Molecular Pharmacology, Vol 10, 999-1003, Copyright © 1974 by the American Society for Pharmacology and Experimental Therapeutics

Limitations in the Use of the 340 nm Absorbance Maximum of Reduced Nicotinamide Adenine Dinucleotide Phosphate for the Determination of Oxidation Rates and Stoichiometry during Rat Hepatic Microsomal Metabolism

¹ Department of Pharmacology, University of Utah, Salt Lake City, Utah 84132

During rat hepatic microsomal oxidative metabolism NADPH not only is oxidized to NADP⁺ but is cleaved to reduced nicotinamide mononucleotide. The 340 nm absorbance maximum of NMNH interferes with the use of the 340 nm absorbance as a measure of NADPH oxidation. The pyrophosphatase responsible for NADPH cleavage can destroy 30% of the added NADPH in the absence of exogenous mixed-function oxidase substrates in microsomes from phenobarbital-treated rats. It is inhibited about 75% by 2 mM 5'-AMP and 25 mM sodium pyrophosphate and about 30% by 10 mM sodium fluoride. Using pyrophosphatase inhibitors, and allowing for the turbidity enhancement of the 340 nm absorbance, the stoichiometry of NADPH oxidized to oxygen consumed approaches 1:1, especially in the presence of exogenous mixed-function oxidase substrates.