Microbial Models of Mammalian Metabolism: Stereoselective Metabolism of Warfarin in the Fungus Cunninghamella elegans

Biotransformation stereoselectivity of warfarin was studied in the fungus Cunninghamella elegans (ATCC 36112) as a model of mammalian metabolism. This organism was previously shown to produce all known phenolic mammalian metabolites of warfarin, including 6-, 7-, 8-, and 4-hydroxywarfarin, and the previously unreported 3-hydroxywarfarin, as well as the diastereomeric warfarin alcohols, warfarin diketone, and aliphatic hydroxywarfarins. Using S-warfarin and R-warfarin as substrates, and an HPLC assay with fluorescence detection to analyze metabolite profiles, the biotransformation of warfarin was found to be highly substrate and product stereoselective. Both aromatic hydroxylation and ketone reduction were found to be stereoselective for R-warfarin. Ketone reduction with the warfarin enantiomers exhibited a high level of product stereoselectivity in that R-warfarin was predominantly reduced to its S-alcohol, while S-warfarin was reduced primarily to the corresponding R-alcohol.     

Characterization of the bone matrix and its contribution to tooth loss in human cadaveric mandibles

Objective. It is uncertain as to what extent the major bone matrix constituents, mineral and collagen, show inter-individual variation and dependence on age and sex in jawbones. The purpose of this study was to clarify this uncertainty using cadaveric mandibles and investigate the association of bone matrix with the number of existing teeth. Materials and methods. Cortical bone samples (1 × 1 cm) collected from the mental of 48 cadaveric mandibles (27 men and 21 women; age range = 56–93 years and 63–103 years, respectively) were used to quantify three bone matrix indices: mineral content, collagen content and extent of lysine hydroxylation of collagen. Associations with age and comparisons by sex were evaluated based on bone matrix indices and the numbers of existing teeth. The numbers of existing teeth were compared between the groups showing low and high bone matrix index values. Results. A great amount of inter-individual variation was seen in all bone matrix indices. No bone matrix indices were associated with age, while the number of existing teeth was negatively associated with age. The bone matrix indices and number of existing teeth did not differ by sex. The number of existing teeth was nearly twice as high in the group showing high collagen content as in the low collagen group; however, an analysis of covariance showed a significant inter-group difference not from bone matrix indices, but rather from age. Interestingly, in comparison to femoral collagen, mandibular collagen showed lower lysine hydroxylation, which can represent an aspect of bone quality. Conclusions. Mandibular bone matrix shows great inter-individual variation and is independent of age and sex, but did not show as strong a relationship with tooth loss as age. Even so, mandibular collagen may represent a unique characteristic of bone matrix and deserves to be further investigated.     

Steroids hydroxylation catalyzed by the monooxygenase mutant 139-3 from Bacillus megaterium BM3

The search of new substrates with pharmaceutical and industrial potential for biocatalysts including cytochrome P450 enzymes is always challenging. Cytochrome P450 BM3 mutant 139-3, a versatile biocatalyst, exhibited hydroxylation activities towards fatty acids and alkanes. However, there were limited reports about its hydroxylation activity towards steroids. Herein, an Escherichia coli–based whole-cell extract containing the recombinant 139-3 protein was used as the biocatalyst to screen 13 steroids. Results revealed that 139-3 was able to specifically hydroxylate androstenedione (1) at 1α-position, generating a hydroxylated steroid 1α-OH-androstenedione (1a). To investigate whether C-1α hydroxylation catalyzed by BM3 mutant 139-3 could be industrially used, an optimization of catalyzing conditions was performed. Accordingly, the BM3 mutant 139-3 enzyme was observed to display maximum activity at 37 °C, under pH 7.0 for 4 h, with 37% transformation rate. Moreover, four 139-3 variants were generated by random mutagenesis with the aim of improving its activity and expanding substrate scope. Surprisingly, these mutants, sharing a common mutated site R379S, lost their activities towards androstenedione (1). These data clearly indicated that arginine residue located at site 379 played key role in the hydroxylation activities of 139-3. Overall, these new findings broadened the substrate scope of 139-3 enzyme, thereby expanding its potential applications as a biocatalyst on steroids hydroxylation in pharmaceutical industry.     

11α-羟基坎利酮生物转化中副产物的影响因素

基于质谱数据推测11α-羟基坎利酮生物转化中的副产物为双羟坎利酮。考察了投料浓度、转化时间、装液量、转速、投料时间和后期转化温度对副产物生成的影响。得到副产物生成量较小的工艺条件为:坎利酮浓度6 g/L,转化时间60 h,转速160 r/min,装液量100 ml/500 ml,后期转化温度28℃,投料时间20 h,11α-羟基坎利酮的转化率为94%。     

In vivo functions of the prolyl‐4‐hydroxylase domain oxygen sensors: direct route to the treatment of anaemia and the protection of ischaemic tissues

The prolyl‐4‐hydroxylase domain (PHD) 1–3 enzymes have been identified based on their ability to regulate the stability of hypoxia‐inducible factor α subunits and thus to modify hypoxia‐inducible gene expression. Transgenic mouse models provided insights into the isoform‐specific functions of these oxygen sensors with physiological implications for angiogenesis, erythropoiesis/oxygen transport, cardiovascular function, metabolism and tissue homeostasis. This knowledge is important for the ongoing development of small molecule PHD inhibitors that are currently tested in preclinical and clinical trials for the treatment of anaemia and for cytoprotection. This review aims at summarizing the insights obtained from key mouse knock‐out models as well as first experiences in the therapeutic application of PHD inhibitors.     

Antitumor Potential of Conjugable Valinomycins Bearing Hydroxyl Sites: In Vitro Studies

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Stereoselective 4'-hydroxylation of phenytoin: relationship to (S)-mephenytoin polymorphism in Japanese

Aims The aim of this study was to clarify whether phenytoin (PHT) stereoselective hydroxylation cosegregates with (S)-mephenytoin phenotype.Methods A single dose of PHT (100 mg) was administered orally to six healthy Japanese subjects in whom the genotype and phenotype of CYP2C19 had been determined previously. The urinary excretion profiles of the metabolites of PHT, (R)- and (S)-p-HPPH [5-(4-hydroxyphenyl)-5-phenylhydantoin] up to 36 h postdose were compared between the two groups of poor metabolizers (PMs, n=3) and extensive metabolizers (EMs, n=3) with respect to CYP2C19. CYP2C9 genotype was also determined.Results All the alleles were found to be wild type (Arg₁₄₄Tyr₃₅₈Ile₃₅₉Gly₄₁₇ ) in each subject. The mean value for cumulative urinary excretion of unchanged PHT was not significantly different between the PMs and the EMs. However, recovery of (R)-p-HPPH at 36 h was 3.5-fold lower and that of (S)-p-HPPH 1.3-fold lower in PMs than in EMs. Although the mean urinary excretion values for both metabolites were significantly lower in the PMs than in the EMs, the difference between the two groups was larger for (R)-p-HPPH. A significant negative correlation was observed between the hydroxylation index of omeprazole (the ratio between the serum concentrations of omeprazole and hydroxyomeprazole in blood samples drawn 3 h after drug intake) and the log₁₀ 0–12 h urinary recovery of (R)-p-HPPH.Conclusions In humans, the 4′-hydroxylation of PHT is highly stereoselective towards formation of the (S)-enantiomer. Thus, (S)-hydroxylation by CYP2C9 might be the major determinant of the disposition of PHT. However, these results support the hypothesis that the stereoselective hydroxylation pathway of PHT to form (R)-p-HPPH cosegregates with the CYP2C19 metabolic polymorphism.     

Protein metabolism in glucagonoma

Summary Although protein wasting and reduced amino acid concentrations are common findings in glucagonoma patients, the mechanisms underlying these alterations are unclear. Therefore, we studied basal postabsorptive leucine, phenylalanine and tyrosine turnover following L-[D₃]-Leucine, L-[D₅]-Phenylalanine and L-[D₂]-Tyrosine i. v. infusions in one male and one female patient with glucagonoma, compared with healthy control volunteers. Plasma amino acid concentrations were reduced (–40 to 80 %, δ > 2 SD vs control subjects) in both patients. Plasma leucine, phenylalanine and tyrosine rates of appearance in patients with glucagonoma were similar to values in the control subjects, except leucine rate of appearence in the female patient with glucagonoma ( + &30 %, d > 2 SD). In contrast, the intracellular leucine rate of appearence, reflecting protein degradation, was considerably increased in both patients ( + 60–80 %, δ > 2 SD). Phenylalanine hydroxylation was moderately higher only in the male patient with glucagonoma ( + &30 %, d > 2 SD). Leucine, phenylalanine and tyrosine clearances ( + 100–300 %), as well as phenylalanine hydroxylative clearance ( + 75–100 %) were also increased in the patients. In conclusion, whole-body protein breakdown is enhanced in patients with glucagonoma compared with healthy control subjects. Phenylalanine hydroxylative clearance is also higher. Reduced plasma amino acid concentrations are probably due, at least in part, to their increased clearance. These alterations could contribute to the determination of the catabolic state of the glucagonoma syndrome. [Diabetologia (1999) 42: 326–329] Received: 3 November 1997 and in final form: 29 September 1998     

Altered mechanisms underlying hypoxic dilation of skeletal muscle resistance arteries of hypertensive versus normotensive Dahl rats

OBJECTIVE: To determine mechanisms underlying hypoxic dilation of skeletal muscle resistance arteries from normotensive (NT) and hypertensive (HT) Dahl salt-sensitive (SS) rats. METHODS: Isolated gracilis arteries (GA) from both rat groups were viewed via television microscopy and vascular responses to a reduction in PO2 from 145 mm Hg to 40 mm Hg were measured with a video micrometer. Responses were determined following endothelium removal and following inhibition of specific biochemical pathways regulating vascular tone. RESULTS: Hypoxic dilation was impaired in HT rats versus NT controls. Endothelium removal abolished hypoxic dilation in NT rats, although a significant dilation to hypoxia remained in vessels from HT animals. Inhibition of cytochrome P450 (CP450) 4A enzymes blunted hypoxic dilation in both groups, while inhibition of epoxyeicosatrienoic acid (EET) production impaired responses in NT rats only. Inhibition of 20-hydroxyeicosatetraenoic acid (20-HETE) production or blockade of membrane receptors for 20-HETE reduced hypoxic dilation in HT rats, with minimal effects in NT animals. Nitric oxide synthase inhibition had no effect on hypoxic dilation in either group, while cyclooxygenase inhibition significantly reduced this response in both groups. CONCLUSIONS: These results suggest that the mechanisms of hypoxic dilation in GA from NT Dahl-SS rats are altered with HT, impairing the response to reduced PO2. While hypoxia induces substantial prostanoid release in both groups, the role of CP450 4A enzymes is shifted from EET production in NT rats toward inhibition of 20-HETE production in HT rats.     

Interest and Limitations of Phenotype Determination of Hydroxylation Ability in Patients Treated with Propafenone

In order to confirm that variations in the plasma levels of propafenone in patients receiving the same daily dose are polyrnorphically distributed in relation to oxydative metabolism, the relationship between propafenone administration and debrisoquine metabolic phenotype was studied in two series of Caucasian patients with chronic supraventricular or ventricular arrhifthmias. In a first series of 16 Caucasian patients treated with propafenone, the poor metabolizer phenotype appeared to be more than 90%. In a second series of 13 Caucasian patients, two debrisoquine tests were performed: the first phenotype determination of debrisoquine hydroxylation ability was done without propafenone administration and the second determination when a steady state concentration of propafenone was reached. Of the 10 extensive metabolizers (determined without propafenone treatment), 7 patients became apparent poor metabolizers while considering the debrisoquine test during propafenone treatment with a general shift of the 13 studied patients to the upper values of log of the metabolic ratiodebrisoquine/4-OH debrisoquine———-suggesting deficiency in metabolizing debrisoquine. This study seems to indicate the presence of a common hydroxylation for propafenone and debrisoquine, which probably compete in the pathway. This metabolic competition should therefore be taken into consideration during phenotypic determination of hydroxylation ability.