Marked elevation in cortical urate and xanthine oxidoreductase activity in experimental bacterial meningitis

Experimental bacterial meningitis due to Streptococcus pneumoniae in infant rats was associated with a time-dependent increase in CSF and cortical urate that was approximately 30-fold elevated at 22 h after infection compared to baseline. This increase was mirrored by a 20-fold rise in cortical xanthine oxidoreductase activity. The relative proportion of the oxidant-producing xanthine oxidase to total activity did not increase, however. Blood plasma levels of urate also increased during infection, but part of this was as a consequence of dehydration, as reflected by elevated ascorbate concentrations in the plasma. Administration of the radical scavenger alpha-phenyl-tert-butyl nitrone, previously shown to be neuroprotective in the present model, did not significantly affect either xanthine dehydrogenase or xanthine oxidase activity, and increased even further cortical accumulation of urate. Treatment with the xanthine oxidoreductase inhibitor allopurinol inhibited CSF urate levels earlier than those in blood plasma, supporting the notion that urate was produced within the brain. However, this treatment did not prevent the loss of ascorbate and reduced glutathione in the cortex and CSF. Together with data from the literature, the results strongly suggest that xanthine oxidase is not a major cause of oxidative stress in bacterial meningitis and that urate formation due to induction of xanthine oxidoreductase in the brain may in fact represent a protective response.

P450 oxidoreductase deficiency - a new form of congenital adrenal hyperplasia

Patients with adrenal insufficiency, genital anomalies and bony malformations resembling the Antley- Bixler syndrome (a craniosynostosis syndrome), are likely to have P450 oxidoreductase (POR) deficiency. Since our first report in 2004, about 26 recessive POR mutations have been identified in 50 patients. POR is the obligate electron donor to all microsomal (type II) P450 enzymes, including the steroidogenic enzymes CYP17A1, CYP21A2 and CYP19A1. POR deficiency may cause disordered sexual development manifested as genital undervirilization in 46,XY newborns as well as overvirilization in those who are 46,XX. This may be explained by impaired aromatization of fetal androgens which may also lead to maternal virilization and low urinary estriol levels during pregnancy. A role for the alternate 'backdoor' pathway of androgen biosynthesis, leading to dihydrotestosterone production bypassing androstenedione and testosterone, has been suggested in POR deficiency but remains unclear. POR variants may play an important role in drug metabolism, as most drugs are metabolized by hepatic microsomal P450 enzymes. However, functional assays studying the effects of specific POR mutations on steroidogenesis showed that several POR variants impaired CYP17A1, CYP21A2 and CYP19A1 activities to different degrees, indicating that each POR variant must be studied separately for each potential target P450 enzyme. Thus, the impact of POR mutations on drug metabolism by hepatic P450s requires further investigation.

Modeling of human P450 oxidoreductase structure by in silico mutagenesis and MD simulation

P450 oxidoreductase (POR) is the obligate electron donor for microsomal cytochrome P450s and mutations in POR cause several metabolic disorders. We have modeled the structure of human P450 oxidoreductase by in silico amino acid replacements in the rat POR crystal structure. The rat POR has 94% homology with human POR and 38 amino acids were replaced to make its sequence identical to human POR. Several rounds of molecular dynamic simulations refined the model and removed structural clashes from side chain alterations of replaced amino acids. This approach has the advantage of keeping the cofactor contacts and structural features of the core enzyme intact which could not be achieved by homology based approaches. The final model from our approach was of high quality and compared well with experimentally determined structures of other PORs. This model will be used for analyzing the structural implications of mutations and polymorphisms in human POR.

P450 oxidoreductase deficiency: a new disorder of steroidogenesis

Microsomal P450 enzymes, which metabolize drugs and catalyze steroid biosynthesis require electron donation from NADPH via P450 oxidoreductase (POR). POR knockout mice are embryonically lethal, but we found recessive human POR missense mutations causing disordered steroidogenesis and Antley-Bixler syndrome (ABS), a skeletal malformation syndrome featuring craniosynostosis. Dominant mutations in exons 8 and 10 of fibroblast growth factor receptor 2 (FGFR2) cause phenotypically related craniosynostosis syndromes and were reported in patients with ABS and normal steroidogenesis. Sequencing POR and FGFR2 exons in 32 patients with ABS and/or hormonal findings suggesting POR deficiency showed complete genetic segregation of POR and FGFR2 mutations. Fifteen patients carried POR mutations on both alleles, four carried POR mutations on 1 allele, nine carried FGFR2/3 mutations on one allele and no mutation was found in three patients. The 34 affected POR alleles included 10 with A287P, 7 with R457H, 9 other missense mutations and 7 frameshifts. These 11 missense mutations and 10 others identified by database mining were expressed in E. coli, purified to apparent homogeneity, and their catalytic capacities were measured in four assays: reduction of cytochrome c, oxidation of NADPH, and support of the 17alpha-hydroxylase and 17,20 lyase activities of human P450c17. As assessed by Vmax/Km, 17,20 lyase activity provided the best correlation with clinical findings. Modeling human POR on the X-ray crystal structure of rat POR shows that these mutant activities correlate well with their locations in the structure. POR deficiency is a new disease, distinct from the craniosynostosis syndromes caused by FGFR mutations.

Diversity and function of mutations in p450 oxidoreductase in patients with Antley-Bixler syndrome and disordered steroidogenesis

P450 oxidoreductase (POR) is the obligatory flavoprotein intermediate that transfers electrons from reduced nicotinamide adenine dinucleotide phosphate (NADPH) to all microsomal cytochrome P450 enzymes. Although mouse Por gene ablation causes embryonic lethality, POR missense mutations cause disordered steroidogenesis, ambiguous genitalia, and Antley-Bixler syndrome (ABS), which has also been attributed to fibroblast growth factor receptor 2 (FGFR2) mutations. We sequenced the POR gene and FGFR2 exons 8 and 10 in 32 individuals with ABS and/or hormonal findings that suggested POR deficiency. POR and FGFR2 mutations segregated completely. Fifteen patients carried POR mutations on both alleles, 4 carried mutations on only one allele, 10 carried FGFR2 or FGFR3 mutations, and 3 patients carried no mutations. The 34 affected POR alleles included 10 with A287P (all from whites) and 7 with R457H (four Japanese, one African, two whites); 17 of the 34 alleles carried 16 "private" mutations, including 9 missense and 7 frameshift mutations. These 11 missense mutations, plus 10 others found in databases or reported elsewhere, were recreated by site-directed mutagenesis and were assessed by four assays: reduction of cytochrome c, oxidation of NADPH, support of 17alpha-hydroxylase activity, and support of 17,20 lyase using human P450c17. Assays that were based on cytochrome c, which is not a physiologic substrate for POR, correlated poorly with clinical phenotype, but assays that were based on POR's support of catalysis by P450c17--the enzyme most closely associated with the hormonal phenotype--provided an excellent genotype/phenotype correlation. Our large survey of patients with ABS shows that individuals with an ABS-like phenotype and normal steroidogenesis have FGFR mutations, whereas those with ambiguous genitalia and disordered steroidogenesis should be recognized as having a distinct new disease: POR deficiency.

P450 oxidoreductase deficiency: a new disorder of steroidogenesis affecting all microsomal P450 enzymes

Combined partial deficiency of 17alpha-hydroxylase and 21-hydroxylase activities was first described in 1985; however the genes for P450c17 and P450c21 in these patients lack mutations. In 1986 we postulated that this disorder might be due to mutations in P450 oxidoreductase (POR), the flavoprotein that donates electron to these and all other microsomal P450 enzymes, but this hypothesis was not tested until the POR gene sequence became available through the genome database. We found five POR missense mutations in our first four patients. In vitro assays of the activities of these mutations showed that the standard assay of POR activity, reduction of cytochrome c, correlated poorly with the patients' phenotypes, but that assays of POR-supported 17alpha-hydroxylase and 17,20 lyase activities correlated well. POR deficiency is a new disorder of adrenal and gonadal steroidogenesis that affects all microsomal cytochrome P450 enzymes, hence may have important implications for genetic differences in drug metabolism.

Mutant P450 oxidoreductase causes disordered steroidogenesis with and without Antley-Bixler syndrome

Deficient activities of multiple steroidogenic enzymes have been reported without and with Antley-Bixler syndrome (ABS), but mutations of corresponding cytochrome P450 enzymes have not been found. We identified mutations in POR, encoding P450 oxidoreductase, the obligate electron donor for these enzymes, in a woman with amenorrhea and three children with ABS, even though knock-out of POR is embryonically lethal in mice. Mutations of POR also affect drug-metabolizing P450 enzymes, explaining the association of ABS with maternal fluconazole ingestion.

Pharmacogenomics of human P450 oxidoreductase

Cytochrome P450 oxidoreductase (POR) supports reactions of microsomal cytochrome P450 which metabolize drugs and steroid hormones. Mutations in POR cause disorders of sexual development. P450 oxidoreductase deficiency (PORD) was initially identified in patients with Antley-Bixler syndrome (ABS) but now it has been established as a separate disorder of sexual development (DSD). Here we are summarizing the work on variations in POR related to metabolism of drugs and xenobiotics. We have compiled mutation data on reported cases of PORD from clinical studies. Mutations found in patients with defective steroid profiles impact metabolism of steroid hormones as well as drugs. Some trends are emerging that establish certain founder mutations in distinct populations, with Japanese (R457H), Caucasian (A287P), and Turkish (399-401) populations showing repeated findings of similar mutations. Most other mutations are found as single occurrences. A large number of different variants in POR gene with more than 130 amino acid changes are now listed in databases. Among the polymorphisms, the A503V is found in about 30% of all alleles but there are some differences across different population groups.

Heterologous expression of equine CYP3A94 and investigation of a tunable system to regulate co-expressed NADPH P450 oxidoreductase levels.

The activity of cytochrome P450 enzymes depends on the enzyme NADPH P450 oxidoreductase (POR). The aim of this study was to investigate the activity of the equine CYP3A94 using a system that allows to regulate the POR protein levels in mammalian cells. CYP3A94 and the equine POR were heterologously expressed in V79 cells. In the system used, the POR protein regulation is based on a destabilizing domain (DD) that transfers its instability to a fused protein. The resulting fusion protein is therefore degraded by the ubiquitin-proteasome system (UPS). Addition of "Shield-1" prevents the DD fusion protein from degradation. The change of POR levels at different Shield-1 concentrations was demonstrated by cytochrome c reduction, Western immunoblot analysis, and immunocytochemistry. The alteration of CYP3A94 activity was investigated using a substrate (BFC) known to detect CYP3A4 activity. Equine CYP3A94 was demonstrated to be metabolically active and its activity could be significantly elevated by co-expression of POR. Cytochrome c reduction was significantly increased in V79-CYP3A94/DD-POR cells compared to V79-CYP3A94 cells. Surprisingly, incubation with different Shield-1 concentrations resulted in a decrease in POR protein shown by Western immunoblot analysis. Cytochrome c reduction did not change significantly, but the CYP3A94 activity decreased more than 4-fold after incubation with 500 nM and 1 µM Shield-1 for 24 hours. No differences were obtained when V79-CYP3A94 POR cells with and without Shield-1 were compared. The basal activity levels of V79-CYP3A94/DD-POR cells were unexpectedly high, indicating that DD/POR is not degraded without Shield-1. Shield-1 decreased POR protein levels and CYP3A94 activity suggesting that Shield-1 might impair POR activity by an unknown mechanism. Although regulation of POR with the pPTuner system could not be obtained, the cell line V79-CYP3A94/DD-POR system can be used for further experiments to characterize the equine CYP3A94 since the CYP activity was significantly enhanced with co-expressed POR.

Control of Steroid 21-oic Acid Synthesis by Peroxisome Proliferator-activated Receptor and Role of the Hypothalamic-Pituitary-Adrenal Axis

A previous study identified the peroxisome proliferator-activated receptor alpha (PPARalpha) activation biomarkers 21-steroid carboxylic acids 11beta-hydroxy-3,20-dioxopregn-4-en-21-oic acid (HDOPA) and 11beta,20-dihydroxy-3-oxo-pregn-4-en-21-oic acid (DHOPA). In the present study, the molecular mechanism and the metabolic pathway of their production were determined. The PPARalpha-specific time-dependent increases in HDOPA and 20alpha-DHOPA paralleled the development of adrenal cortex hyperplasia, hypercortisolism, and spleen atrophy, which was attenuated in adrenalectomized mice. Wy-14,643 activation of PPARalpha induced hepatic FGF21, which caused increased neuropeptide Y and agouti-related protein mRNAs in the hypothalamus, stimulation of the agouti-related protein/neuropeptide Y neurons, and activation of the hypothalamic-pituitary-adrenal (HPA) axis, resulting in increased adrenal cortex hyperplasia and corticosterone production, revealing a link between PPARalpha and the HPA axis in controlling energy homeostasis and immune regulation. Corticosterone was demonstrated as the precursor of 21-carboxylic acids both in vivo and in vitro. Under PPARalpha activation, the classic reductive metabolic pathway of corticosterone was suppressed, whereas an alternative oxidative pathway was uncovered that leads to the sequential oxidation on carbon 21 resulting in HDOPA. The latter was then reduced to the end product 20alpha-DHOPA. Hepatic cytochromes P450, aldehyde dehydrogenase (ALDH3A2), and 21-hydroxysteroid dehydrogenase (AKR1C18) were found to be involved in this pathway. Activation of PPARalpha resulted in the induction of Aldh3a2 and Akr1c18, both of which were confirmed as target genes through introduction of promoter luciferase reporter constructs into mouse livers in vivo. This study underscores the power of mass spectrometry-based metabolomics combined with genomic and physiologic analyses in identifying downstream metabolic biomarkers and the corresponding upstream molecular mechanisms.

Restoration of mutant cytochrome P450 reductase activity by external flavin

Cytochrome P450 oxidoreductase (POR) supplies electrons from NADPH to steroid and drug metabolizing reactions catalyzed by the cytochrome P450s located in endoplasmic reticulum. Mutations in human POR cause a wide spectrum of disease ranging from disordered steroidogenesis to sexual differentiation. Previously we and others have shown that POR mutations can lead to reduced activities of steroidogenic P450s CYP17A1, CYP19A1 and CYP21A1. Here we are reporting that mutations in the FMN binding domain of POR may reduce CYP3A4 activity, potentially influencing drug and steroid metabolism; and the loss of CYP3A4 activity may be correlated to the reduction of cytochrome b(5) by POR. Computational molecular docking experiments with a FMN free structural model of POR revealed that an external FMN could be docked in close proximity to the FAD moiety and receive electrons donated by NADPH. Using FMN supplemented assays we have demonstrated restoration of the defective POR activity in vitro.

One-Step (18)F-Labeling of Carbohydrate-Conjugated Octreotate-Derivatives Containing a Silicon-Fluoride-Acceptor (SiFA): In Vitro and in Vivo Evaluation as Tumor Imaging Agents for Positron Emission Tomography (PET)

The synthesis, radiolabeling, and initial evaluation of new silicon-fluoride acceptor (SiFA) derivatized octreotate derivatives is reported. So far, the main drawback of the SiFA technology for the synthesis of PET-radiotracers is the high lipophilicity of the resulting radiopharmaceutical. Consequently, we synthesized new SiFA-octreotate analogues derivatized with Fmoc-NH-PEG-COOH, Fmoc-Asn(Ac?AcNH-?-Glc)-OH, and SiFA-aldehyde (SIFA-A). The substances could be labeled in high yields (38 ± 4%) and specific activities between 29 and 56 GBq/?mol in short synthesis times of less than 30 min (e.o.b.). The in vitro evaluation of the synthesized conjugates displayed a sst2 receptor affinity (IC?? = 3.3 ± 0.3 nM) comparable to that of somatostatin-28. As a measure of lipophilicity of the conjugates, the log P(ow) was determined and found to be 0.96 for SiFA-Asn(AcNH-?-Glc)-PEG-Tyr³-octreotate and 1.23 for SiFA-Asn(AcNH-?-Glc)-Tyr³-octreotate, which is considerably lower than for SiFA-Tyr³-octreotate (log P(ow) = 1.59). The initial in vivo evaluation of [¹?F]SiFA-Asn(AcNH-?-Glc)-PEG-Tyr³-octreotate revealed a significant uptake of radiotracer in the tumor tissue of AR42J tumor-bearing nude mice of 7.7% ID/g tissue weight. These results show that the high lipophilicity of the SiFA moiety can be compensated by applying hydrophilic moieties. Using this approach, a tumor-affine SiFA-containing peptide could successfully be used for receptor imaging for the first time in this proof of concept study.