Abnormal glutathione conjugation in patients with tyrosinaemia type I.

Previous studies have suggested that tyrosinaemia type I may be associated with reduced glutathione availability due to conjugation of tyrosinaemia-associated reactive intermediates with glutathione. In the present study, the glutathione/ glutathione S-transferase system of two tyrosinaemia patients and three healthy controls were characterized by administering the racemic sedative drug bromisoval, a probe drug for assessing glutathione conjugation activity in vivo. Furthermore, concentrations of glutathione and glutathione S-transferase class alpha (GSTA) isoenzymes as well as the glutathione S-transferase class mu phenotype were assessed in the blood of six tyrosinaemia patients. The excretion of bromisoval mercapturates in healthy children was comparable to that observed in healthy adults. Tyrosinaemia patients were found to have a very high urinary recovery of bromisoval mercapturates (> or = 60% of the dose compared to about 30% for healthy, age-matched children and adults), which could be attributed mainly to a higher urinary excretion of the mercapturate derived from S-bromisoval. Healthy children and adults predominantly excrete the (R)-bromisoval mercapturate. The differences in amount excreted as well as in stereoselectivity of the urinary excretion of bromisoval mercapturates in tyrosinaemia patients are possibly related to an increased activity of specific glutathione S-transferase isoenzymes. Plasma glutathione and blood cell glutathione disulphide concentrations in tyrosinaemia patients were normal. Low blood cell glutathione concentrations were in general found only in two patients with a poor clinical condition. These results indicate that, in contrast to previous suggestions, reduced glutathione availability is not a generalized problem in (stabilized) tyrosinaemia patients.

Effect of ifosfamide treatment on glutathione and glutathione conjugation activity in patients with advanced cancers.

Several studies have suggested that the glutathione/glutathione S-transferase (GSH/GST) system is involved in resistance of tumors toward ifosfamide and other cytostatic agents. Besides, ifosfamide metabolites (in vitro) as well as ifosfamide treatment (in vivo) have been shown to decrease cellular GSH availability. In the present study, the in vivo effects of three different ifosfamide treatment schedules on the GSH/GST system were studied in patients with advanced cancers (n = 24): continuous i.v. infusions of 1300 mg/m2 daily for 10 days and 5000 mg/m2/day for 24 h, as well as a 4-h infusion of 3000 mg/m2 daily for 3 days. The GSH/GST system was characterized by administering bromisoval, a probe drug to assess GSH conjugation activity in vivo, as well as by daily monitoring of GSH concentrations in blood cells and plasma. Bromisoval pharmacokinetics was assessed before and at the end of the ifosfamide treatment. Blood cell GSH levels decreased significantly (P < 0.05) during the 3- and 10-day ifosfamide treatment schedules; the 24-h treatment had no effect. The ifosfamide treatment schedules had only minimal effects on bromisoval pharmacokinetics. Assuming that the kinetics of the probe drug provide an accurate reflection of enzyme activity, this suggests that GST activity remains unchanged. Because GSH conjugation of bromisoval enantiomers requires both GST activity and GSH availability, these results also indicate that, despite the 35% decrease in GSH in blood cells of two patient groups, the GSH availability of the cancer patients was not rate-limiting for GSH conjugation of bromisoval enantiomers. If GSH levels in blood cells reflect those in tumors/other tissues, the present results indicate that ifosfamide may be used clinically to decrease GSH levels. However, whether a 35% decrease is sufficient to increase tumor sensitivity toward (other) cytostatics remains uncertain.

Characterization of glutathione conjugation in humans: stereoselectivity in plasma elimination pharmacokinetics and urinary excretion of (R)- and (S)-2-bromoisovalerylurea in healthy volunteers.

Characterization of glutathione conjugation in vivo was performed in 12 healthy male volunteers by use of the racemic drug bromisovalum (bromisoval; 2-bromoisovalerylurea) as a model substrate. To study whether the pharmacokinetics of both bromisovalum enantiomers was related to the glutathione S-transferase class Mu phenotype, six subjects who were class Mu deficient and six subjects who were not class Mu deficient participated. After oral administration of 600 mg racemic bromisovalum, enantioselective measurement of unchanged bromisovalum (plasma and saliva) and the diastereomeric bromisovalum mercapturates (urine) showed a pronounced stereoselectivity in all subjects. The plasma clearance of R-bromisovalum was about 12 times higher than that of S-bromisovalum (9.3 +/- 3.7 and 0.78 +/- 0.38 L/min, respectively), which was in agreement with the higher urinary cumulative excretion for the mercapturate derived from R-bromisovalum: 26% +/- 4% of the dose versus 8% +/- 3% of the dose for the mercapturate derived from S-bromisovalum. Both the bromisovalum pharmacokinetics in general and the stereoselectivity in bromisovalum pharmacokinetics were not different for the subjects who were glutathione S-transferase class Mu deficient and the subjects who were not glutathione transferase class Mu deficient.

Enantioselective determination of R- and S-(alpha-bromoisovaleryl)urea in plasma using high-performance liquid chromatography after solid-phase extraction.

A stereoselective method has been developed for the determination of R- and S-(alpha-bromoisovaleryl)urea in plasma and saliva after oral administration. The chiral separation was carried out on Chiralcel OJ or OD columns with hexane--2-propanol as the mobile phase. The poor detection properties of the analyte required the development of an effective sample pretreatment procedure to enable ultraviolet detection at 210 nm. Solid-phase extraction using hydrophobic Amberlite XAD-2 in combination with washing steps at alkaline and acidic pH completely removed interfering components of the biological matrix and allowed the detection of the optical isomers at concentrations down to 10 ng/ml (0.05 microM). The method was validated by determining the recovery, linearity, accuracy and within-day and between-day repeatability at 50, 200 and 2000 ng/ml. Application to the analysis of plasma and saliva samples is demonstrated.

Stereoselectivity of glutathione conjugation: blood elimination of alpha-bromoisovalerylurea enantiomers and biliary excretion of the conjugates in unanesthetized normal or congenitally jaundiced rats.

Stereoselectivity of glutathione conjugation was studied in unanesthetized normal and congenitally jaundiced rats (Groningen Yellow), using the separate enantiomers of alpha-bromoisovalerylurea (BIU) as substrates. The blood elimination half-lives of (R)- or (S)-BIU were 8 and 38 min, respectively. The excretion half-lives of the GSH conjugates in bile in normal rats showed a similar difference: (R)-BIU yielded exclusively (S)-IU-S-G with a T1/2 of 12 min, and (S)-BIU yielded only (R)-IU-S-G with a T1/2 of 36 min. In normal rats 45-47% of the dose of (R)-BIU and (S)-BIU was found in bile as glutathione (GSH) conjugate, and 19-25% was excreted in urine as mercapturates. Similar values in the mutant rats indicated that BIU elimination by GSH conjugation was unimpaired, but the GSH conjugates were absent from bile. In the urine twice as much mercapturates was found as in normal rats. The GSH content and the activity of the glutathione-S-transferases in the liver were similar in mutant and controls. The data on blood elimination of the BIU enantiomers and biliary excretion of the GSH conjugates suggest that for (S)-BIU the conjugation step is rate-limiting, whereas for (R)-BIU a transport step into bile may be rate-limiting.

Stereoselective glutathione conjugation and amidase-catalyzed hydrolysis of alpha-bromoisovalerylurea enantiomers in isolated rat hepatocytes.

alpha-Bromoisovalerylurea (BIU) is used as model substrate for studies on the pharmacokinetics of glutathione conjugation in vivo. Its metabolism in isolated rat hepatocytes is presently studied. A major part of the substrate was conjugated with glutathione, but also amidase-catalyzed hydrolysis occurred, resulting in the products urea and alpha-bromoisovaleric acid (BI). The amidase activity was located in the microsomal fraction of the rat liver. The product of hydrolysis, BI, also was conjugated efficiently with glutathione. In glutathione-depleted hepatocytes, no glutathione conjugates but only urea and BI were formed. A pronounced stereoselectivity in the metabolism of the BIU enantiomers was observed: (R)-BIU was conjugated with glutathione much faster than (S)-BIU. (S)-BIU was hydrolyzed substantially in the cells and the glutathione conjugate of the hydrolytic product, (S)-BI, could be detected. At high BIU concentrations (500 microM of the racemate) intracellular glutathione was seriously depleted; then, the cosubstrate availability most likely was the rate-limiting factor in the conjugation of BIU with glutathione. More urea was formed from (racemic) BIU in isolated rat hepatocytes in the present study than in the perfused liver and the intact rat in previous studies. This in vivo-in vitro difference is tentatively assigned to differences in glutathione availability in these systems. The results suggest that BI may also be a useful model substrate to study the kinetics of glutathione conjugation in vivo and in vitro.