Correlation Between Red Blood Cell Survival and Cytochrome P450 1A2 Enzyme Activity

Autologous red blood cell (RBC) 24h recovery in healthy volunteers is known to be highly variable between subjects, but repeatable within subjects. We previously performed metabolic screening on 2 subjects (HR) with consistently high historical 24h recoveries (85%, 88%) and 2 subjects (LR) with lower recoveries (74%, 74%). We observed that the HR subjects had lower RBC caffeine levels than the grand average for the study, while LR subjects had higher levels. Because these differences were not explained by the subjects’ self-reported caffeine consumption, we hypothesized that they may be due to differences in caffeine metabolism and clearance that correlate with RBC survival following storage and transfusion Caffeine (1,3,7-trimethylxanthine - 137X ) is demethylated by cytochrome P450 1A2 (CYP1A2) to form 81.5% paraxanthine (1,7-dimethylxanthine -17X), 5.4% theophylline, and 10.8% theobromine. Single nucleotide polymorphisms (SNPs) in CYP1A2 results in variation in enzyme activity. Polymorphisms in the aromatic hydrocarbon receptor (AHR) may also affect caffeine clearance. Caffeine clearance is the gold standard assay for in vivo determination of CYP1A2 activity, and a 4 hour point estimate may be obtained using the plasma 17X/137X ratio following caffeine challenge.

We performed focused genotyping of the 4 index subjects for 3 CYP1A2 SNPs (rs762551, rs24708932, and rs2472297) and 2 AHR SNPs (rs6968865 and rs4410790), all previously reported as affecting caffeine metabolism. DNA was extracted from whole blood and SNP genotyping performed using real time PCR with Taqman probes on the AB 7500 FAST system. Caffeine metabolic clearance was determined in 3 of 4 index subjects by a controlled challenge. 100mg caffeine PO was administered in apple juice to subjects that had abstained from caffeine for 24h. Venous blood was obtained pre and 4h post caffeine administration. Plasma was separated and frozen within 2h of collection. Caffeine and metabolites were quantified by liquid chromatography-mass spectrometry (LC-MS) with isotopically labeled internal standards. The single point clearance rate of caffeine was estimated as the ratio of the 4h difference from baseline in (17x+theophiline):caffeine.

The one HR who could be recalled for caffeine challenge had the highest 4h caffeine metabolic clearance ratio; the two LR subjects had lower ratios (see Table). HR were homozygous (A/A) for CYP1A2 rs762551, while the 2 LR subjects were heterozygous (A/C) for CYP1A2 rs762551. The correlations between SNP results and caffeine clearance agree with known CYP1A2 activities for these genotypes. Other CYP1A2 and AHR SNPs did not partition with the RBC recoveries, caffeine levels or caffeine metabolic ratios.

In a small number of subjects (n-=2 from each group), historical in vivo RBC recovery was observed to be associated with peripheral blood caffeine levels, caffeine metabolic clearance, and known functional polymorphisms in the CYP1A2 gene. In the event that these associations persist upon subsequent studies with more participants, then additional trials will be needed to test if the caffeine (or its metabolites) has a direct effect upon RBCs or if caffeine clearance is a surrogate marker of CYP1A2 function or activity of other metabolic pathways that can regulate RBC survival after storage and transfusion.

Zimring:Immucor Inc.: Research Funding; Terumo: Research Funding; Haemonetics: Consultancy; Cerus: Honoraria.