Rabbit 3-hydroxyhexobarbital dehydrogenase is a NADPH-preferring reductase with broad substrate specificity for ketosteroids, prostaglandin D2, and other endogenous and xenobiotic carbonyl compounds.

3-Hydroxyhexobarbital dehydrogenase (3HBD) catalyzes NAD(P)⁺-linked oxidation of 3-hydroxyhexobarbital into 3-oxohexobarbital. The enzyme has been thought to act as a dehydrogenase for xenobiotic alcohols and some hydroxysteroids, but its physiological function remains unknown. We have purified rabbit 3HBD, isolated its cDNA, and examined its specificity for coenzymes and substrates, reaction directionality and tissue distribution. 3HBD is a member (AKR1C29) of the aldo-keto reductase (AKR) superfamily, and exhibited high preference for NADP(H) over NAD(H) at a physiological pH of 7.4. In the NADPH-linked reduction, 3HBD showed broad substrate specificity for a variety of quinones, ketones and aldehydes, including 3-, 17- and 20-ketosteroids and prostaglandin D2, which were converted to 3α-, 17ß- and 20α-hydroxysteroids and 9α,11ß-prostaglandin F2, respectively. Especially, α-diketones (such as isatin and diacetyl) and lipid peroxidation-derived aldehydes (such as 4-oxo- and 4-hydroxy-2-nonenals) were excellent substrates showing low K(m) values (0.1-5.9 µM). In 3HBD-overexpressed cells, 3-oxohexobarbital and 5ß-androstan-3α-ol-17-one were metabolized into 3-hydroxyhexobarbital and 5ß-androstane-3α,17ß-diol, respectively, but the reverse reactions did not proceed. The overexpression of the enzyme in the cells decreased the cytotoxicity of 4-oxo-2-nonenal. The mRNA for 3HBD was ubiquitously expressed in rabbit tissues. The results suggest that 3HBD is an NADPH-preferring reductase, and plays roles in the metabolisms of steroids, prostaglandin D2, carbohydrates and xenobiotics, as well as a defense system, protecting against reactive carbonyl compounds.

Ozonolysis of geraniol-trans, 6-methyl-5-hepten-2-one, and 6-hydroxy-4-methyl-4-hexenal: kinetics and mechanisms.

A combined density functional theory and transition-state theory study of the mechanisms and reaction coefficients of gas-phase ozonolysis of geraniol-trans, 6-methyl-5-hepten-2-one, and 6-hydroxy-4-methyl-4-hexenal is presented. The geometries, energies, and harmonic vibrational frequencies of each stationary point were determined by B3LYP/6-31(d,p), MPW1K/cc-pVDZ, and BH&HLYP/cc-pVDZ methods. According to the calculations, the ozone 6-methyl-5-hepten-2-one reaction is faster than the ozone 6-hydroxy-4-methyl-4-hexenal reaction, but both are slower than the ozone geraniol-trans reaction. By using the BH&HLYP/cc-pVDZ data, a global rate coefficient of 5.9 x 10(-16) cm(3) molecule(-1) s(-1) was calculated, corresponding to the sum of geraniol-trans, 6-methyl-5-hepten-2-one, and 6-hydroxy-4-methyl-4-hexenal reactions with the ozone. These results are in good agreement with the experimental studies.

Short and simple syntheses of 4-oxo-(E)-2-hexenal and homologs: pheromone components and defensive compounds of Hemiptera.

One-step syntheses of 4-oxo-(E)-2-hexenal and 4-oxo-(E)-2-octenal from commercially available 2-ethyl- and 2-butylfuran are described. A two-step synthesis of the homolog 4-oxo-(E)-2-decenal from furan is also reported. These compounds are common components of true bug defensive secretions, and recently have been identified as pheromone components for several species. The simple syntheses reported here will make these compounds readily available for further research.

cis-3-Hexenal production in tobacco is stimulated by 16-carbon monounsaturated fatty acids.

Transgenic tobacco plants O9 and T16 expressing the yeast acyl-CoA Delta9 desaturase and an insect acyl-CoA Delta11 desaturase, respectively, displayed altered profiles of fatty acids compared to wild-type tobacco plants and marked increases in cis-3-hexenal, a major leaf volatile derived from alpha-linolenic acid (18:3). As expected, O9 and T16 plants had increased levels of the major unsaturated fatty acid products formed by the transgenic desaturases they expressed, viz., palmitoleic acid (16:1(Delta9)) and palmitvaccenic acid (16:1(Delta11)), respectively. In addition, levels of 18:3 lipid declined slightly and the pool of free 18:3, which accounts for about 30% of free fatty acids in wild-type plants, disappeared completely in both transgenics. Both O9 and T16 plants were found to have a two-fold increase in 13-lipoxygenase (13-LOX) activity, which catalyzes the first of two steps leading to hexenal production from 18:3. In O9 and T16 plants, the activity of 9-lipoxygenase and hydroperoxide lyase, the latter catalyzing the formation of cis-3-hexenal from alpha-linolenic acid hydroperoxide, was significantly different from that of the wild-type plants. Although 16:1(Delta9) and 16:1(Delta11) had no direct effects on 13-LOX activity in vitro, cis-3-hexenal production increased in tobacco leaves treated with these fatty acids, suggesting that they may act in vivo by stimulating 13-LOX gene expression.

Stereoselective disposition of hexobarbital and its metabolites: relationship to the S-mephenytoin polymorphism in Caucasian and Chinese subjects.

In vitro studies with human liver preparations suggest that the metabolism of hexobarbital involves CYP2CMP--the determinant of the S-mephenytoin 4'-hydroxylation polymorphism, but no in vivo evidence of interphenotypic differences exist. The pharmacokinetics and urinary excretion of hexobarbital and its metabolites were, therefore, investigated following oral administration of a differentially labelled pseudoracemate that allowed determination of the fate of the individual enantiomers. Studies were undertaken in 10 Caucasian and nine Chinese healthy subjects known to be either extensive (EM) or poor (PM), metabolizers of mephenytoin. No inter-racial differences were observed in any of the measured parameters within a given phenotype. However, pronounced stereoselectivity in disposition was noted in EMs with R-(-)-hexobarbital's oral clearance being five- to six-fold greater than that for the S-(+)-enantiomer. By contrast, the S-(+)-isomer was eliminated twice as fast as R-(-) hexobarbital in PMs and, in addition, the oral clearances of both enantiomers were significantly reduced compared with their values in EMs. Formation of 3'-hydroxy- and 3'-ketohexobarbital and 1,5-dimethylbarbituric acid were the major identified routes of metabolism for each enantiomer in both phenotypes. Furthermore, these pathways were found to co-segregate with the mephenytoin polymorphism and in EMs they were primarily responsible for the observed stereoselectivity in disposition. These findings, therefore, confirm the stereoselectivity in hexobarbital's disposition in humans and identify the major pathways of metabolism involved. Additionally, the results indicate that CYP2CMP is a major determinant of the in vivo metabolism of both of hexobarbital's enantiomers but especially that of the R-(-)-enantiomer.

Hexobarbital metabolism: a new metabolic pathway to produce 1,5-dimethylbarbituric acid and cyclohexenone-glutathione adduct via 3'-oxohexobarbital.

1. In the presence of glutathione under physiological conditions, 3'-oxohexobarbital was non-enzymically converted to 1,5-dimethylbarbituric acid and a cyclohexenone-glutathione adduct. 2. The two reaction products were characterized by mass spectrometry, 1H- and 13C-n.m.r. spectrometry, and UV spectral analyses. 3. 1,5-Dimethylbarbituric acid was excreted in urine of rat given hexobarbital, 3'-oxohexobarbital, or 1',2'-epoxyhexobarbital, and accounted for 13.4, 14.5 and 4.7% of dose, respectively. 4. The cyclohexenone-glutathione adduct, a novel metabolite of hexobarbital, was excreted in the bile of rat given hexobarbital. 5. The route of 1,5-dimethylbarbituric acid formation via 3'-oxohexobarbital in the metabolism of hexobarbital was discussed in comparison with the epoxide-diol pathway.

Disposition of allylic oxidation pathway metabolites of racemic hexobarbital in the rat.

The pharmacokinetics in blood of the major metabolites of hexobarbital (HB), 3'-hydroxyhexobarbital (OH-HB) and 3'-ketohexobarbital (K-HB) were studied in rats. In addition urinary excretion of OH-HB and K-HB and 1,5-dimethylbarbituric acid (DMBA) was determined. Half-lives of OH-HB and K-HB were slightly longer than that of the parent drug. Urinary recovery of OH-HB, K-HB and DMBA following i.a. administration of OH-HB (75%) was more complete than the recovery following i.a. administration of K-HB (52%). Most probably further metabolism of K-HB takes place. Of K-HB, 41% was excreted renally, and 3.4% of K-HB reverted back to OH-HB. Of OH-HB, about 45% was excreted renally, following p.o. or i.a. administration. Since about 10% of both OH-HB and K-HB was converted to DMBA, it seems that the epoxide-diol pathway as proposed for HB also plays a minor role in the metabolism of OH-HB and K-HB. It is further concluded that measuring allylic pathway oxidation metabolites of HB does not improve the usefulness of HB as a model compound in the assessment of the activity of oxidative drug metabolizing activity.

Biotransformation of N-methylcyclobarbital in vivo in rabbit and rat.

Metabolism of N-methylcyclobarbital in the rabbit and rat has been studied in vivo for the purpose of comparison with the C5-methylated analogue, hexobarbital. In the rabbit, the main route of the metabolism of N-methylcyclobarbital is glucuronide formation after hydroxylation at the 3'-position of the parent compound. Dehydrogenation of the 3'-hydroxy product, a major pathway in the metabolism of hexobarbital, was a minor route in the case of N-methylcyclobarbital. In addition, a new type of metabolite, thought to be dihydroxylated products from spectral studies, was isolated. In the rat, there were almost no differences in the metabolic fates of N-methylcyclobarbital and hexobarbital. Profiles of metabolism of four analogous barbiturates (N-methylcyclobarbital, hexobarbital, cyclobarbital and norhexobarbital), which have a cyclohexene ring on the 5-carbon, reveal the contribution of alkyl substituents in the barbiturate ring on the bioavailability and metabolism of these compounds.

Disposition of hexobarbitone in healthy man: kinetics of parent drug and metabolites following oral administration.

1 Hexobarbitone plasma kinetics were determined in six healthy volunteers, who received 500 mg hexobarbitone orally. In addition urinary excretion rate and cumulative excretion were measured of its three major metabolites: 3'-hydroxyhexobarbitone, 3'-ketohexobarbitone and 1,5-dimethylbarbituric acid. 2 The mean plasma elimination half-life of hexobarbitone was 3.7 +/- 0.9 h (n = 6). Assuming complete absorption, the volume of distribution and the metabolic clearance were 81.3 +/- 20.5 1 and 16.4 +/- 2.9 1/h, respectively. The mean maximal plasma concentration was 7.1 +/- 2.1 micrograms/ml and was reached 1.2 +/- 0.4 h after drug administration. 3 3'-Hydroxyhexobarbitone and 3'-ketohexobarbitone, which are products of allylic side-chain oxidation of hexobarbitone, were excreted in 24 h to the extent of 4.7 +/- 1.3 and 32.1 +/- 11.9% of the dose, respectively. In the same period, 1,5-dimethylbarbituric acid, which is the end product of the epoxide-diol pathway, was excreted to 18.0 +/- 7.8% of the dose. The ratio of the sum of 3'-hydroxy- and 3'-ketohexobarbitone vs 1,5-dimethylbarbituric acid excreted varied with time and amounted ultimately in 24 h urine to 2.3 +/- 1.0. 4 The half-lives of 3'-hydroxyhexobarbitone and 1,5-dimethylbarbituric acid, calculated from their renal excretion rate curves, amounted 5.2 +/- 0.9 and 6.6 +/- 1.3 h and were significantly longer than the half-life of hexobarbitone in plasma. The half-life of 3'-ketohexobarbitone was 4.2 +/- 0.8 h. The maximum excretion rate of 1,5-dimethylbarbituric acid was reached at 7.7 +/- 1.0 h after administration of hexobarbitone. 3'-Hydroxy- and 3'-ketohexobarbitone were excreted with a maximal rate at 2.2 +/- 0.8 and 2.8 +/- 0.4 h respectively.

The metabolic fate of 1',2'-epoxyhexobarbital in the rat.

1. In urine of rats treated with 1',2'-epoxyhexobarbital, unchanged compound and six metabolites were identified: 1,5-dimethylbarbituric acid, which is the end product of an epoxide-diol pathway, two stereochemically different 3'-hydroxy-1',2'-epoxyhexobarbitals, a hydroxyfuropyrimidine, 3'-hydroxyhexobarbital and 3'-ketohexobarbital. 2. The analytical methods used were based on capillary g.l.c. with nitrogen-selective or mass spectrometric detection. Identification was by electron impact and chemical ionization mass spectrometry. All the reference compounds needed for comparison were synthesized. 3. The mean plasma elimination half-life of 1',2'-epoxyhexobarbital after intra-arterial administration to the rat was 13.7 +/- 1.5 min (mean +/-S.D.; n = 3). A total body clearance of 35.2 +/- 9.6 ml/min (mean +/- S.D.) was calculated, which includes renal clearance of unchanged epoxide. 4. In rat liver microsomal preparations it was demonstrated that 1',2'-epoxyhexobarbital is hydrated by epoxide hydratase. With 1 mM 1,1,1,-trichloropropene-2,3-oxide (TCPO) this enzymic reaction could be inhibited completely. 5. On administration of the individual metabolites of the epoxide to rats, no evidence was found for their possible intermediacy in the formation of 3'-hydroxy- or 3'-ketohexobarbital, which are major metabolites of hexobarbital.

Stereoselective formation of glucuronides in metabolism of hexobarbital enantiomers in vivo: isolation and quantitation of glucuronides in rabbit urine.

An improved column-chromatographic method was described for isolation and purification on preparative scale of a glucuronide from urine of rabbits administered (RS)-hexobarbital. The analytically pure preparation obtained was found to be a mixture of two glucuronides of diastereomeric 3'-hydroxyhexobarbitals. Rates of hydrolysis of the glucuronides were dependent on the enzyme preparations used as well as on the configuration of the substrate. For quantitative determination, the glucuronides were hydrolyzed completely by beta-glucuronidases from either Escherichia coli or abalone entrails under the conditions used. In vivo studies on the metabolism of (R)-(-)- and (S)-(+)-hexobarbital in the rabbit showed that the glucuronides excreted in 24-hr urine accounted for about 30% of the dose of each enantiomer, and that conjugation of the hydroxy isomers with glucuronic acid was so stereoselective that the isomers with S-configuration at the 3'-position were preferentially conjugated. There were almost no differences in the urinary metabolite profile between normal an PB-treated rabbits; however, a noticeable change was observed in recovery of unchanged (S)-hexobarbital after phenobarbital treatment.

Preparation of four optical isomers of hydroxylated hexobarbital and activities of 3-hydroxyhexobarbital dehydrogenase from guinea pig and rabbit liver.

The preparation of four optical isomers of 3'-hydroxyhexobarbital [5-(3'-hydroxy-1'-cyclohexen-1'-yl)-1,5-dimethylbarbituric acid] is described. The absolute configuration of the four isomers were assigned as (3'S, 5 R) and (3'R, 5R) for alpha- and beta-isomers from (R)-(--)-hexobarbital, respectively. Some of these isomeric 3'-hydroxyhexobarbitals, which are formed in the reaction of hexobarbital with liver microsomal mono-oxygenase, are oxidized to 3'-oxohexobarbital [5-(3'-oxo-1'-cyclohexen-1'-yl)-1,5-dimethylbarbituric acid] by dehydrogenase localized in the soluble fraction of liver homogenates. Comparison of activities among the four isomers for 3-hydroxyhexobarbital dehydrogenase was made by use of enzymes from guinea pig and rabbit liver. It became evident that either enzyme had a quite different activity towards each optical isomer, and the configuration of the 3'-position of hydroxyhexobarbital was an important factor affecting the reactivity of enzyme; isomers with 3'S-configuration were preferentially dehydrogenated to enantiomers with 3'R-configuration. Both enzymes had the highest activity towards (3'S, 5R)-3'-(--)-hydroxy-(--)-hexobarbital, irrespective of their having specificity towards different types of substrates.

Dehydrogenation of indanol by rabbit liver 3-hydroxyhexobarbital dehydrogenase.

1. Among the several enzyme activities in rabbit liver cytosol able to dehydrogenate 1-indanol, only the main activity was not separable from 3-hydroxyhexobarbital dehydrogenase during purification including polyacrylamide gel disc electrophoresis. 2. Results of mixed substrate method indicated that the same enzyme catalyses the dehydrogenation of 1-indanol and 3-hydroxyhexobarbital. The ratio between the two dehydrogenation activities was almost constant as the enzyme underwent thermal inactivation. The Ki values of p-chloromercuribenzoate, the Km values for NAD+, and the Km values for NADP+ were very similar for the two dehydrogenations. These results lead to the conclusion that the same enzyme catalyses the dehydrogenation of 3-hydroxyhexobarbital and 1-indanol. 3. 1-Tetralol, 1-acenaphthenol, 9-fluorenol, thiochroman-4-ol and 4-chromanol also served as substrate of the enzyme, but 2-indanol, 2-tetralol, and trans- and cis-indan-1,2-diol were not oxidized. 4. Reversibility of the reaction was also confirmed using 1-indanone as substrate.

Guinea pig liver 3-hydroxyhexobarbital dehydrogenase. Purification and properties.

3-Hydroxyhexobarbital dehydrogenase, which catalyzes the reversible oxidation of 3-hydroxyhexobarbital to 3-oxohexobarbital, has been purified 470-fold from the soluble fraction of guinea pig liver with a yield of 47%. The specific activity of the purified enzyme is 9.4 units/mg of protein. Results of polyacrylamide gel disc electrophoresis and isoelectric focusing indicated that the purified enzyme preparation is a single and homogeneous protein. NADP+ served as preferred co-factor, but NAD+ is also utilized in the presence of phosphate ion. The guinea pig liver enzyme possessed a relatively narrow substrate specificity in comparison with the rabbit liver enzyme. It is very distinctive that guinea pig liver 3-hydroxyhexobarbital dehydrogenase catalyzes the dehydrogenation of 17beta-hydroxysteroids such as testosterone, 4-androstene-3beta,17beta-diol, 5alpha-androstane-3alpha,17beta-diol, 5alpha-androstane-3beta,17beta-diol, 5alpha-androstan-17beta-ol-3-one, and 5beta-androstane-3alpha,17beta-diol.