Effects of Diminished NADPH:cytochrome P450 Reductase in Human Hepatocytes on Lipid and Bile Acid Homeostasis

NADPH:cytochrome P450 oxidoreductase (POR) is the obligate electron donor for microsomal cytochrome P450 (CYP) enzymes involved in the biosynthesis of endogenous substances like bile acids and other steroids as well as in the oxidative metabolism of xenobiotics. P450 oxidoreductase also supports other redox enzymes in fatty acid and cholesterol pathways. Recently, we have established CRISPR/Cas9-mediated POR knockdown in a human hepatic cell model, HepaRG, and demonstrated the differential effects of limited POR expression on CYP activity. The aim of the present work was to systematically investigate the impact of POR knockdown with a focus on the expression of ADME (absorption, distribution, metabolism, and excretion) genes and related regulators. Functional consequences have been assessed using quantitative mass spectrometry for targeted metabolomics covering bile acids, and cholesterol and its precursors, and for untargeted proteomics. In addition to the previously described alteration of RNA expression of CYP genes, we showed significant downregulation of transcriptional regulators of drug metabolism and transport, including NR1I3 (CAR), NR1I2 (PXR), NR1H4 (FXR), and NR1H3 (LXRα) in cells with POR gene disruption. Furthermore, POR knockdown resulted in deregulated bile acid and cholesterol biosynthesis demonstrated by low levels of cholic acid derivates and increased concentrations of chenodeoxycholic acid derivates, respectively. Systemic effects of POR knockdown on global protein expression were indicated by downregulation of several metabolic pathways including lipid metabolism and biological oxidation reactions. The deduced protein network map corroborates CYP enzymes as direct interaction partners, whereas changes in lipid metabolism and homeostasis are the result of indirect effects. In summary, our results emphasize a widespread role of POR in various metabolic pathways and provide the first human data on the effects of diminished POR expression on drug and endogenous metabolism in a genomeedited HepaRG cell model.

Study on Changes of 19 Steroids in Plasma From 9 Diagnosed Patients With P450 Oxidoreductase Deficiency

Purpose: Diagnostic of congenital adrenal hyperplasia (CAH) patients with P450 oxidoreductase deficiency (PORD) subtype is a difficult task as biochemical monitoring criteria is not well defined. For the identification of steroid hormone biomarkers in plasma to diagnose PORD. Methods: Study of the steroid hormone metabolic pathway, a novel method for the determination of 19 steroid hormone were established based on liquid chromatography–tandem mass spectrometry (LC-MS/MS). The measurement of 19 steroid hormones in plasma samples of clinical patients and normal reference population was carried out. A total of 9 clinically diagnosed patients (3 males, 6 females) with PORD were enrolled in the study. Results: In 9 patients with PORD, plasma pregnenolone, 17-hydroxyprogesterone, corticosterone, 11-deoxycorticosterone, 11-deoxycortisol, and 21-deoxycortisol, were significantly (P<0.001) increased when compared with those of the normal control group at the initial diagnosis of PORD. The concentrations of androstenedione, testosterone, dihydrotestosterone, dehydroepiandrosterone sulfate and estradiol in male patients with PORD were significantly (P<0.001) lower than those in control group. There were significant (P<0.001) differences between the female patients with PORD and the control group in the concentration of dihydrotestosterone and dehydroepiandrosterone sulfate. Conclusion: Pregnenolone, 17-hydroxyprogesterone, corticosterone, 11-deoxycorticosterone, 11-deoxycorticosteroid, 21-deoxycorticosteroid, androstenedione, testosterone, dihydrotestosterone, dehydroepiandrosterone, estradiol, and the ratio of testosterone to dihydrotestosterone are important markers in the diagnosis of PORD.

Biased cytochrome P450-mediated metabolism via small-molecule ligands binding P450 oxidoreductase

Metabolic control is mediated by the dynamic assemblies and function of multiple redox enzymes. A key element in these assemblies, the P450 oxidoreductase (POR), donates electrons and selectively activates numerous (>50 in humans and >300 in plants) cytochromes P450 (CYPs) controlling metabolism of drugs, steroids and xenobiotics in humans and natural product biosynthesis in plants. The mechanisms underlying POR-mediated CYP metabolism remain poorly understood and to date no ligand binding has been described to regulate the specificity of POR. Here, using a combination of computational modeling and functional assays, we identify ligands that dock on POR and bias its specificity towards CYP redox partners, across mammal and plant kingdom. Single molecule FRET studies reveal ligand binding to alter POR conformational sampling, which results in biased activation of metabolic cascades in whole cell assays. We propose the model of biased metabolism, a mechanism akin to biased signaling of GPCRs, where ligand binding on POR stabilizes different conformational states that are linked to distinct metabolic outcomes. Biased metabolism may allow designing pathway-specific therapeutics or personalized food suppressing undesired, disease-related, metabolic pathways.

Successful live birth in a Chinese woman with P450 oxidoreductase deficiency through frozen-thawed embryo transfer: a case report with review of the literature

Background Congenital adrenal hyperplasia (CAH) caused by P450 oxidoreductase deficiency (PORD) in 46, XX patients is characterized by genital ambiguity, primary amenorrhea, absent or incomplete sexual maturation, infertility, skeletal malformations and so on. But few pregnancies have been reported from these female patients with PORD. Case description A 29-year-old Chinese woman with PORD due to the compound heterozygous mutation (c.1370G > A/c.1196_1204del) in the P450 oxidoreductase (POR) gene had suffered from primary amenorrhea and infertility. She had one cancelled cycle of ovulation induction due to low serum estradiol(E2), high progesterone(P) levels and thin endometrium, then in vitro fertilization (IVF) was recommended. At the first IVF cycle, 4 oocytes were retrieved and 4 viable embryos were cryopreserved due to thin endometrium associated with low E2 and prematurely elevated P after ovarian stimulation, even though oral dexamethasone were used to control adrenal P overproduction at the same time. When basal P fell to < 1.5 ng/ml after the therapy of oral dexamethasone, artificial endometrial preparation and frozen embryo transfer were performed, resulting in a twin pregnancy. She delivered a healthy boy and a healthy girl by caesarean section at 37 weeks and 2 days of gestation. After the literature search in PORD women, no spontaneous pregnancy has been reported and only two previous case reports of 3 successful pregnancies through IVF were summarized. Conclusions It is the third report that successful pregnancy was achieved in a CAH woman caused by a compound heterozygous POR mutation, with primary amenorrhea and disorders of steroidogenesis. It seemed that disorders of steroidogenesis caused by PORD didn’t impair the developmental potential of oocytes. IVF and frozen embryo transfer after adequate hormonal control and endometrial preparation should be an effective infertility treatment for PORD women.