Assay of caffeine metabolism in vitro by human liver microsomes using radio-high-performance liquid chromatography

doi:10.1016/0731-7085(90)80121-5

Journal of Pharmaceutical and Biomedical Analysis

Volume 8, Issues 8–12, 1990, Pages 783-787

https://doi.org/10.1016/0731-7085(90)80121-5 Get rights and content

Abstract

The low turnover of caffeine in vitro by human liver microsomes makes the study of the metabolic pathways of this compound difficult. Analytical methods with high sensitivity and specificity are needed for the detection of its metabolic products. A method based on the on-line radiometric determination of [8C-³H]caffeine and its principal metabolite (paraxanthine) in man has been developed using reversed-phase high-performance liquid chromatography. The method has been successfully employed in preliminary studies of the kinetics of this reaction.

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Changes in CYP1A2 activity in humans after 3,4-methylenedioxymethamphetamine (MDMA, Ecstasy) administration using caffeine as a probe drug
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3,4-Methylenedioxymethamphetamine (MDMA; ecstasy) is a ring-substituted amphetamine widely used for recreational purposes. MDMA is predominantly O-demethylenated in humans by cytochrome P450 (CYP) 2D6, and is also a potent mechanism-based inhibitor of the enzyme. After assessing the inhibition and recovery of CYP2D6 in a previous study, the aim of this work was to study in humans the activity of CYP1A2 in vivo after CYP2D6 had been inhibited by MDMA, using caffeine as a probe drug. Twelve male and nine female recreational MDMA users were included. In session 1, 100 mg of caffeine was given at 0 h. In session 2, a 1.5 mg/kg MDMA dose (range 75–100 mg) was given at 0 h followed by a 100 mg dose of caffeine 4 h later. Aliquots of plasma were assayed for caffeine (137X) and paraxanthine (17X) and statistically significant differences were assessed with a one-way ANOVA. There were significant gender differences at basal condition, which persisted after MDMA administration. CYP1A2 activity was higher in both genders after drug administration, with an increase in 40% in females and 20% in males. Results show an increase in CYP1A2 activity when CYP2D6 is inhibited by MDMA in both genders, being more pronounced in females.
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The reactions catalysed by the various cytochrome P-450 enzymes are reviewed with respect to the analysis of products by high-performance liquid chromatography (HPLC). Especially biotransformation reactions of purified cytochrome P-450 enzymes in a reconstituted system and in microsomes mainly of rat liver origin are considered. Emphasis is put on the specificity of product formation due to the individual isozymes of cytochrome P-450. It is shown that the presence of eight cytochrome P-450 isozymes can be monitored and determined by specific product formation after HPLC analysis, which is an important parameter in toxicological studies.
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^☆: Presented at the “Second International Symposium on Pharmaceutical and Biomedical Analysis”, April 1990, York, UK.

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Assay of caffeine metabolism in vitro by human liver microsomes using radio-high-performance liquid chromatography☆

Abstract

Toxicol. Lett.

Biochem. Pharmacol.

Hepatology

Farm. Clin.

Clin. Pharmacol. Ther.