Fig. 2. Determining the role of CYP1B1 in the biochemistry of t-RA synthesis and breakdown. (A) Reverse phase HPLC profile of the metabolism of t-retinol by CYP1B1 (0.025 nM CYP1B1 in 0.1 M potassium phosphate buffer pH 7.4, plus NADPH-cytochrome p450 reductase; 20 minutes at 37°C). Black arrow, input retinol; blue arrow, output retinal. (B) Reverse phase HPLC profile of the metabolism of t-retinal by CYP1B1 under the same conditions for differing lengths of time (20, 60 and 240 minutes). Black arrow, input retinal; blue arrow, output RA. The rates of synthesis of RA are given in Table 2. (C) Reverse phase HPLC profile of the metabolism of t-RA by CYP1B1 under the same conditions. All incubations were repeated in the absence of NADPH or CYP1B1, and no metabolic conversion of the input substrate was recorded in either case (data not shown). The data support a model in which CYP1B1 can actively convert retinol to retinal and then to RA, but cannot participate in the breakdown of either the newly formed RA or RA from other anabolic sources.