High-performance liquid chromatographic assays for bufuralol 1'-hydroxylase, debrisoquine 4-hydroxylase, and dextromethorphan O-demethylase in microsomes and purified cytochrome P-450 isozymes of human liver.

Bufuralol, debrisoquine, and dextromethorphan are three prototype substrates of the common genetic deficiency of oxidative drug metabolism in man known as debrisoquine/sparteine-type polymorphism. We describe assays for the in vitro metabolism of (+)- and (-)-bufuralol, debrisoquine, and dextromethorphan in human liver microsomes and reconstituted purified cytochrome P-450 isozymes. These assays combine nonextractive sample preparation by precipitation of protein with perchloric acid with reversed-phase inorganic ion-pair HPLC and fluorescence detection. The minimal detectable levels of the major metabolites formed are 1'-hydroxybufuralol, 0.1 ng/ml; 4-hydroxydebrisoquine, 0.8 ng/ml; and dextrorphan, 0.1 ng/ml. Formation of these metabolites is linear for at least 45 min and between 1 and 100 micrograms of microsomal protein. Comparative kinetic analysis of the three monooxygenase reactions in human liver microsomes revealed an apparent biphasicity of (+)- and (-)-bufuralol 1'-hydroxylation and dextromethorphan O-demethylation but monophasic formation of 4-hydroxydebrisoquine in the substrate concentration range (less than 1 mM) studied. These data, in combination with those obtained by purified human cytochrome P-450 isozymes indicate the involvement of the same enzyme in the metabolism of all three substrates investigated. However, additional and distinct activities contribute to the metabolism of (+)- and (-)-bufuralol and dextromethorphan.

Mephenytoin-type polymorphism of drug oxidation: purification and characterization of a human liver cytochrome P-450 isozyme catalyzing microsomal mephenytoin hydroxylation.

A genetic polymorphism causing deficient metabolism of the anticonvulsant drug mephenytoin occurs in 5% of the Caucasian and 23% of the Japanese population. By monitoring the activities of the two major oxidative pathways of mephenytoin metabolism in the column eluates, we have purified from human livers a cytochrome P-450 isozyme, P-450 meph, which exclusively and stereoselectively catalyzes the 4-hydroxylation of (S)-mephenytoin, the major pathway affected by the polymorphism, whereas P-450 meph was virtually devoid of catalytic activity for N-demethylation of mephenytoin, the pathway remaining unaffected by the genetic deficiency. P-450 meph had an apparent Mr of 55 000 and a lambda max in the reduced CO-binding spectrum of 450 nm. Polyclonal rabbit antibodies against purified human P-450 meph almost completely inhibited the 4-hydroxylation of mephenytoin but had little effect on N-demethylation in human liver microsomes. In microsomes of liver biopsies of two subjects characterized in vivo as 'poor metabolizers' of mephenytoin, immunocrossreactive and immunoinhibitable material was observed with similar or identical properties to those of P-450 meph. There was no difference in the extent of the immunochemical reaction between microsomes of in vivo phenotyped poor metabolizers and extensive metabolizers of mephenytoin. These data suggest that P-450 meph is the target of the genetic deficiency and support the concept that a functionally altered variant form of P-450 meph causes this polymorphism.

Debrisoquine/sparteine-type polymorphism of drug oxidation. Purification and characterization of two functionally different human liver cytochrome P-450 isozymes involved in impaired hydroxylation of the prototype substrate bufuralol.

The debrisoquine/sparteine-type polymorphism of drug oxidation presumably is caused by the absence or deficiency of cytochrome P-450 (P-450) isozyme(s). Using bufuralol 1'-hydroxylation as a prototype reaction of this polymorphism, two functionally distinct forms, P-450 buf I and P-450 buf II, with identical apparent Mr of 50,000 were purified from liver microsomes of three different human livers. P-450 buf I exhibited a marked selectivity for the (+)-enantiomer of bufuralol ((-)/(+) ratio = 0.15), P-450 buf II was nonstereoselective((-)/(+) ratio = 1.03). The Km values for (-)- and (+)-bufuralol were 31 and 54 microM with P-450 buf I and 314 and 245 microM with P-450 buf II. P-450 buf II generated two other metabolites in addition to 1'-OH-bufuralol which were not observed with P-450 buf I. Using the inhibitor quinidine, a Ki of 0.06 microM was observed with P-450 buf I as opposed to 80 microM with P-450 buf II for bufuralol 1'-hydroxylation. A strong immunochemical relatedness of P-450 buf I and P-450 buf II was found since polyclonal antibodies against either form recognized the heterologous antigen to the same extent as the homologous antigen on Western blots and in immunoinhibition and in immunoprecipitation experiments. Cross-reactivity of these antibodies with a microsomal nonheme protein of unknown function (apparent Mr 50,000) also was noted. Western blots of microsomes of in vivo and in vitro phenotyped extensive and poor metabolizer individuals revealed no correlation of in vivo-determined metabolic ratio, microsomal activity, and amount of immunoreactive material. Antibodies against P-450 buf I and P-450 buf II inhibited bufuralol 1'-hydroxylation in microsomes of in vivo and in vitro phenotyped poor metabolizer individuals demonstrating that the residual activities are immunochemically related to the activities in extensive metabolizers.

The molecular mechanisms of two common polymorphisms of drug oxidation--evidence for functional changes in cytochrome P-450 isozymes catalysing bufuralol and mephenytoin oxidation.

Using the stereospecific metabolism of (+)- and (-)-bufuralol and (+)- and (-)-metoprolol as model reactions, we have characterized the enzymic deficiency of the debrisoquine/sparteine-type polymorphism by comparing kinetic data of subjects in vivo with their microsomal activities in vitro and with reconstituted activities of cytochrome P-450 isozymes purified from human liver. The metabolism of bufuralol in liver microsomes of in vivo phenotyped 'poor metabolizers' of debrisoquine and/or sparteine is characterized by a marked increase in Km, a decrease in Vmax and a virtual loss of the stereoselectivity of the reaction. These parameters apparently allow the 'phenotyping' of microsomes in vitro. A structural model of the active site of a cytochrome P-450 for stereospecific metabolism of bufuralol and other polymorphically metabolized substrates was constructed. Two cytochrome P-450 isozymes, P-450 buf I and P-450 buf II, both with MW 50,000 Da, were purified from human liver on the basis of their ability to metabolize bufuralol to 1'-hydroxy-bufuralol. However, P-450 buf I metabolized bufuralol in a highly stereoselective fashion ((-)/(+) ratio 0.16) as compared to P-450 buf II (ratio 0.99) and had a markedly lower Km for bufuralol. Moreover, bufuralol 1'-hydroxylation by P-450 buf I was uniquely characterized by its extreme sensitivity to inhibition by quinidine. Antibodies against P-450 buf I and P-450 buf II inhibited bufuralol metabolism in microsomes and with the reconstituted enzymes. Immunochemical studies with these antibodies with microsomes and translations in vitro of RNA from livers of extensive and poor metabolizers showed no evidence for a decrease in the recognized protein or its mRNA. Because the antibodies do not discriminate between P-450 buf I and P-450 buf II, both a decreased content of P-450 buf I or its functional alteration could explain the polymorphic metabolism in microsomes. The genetically defective stereospecific metabolism of mephenytoin was determined in liver microsomes of extensive and poor metabolizers of mephenytoin phenotyped in vivo. Microsomes of poor metabolizers were characterized by an increased Km and a decreased Vmax for S-mephenytoin hydroxylation as compared to extensive metabolizers and a loss of stereospecificity for the hydroxylation of S-versus R-mephenytoin. A cytochrome P-450 with high activity for mephenytoin 4-hydroxylation was purified from human liver. Immunochemical studies with inhibitory antibodies against this isozyme suggest the presence in poor-metabolizer microsomes of a functionally altered enzyme.

Debrisoquine-type polymorphism of drug oxidation: purification from human liver of a cytochrome P450 isozyme with high activity for bufuralol hydroxylation.

Indirect evidence suggests that the genetically defective metabolism of drugs such as debrisoquine and bufuralol observed in up to 10% of the population (poor metabolizers) is caused by the absence or functional deficiency of a cytochrome P450 isozyme. Using bufuralol-1'-hydroxylation to carbinol to optimize the procedure, 3 cytochrome P450 isozymes (P450A, P450buf, P450C) were purified to apparent electrophoretic homogeneity from human liver microsomes. P450buf had a specific activity of 20.3 nmol carbinol X nmol P450-1 X 15 min-1 as compared to microsomes (10.0 nmol carbinol X nmol P450(-1) X 15 min-1) when (+)-bufuralol was used as substrate. The stereoselective metabolism of (-)- and (+)-bufuralol to carbinol by purified P450buf [(-)/(+) ratio: 0.13] was strikingly different from that in the microsomes of either an extensive [(-)/(+) ratio: 0.4] or poor metabolizer [(-)/(+) ratio: 0.83] of bufuralol. We propose that this isozyme is the major bufuralol and debrisoquine hydroxylating species and is the target of the genetic deficiency.