Purpose

Perillyl alcohol (POH), a flavoring terpenoid agent, is now being evaluated as a chemotherapeutic and chemopreventive agent. The mechanism of action is thought to be related to its phase I and phase II metabolites, which have only been partially elucidated. The objective of this study is to characterize its metabolic profiles in humans and in human intestinal cells and cryopreserved hepatocytes.

Methods

Following a single oral dose of 500 mg POH, the cumulative 24-hr urine was collected. POH was also incubated separately in human cryopreserved hepatocytes and human CaCO-2 cells for 2 hrs. POH metabolism was profiled in urine and in incubates. A tandem LC/MS/MS method using a LUNA C-18 column eluted with methanol-water-formic acid mobile phase system was used for metabolite profiling. While the urine samples and its blank were directly injected into LC/MS/MS system, the cell incubates and their blanks were treated with addition of acetonitrile or methanol, and the supernatants were lyophilized. The lyophilized powder was reconstituted with an appropriate volume of MeOH:H₂O (1:1) before LC/MS/MS analysis. The analysis was carried out under positive-ion electrospray mode. To fully capture all the Phase I and Phase II metabolites produced by POH, the following LC/MS/MS scanning strategies were used: 1) a full scan was run first in both the blanks and samples to generate total ion chromatograms (TIC); 2) extracted ion chromatograms (XICs) were then generated, based on data obtained from step 1); 3) XICs blanks and samples were then compared to select the potential metabolites; 4) product scans were performed for the [M+1] ions of the potential metabolites found from step 3); 5) product scan mass spectra were elucidated to resolve the structures of the potential metabolites; 6) in some cases, the precursor scan and neutral loss scan were used to confirm the proposed metabolite structures; 7) β-glucuronidase enzymatic hydrolysis of the samples was conducted to confirm the existence of the glucuronide conjugates.

Results

POH was found to be extensively metabolized by Phase I and Phase II metabolic enzymes. Perillic acid (PA), PA-glucuronide and POH-glucuronide conjugates were detected in CaCO-2 cell incubates. PA, PA-isomers, cis-/trans-dihydroperillic acid (DHPA), two forms of POH-cysteine conjugates, two forms of POH-glutathione conjugates, POH-glucuronide conjugate, glucuronide conjugates of PA and PA-isomers were observed in human cryopreserved hepatocytes. PA-glycine conjugate, two forms of POH cysteine conjugates, POH-acetylcysteine conjugates, glucuronide conjugates of PA- and its isomers, DHPA-glucuronide and POH-glucuronide conjugates were found in human urine samples.

Conclusion

The unique chemical structure of POH makes it highly susceptible to multiple Phase I and Phase II enzymes. POH may be useful as a model compound for screening for profiling metabolic activity in a cell and enzyme system.