Evaluation of 4β-Hydroxycholesterol and 25-Hydroxycholesterol as Endogenous Biomarkers of CYP3A4: Study with CYP3A-Humanized Mice

Abstract

Cytochrome P450 3A (CYP3A) enzymes metabolize approximately half of all drugs on the market. Since the endogenous compounds 4β-hydroxycholesterol (4β-HC) and 25-hydroxycholesterol (25-HC) are generated from cholesterol via CYP3A enzymes, we examined whether the plasma levels of 4β-HC and 25-HC reflect hepatic CYP3A4 activity by using a CYP3A-humanized mouse model, in which the function of endogenous Cyp3a was genetically replaced by human CYP3A. CYP3A-humanized mice have great advantages for evaluation of the relationship between hepatic CYP3A protein levels and plasma and hepatic levels of 4β-HC and 25-HC. Levels of CYP3A4 protein in the liver microsomes of CYP3A-humanized mice were increased by treatment with pregnenolone-16α-carbonitrile, a CYP3A inducer. Hepatic and plasma levels of 4β-HC and 25-HC normalized by cholesterol were significantly correlated with hepatic CYP3A4 protein levels. In addition, in vitro studies using human liver microsomes showed that the formation of 4β-HC was strongly inhibited by a CYP3A inhibitor, while the inhibitory effect of the CYP3A inhibition on the formation of 25-HC was weak. These results suggested that CYP3A mainly contributed to the formation of 4β-HC in human liver microsomes, whereas other factors may be involved in the formation of 25-HC. In conclusion, the in vivo studies using CYP3A-humanized mice suggest that plasma 4β-HC and 25-HC levels reflect hepatic CYP3A4 activity. Furthermore, taking the results of in vitro studies using human liver microsomes into consideration, 4β-HC is a more reliable biomarker of hepatic CYP3A activity.

Elevated 4β‐hydroxycholesterol/cholesterol ratio in anorexia nervosa patients

“…An advantage with 4βOHC as biomarker is its selectivity; CYP3A4 and CYP3A5 convert cholesterol to 4βOHC, while only negligible amounts of 4βOHC were produced by seven other CYP enzymes . Furthermore, 4βOHC has an unusually long half‐life of up to 17 days, which leads to low intra‐individual variability .…”

“…CYP3A4 and CYP3A5 convert cholesterol to 4βOHC, while only negligible amounts of 4βOHC were produced by seven other CYP enzymes. 3,4 Furthermore, 4βOHC has an unusually long half-life of up to 17 days, which leads to low intra-individual variability. 27 Since 4βOHC is sensitive to CYP3A induction, 27 we consider it a suitable biomarker for the present study where anorexic patients displayed elevated 4βOHC levels.…”

“…Substantial inter‐individual variability in CYP3A activity exists due to a combination of genetic, environmental, and endogenous factors . CYP3A phenotype can be measured by the endogenous biomarker 4β‐hydroxycholesterol (4βOHC), which is metabolized from cholesterol by CYP3A4 and CYP3A5, the two most important CYP3A enzymes in humans . Variations in cholesterol concentration have been found to explain about 10% of 4βOHC variation, and 4βOHC/cholesterol (4βOHC/C) ratio is preferable to 4βOHC as CYP3A biomarker in patient groups where cholesterol levels are abnormal …”

“…2 CYP3A phenotype can be measured by the endogenous biomarker 4βhydroxycholesterol (4βOHC), which is metabolized from cholesterol by CYP3A4 and CYP3A5, the two most important CYP3A enzymes in humans. 3,4 Variations in cholesterol concentration have been found to explain about 10% of 4βOHC variation, 5 and 4βOHC/cholesterol (4βOHC/C) ratio is preferable to 4βOHC as CYP3A biomarker in patient groups where cholesterol levels are abnormal. 6 Recent studies have shown that body weight is negatively correlated with 4βOHC/C ratio in healthy volunteers, 7 and that obese patients have lower 4βOHC levels than healthy controls.…”

Localisation of oxysterols at the sub-cellular level and in biological fluids

“…Non-heme iron-containing oxidoreductase, cholesterol 25-hydroxylase (CH25H), has recently received attention due to the involvement of its product, 25-OHC, in immunity control [14]. However, 25-OHC has been reported to be produced through other cytochromes (CYP3A4, CYP27, and CYP46A1) and also through free-radical reactions [15][16][17]. Cholesterol CYP46A1 24S-OHC Cholesterol CH25H 25-OHC Cholesterol CYP27 27-OHC 7α-OHC CYP27 7α,26-diOHC 22R-OHC CYP11A1 20R,22R-diOHC 24S-OHC CYP39A1 7α,24S-diOHC 25-OHC CYP7B1 7α,25-diOHC 27-OHC CYP7B1 7α,27-diOHC Table 1: Oxidoreductase enzymes involved in the metabolism of oxysterols Similar to cholesterol, oxysterols can also be sulfated by sulfotransferases (SULT2B1b, SULT2B1a and SULT2A1) in the position 3 of the steroid ring A.…”