activated oxygen species, singlet oxygen, in this system.

Increases in cholesterol 7-hydroperoxides in lipids of human skin by sunlight exposure.

Free and ester forms of cholesterol 7alpha- and 7beta-hydroperoxides (Ch 7-OOHs) in skin lipids of humans were separated and determined by high performance liquid chromatography with a chemiluminescence detector. We first demonstrated the presence of Ch 7-OOHs in lipids of human skin. The levels of Ch 7-OOHs found in skin lipids of healthy Japanese volunteers (n = 5) ranged from 2.78 to 25.2 pmol/cm2 skin, indicating large inter-individual differences. However, the intra-individual differences of Ch 7-OOHs levels in skin lipids between right and left arms were less than 25% (-16.4% to 24.0%). Inter-day differences of Ch 7-OOHs in 5 subjects at 1 week interval were also small (-36.7% to 47.7%). Additionally, we investigated effects of sunlight exposure on the levels of Ch 7-OOHs in skin lipids of healthy Japanese volunteers (n = 24). The levels of Ch 7-OOHs in skin lipids significantly increased from 10.0+/-6.7 to 38.9+/-38.0 pmol/cm2 skin by sunlight exposure (10-40 mJ/cm2/min) for 3 h. Therefore, natural sunlight exposure causes lipid peroxidation in skin lipids of humans. These results suggest that the level of Ch 7-OOHs is a good marker for lipid peroxidation in human skin.

Quantitative determination of cholesterol 5alpha-, 7alpha-, and 7beta-hydroperoxides in rat skin.

An assay method for determination of cholesterol 5alpha-, 7alpha-, and 7beta-hydroperoxides (ChOOHs) in rat skin using high-performance liquid chromatography (HPLC) with a chemiluminescence detector has been developed. In the assay method, free form and free plus ester forms of ChOOHs could be separately determined by HPLC in combination with the treatment of a tissue extract by cholesterol esterase. Lower limits of quantitation for cholesterol 5alpha-, 7alpha-, and 7beta-hydroperoxides were 0.2, 0.1, and 0.5 nmol/g skin, respectively. This assay method showed that (i) good absolute recoveries of ChOOHs from rat skin (80-90% of radiolabeled ChOOHs added to rat skin); (ii) negligible autoxidation of cholesterol caused by the assay procedure (<9.4x10(-5)% of radiolabeled cholesterol added to rat skin); and (iii) good correlation between ChOOHs added to rat skin and ChOOHs determined, indicating this assay method is applicable to quantify ChOOHs in rat skin. By using this assay method, we observed that (i) cholesterol 5alpha-hydroperoxide was detected in skin of rats pretreated with oral doses of pheophorbide a and subsequent visible irradiation; (ii) concentrations of cholesterol 7-hydroperoxides in skin of rats in an ambient light room were not significantly different from those in a dark room for 12 weeks; and (iii) ultraviolet light B irradiation markedly enhanced the concentrations of cholesterol 7-hydroperoxides in the skin of rats.

Inversion of enantioselectivity in glutathione conjugation of 9,10-dihydrobenzo[a]pyrene 7,8-oxide in hepatic cytosol of rats following induction of hepatic hyperplastic nodules by chemical carcinogens.

Enantiomers of 9,10-dihydrobenzo[a]pyrene 7,8-oxide (DBPO) were stereoselectivity conjugated with glutathione (GSH) specifically at benzylic carbon (C7) in normal Sprague--Dawley (SD) rat liver cytosol: (7R,8S)-(+)- greater than (7S,8R)-(-)-DBPOs. In contrast, in liver cytosol of SD rats bearing hepatic hyperplastic nodules induced with chemical carcinogens, (7S,8R)-(-)-DBPO was preferentially conjugated with GSH to (7R,8S)-(+)-DBPO. GSH S-transferases (GSTs) having sub-unit protein 4 were strongly suggested to play an important role in the preferential conjugation of (7R,8S)-(+)-DBPO in the normal rat liver cytosol, while the preferential conjugation of (7S,8R)-(-)-DBPO in the liver cytosol of the rat bearing hepatic hyperplastic nodules, was most likely to be attributable to GST 7-7, a characteristically induced protein in the hepatic hyperplastic nodules.

Regiospecific and diastereoselective inactivation of mutagenic 9,10-dihydrobenzo[a]-pyrene 7,8-oxide by hepatic cytosolic glutathione S-transferase.

Racemic, (7R,8S)-(+)-, and (7S,8R)-(-)-9,10-dihydrobenzo[a]pyrene 7,8-oxides (DBPOs) showed markedly different mutagenicity towards Salmonella typhimurium TA 98 in the order of (7R,8S)-(+)- greater than racemic greater than (7S,8R)-(-)-DBPOs. The enantiomeric epoxides were inactivated at significantly different rates by preincubating with rat liver cytosol fortified with glutathione (GSH) in the order of (7S,8R)-(-)- greater than racemic greater than (7R,8S)-(+)-DBPOs. Two non-mutagenic water-soluble metabolites were isolated from the preincubation mixture containing racemic DBPO as a substrate, separated by hplc, and identified by 13C nmr and uv absorption spectroscopy as diastereoisomers of S-(8-hydroxy-7,8,9,10-tetrahydrobenzo[a]pyren-7-yl)glutathione (conjugates I and II). Conjugates I and II were specifically yielded from (7R,8S)-(+)- and (7S,8R)-(-)-DBPOs, respectively, at different rates by rat liver cytosol; apparent values of Km were 20.1 and 15.6 microM and of Vmax 17.2 and 26.7 nmole/mg protein/min for (7R,8S)-(+)- and (7S,8R)-(-)-DBPOs, respectively. Conjugates I and II, therefore, were reasonably assigned to have (7S,8S)- and (7R,8R)-configurations, respectively. Conjugate II was yielded preferentially to conjugate I from racemic DBPO at an early stage of the enzyme reaction.

Studies on metabolism and toxicity of styrene--VI. Regioselectivity in glutathione S-conjugation and hydrolysis of racemic, R- and S-phenyloxiranes in rat liver.

Rat liver cytosol converted phenyloxirane enantiomers regioselectively to glutathione S-conjugates. R-(+)-Phenyloxirane was converted to S-(1-phenyl-2-hydroxyethyl)glutathione (conjugate 1) and S-(2-phenyl-2-hydroxyethyl)glutathione (conjugate 2) (ratio 6.1:1), and S-(-)-phenyloxirane to conjugates 1 and 2 (ratio 1:32). Racemic phenyloxirane was converted to conjugates 1 and 2 (ratio 1.8:1). The conjugates were separated by HPLC on an octadecylsilicone column and identified with synthetic specimens whose structures were assigned by 13C NMR spectrometry. R-(+)-, S-(-)- and racemic phenyloxiranes were hydrolyzed to R-(-)-, S-(+)- and racemic phenylethanediols by microsomal epoxide hydrolase without inversion of absolute configurations of their benzylic carbons. R-(+)-Phenyloxirane had much smaller Km and Vmax than the S-(-)-oxirane did. The R-(+)-oxirane potentially inhibited the microsomal hydrolysis of the S-(-)-oxirane and was preferentially hydrolyzed when the racemic oxirane was used as the substrate. Microsomal monooxygenase oxidized styrene to R-(+)- and S-(-)-phenyloxiranes (ratio 1.3:1), and the ratio was little changed by the pretreatment of the animal with phenobarbital, 3-methylcholanthrene and polychlorinated biphenyls.

Cholesterol 7-hydroperoxides in rat skin as a marker for lipid peroxidation.

Concentrations of cholesterol 7alpha- and 7beta-hydroperoxides (Ch 7-OOHs) in the skin of rats were determined by HPLC with a chemiluminescence detector. We demonstrated that (a) the concentrations of Ch 7-OOHs in rat skin were highly correlated with rat age (r = 0.929; N = 51, 1 to 55 weeks old), (b) the concentrations of Ch 7-OOHs in the skin of rats in an ambient light room were not significantly different from those found in rats kept in a dark room for 12 weeks, and (c) lipid peroxidation in vitro induced by ADP-Fe2+ caused an increase in the concentrations of Ch 7-OOHs in homogenates of rat skin. These results indicated that levels of Ch 7-OOHs in skin might be a good marker for aging of rats and might be independent of housing illumination, thus a good marker for endogenous lipid peroxidation. Furthermore, we observed that ultraviolet light B (UVB) irradiation markedly enhanced the concentrations of Ch 7-OOHs in the skin of rats in vivo depending on the duration of the irradiation, and the increases in Ch 7-OOHs were inhibited by radical scavengers, i.e. tocopherols. Therefore, it was suggested that the levels of Ch 7-OOHs in the skin could also be a good marker for UVB-dependent lipid peroxidation.

7-Glycidoxycoumarin (GOC): a fluorophotometric epoxide substrate for the assay of glutathione S-transferase activity.

7- Glycidoxycoumarin ( GOC ), a new fluorophotometric epoxide substrate for glutathione S-transferase (GSH TFase ), was conjugated regiospecifically with GSH at pH 6.5 in rat liver cytosol to yield S-(2-hydroxy-3-(7'- coumaroxy )-1-propyl)glutathione which was isolated by HPLC and identified with an authentic specimen by 13C NMR spectroscopy. The conjugation product formed in the incubation media consisting of GOC , GSH, and 9000 g supernatant fractions from various tissues of the rat, was directly determined by photometry of fluorescence emission at 388 nm at an excitation wavelength of 328 nm after removal of the unreacted substrate and its enzymic hydrolysis product, 7-(1',2'-dihydroxy-3'-propoxy)coumarin, by simple extraction with isobutyl alcohol in the presence of a saturating amount of sodium chloride. Stability of GOC at pH 6.5 markedly retarded its autoconjugation with GSH and made the fluorophotometric method sensitive enough to assay small GSH TFase activities in gel column chromatographic fractions as well as in various tissues of the animals. Apparent Km and Vmax for GOC in rat liver cytosol were 55 microM and 7.41 nmole/mg protein/min, respectively. GSH conjugation of GOC was catalyzed by at least two isozymes, E and AA, of hepatic GSH TFases .

Glutathione conjugation of styrene 7,8-oxide enantiomers by major glutathione transferase isoenzymes isolated from rat livers.

Male Sprague-Dawley rat liver cytosol mediated regioselective conjugation of styrene 7,8-oxide (STO) enantiomers with glutathione in completely trans-ring-opening manner to afford (1S)-S-(1-phenyl-2-hydroxyethyl)glutathione and (2R)-S-(2-phenyl-2-hydroxyethyl)glutathione in the ratio 22:1 for (R)-STO and also to afford (1R)-S-(1-phenyl-2-hydroxyethyl)glutathione and (2S)-S-(2-phenyl-2-hydroxyethyl)glutathione in the ratio 12:1 for (S)-STO. In the above cytosolic reactions, (R)-STO was conjugated 1.8 times faster than (S)-STO, while the (R)- to (S)-ratio in rate of the conjugation was 2.7 when racemic STO was used as a substrate. A kinetic study, carried out by using six major glutathione transferase (GST) isoenzymes isolated from the cytosol, indicated that GSTs 3-3, 3-4 and 4-4 (class mu enzymes) had much higher Kcat/Km values towards both STO enantiomers than the other three major isoenzymes, GSTs 1-1, 1-2 and 2-2 (class alpha enzymes). All the class mu enzymes mediated preferential glutathione conjugation of (R)-STO to (S)-STO. On the contrary, the class alpha enzymes catalysed the conjugation of (S)-STO preferentially to (R)-STO. The kinetic study strongly suggested that GSTs determining the higher enantioselectivity towards (R)-STO in the rat liver cytosol were the class mu enzymes, especially GST 3-3, which had the highest Kcat/Km value towards (R)-STO as well as the highest (R) to (S) ratio in the enantioselectivity among the six isoenzymes examined. GST 7-7, isolated as a major enzyme from the liver cytosol of the animals bearing hepatic hyperplastic nodules which were induced by chemical carcinogens, catalysed preferential GSH conjugation of (S)-STO to (R)-STO.

Glutathione conjugation of the fluorophotometric epoxide substrate, 7-glycidoxycoumarin (GOC), by rat liver glutathione transferase isoenzymes.

The fluorophotometric substrate, 7-glycidoxycoumarin (GOC), was examined for the assay of epoxide-glutathione (GSH)-conjugating activities of seven major GSH transferases (GSTs) isolated from rat liver cytosols. GST 7-7 (GST-P), isolated from the liver cytosol of rats bearing hepatic hyperplastic nodules, catalysed the GSH conjugation of GOC at a higher rate than any other examined GST isolated from the normal rat liver cytosol. GSTs 3-3, 3-4 and 4-4 (group 3-4 enzymes) had specific activities towards GOC by one fifth to one third of that of GST 7-7. GSTs 1-1, 1-2 and 2-2 (group 1-2 enzymes) had very low activities towards this epoxide. A kinetic study indicated that GST 7-7 showed the largest kappa cat/Km value for the catalytic reaction of GOC-GSH conjugation among the GSTs. In spite of their much smaller kappa cat values, group 3-4 enzymes showed much larger kappa cat/Km values for GOC than the group 1-2 enzymes, because GOC had a much higher affinity for group 3-4 enzymes than for group 1-2 enzymes. A comparative study was also done with GSH conjugations of styrene 7,8-oxide (STO) and 1-chloro-2,4-dinitrobenzene by the GSTs. Unlike GOC, the conjugation of STO was mediated at rates about twice as high by group 3-4 enzymes than by GST 7-7. STO was also a very poor substrate for group 1-2 enzymes.

The demethylenation of methylenedioxymethamphetamine ("ecstasy") by debrisoquine hydroxylase (CYP2D6).

The metabolism of methylenedioxymethamphetamine (MDMA, "ecstasy") was examined in a microsomal preparation of the yeast Saccharomyces cerevisiae expressing human debrisoquine hydroxylase, CYP2D6. Only one product, dihydroxymethylamphetamine (DHMA), was detected in the incubation mixture, and this product accounted for all of the substrate consumption at low concentration (10 microM). Mean +/- SD values of apparent Km(microM) and Vmax (nmol/min per nmol P450) for the demethylenation of (+) and (-)-MDMA at low concentrations (1-100 microM) were 1.72, 0.12 and 6.45, 0.10 and 2.90, 0.10 and 7.61, 0.06, respectively. At high concentrations (> 1000 microM) substrate inhibition was noted, with Ki values of 14.2 and 28.2 mM, respectively, for the (+) and (-) enantiomers. Incubation of MDMA isomers with human liver microsomes indicated that their demethylenation is deficient in the poor metabolizer phenotype. Thus, MDMA is converted to the catecholamine DHMA by CYP2D6, and this may give rise to genetically-determined differences in toxicity.

A questionnaire for neurological symptoms in patients with diabetes--cross-sectional multicenter study in Saitama Prefecture, Japan.

This study was designed to investigate the prevalence of neurological symptoms in diabetic patients living in Saitama Prefecture, Japan using 13-item questionnaire. A total of 6472 outpatients with diabetes (3417 men and 3055 women) were recruited from 100 centers. Mean age and mean disease duration were 60.9-year old and 10.4 years, respectively. The questionnaire for monitoring of neurological symptoms was completed at the clinic or hospital visited, and Achilles' tendon reflex, ophthalmologic, blood and urinary examinations were also performed. Of the 6472 patients, 84.8% suffered from a mean of 3.3+/-2.2 neurological symptoms. However, half of these symptoms were not considered to be those of diabetic neuropathy by attending physicians. "Feeling as if a piece of paper is attached to the sole of the foot," "stinging and prickling sensations in feet," and "pain in feet" were the most common symptoms of diabetic neuropathy. The prevalence of diabetic neuropathy as determined by attending physician increased with disease duration and worse control of diabetes. This study found that the majority of diabetics were suffered from neurological symptoms, although half of such symptoms were not considered to be those of diabetic neuropathy by physicians. Furthermore, it is important for diabetics to be diagnosed and treated earlier to prevent progression to severe neuropathic complications by means of optimal glycemic control and use of some chemicals such as aldose reductase inhibitor, and to develop this study to evaluate the efficacy of treatments.

Major hydroxysteroid sulfotransferase STa in rat liver cytosol may consist of two microheterogeneous subunits.

The possible existence of two microheterogeneous subunits, designated ST-40P and ST-41P, of hydroxysteroid sulfotransferases in female Sprague-Dawley rat liver cytosol was demonstrated by cloning and sequencing of cDNAs, both isolated from two rat liver cDNA libraries. These subunits consisted of an equal number of amino acid residues with only one amino acid substitution. ST-40P and ST-41P expressed as homodimers from the ST-40 and ST-41 cDNAs in Escherichia coli had enzyme activities toward all of the examined 20 hydroxysteroids, 13 bile acids, and the carcinogen 5-hydroxymethylchrysene (5-HCR), with formation of the reactive metabolite 5-HCR sulfate, at rates very similar to those by STa, the major hydroxysteroid sulfotransferase in rat liver cytosol. This strongly suggested that they are essential components of STa. The present study carried out by using the recombinant enzymes provides the first direct evidence for the identity of sulfotransferases catalysing the sulfation of hydroxysteroids and bile acids and proposes that the current nomenclature system used for distinguishing hydroxysteroid sulfotransferases from bile acid sulfotransferases should be improved.

Molecular cloning and functions of rat liver hydroxysteroid sulfotransferases catalysing covalent binding of carcinogenic polycyclic arylmethanols to DNA.

Three sulfotransferases (STs) catalysing the metabolic activation of potent carcinogenic polycyclic arylmethanols were purified from female Sprague-Dawley (SD) rat liver cytosol without loss of their enzyme activities in the presence of Tween 20 used for preventing the enzymes from aggregation during purification and identified as hydroxysteroid sulfotransferases (HSTs). All the purified HSTs, STa, STb, and STc, with different electric charges had an apparently equal size of subunit (30.5 kDa) and cross-reacted with polyclonal antibody raised against STa. Our study on molecular cloning of cDNA libraries from two female SD rat livers indicated that both contained cDNA inserts coding for 5 different HST subunits, consisting of 284-285 amino acid residues (M(r), 33,084-33,535) and sharing strong amino acid sequence identity (> 83%). Of the 5 HST subunits, two had an identical amino acid sequence except for only one amino acid residue, and the other two contained only 6 amino acid substitutions in their sequences.

Studies on metabolism and toxicity of styrene. IV. 1-Vinylbenzene 3, 4-oxide, a potent mutagen formed as a possible intermediate in the metabolism in vivo of styrene to 4-vinylphenol.

1-Vinylbenzene 3,4-oxide, a putative intermediate in the metabolism of styrene to 4-vinylphenol, was synthesized and examined for its obligatory intermediacy to the phenol, its physical properties, and its mutagenicity toward Salmonella typhimurium TA98 and TA100. The 3,4-oxide had a half-life of 4.3 sec at pH 7.4 in an aqueous solution, and yielded 4-vinylphenol quantitatively without concomitant formation of any trace amount of 3-vinylphenol. The 3,4-oxide had a potent mutagenicity toward the TA100 bacteria but not toward the TA98 strain, whereas it showed a potent cytotoxicity to both of them His+ revertant colonies induced by the 3,4-oxide were 7233/plate at a total dose of 1.0 micromole/plate when it was applied in a sequential manner to the bacterial suspension during the pre-incubation of the testing system. Under the same conditions, benzo[a]pyrene 4,5-oxide and phenyloxirane showed 1283 and 1657 of His+ revertant colonies/plate at 19 nmoles and 10 micromoles/plate, respectively, as the maximal activities. The isomeric arene oxide, 1-vinylbenzene 1,2-oxide, had a longer half-life (1.63 min) than the 3,4-oxide at pH 7.4 in aqueous solution and was specifically rearranged to 2-vinylphenol. The 1,2-oxide also showed more potent mutagenicity to the TA100 strain bacteria than phenyloxirane but weaker than the 3, 4-oxide. 4- and 2-vinylphenols were neither mutagenic nor cytotoxic to the bacteria at concentrations ranging up to 4 micromoles/plate.

Changes in the hepatic glutathione peroxidase redox system produced by coplanar polychlorinated biphenyls in Ah-responsive and -less-responsive strains of mice: mechanism and implications for toxicity.

The alteration in hepatic glutathione peroxidase (GPx) produced by polychlorinated biphenyls (PCBs) was studied in vivo in aryl hydrocarbon (Ah)-responsive C57BL and -less-responsive DBA strains of mice. 3,3',4,4',5-Pentachlorobiphenyl (PCB 126), one of the high-affinity ligands for the Ah receptor, significantly reduced Se-dependent GPx activity in C57BL mice, but not in DBA mice. A reduction in activity in C57BL mice was also observed following treatment with a high dose of 3,3',4,4'-tetrachlorobiphenyl with lesser affinity for the Ah receptor than PCB 126, but not by 2,2',5,5'-tetrachlorobiphenyl, a low-affinity ligand. To assess the effects on GPx in the liver, the content of reduced glutathione (GSH), an obligate co-factor for GPx, and the activity of two enzymes, γ-glutamyl transpeptidase (γ-GTP) and glutathione reductase (GR), which play a role in supplying GSH were determined after PCB treatment. The results showed that although the hepatic activity of γ-GTP and GR was affected differently by PCB 126, the content of GSH was slightly increased rather than reduced in both strains of mice. The activity of non-Se-dependent GPx, which is due to the catalysis by some isozymes of glutathione S-transferase (GST), was significantly increased only in C57BL mice by PCB 126 treatment. Immunoblot analysis demonstrated that the induction of the class θ GST, which is a potent reducer of peroxides (Hiratsuka et al., 1995. Biochem. Biophys. Res. Commun. 212, 743) reflects the enhancement of the above activity. These results suggest that (i) the PCB-induced reduction in Se-dependent GPx activity is mediated by a mechanism involving the Ah receptor; and (ii) a concomitant increase in the class θ GST partially rescues the Ah-responsive mice from coplanar PCB-induced oxidative stress.

[Evaluation of the effective use of the "health notebook"].

Since 1983, with the institution of the "Health Service Law for the Aged", the "health notebook" has been issued to people aged 40 years and over in order to aid in management of their health. Few people actually fill their health data in notebook by themselves. In order to develop effective use of the health notebook by residents and health professionals, the uses of the health notebooks by residents aged 40 years and over, public health nurses, and physicians were investigated. Three hundred and fifty four residents aged 40 and over, 41 public health nurses, and 18 physicians were studied in 1990, in Yamagata city. A majority of residents took their health notebooks with them to health consultations, and public health nurses used the notebooks to provide advice to them. Public health nurses effectively issued the health notebooks to residents using occasions where residents gathered. Some physicians reported that health notebooks were useful for motivating the people to maintain their health, while others preferred using a health card media. When comparing the health notebooks to the maternity passbooks, health notebooks need to be more easily utilized by users for recording information, and their value should be effectively explained to them. Furthermore, in order to promote self-care behaviors, greater use of health notebooks by all health professionals in indicated.

Stable isotope dilution mass spectrometric assay for PRPP using enzymatic procedures.

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.