Microsome-catalyzed NADH reduction

Figure 10. Microsome-catalyzed NADH reduction of Cl(NHo)r,Ru(lll) cmd subsequent formation of (Isonicotinamide)(NH3)sRu(II) A, reaction run in air B, reaction run under Ar C, reaction run under Ar in presence of metyrapone D, reaction run under N2 (3S)...

Indicine IV-oxide (169) (Scheme 36) is a clinically important pyrrolizidine alkaloid being used in the treatment of neoplasms. The compound is an attractive drug candidate because it does not have the acute toxicity observed in other pyrrolizidine alkaloids. Indicine IV-oxide apparently demonstrates increased biological activity and toxicity after reduction to the tertiary amine. Duffel and Gillespie (90) demonstrated that horseradish peroxidase catalyzes the reduction of indicine IV-oxide to indicine in an anaerobic reaction requiring a reduced pyridine nucleotide (either NADH or NADPH) and a flavin coenzyme (FMN or FAD). Rat liver microsomes and the 100,000 x g supernatant fraction also catalyze the reduction of the IV-oxide, and cofactor requirements and inhibition characteristics with these enzyme systems are similar to those exhibited by horseradish peroxidase. Sodium azide inhibited the TV-oxide reduction reaction, while aminotriazole did not. With rat liver microsomes, IV-octylamine decreased... 

This mechanism is now considered to be of importance for the protection of LDL against oxidation stress, Chapter 25.) The antioxidant effect of ubiquinones on lipid peroxidation was first shown in 1980 [237]. In 1987 Solaini et al. [238] showed that the depletion of beef heart mitochondria from ubiquinone enhanced the iron adriamycin-initiated lipid peroxidation whereas the reincorporation of ubiquinone in mitochondria depressed lipid peroxidation. It was concluded that ubiquinone is able to protect mitochondria against the prooxidant effect of adriamycin. Inhibition of in vitro and in vivo liposomal, microsomal, and mitochondrial lipid peroxidation has also been shown in studies by Beyer [239] and Frei et al. [240]. Later on, it was suggested that ubihydroquinones inhibit lipid peroxidation only in cooperation with vitamin E [241]. However, simultaneous presence of ubihydroquinone and vitamin E apparently is not always necessary [242], although the synergistic interaction of these antioxidants may take place (see below). It has been shown that the enzymatic reduction of ubiquinones to ubihydroquinones is catalyzed by NADH-dependent plasma membrane reductase and NADPH-dependent cytosolic ubiquinone reductase [243,244]. 

Heme oxygenase, which catalyzes the conversion of free heme groups to biliverdin and CO, functions as part of a microsomal electron transport system similar to that of cytochrome 1 450-(FP = NADPH-cytochrome P450 reductase.) Heme oxygenase requires 3 02 and 5 NADPH. Biliverdin reductase can use NADPH or NADH as a reductant. 

The enzymes necessary for the conversion of famesyl pyrophosphate to squalene are called squalene synthetase . The enzymes necessary for the two reactions have not been resolved nor has either been purified to a significant extent, and it is not yet certain if the two reactions are catalyzed by two discrete entities. Squalene synthetase has an absolute requirement for a divalent cation, Mg " and Mn " being the best. A reduced pyridine nucleotide (NADH or NADPH) is required for the reduction of presqualene pyrophosphate to squalene. Yeast microsomes with Mn " and no reduced pyridine nucleotide will form dehydrosqualene instead [70]. The conversion of famesyl pyrophosphate to presqualene pyrophosphate is enhanced several-fold by the reduced pyridine nucleotide [71]. Also, some organic solvents as well as detergents increase this activity. 

Studies on metabolic stability using hepatocyte suspensions are not feasible for automation/HTS, but these studies do provide rather complete profiles of hepatic biotransformation without the supplements of cofactors and cosubstrates. The use of S9 in metabolic stability studies can be evaluated in a manner similar to that used for the microsomal assays, but with the possible addition of a broader panel of cofactors or cosubstrates. These include NADPH for CYP/FMO-mediated reactions, NADH for xanthine oxidoreductase and quinone oxidoreductase 2, NADPH-dependent reductions by carbonyl reductases, and NADPH/NADH-dependent reductions catalyzed by aldo-keto reductases, uridine 5 -diphosphate... 

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