Riboflavin and pyrroloquinoline quinone generate carbon monoxide in the presence of tissue microsomes or recombinant human cytochrome P-450 oxidoreductase: implications for possible roles in gasotransmission.

Carbon monoxide (CO), an endogenously produced gasotransmitter, regulates inflammation and vascular tone, suggesting that delivery of CO may be therapeutically useful for pathologies like preeclampsia where CO insufficiency is implicated. Our strategy is to identify chemicals that increase the activity of endogenous CO-producing enzymes, including cytochrome P-450 oxidoreductase (CPR). Realizing that both riboflavin and pyrroloquinoline quinone (PQQ) are relatively nontoxic, even at high doses, and that they share chemical properties with toxic CO activators that we previously identified, our goal was to determine whether riboflavin or PQQ could stimulate CO production. Riboflavin and PQQ were incubated in sealed vessels with rat and human tissue extracts and CO generation was measured with headspace-gas chromatography. Riboflavin and PQQ increased CO production ∼60% in rat spleen microsomes. In rat brain microsomes, riboflavin and PQQ increased respective CO production approximately fourfold and twofold compared to baseline. CO production by human placenta microsomes increased fourfold with riboflavin and fivefold with PQQ. In the presence of recombinant human CPR, CO production was threefold greater with PQQ than with riboflavin. These observations demonstrate for the first time that riboflavin and PQQ facilitate tissue-specific CO production with significant contributions from CPR. We propose a novel biochemical role for these nutrients in gastransmission.

Establishment of LC-MS/MS method for quantifying chlorpromazine metabolites with application to its metabolism in liver and placenta microsomes.

Chlorpromazine has sedative and antiemetic pharmacological effects and is widely used in clinic. Its main metabolites include 7-hydroxychlorpromazine, N-monodesmethylchlorpromazine and chlorpromazine sulfoxide, which affect the therapeutic efficacy. To support metabolism research, the quantitative analysis method of 7-hydroxychlorpromazine, N-monodesmethylchlorpromazine and chlorpromazine sulfoxide in microsomal enzymes was established for the first time by LC-MS/MS. This method has been fully validated in rat liver microsomes, and partially verified in human liver microsomes and human placenta microsomes. The intra-day and inter-day accuracy and precision of the analytes were all within ± 15%. The extraction recovery was good, and no matrix effect was detected. This accurate and sensitive method was successfully applied to chlorpromazine metabolism in different microsomal enzymes. In particular, the biotransformation of chlorpromazine in human placenta microsomes was detected for the first time. The metabolites detected in human liver and placenta microsomes presented different formation rates, indicating the wide distribution and different activities of drug-metabolizing enzymes.