Calcium alginate-Immobilized hepatic microsomes: Effect of NADPH cofactor on oxidation rates

Abstract

An important function of the liver is detoxification of drugs, toxins and foreign compounds. Within the liver cell, the endoplasmic reticulum, isolated as the microsomal fraction, is especially active. Microsomal oxidation is the major oxidation pathway for many compounds, and the requirement for NADPH, an expensive cofactor, is an important consideration in bioreactor design. This paper presents design information for NADPH- and substrate-dependent oxidation rates for free and immobilized microsomes. The primary goal of this paper is determining NADPH requirements for oxidation. The effect of various initial levels of nicotinamide adenine dinucleotide phosphate (NADPH) on chlorpromazine oxidation rate has been studied for a crude hepatic microsomal fraction immobilized in calcium alginate gel. At an initial NADPH concentration of 600 nmoles/ml, immobilized microsomes accelerate to a maximal velocity of ≈ 240 nmoles min −1 ml −1 of oxygen consumption. In comparison, free microsomes reach a maximal velocity of approximately 150 nmoles min −1 ml −1 at an initial NADPH concentration of 220 nmoles/ml. By fitting the “initial” rate as a function of NADPH concentration to Michaelis-Menten kinetics, the apparent half-saturation coefficients ( K m) app are 3.5 nmole/ml for free microsomes and 134.4 nmole/ml for immobilized microsomes, however the maximum reaction velocity, V max, for immobilized microsomes is calculated to be 322 nmoles min −1 ml −1 compared with 145 nmols min −1 ml −1 for free microsomes. Preliminary studies indicate that is is possible to obtain significant reaction rates using calcium alginate immobilized microsomes and that this system may offer advantages due to its simplicity and lower cost.