Method for screening and quantitative determination of serum levels of salicylic Acid, acetaminophen, theophylline, phenobarbital, bromvalerylurea, pentobarbital, and amobarbital using liquid chromatography/electrospray mass spectrometry.

We investigated a method for the simultaneous screening, identification, and quantitative determination of salicylic acid, acetaminophen, theophylline, barbiturates, and bromvalerylurea, drugs that frequently cause acute poisoning in Japan and therefore require rapid analysis for effective treatment in the clinical setting. The method employs liquid chromatography/electrospray mass spectrometry (LC/MS) of solid-phase extracted serum samples. For LC/MS ionization, the electrospray-ionization method was used, with acetaminophen in the positive-ion mode, and salicylic acid, theophylline, phenobarbital, bromvalerylurea, pentobarbital, amobarbital, and o-acetamidophenol (internal standard) in the negative-ion mode, the base ions were used in each case for quantitative analysis. Quantitation was possible for the following sample concentration ranges: salicylic acid and acetaminophen, 100 to 5 microg/ml; theophylline, 100 to 0.5 microg/ml; and phenobarbital, bromvalerylurea, pentobarbital, and amobarbital, 100 to 1 microg/ml. Using full-scan mass spectrometry, the lower detection limits of 1 microg/ml for salicylic acid and acetaminophen, 0.1 microg/ml for theophylline, and 0.5 microg/ml for phenobarbital, bromvalerylurea, pentobarbital, and amobarbital were adequate for identifying acute poisoning. When each compound was added to serum to a final concentration of 5 microg/ml and solid-phase extraction was performed using Oasis HLB 1-cc (30-mg), the mean recovery rate of each compound was 89.2 to 96.1% (n=5), and the coefficients of variation of the intraday and interday assays were 3.55 to 6.05% (n=5) and 3.68 to 6.38% (n=5), respectively, which are acceptable. When this method of analysis was applied in testing the sera of a female patient who had consumed a large amount of an unknown commercial drug, salicylic acid and bromvalerylurea were identified, and the treatment strategy could be determined in accordance with the serum concentration of those drugs.

Determination of bromvalerylurea and its metabolites in biological samples by frit-fast atom bombardment liquid chromatography-mass spectrometry.

A simple, rapid, and sensitive method which allows us to simultaneously determine bromvalerylurea (BVU) and its three metabolites (3-methylbutyrylurea [MVU], alpha-(cystein-S-yl)isovalerylurea [CVU], and alpha-(N-acetylcystein-S-yl)isovalerylurea [AcCVU]) was investigated by frit-fast atom bombardment liquid chromatography-mass spectrometry (frit-FAB LC-MS). The LC-MS analysis was performed after the solid-phase extraction from tissue and urine samples with a Sep-Pak C18 cartridge. Tissue homogenates and urine were adjusted to pH 4.0 and applied to the cartridges. The retained BVU and its metabolites were eluted from the cartridge with 2 mL of acetonitrile/10 mM ammonium acetate buffer (pH 3.5, 50:50, v/v). The eluate was analyzed by LC-MS, which employs a semimicro type L-column ODS column. The proposed conditions are as follows: mobile phase A, 0.4% glycerol in acetonitrile/10 mM ammonium acetate buffer (pH 3.5) (5:95, v/v); mobile phase B, 0.4% glycerol in acetonitrile; elution mode, linear gradient, 100% A (5 min) to 100% B in 15 min; flow rate, 0.2 mL/min; split ratio, 1:40. Extraction recoveries of BVU and its metabolites were 91.90-97.79% from the spiked liver homogenate and 89.68-96.13% from the spiked urine. The detection limits ranged from 10 to 25 ng/g in selected ion monitoring mode.

Involvement of a cytochrome P450 system in microsomal debromination of alpha-(bromisovaleryl)urea.

The reductive debromination of a hypnotic, (alpha-bromisovaleryl)urea to (3-methylbutyryl)urea by rat liver microsomes was studied. Pretreatment of rats with cytochrome P450 inducers such as phenobarbitone, 3-methylcholanthrene, acetone and pregnenolone-16 alpha-carbonitrile enhanced the debromination of (alpha-bromisovaleryl)urea by liver microsomes. Microsomal debromination was inhibited by cytochrome P450 inhibitors such as metyrapone, alpha-naphthoflavone, SKF 525-A and carbon monoxide. Microsomal debromination was enhanced by addition of NADPH-cytochrome P450 reductase and inhibited by addition of an antibody against the flavo enzyme to the liver microsomes. A reconstituted cytochrome P450 system containing NADPH-cytochrome P450 reductase, and cytochrome P450 1AI or P450 2BI exhibited debrominating activity toward the hypnotic. These results indicated that a cytochrome P450 system plays an essential role in the microsomal debromination of (alpha-bromisovaleryl)urea.

Stereoselectivity of human liver and intestinal cytosolic fractions as well as purified human glutathione S-transferase isoenzymes towards 2-bromoisovalerylurea enantiomers.

Glutathione (GSH) conjugation of 2-bromoisovalerylurea (BIU) enantiomers is stereoselective in humans in vivo. Administration of racemic BIU results in a higher plasma elimination and urinary excretion of R-BIU and its mercapturate, respectively, than of S-BIU and its mercapturate. In order to relate the in vivo BIU pharmacokinetics to the activity of glutathione S-transferase (GST) isoenzymes, the GSH conjugation of BIU enantiomers was studied with human liver and intestinal cytosolic fractions as well as purified human class alpha (GSTA1-1, GSTA2-2), mu (GSTM1a-1a) and pi (GSTP1-1) GST isoenzymes. Stereoselective GSH conjugation of BIU enantiomers was observed for most human liver and intestinal cytosolic fraction. In general, the cytosolic fractions preferentially conjugated S-BIU. Stereoselective preference for GSH conjugation of S-BIU was also observed for GSTA2-2 and GSTM1a-1a, whereas GSTA1-1 was not selective for either of the BIU enantiomers. GSTP1-1 did not catalyse conjugation of R- and S-BIU. Quantification of the GST isoenzymes in the liver cytosolic fractions showed that the stereoselectivity towards S-BIU was related to the profile and amount of GST subunits in the cytosolic fractions. The discrepancy in stereoselectivity between the BIU pharmacokinetics in vivo and the GSH conjugation of BIU enantiomers in vitro is discussed. In addition, since in contrast to human GSTM1a-1a, rat class Mu isoenzymes prefer R-BIU, the present results indicate that related isoenzymes in different species may have a different stereoselectivity.

Stereoselectivity in glutathione conjugation and amidase-catalyzed hydrolysis of the 2-bromoisovalerylurea enantiomers in the single-pass perfused rat liver.

Stereoselective glutathione conjugation and amidase-catalyzed hydrolysis of [(R)- and (S)-]2-bromoisovalerylurea (BIU), yielding bromoisovaleric acid (BI) and urea, have been observed in the rat in vivo and in isolated rat hepatocytes. The metabolism of enantiomeric (R)- and (S)-BIU was presently examined in the single-pass perfused rat liver with varying input concentrations (8-250 microM). Steady-state hepatic extraction ratios for (R)-BIU (0.6) were constant and higher than those for (S)-BIU, whose extraction ratio decreased from 0.36 (8 microM) to 0.23 (236 microM). (R)- and (S)-BIU were excreted unchanged only minimally into bile. [14C-Urea](R)-BIU underwent amidase-catalyzed hydrolysis to yield [14C]urea (15-24% of rate in) and conjugation to form the (S)-glutathionyl conjugate (31-35% of rate in); two metabolites, most likely the cysteinyl and dipeptide conjugates of BIU (10% of rate in), were found. [3H-Isovaleryl](S)-BIU formed much less amidase-hydrolyzed product, [3H]BI (1-2% of rate in) less (R)-glutathionyl conjugate (9-18% of rate in), but appreciable amounts (14-17% of rate in) of three other metabolites, of which two were most likely the cysteinyl and glycinylcysteinyl conjugates of BIU. When the glutathione conjugation products (glutathione, cysteine and cysteinylglycine conjugates) were summed, the total glutathione conjugation rate for (R)-BIU (44% of rate in) exceeded that for (S)-BIU (34 to 24% of rate in). Fitting of data to the Michaelis-Menten equation revealed similar Km for glutathione conjugation, but a 2-fold higher Vmax for (R)-BIU.(ABSTRACT TRUNCATED AT 250 WORDS)

Stereoselective conjugation of 2-bromocarboxylic acids and their urea derivatives by rat liver glutathione transferase 12-12 and some other isoforms.

Glutathione (GSH) conjugation of the separate enantiomers of five 2-bromocarboxylic acids and some of their urea derivatives by rat liver GSH transferases (GSTs) was studied. The liver cytosolic fraction conjugated all compounds, except for (R)-2-bromoisovaleric acid, with a variable degree of stereoselectivity. A GST pool, prepared by S-hexyl-GSH affinity chromatography, conjugated the urea derivatives at a somewhat higher rate but had very little activity towards the carboxylic acids, indicating that much activity towards the latter substrates was due to transferases not bound by the affinity column. Therefore, the activity was studied of some pure GSTs that are bound only slightly by the affinity column towards the separate enantiomers of 2-bromovaleric acid (BV), its urea derivative and 2-bromo-3-phenylpropionic acid (BPP). No activity was detected with transferases 5-5 and 8-8. Transferase 1-1 was active towards all compounds with high activity towards the urea derivatives. Transferase 12-12 showed high, stereospecific activity towards the R enantiomers of BV, its urea derivative and BPP.

Relationship between glutathione content in liver and glutathione conjugation rate in the rat in vivo. Effect of buthionine sulphoximine pretreatment on conjugation of the two 2-bromoisovalerylurea enantiomers during intravenous infusion.

The relationship between hepatic glutathione content and hepatic glutathione conjugation rate in the rat in vivo was investigated. As substrate for glutathione conjugation, racemic (R,S)-2-bromoisovalerylurea (BIU) was used which gives rise to the biliary excretion of two diastereoisomeric glutathione conjugates and the urinary excretion of two diastereoisomeric mercapturates. The excretion rate of the glutathione conjugate in bile reflects hepatic conjugation exclusively. An intravenous infusion of BIU was given and the excretion rates of the metabolites in bile and urine were determined. The glutathione concentration in the liver was followed by taking biopsies every hour. Glutathione was depleted by the infused substrate; in rats that were pretreated with the inhibitor of glutathione biosynthesis, buthionine sulphoximine (BSO), the depletion of the glutathione content was more rapid. The rate of excretion of the glutathione conjugate in bile was plotted against hepatic glutathione content. These results indicate that the 'organ Km' for glutathione in the liver is approximately 0.5 mumol/g of liver, so that the hepatic glutathione conjugation rate is decreased only at severe glutathione depletion.

Stereospecific glutathione conjugation of (R)- and (S)-2-bromoisovalerylurea in freshly isolated rat kidney proximal tubular cells.

The glutathione conjugation of 2-bromoisovalerylurea (BIU) was studied in isolated proximal tubular kidney cells of the rat. Racemic (R,S)-BIU was incubated with the cell suspension, and the incubation medium was analysed for the diastereomeric glutathione (GSH) conjugates, cysteine conjugates and mercapturates that can be formed from (R)- and (S)-BIU. Only the mercapturate formed from (R)-BIU was found, as well as its cysteine precursor. No GSH conjugates were detected. These results indicate that these cells conjugate only the (R)-BIU enantiomer, and that the GSH conjugate is immediately further metabolized to its cysteine conjugate and mercapturate.

The fate of diastereomeric glutathione conjugates of alpha-bromoisovalerylurea in blood in the rat in vivo and in the perfused liver. Stereoselectivity in biliary and urinary excretion.

The mixture of the two diastereomeric glutathione (GSH) conjugates of alpha-bromoisovalerylurea, (RS)-IU-G, was administered i.v. to anesthetized rats. Bile and urine were collected for 6 hr. Some 70 to 75% was recovered in urine as mercapturates. The half-lives of the urinary excretion were the same for the two mercapturates: 18 min and approximately 130 min, respectively, for the rapid and the slow phase. In bile only 1.5% of the dose of (R) and (S)-IU-G was found; two unidentified metabolites were also found. In rats with ligated kidneys, 4% of the dose of each glutathione conjugate was excreted in bile. Again, the two unidentified metabolites were found. In the isolated recirculating liver perfusion experiments, 1.4% of the administered GSH conjugates was found in bile. The concentration of the GSH conjugates in the perfusion medium remained constant and no other metabolites were formed. When (RS)-alpha-bromoisovalerylurea itself was added to the perfusate, the GSH conjugates in bile increased rapidly. The results show that the GSH conjugate in blood is little excreted in bile due to a slow uptake of the conjugate by the liver. The diastereomeric GSH conjugates show no stereoselectivity in their pharmacokinetics, indicating that the rate limiting step in this process is not stereoselective.

Stereoselectivity of rat liver glutathione transferase isoenzymes for alpha-bromoisovaleric acid and alpha-bromoisovalerylurea enantiomers.

The stereoselectivity of purified rat GSH transferases towards alpha-bromoisovaleric acid (BI) and its amide derivative alpha-bromoisovalerylurea (BIU) was investigated. GSH transferase 2-2 was the only enzyme to catalyse the conjugation of BI and was selective for the (S)-enantiomer. The conjugation of (R)- and (S)-BIU was catalysed by the isoenzymes 2-2, 3-3 and 4-4. Transferase 1-1 was less active, and no catalytic activity was observed with transferase 7-7. Isoenzymes 1-1 and 2-2 of the Alpha multigene family preferentially catalysed the conjugation of the (S)-enantiomer of BIU (and BI), whereas isoenzymes 3-3 and 4-4 of the Mu multigene family preferred (R)-BIU. The opposite stereoselectivity of conjugation of BI and BIU previously observed in isolated rat hepatocytes and the summation of activities of enzymes known to be present in hepatocytes on the basis of present data are in accord.

Stereoselective glutathione conjugation of the separate alpha-bromoisovalerylurea and alpha-bromoisovaleric acid enantiomers in the rat in vivo and in rat liver subcellular fractions.

Glutathione (GSH) conjugation of the separate alpha-bromoisovalerylurea (BIU) enantiomers was studied in the rat. Administration of (R)-BIU resulted in excretion of a single glutathione conjugate in bile (IU-S-G/I) and a single mercapturate in urine (IU-S-MA/B). The other enantiomer, (S)-BIU, was exclusively metabolized to the other diastereomeric conjugates, IU-S-G/II and IU-S-MA/A. Thus, the conjugation of BIU with glutathione was completely stereospecific. Both the GSH conjugate and mercapturate derived from (R)-BIU were excreted two to three times more rapidly than their diastereomeric (S)-BIU counterparts. The enantiomers did not influence each others metabolism as reflected by identical metabolite excretion rates when the BIU enantiomers were administered either separately or as the racemic mixture. A similar rate difference for GSH conjugation of the separate BIU enantiomers was observed in incubations with rat liver cytosol as source of GSH transferases, suggesting that the stereoselectivity in vivo was due to glutathione conjugation properly. Similar results were obtained with a rat liver microsomal fraction, indicating that microsomal GSH transferases are active towards BIU and have a similar stereoselectivity as the cytosolic enzymes. Comparison of the GSH conjugation of BIU with that of its analogue alpha-bromoisovaleric acid (BI, which lacks the amide-linked urea group) revealed an opposite stereoselectivity: while (R)-BIU was conjugated faster than (S)-BIU, the (R) enantiomer of the acid was conjugated more slowly than (S)-BI. The alpha-bromocarbonyl compounds BI and BIU present a new type of substrate for the GSH transferases and allow studies of these enzymes in intact organisms as well as investigations on the stereoselectivity of GSH conjugation.

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.

Three cases of chronic bromisoval intoxication: clinical symptoms and application of energy dispersive X-ray analysis (EDX) to detect bromine in serum, urine and cerebrospinal fluid.

Bromisoval has been used as a hypnotic for the past several decades, and its abuse was known to cause various neurological as well as psychiatric symptoms. Three patients showed a variety of symptoms which could not be explained neuroanatomically: nystagmus, gait disturbance and hyperreflexia of the limbs in all the cases, dysarthria, double vision, hypotonia, ataxic gait and disturbance of consciousness occasionally and auditory agnosia in one case. For the purpose of determining the diagnosis, an energy dispersive X-ray microanalysis (EDX) was used to detect bromine. Five microliters of specimens were placed on the carbon-coated mesh, and using a TN-2000 analyzer, characteristic X-ray peaks of bromine were detected in the serum, urine and cerebrospinal fluid. The sensitivity to detect bromine in the serum was 30 micrograms/ml.

alpha-Bromoisovalerylurea as model substrate for studies on pharmacokinetics of glutathione conjugation in the rat. I. (Bio-) synthesis, analysis and identification of diastereomeric glutathione conjugates and mercapturates.

In order to find a model substrate for kinetic characterization of glutathione conjugation in vivo alpha-bromoisovalerylurea (BIU) was studied. After administration of racemic [14C]urea BIU to rats, two radioactive metabolites were found in bile by high-performance liquid chromatography. The identity of these metabolites was established by various methods. Based on the hydrolytic activity of gamma-glutamyltranspeptidase (presence of the gamma-glutamyl moiety), high resolution nuclear magnetic resonance (isovaleryl and glutathionyl moieties) and fast atom bombardment mass spectrometry (molecular weight and fragmentation pattern), they were identified as glutathione conjugates of BIU. Because both conjugates in bile had these characteristics in common they must be diastereomers. Incubation of BIU with glutathione in the presence of rat liver cytosol resulted in formation of the same diastereomeric glutathione conjugates. Chemical synthesis of the diastereomers confirmed their identity. The major urinary excretion products of [14C]urea BIU in the rat were identified as diastereomeric mercapturates. A convenient chromatographic separation of the diastereomeric glutathione conjugates and the mercapturic acids is described. Electrochemical detection was used to determine the presence of the thioethers in both urine and bile. Pharmacokinetic results on BIU conjugation are described in the accompanying paper.

alpha-Bromoisovalerylurea as model substrate for studies on pharmacokinetics of glutathione conjugation in the rat. II. Pharmacokinetics and stereoselectivity of metabolism and excretion in vivo and in the perfused liver.

The hypnotic drug alpha-bromoisovalerylurea (BIU) has been studied in the rat with respect to its potential use as model substrate to investigate the pharmacokinetics of glutathione conjugation in vivo. The major metabolites of racemic BIU are the diastereomeric glutathione conjugates (bile) and mercapturates (urine). BIU was metabolized mainly by glutathione conjugation: after i.v. administration of [14C]BIU to freely moving rats, 89% of the dose was recovered in urine within 24 hr, mostly as mercapturates. The rate-limiting step in the clearance of BIU from blood most likely is glutathione conjugation as it was shown that rate-limitation is not due to flow-limited clearance in the liver (the initial extraction ratio of BIU in the perfused liver preparation was low: hepatic extraction ratio = 0.23), protein binding (60% was unbound in plasma) or enzyme saturation (linear pharmacokinetics in the dose range studied: 22-270 mumol/kg). Water solubility of BIU was sufficient to allow its i.v. administration, whereas the absence of toxic effects enables animal as well as human studies. Thus, BIU is a promising model substrate for studies of glutathione conjugation in vivo. In pentobarbital-anesthetized rats with a bile duct catheter, equal amounts of metabolites were excreted in bile (almost exclusively as the two diastereomeric BIU glutathione conjugates) and urine (mostly as the two diastereomeric mercapturates). Based on similar experiments with bile duct-ligated rats, it was concluded that the appearance of the mercapturates in urine could also occur without biliary excretion and subsequent gut metabolism of the BIU glutathione conjugates. The ability of the liver to metabolize BIU was studied in a hemoglobin-free, recirculating liver perfusion system. Of the recovered radioactivity 40% was excreted in bile within 2 hr, almost exclusively in the form of the two BIU glutathione conjugates. Also, glutathione conjugates were found in the perfusate (16% of the radioactivity present in the perfusate after 2 hr). A distinct stereoselectivity was observed in the metabolite excretion rates. The excretion half-lives of the two diastereomeric glutathione conjugates in bile differed 2- to 3-fold, both in anesthetized rats and in the perfused liver preparation. A similar difference in excretion half-lives was found for the urinary excretion of the diastereomeric mercapturates. Thus, BIU can be used to investigate in vivo the stereoselectivity of glutathione conjugation.