Why Dried Blood Spots Are an Ideal Tool for CYP1A2 Phenotyping

Abstract

Background and ObjectiveDried blood spot (DBS) sampling has gained wide interest in bioanalysis during the last decade. Also in pharmacokinetic and phenotyping studies, DBS-based sampling has already been successfully applied. However, all available phenotyping studies used small data sets and did not include a systematic evaluation of DBS-specific parameters. The latter is important since several of these factors still challenge the breakthrough of DBS in routine practice. In this study, caffeine and paraxanthine are determined in capillary DBS, venous DBS, whole blood and plasma for CYP1A2 phenotyping (CYP; cytochrome P450). The aim of this study was to explore the usefulness of DBS as a tool for CYP1A2 phenotyping. MethodsA CYP1A2 phenotyping study was conducted in seventy-three healthy volunteers, who received a 150 mg oral dose of caffeine. Six hours post-administration, caffeine and paraxanthine concentrations and paraxanthine:caffeine molar concentration ratios, i.e. the actual CYP1A2 phenotyping indices, were determined in capillary DBS (obtained by non-volumetric application, direct from the fingertip), venous DBS, whole blood and plasma. Furthermore, the impact of DBS-specific parameters, including hematocrit, volume spotted and punch location, was evaluated. ResultsConcentrations of caffeine and paraxanthine in capillary DBS were on average 12.7 respectively 13.8 % lower than those in venous DBS and 31.5 respectively 33.1 % lower than those in plasma. While these differences were statistically significant (p < 0.001), no significant difference was observed between the paraxanthine:caffeine molar ratios in the distinct evaluated matrices (p ≥ 0.053). This ratio also alleviated the impact of hematocrit and volume spotted. ConclusionsUsing the largest DBS-based phenotyping study to date, we have demonstrated that CYP1A2 phenotyping in capillary DBS is a valid and convenient alternative for the classical plasma-based approach. Additionally, we have provided an objective basis as to why DBS are an ideal tool for CYP1A2 phenotyping.  While capillary DBS concentrations of caffeine and paraxanthine differed significantly from those in venous blood and plasma and were impacted by hematocrit and blood volume spotted, the paraxanthine:caffeine molar ratio essentially remained unaffected. Capillary DBS-based CYP1A2 phenotyping proved to be a valid and convenient alternative for the classical plasma-based approach.4

Paraxanthine/Caffeine Concentration Ratios in Hair: An Alternative for Plasma-Based Phenotyping of Cytochrome P450 1A2?

“…Linear calibration curves for caffeine and paraxanthine ranged from 20 to 500 pg/mg, with 20 pg/mg being the lower limit of quantification (LLOQ) for both compounds. Intra-and interbatch precision was below 12% (% relative standard deviation, %RSD) and accuracy below 7% (%bias) for all QCs (20,60,200 and 400 pg/mg).…”

“…For 4 volunteers, a segmental analysis of 6 consecutive 3-cm hair segments was performed as well. At the day of hair sampling, a standard CYP1A2 phenotyping procedure was conducted in the same study population, as described elsewhere [20]. Briefly, caffeine and paraxanthine concentrations were determined with a validated method in plasma samples from volunteers that had taken a 150 mg caffeine capsule 6 hours (± 5 minutes) before sample collection [20,22].…”

“…At the day of hair sampling, a standard CYP1A2 phenotyping procedure was conducted in the same study population, as described elsewhere [20]. Briefly, caffeine and paraxanthine concentrations were determined with a validated method in plasma samples from volunteers that had taken a 150 mg caffeine capsule 6 hours (± 5 minutes) before sample collection [20,22]. By means of a written questionnaire, data on sex, age, general health status, smoking habits, caffeine intake, alcohol consumption, intake of drugs (including hormonal contraception) and cosmetic hair colouring were obtained.…”

“…In typical phenotyping approaches the paraxanthine/caffeine concentration ratio is determined 4-6 h post-administration in serum or plasma samples [19], although dried blood spots [20] and saliva [21] have proven to be appropriate matrices for this purpose as well. A CYP1A2 phenotyping procedure based on hair analysis would eliminate the need to administer a caffeine test dose, since it is expected that caffeine and paraxanthine will readily be detected in hair samples of a large part of the population given the widespread consumption of caffeine-containing beverages (e.g.…”

Alternative Sampling Strategies for Cytochrome P450 Phenotyping

“…As a consequence, Bland-Altman and Passing-Bablok plots showed that paraxanthine/caffeine ratios, i.e. the CYP1A2 phenotyping metric, were highly comparable in all matrices [41].…”