Monohydroxylated Metabolites Research Articles

Difference in epoxides formation and their further metabolism between delta 9- and delta 8-tetrahydrocannabinols by human liver microsomes.

In vitro metabolism of delta 9- and delta 8-tetrahydrocannabinols (THCs) was studied using human liver microsomes. Delta 9- or delta 8-THC was incubated with microsomes in the presence of an NADPH-generating system. The metabolites formed were extracted with ethyl acetate, separated by preparative thin-layer chromatography, and identified as trimethylsilyl derivatives by gas chromatography-mass spectrometry. 9 alpha, 10 alpha-Epoxyhexahydrocannabinol (EHHC) together with four monohydroxylated metabolites was formed from delta 9-THC. The epoxide was found to resist the hydrolysis by epoxide hydrolase, and was further converted to several metabolites by monooxygenase system involving cytochrome P-450. On the other hand, 8 beta, 9 alpha-dihydroxyhexahydrocannabinol (diOH-HHC) instead of epoxy metabolites was formed from delta 8-THC under the conditions for monooxygenase. When 1,1,1-trichloropropene-2,3-oxide was further added to the incubation mixture, both of 8 alpha, 9 alpha-EHHCs were found to be formed from delta 8-THC. These epoxides of delta 8-THC were preferentially hydrolyzed to 8 beta, 9 alpha-diOH-HHC by epoxide hydrolase. These results indicate that 9 alpha, 10 alpha-EHHC formed from delta 9-THC is further metabolized not by epoxide hydrolase but by monooxygenase system involving cytochrome P-450, and that, on the contrary, 8 alpha, 9 alpha- and 8 beta, 9 beta-EHHCs derived from delta 8-THC may be metabolized by epoxide hydrolase rather than cytochrome P-450 in the human liver, forming 8 beta, 9 alpha-diOH-HHC.

Simultaneous determination of 25-hydroxy- and 1,25-dihydroxyvitamin D from a single sample by dual-cartridge extraction.

With this dual-cartridge system we extract 25-hydroxyvitamin D [25(OH)D] and 1,25-dihydroxyvitamin D [1,25(OH)2D] from a single serum sample by using a nonpolar octadecylsilanol silica cartridge to adsorb the vitamin D metabolites and other nonpolar substances; the polar substances wash through the cartridge. The eluted material is then applied to a second alkylamine cartridge, which adsorbs the relatively polar hydroxylated metabolites; the less-polar substances are washed from the second cartridge. Elution from the second cartridge purifies and also separates 25(OH)D and 1,25(OH)2D with analytical recoveries near 90%. The monohydroxyl metabolites are determined by "high-performance" liquid chromatography (HPLC); the dihydroxyl metabolites are further purified by HPLC and determined by radioreceptor assay according to established procedures. Mean (+/- SD) winter normal values (34 subjects of both sexes; blood drawn in mid-April) were 18 +/- 5 micrograms/L for 25(OH)D and 25 +/- 7 ng/L for 1,25(OH)2D. In nine laboratory volunteers, the mean increase in the serum 25(OH)D3 value 5 h after ingestion of 50 micrograms of 25-hydroxycholecalciferol (Calderol) was 9 (SD 4) micrograms/L.

Isomodal column switching high-performance liquid chromatographic technique for the analysis of ciglitazone and its metabolites in human serum

The application of isomodal column switching high-performance liquid chromatography as an alternative to gradient elution was investigated for the analysis of ciglitazone, a potential oral antidiabetic agent, and its monohydroxyl metabolites in human serum. A high-performance liquid chromatographic apparatus was designed to perform on-line fractionation of the serum extract into non-polar (drug) and polar (metabolite) fractions which were then automatically routed into individually optimized, isocratic, reversed-phase high-performance liquid chromatographic systems for simultaneous analysis. Sample fractionation was performed with a reversed-phase guard column, and solvent routing was accomplished with microprocessor-controlled switching valves. Serum was extracted for analysis by a one-step mode sequencing procedure using disposable bonded-phase columns, and quantitation was accomplished with spiked serum standards. Performance specifications of the method were defined for precision, accuracy, linearity, and sensitivity. The column switching method was found to be both expedient and reliable, and it may have general utility for the routine, quantitative analysis of drug/metabolite mixtures that cannot be assayed by simple isocratic elution methods.

Modification of CCNU pharmacokinetics by misonidazole--a major mechanism of chemosensitization in mice.

We have investigated the effect of misonidazole (MISO) on the pharmacokinetics of 1-(2-chloroethyl)-3-cyclohexyl-1-nitrosourea (CCNU) in mice. CCNU and its monohydroxylated metabolites were measured using a high performance liquid chromatography (HPLC) method. In the absence of MISO the plasma disappearance of CCNU was biphasic with a t 1/2 alpha of 2.3 min and a t 1/2 beta of 53 min. The monohydroxylated metabolites of CCNU also followed biphasic clearance kinetics. A large single dose of MISO (0.5 mg g-1), given i.p. 30 min prior to CCNU, prolonged the t 1/2 alpha by a factor of 2.6 but had no effect on t 1/2 beta. In addition, the apparent volume of distribution was decreased by a factor of 1.6. Consequently, the plasma area under the curve (AUC0 - infinity) was increased by a factor of 1.7 for CCNU and by a factor of 2.0 for total nitrosourea (CCNU + monohydroxylated metabolites). The effects of MISO on CCNU kinetics were dependent on MISO dose and plasma concentration and on the interval between MISO and CCNU administration. The concentration of CCNU was measured in 4 tumours: the KHT, RIF-1 and EMT6 mouse tumours, and the HT29 xenograft. For all 4 tumours, 0.5 mg g-1 MISO raised the tumour concentrations of CCNU and total nitrosourea by a considerable amount (2-2.5 times). More detailed studies in the KHT tumour demonstrated that there was a significant lag period before peak tumour CCNU concentrations were reached, and that MISO increased the peak concentrations by a factor of about 2.4. In contrast, there was no such lag period for the plasma and MISO did not increase the plasma peak CCNU concentrations. These data strongly suggest that modification of the pharmacokinetics may be a major contributory factor in the enhancement of CCNU cytotoxicity by large single doses of MISO in vivo.

Human liver microsomal oxidation of delta 8-tetrahydrocannabinol.

Human Liver microsomal oxidation products of Δ8-tetrahydrocannabinol (THC) were isolated and identified by thin-layer chromatography, gas chromatography and gas chromatography-mass spectrometry. As the results, five monohydroxylated metabolites including 11-hydroxy-Δ8-THC, and 8β, 9α-dihydroxyhexahydrocannabinol were identified as trimethyl-silyl derivatives. Isolation of the dihydroxylated metabolite suggests that epoxy metabolite (s) may be formed from Δ8-THC in humans.

Gas chromatographic mass spectrometric identification of O-demethylated and mono-hydroxylated metabolites of levomepromazine in blood from psychiatric patients by selected ion recording with high resolution.

An O-demethylated and two mono-hydroxylated metabolites of levomepromazine have previously been identified as conjugates in the urine from psychiatric patients. The three metabolites were identified in hydrolysed and nonhydrolysed plasma and erythrocytes from three patients by selected ion monitoring of the trimethylsilyl derivatives, with a mass spectrometric resolution of 5000. The three metabolites were mainly found as conjugates in the plasma, and smaller amounts were found unconjugated in the plasma and the erythrocytes.

Separation of epimeric monohydroxylated metabolites of testosterone, androstenedione and thep-nitrobenzyl esters of hydroxylated 5β-cholanic acid using the same HPLC conditions

The separation of authentic reference standards of epimeric monohydroxylated derivatives of both testosterone and lithocholic acidp-nitrobenzyl esters on a 10 μm Partisil-10 column with a single mobile phase of hexane-tetrahydrofuran-isopropanol (80∶15∶5) is reported. Baseline separation of all epimeric derivatives of the bile acid PNB-esters was achieved, and the principal testosterone metabolites (6β-, 7α-, 16β-, and 15β- and 16α-hydroxy-testosterones) were separated from one another and the parent compound. This system was used to separate metabolites of both androstene-dione and [14C]-lithocholic acid.

Stereochemical aspects of the metabolism of phenprocoumon in rat liver microsomes

The oxidation of the enantiomers of phenprocoumon catalyzed by noninduced rat hepatic microsomes was determined, and the formation of products was found to be either nonstereoselective or selective for the (S)-enantiomer. Of the seven possible aromatic monohydroxylated metabolites, only the 4′-, 6-, 7- and 8-hydroxy derivatives were detected. These hydroxylation reactions were shown to be cytochrome P-450-dependent by decreased product formation in the presence of the inhibitors CO, metyrapone and SKF-525A (β-diethylaminoethyl 2,2-diphenylvalerate) and by decreased product formation when NADH was substituted for NADPH. Comparison of these results to those obtained earlier for warfarin reveals that major regioselective and stereoselective differences exist in the in vitro metabolism of these two similar coumarin anticoagulants.

Metabolism of polyunsaturated fatty acids by mouse peritoneal macrophages: the lipoxygenase metabolic pathway.

When resident macrophages from mice are incubated with exogenous polyunsaturated fatty acids, they produce lipoxygenic metabolites. To delineate this metabolic chart we used high pressure liquid chromatography and gas chromatography prior to mass spectrometry-computer system. The lipoxygenic activity of these cells leads to many compounds. Among them we describe the monohydroxylated metabolites and vicinal hydroxyepoxyenes. In the mechanism of formation of the latter unstable cyclic precursors might occur as intermediates between hydroperoxides and them. Dihydroxy compounds could arise from hydrolysis of unstable epoxide precursor which could be the second substrate of the glutathione transferase system and could lead to thioaminolipids.

Effects of oral perhexiline on canine myocardial flow distribution.

Perhexiline maleate at a dose of 400 mg three times daily for 2 days administered to normal dogs altered the relative regional transmural resistance so that during reduced coronary flow the endocardium:epicardium (endo:epi) ratio is increased. In the presence of acute myocardial infarction heart rate was significantly lower and the endo:epi ratio of perfused areas was increased when coronary flow was normal. A linear relationship was observed between the endo:epi ratio and the concentration of perhexiline in plasma, and its monohydroxyl metabolite in plasma. The results suggest that the mechanism of action of the drug is due to redistribution of a limited coronary flow.

The pharmacokinetic fate of cannabidiol and its relationship to barbiturate sleep time

Cannabidiol (CBD) is a known inhibitor of a number of hepatic drug metabolism reactions. Its pharmacokinetics in the liver were studied to determine the relationship between the amount of CBD in this organ and the effect on barbiturate sleep time, which has been shown by others to reflect an inhibition of drug metabolism. A methanol extract of liver was subjected to thin-layer chromatography which yielded three distinct fractions: CBD, the monohydroxylated metabolites and a relatively polar, unidentified fraction. The quantitative analysis of these fractions, as a function of time after drug administration, indicated that CBD is metabolized rapidly: the apparent half life is only about 52 min, which is approximately the same value obtained for the monohydroxylated metabolites; both of these fractions have virtually disappeared from the liver at 4 hr, at which time a significant effect on sleep time still persists. In contrast, the unidentified metabolite fraction contains a substantial amount of cannabinoid throughout the course of the effect on sleep time. These data suggest that the inhibition of hepatic drug metabolism is not caused by either CBD or one of its monohydroxylated metabolites; rather, the effect correlates with the persistence of more polar metabolites.

Combined high-pressure liquid chromatography and radio-immunoassay method for the quatitatin of Δ 9-tetrahydrocannabinol and some of its metabolites in human plasma

A high-pressure liquid chromatography-radiimmunoassay (HPLC-RIA) method for the measurement of cannabinoid levels in plasma is described. The method is capable of quantifying 0.1 ng of a cannabinoid in 1 ml of plasma. The experimental procedure consits of an initial separation of cannabinoids in a plasma extract by HPLC followed by collection of the HPLC eluate and RIA. A chromatogram consisting of the cross-reacting cannabinoids in plasma may then be constructed. The plasma concentrations of cannabinoids with retention volumes equivalent to those f Δ 9-tetrahydrocannabinol, cannabinol and mono-hydroxylated metabolites have been measured by this technique.

Metabolism of 2,4,5,2',5'-Pentachlorobiphenyl by lung and liver microsomes.

Earlier studies on distribution, excretion and metabolism of 14C-2,4, 5,2′,5′-pentachlorobiphenyl (PCB) in mouse and monkey have shown the presence of two mono-hydroxylated metabolites in feces, a high concentration of radioactivity in liver and an accumulation of radioactivity in the lung, especially in the bronchial epithelium. An investigation has therefore been made to study the metabolism on the microsomal fractions of lung and liver from mouse, rat, monkey and man and on the microsomal fractions of lung and liver from rats pretreated with methylcholanthrene (MC) or phenobarbital (PB).

Identification of monohydroxylated metabolites of cannabidiol formed by rat liver.

Cannabidiol (CBD) was metabolized in vitro by rat liver enzymes. Unchanged CBD and eight monohydroxylated metabolites were isolated and positively identified. As previously reported, 7-hydroxy-CBD was the major metabolite. The second most abundant metabolite was 6alpha-hydroxy-CBD; whereas only a trace amount of 6beta-hydroxy-CBD was found. In addition hydroxylation occurred in all positions of the pentyl side chain, 4 inches-hydroxy-CBD being most abundant. 3 inches-Hydroxy-CBD was formed in half of the yield of 4 inches-hydroxy-CBD, while 1 inches-, 2 inches-, 5 inches-hydroxy-CBD were each formed in approximately one fourth of the yield of 4 inches-hydroxy-CBD.