RT Journal Article SR Electronic T1 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 JF Molecular Pharmacology JO Mol Pharmacol FD American Society for Pharmacology and Experimental Therapeutics SP 999 OP 1003 VO 10 IS 6 A1 MILDRED K. BUENING A1 MICHAEL R. FRANKLIN YR 1974 UL http://molpharm.aspetjournals.org/content/10/6/999.abstract AB 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.