NADPH-fortified human liver microsomes

Figure 11 Irreversible inhibition of CYP2A6 by 8-methoxypsoralen at two concentrations with and without a dilution step. The overall design of the experiment is discussed in section II.C.7.b. Panels A and B show the effects of preincubating 8-methoxypsoralen (0.05 and 1.25 pM) for 30 minutes with NADPH-fortified human liver microsomes (0.0125 mg/mL) without a dilution prior to the incubation with substrate (coumarin). Panels B and D show the effects of preincubating 8-methoxypsoralen (0.05 and 1.25 pM) for 30 minutes with NADPH-fortified human liver microsomes (0.3125 mg/mL) with a 25-fold dilution prior to the incubation with substrate (coumarin). Panel E shows the effects of preincubating 8-methoxypsoralen (1.25 pM) for 30 minutes with pooled human liver microsomes (0.0125 mg/mL) in the absence of NADPH without a dilution step prior to the incubation with substrate (coumarin). Inhibition in the latter case is caused by inactivation of CYP2A6 during the substrate incubation step (5 minutes) because it occurs to the same extent in both the 0- and 30-minute preincubation samples.

A metabolite with +16 amu is generally suspected of forming by hydrox-ylation (or by some other reaction involving the addition of oxygen). However, a metabolite with +14 amu is often suspected of forming by methylation (+CH2), not by a combination of the addition of oxygen (+16) and dehydrogenation (—2). NADPH-fortified human liver microsomes cannot catalyze the methylation of dmg candidates (such reactions are catalyzed by cytosolic enzymes in the presence of. S -adenosylmethionine). However, methylation can sometimes occur as an artifact when mass spectrometry is conducted in the presence of methanol (164), and [M + 12] adducts can form from condensation reactions with formaldehyde, which is a microsomal metabolite of methanol (165). A metabolite with +30 amu is indicative of either formation of a carboxylic acid metabolite or a combination of hydroxylation (+16) and methylation (+14). Only the former can be catalyzed by NAPDH-fortified liver microsomes. 

The FDA-approved and acceptable chemical inhibitors for reaction phenotyping are included in Table 2. Many of the inhibitors listed in Table 2 are metabolism-dependent inhibitors that, in order to inhibit CYP, require preincubation with NADPH-fortified human liver microsomes for 15 minutes or more. In the absence of the metabolism-dependent inhibitor, this preincubation of microsomes with NADPH can result in the partial, spontaneous loss of several CYP enzyme activities (see sec. II.C.7.c). Furthermore, the organic solvents commonly used to dissolve chemical inhibitors can themselves inhibit (or possibly activate) certain CYP enzymes, as discussed in section II.C.4. Therefore, appropriate solvent and preincubation controls should be included in all chemical inhibition experiments. 

The reaction is demonstrated in NADPH-fortified human liver microsomes (if the reaction of interest is restricted to another tissue, then this tissue would be used instead). The effects of selective inhibitors on the reaction are examined. A list of some of the inhibitors that have been used is presented elsewhere in this volume by Correia (Chapter 7)6 . 

In the 5a series allochenodeoxycholate is 12a-hydroxylated to allocholate in the bile fistula rat [131] or with a hepatic microsomal preparation from rat, rabbit or human liver fortified with NADPH [152,153]. Kallner [133] noted that small amounts of more polar derivatives were present in rat bile, with largely unchanged allochenodeoxycholate. AUohyocholate was identified as a minor metabolite [154]. With rabbit liver microsomal preparations, allochenodeoxycholate is a competitive inhibitor for 12a-hydroxylation of 7a-hydroxy-cholest-4-en-3-one and 5a-choles-tane-3a,7a-diol, precursor of cholic and allocholic acids, respectively [155]. Allo-cholic acid has also been characterized as a metabolite of 3 8,7a-dihydroxy-5-cholenic acid after intraperitoneal injection into carp [156]. 

The reaction sequence in the conversion of cholesterol into 5/5-choles-tane-3a,7a,12a-triol and 5/5-cholestane-3a,7a-diol has been shown to be the same in human and rat liver homogenates (3). All of these reactions occur with the 20,000g supernatant fluid of homogenates of human liver. The 12a-hydroxylation of 7a-hydroxycholest-4-en-3-one proceeds efficiently in the presence of a microsomal fraction fortified with NADPH (3). 

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