Biotransformation of caffeine by cDNA-expressed human cytochromes P-450

Eur J Clin Pharmacol. 1996;49(4):309-15. doi: 10.1007/BF00226333.


Objectives: The biotransformation of caffeine has been studied in vitro using human cytochrome P-450 isoenzymes (CYPs) expressed in human B-lymphoblastoid cell lines, namely CYP1A1, 1A2, 2A6, 2B6, 2D6-Val, 2E1 and 3A4, and microsomal epoxide hydroxylase (EH). In addition, CYP2D6-Met was also studied, in which a valine in the wild type (CYP2D6-Val) has been replaced by a methionine due to a G to A mutation in position 112.

Results: At caffeine 3 mmol center dot l-1, five CYPs (1A1, 1A2, 2D6-Met, 2E1 and 3A4) catalysed the biotransformation of caffeine. Among the enzymes studied, CYP1A2, which predominantly catalysed paraxanthine formation, had the highest intrinsic clearance (160 l center dot h-1 center dot mmol-1 CYP). Together with its high abundance in liver, it should be considered, therefore, to be the most important isoenzyme in caffeine metabolism. The affinity of caffeine for CYP1A1 was comparable to that of its homologue 1A2. CYP2D6-Met, which catalysed caffeine metabolism by demethylation and 8-hydroxylation, also had a relatively high intrinsic clearance (3.0 l center dot h-1 mmol-1 CYP), in particular for theophylline and paraxanthine formation, with kM values between 9-16 mmol center dot l-1. In contrast, the wild type, CYP2D6-Val, had no detectable activity. In comparison, CYP2E1 played a less important role in in vitro caffeine metabolism. CYP3A4 predominantly catalysed 8-hydroxylation with a kM value of 46 mmol center dot l-1 and an intrinsic clearance of 0.60 l center dot h-1 center dot mmol-1 CYP. Due to its high abundance in human liver, the latter CYP may contribute significantly to the in vivo formation of TMU.

Conclusion: The findings of this study indicate that i) microsomes from transfected human B-lymphoblastoid cell lines give results close to those obtained with microsomes isolated from human liver, ii) at least four CYP isoforms are involved in caffeine metabolism, iii) at a substrate concentration <0.1 mmol center dot l-1, CYP1A2 and 1A1 are the most important isoenzymes, iv) at higher concentrations the participation of other isoenzymes, in particular CYP3A4, 2E1 and possibly also CYP2D6-Met, are important in caffeine metabolism, and v) the nucleotide composition at position 1120 of CYP2D6 determines the activity of this isoenzyme in caffeine metabolism.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Biotransformation
  • Caffeine / pharmacokinetics*
  • Cell Line
  • Central Nervous System Stimulants / pharmacokinetics*
  • Cytochrome P-450 Enzyme System / metabolism*
  • Microsomes, Liver / enzymology


  • Central Nervous System Stimulants
  • Caffeine
  • Cytochrome P-450 Enzyme System