HPLC Determination of Caffeine and Paraxanthine in Urine: An Assay Cytochrome P450 1A2 Activity
Cytochrome P450 enzymes are a family of heme-containing proteins located throughout the body with roles in metabolism of endogenous and exogenous compounds. Among exogenous compounds, clinically relevant pharmaceutical agents are nearly all metabolized by P450 enzymes. The activity ofhuman P450 1A2, which converts caffeine into paraxanthine, can be investigated by measuring the change in caffeine and paraxanthine concentrations in urine over time following a single dose of caffeine. The goal of this study was to assess P450 1A2 metabolic activity using an HPLC method capable of determining levels of caffeine and paraxanthine present in urine from 10 study participants over a six hour time period. Paraxanthine was detected in all collected urine samples and ranged in concentration from 1.93 ng/mL to 51.98 |ug/mL. Caffeine was detected in 23 of the 30 urine samples collected and ranged in concentration from 0.70 |ig/mL to 77.17 |iig/mL. The metabolic ratio of each urine sample at each time point was determined using the relationship MR = [paraxanthine]/[caffeine]. The range for MR values found was 0.34 to 26.34. These values are within the ranges described in the literature by other groups with much larger sample size. The largest percentage of the study participants (4 out of 10) were determined to have the greatest value for MR at the 3-hour time point, consistent with rapid caffeine metabolism by P450 1A2. In summary, the data presented here demonstrates that HPLC analysis of urine samples utilizing a caffeine probe is a reliable method for mapping the metabolic activity of P450 1A2.