Liquid chromatography-tandem mass spectrometry analysis of oxidation of 2′-, 3′-, 4′- and 6-hydroxyflavanones by human cytochrome P450 enzymes

Abstract

1. 2′-Hydroxyflavanone (2′OHFva), 3′OHFva, 4′OHFva, and 6OHFva, the major oxidative products of flavanone by human cytochrome P450 (P450, CYP) enzymes, were studied in regard to further oxidation by human CYP1A1, 1A2, 1B1.1, 1B1.3, and 2A6. The products formed were analyzed with LC-MS/MS and characterized by their positive ion fragmentations on mass spectrometry.

2. Several di-hydroxylated flavanone (diOHFva) and di-hydroxylated flavone (diOHFvo) products, detected by analyzing parent ions at m/z 257 and 255, respectively, were found following incubation of these four hydroxylated flavanones with P450s. The m/z 257 products were produced at higher levels than the latter with four substrates examined. The structures of the m/z 257 products were characterized by LC-MS/MS product ion spectra, and the results suggest that 3′OHFva and 4′OHFva are further oxidized mainly at B-ring by P450s while 6OHFva oxidation was at A-ring.

3. Different diOHFvo products (m/z 255) were also characterized by LC-MS/MS, and the results suggested that most of these diOHFvo products were formed through oxidation or desaturation of the diOHFva products (m/z 257) by P450s. Only when 4′OHFva (m/z 241) was used as a substrate, formation of 4′OHFvo (m/z 239) was detected, indicating that diOHFvo might also be formed through oxidation of 4′OHFvo by P450s.

4. Finally, our results indicated that CYP1 family enzymes were more active than CYP2A6 in catalyzing the oxidation of these four hydroxylated flavanones, and these findings were supported by molecular docking studies of these chemicals with active sites of P450 enzymes.

Keywords:

• Hydroxy flavanones
• LC-MS/MS
• oxidation
• human
• cytochrome P450

Disclosure statement

No potential conflict of interest was reported by the author(s).

Additional information

Funding

This study was supported in part by JSPS KAKENHI [16K21710] (to H. N), [15K07770] (to S. T.), [17K08630] (to J. K.), [JP18K0600] (to M. K.), [17K08426] (to N. M.), and [17K08425] (to H. Y.), National Research Foundation of Korea [NRF-2019R1A2C1004722] (to D. K.), and United States Public Health Service grant [R01 GM118122] (to F. P. G.). The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.