Covariation of human microsomal protein per gram of liver with age: absence of influence of operator and sample storage may justify interlaboratory data pooling.

Scaling of metabolic clearance values from liver microsomal data or recombinantly expressed cytochrome P450 enzymes to predict human hepatic clearance requires knowledge of the amount of microsomal protein per gram of liver (MPPGL). Identification of physiological covariates of MPPGL requires analysis of values from large diverse populations, which necessitates pooling of data from numerous sources. To ensure compatibility between results obtained within and between studies, the impact of interoperator differences and sample storage on values of MPPGL was investigated. With use of triplicate samples from one liver (HL86), no statistically significant difference was detected between values of MPPGL prepared from samples stored at -80 degrees C (23.5 +/- 1.2 mg g(-1)) and those determined using fresh tissue (21.9 +/- 0.3 mg g(-1)). Although there was a significant difference in the yield of microsomal protein obtained from another liver sample (HL43) by three different operators (17 +/- 1, 19 +/- 2, and 24 +/- 1 mg g(-1); p = 0.004, analysis of variance), no difference was observed in the estimated MPPGL after application of appropriate correction factors for each operator (28 +/- 1, 30 +/- 5, and 31 +/- 4 mg g(-1)). The result provided justification for pooling reported values of MPPGL for use in covariate analysis. Investigation of the relationship between age and MPPGL provided preliminary evidence that MPPGL values increase from birth to a maximum of 40 mg g(-1) [95% confidence interval for the geometric mean (95% CI mean(geo)): 37-43 mg g(-1) at approximately 28 years followed by a gradual decrease in older age (mean of 29 mg g(-1) at 65 years; 95% CI mean(geo): 27-32 mg g(-1)). Accordingly, appropriate age-adjusted scaling factors should be used in extrapolating in vitro clearance values to clinical studies.

A discordance between cytochrome P450 2D6 genotype and phenotype in patients undergoing methadone maintenance treatment.

AIMS: To assess CYP2D6 activity and genotype in a group of patients undergoing methadone maintenance treatment (MMT). METHODS: Blood samples from 34 MMT patients were genotyped by a polymerase chain reaction-based method, and results were compared with CYP2D6 phenotype (n = 28), as measured by the molar metabolic ratio (MR) of dextromethorphan (DEX)/dextrorphan (DOR) in plasma. RESULTS: Whereas 9% of patients (3/34) were poor metabolizers (PM) by genotype, 57% (16/28) were PM by phenotype (P < 0.005). Eight patients, who were genotypically extensive metabolizers (EM), were assigned as PM by their phenotype. The number of CYP2D6*4 alleles and sex were significant determinants of CYP2D6 activity in MMT patients, whereas other covariates (methadone dose, age, weight) did not contribute to variation in CYP2D6 activity. CONCLUSIONS: There was a discordance between genotype and in vivo CYP2D6 activity in MMT patients. This finding is consistent with inhibition of CYP2D6 activity by methadone and may have implications for the safety and efficacy of other CYP2D6 substrates taken by MMT patients.

In-vitro analysis of the contribution of CYP2D6.35 to ultra-rapid metabolism.

From 10 to 30% of CYP2D6 ultra-rapid metabolizers of Caucasian origin harbor alleles with duplicated or amplified functional CYP2D6 genes. Recently, the CYP2D6*35 allele has been reported to be more frequent in ultra-rapid metabolizing subjects than in extensive metabolizers, suggesting a possible role of this variant in CYP2D6 duplication-negative ultra-rapid metabolizing subjects. In this study, we examined the functional consequences of the Val11Met, Arg296Cys and Ser486Thr amino acid substitutions associated with the CYP2D6*35 on the expression and catalytic activity of the variant enzyme, heterologously expressed in yeast. Our results indicate that the functional activity and level of expression of recombinant CYP2D6.35 are comparable with those of the wild-type enzyme, thus precluding the hypothesis that the high level of enzyme activity in CYP2D6 duplication-negative ultra-rapid metabolizing subjects is a consequence of the expression of a more catalytically effective CYP2D6.35 enzyme.

Influence of phenylalanine-481 substitutions on the catalytic activity of cytochrome P450 2D6.

Homology models of the active site of cytochrome P450 2D6 (CYP2D6) have identified phenylalanine 481 (Phe(481)) as a putative ligand-binding residue, its aromatic side chain being potentially capable of participating in pi-pi interactions with the benzene ring of ligands. We have tested this hypothesis by replacing Phe(481) with tyrosine (Phe(481)-->Tyr), a conservative substitution, and with leucine (Phe(481)-->Leu) or glycine (Phe(481)-->Gly), two non-aromatic residues, and have compared the properties of the wild-type and mutant enzymes in microsomes prepared from yeast cells expressing the appropriate cDNA-derived protein. The Phe(481)-->Tyr substitution did not alter the kinetics [K(m) (microM) and V(max) (pmol/min per pmol) respectively] of oxidation of S-metoprolol (27; 4.60), debrisoquine (46; 2.46) or dextromethorphan (2; 8.43) relative to the respective wild-type values [S-metoprolol (26; 3.48), debrisoquine (51; 3.20) and dextromethorphan (2; 8.16)]. The binding capacities [K(s) (microM)] of a range of CYP2D6 ligands to the Phe(481)-->Tyr enzyme (S-metoprolol, 22.8; debrisoquine, 12.5; dextromethorphan, 2.3; quinidine, 0.13) were also similar to those for the wild-type enzyme (S-metoprolol, 10.9; debrisoquine, 8.9; dextromethorphan, 3.1; quinidine, 0.10). In contrast, the Phe(481)-->Leu and Phe(481)-->Gly substitutions increased significantly (3-16-fold) the K(m) values of oxidation of the three substrates [S-metoprolol (120-124 microM), debrisoquine (152-184 microM) and dextromethorphan (20-31 microM)]. Similarly, the K(s) values of the ligands to Phe(481)-->Leu and Phe(481)-->Gly mutants were also increased 3 to 10-fold (S-metoprolol, 33.2-41.9 microM; debrisoquine, 85-90 microM; dextromethorphan, 15.7-18.8 microM; quinidine 0.35-0.53 microM). However, contrary to a recent proposal that Phe(481) has the dominant role in the binding of substrates that undergo CYP2D6-mediated N-dealkylation routes of metabolism, the Phe(481)-->Gly substitution did not substantially decrease the capacity of the enzyme to N-deisopropylate metoprolol (wild-type, 1.12 pmol/min per pmol of P450; Phe(481)-->Gly, 0.71), whereas an Asp(301)-->Gly substitution decreased the N-dealkylation reaction by 95% of the wild-type rate. Overall, our results are consistent with the proposal that Phe(481) is a ligand-binding residue in the active site of CYP2D6 and that the residue interacts with ligands via a pi-pi interaction between its phenyl ring and the aromatic moiety of the ligand. However, the relative importance of Phe(481) in binding is ligand-dependent; furthermore, its importance is secondary to that of Asp(301). Finally, contrary to predictions of a recent homology model, Phe(481) does not seem to have a primary role in CYP2D6-mediated N-dealkylation.

Influence of genetic polymorphisms in the beta2-adrenoceptor on desensitization in human lung mast cells.

The beta-adrenoceptor agonist, isoprenaline, inhibited the immunoglobulin E-mediated release of histamine from human lung mast cells (HLMC). Long-term (24 h) exposure of HLMC to isoprenaline reduced the subsequent effectiveness of isoprenaline to inhibit histamine release. The extent of this functional desensitization was variable with some HLMC preparations resistant and others highly susceptible. We sought to determine whether the variability in the degree of functional desensitization was influenced by genetic polymorphisms in the beta2-adrenoceptor. HLMC preparations were genotyped at two polymorphic loci, positions 16 (arg to gly) and 27 (gln to glu), and the effect of desensitizing conditions (24 h with 10(-6) M isoprenaline) on the subsequent ability of isoprenaline (10(-7) M) to inhibit histamine release from HLMC was determined (n = 72). In HLMC preparations expressing beta2-adrenoceptors with arg (wild-type) or gly (mutant) at position 16, desensitization was 71 +/- 5% (n = 18) or 43 +/- 5%, (n = 26), respectively, whereas the desensitization was 59 +/- 6% (n = 28) for heterozygotes at this position. In HLMC preparations expressing beta2-adrenoceptors with gln (wild-type) or glu (mutant) at position 27, desensitization was 65 +/- 5% (n = 25) or 28 +/- 7% (n = 17), respectively, whereas the desensitization was 61 +/- 5% (n = 30) for heterozygotes at this position. These data suggest that mutant (gly16 and glu27) forms of the receptor are resistant to desensitization compared to wild-type (arg16 and gln27) forms. However, analyses to determine the relative contributions of positions 16 and 27 suggest that position 27 is more important in influencing the degree of functional desensitization.

Study of the association between cytochromes P450 2D6 and 2E1 genotypes and the risk of drug and chemical induced idiosyncratic aplastic anaemia.

A genetic susceptibility to drug or chemical toxicity may provide a basis for an increased risk of idiosyncratic aplastic anaemia (AA). The cytochrome P450 enzymes are responsible for the metabolism of many drugs, some of which have been linked to AA. Mutations in the cytochrome P450 CYP2D6 gene result in absent or impaired enzyme activity in about 7% of Caucasians, whereas a specific mutation in the 5'-regulatory region of the CYP2E1 gene causes overexpression of the gene. We evaluated the frequency of allelic variants of CYP2D6 and CYP2E1 using allele-specific PCR amplification and restriction enzyme analysis of blood mononuclear cell DNA among 54 Caucasian AA patients. CYP2D6 and CYP2E1 were chosen because of the link between AA and the antipsychotic drug remoxipride (CYP2D6 substrate) and benzene (CYP2E1 substrate), respectively. Results were compared with 53 controls matched for age, sex and ethnicity. The percentage of AA patients homozygous for the CYP2D6*3, CYP2D6*4 alleles (poor metabolizer phenotype) and the CYP2E1 mutant allele (overexpression) was 0%, 4% and 0%, respectively, and the percentage of heterozygotes was 2%, 28% and 15%, respectively. For normal controls the corresponding results for homozygous mutants were 0%, 4% and 0% and for heterozygotes 4%, 25% and 6%, respectively. We concluded that there were no major differences in the frequencies of the genetic polymorphisms between this series of AA patients and controls, but due to the low number of cases with the poor metabolizer phenotype and those with a history of drug exposure, the power of the study was too low to disprove an interaction.

Renal function and morphometry in the dwarf rat following a reduction in renal mass.

BACKGROUND: The compensatory increase in glomerular filtration rate (GFR) and effective renal plasma flow (ERPF) which follows a reduction in renal mass may be mediated by growth hormone, a renal vasodilator. METHODS: GFR, ERPF and glomerular morphometry were assessed in the dwarf rat, selectively deficient in GH, and compared its Lewis base strain. Studies were performed 21-days after sham-operation, unilateral nephrectomy or subtotal nephrectomy in age-matched animals. GFR and ERPF were assessed from the renal clearance of inulin and p-aminohippurate measured under barbiturate anaesthesia. RESULTS: The dwarf rat had a lower GFR and ERPF than the Lewis rat, in proportion to its lower body weight and lower kidney weight. Kidneys from the dwarf rat had a similar number of glomeruli to the Lewis, but smaller glomerular components in proportion to a lower kidney weight. Following unilateral nephrectomy, GFR (dwarf + 58%, Lewis + 53%) and ERPF (dwarf + 58%, Lewis + 52%) increased to a similar degree in both rat strains. Glomerular diameter, volume and capillary surface area increased in proportion to kidney growth, although compensatory renal growth (dwarf + 62%, Lewis + 78%) was somewhat lower in the dwarf. Following 5/6 subtotal nephrectomy, GFR (dwarf + 143%, Lewis + 171%) increased to a similar degree in both rat strains while ERPF (dwarf + 108%, Lewis + 48%) and compensatory renal growth (dwarf + 115%, Lewis + 86%) were greater in the dwarf than the Lewis rat. Subtotal nephrectomy was also associated with an increase in the thickness of the glomerular basement membrane in both rat strains. CONCLUSIONS: The results do not support a role for GH in the compensatory increase in renal function or hypertrophy which follows a reduction in renal mass, excluding this as a potential mechanism for GH-dependent renal scarring.