18] Determining NAD synthesis in erythrocytes

Abstract

This chapter describes methods for the study of nicotinamide adenine dinucleotide (NAD) synthesis in human erythrocytes, developed according to two approaches— the determination of intracellular concentrations of pyridine compounds and the rate of conversion of radiolabeled precursors in intact cells, and enzyme activity assays in crude lysates. All determinations are performed by methods involving high-performance liquid chromatography (HPLC). Different methods of obtaining protein-free extracts suitable for HPLC processing are examined. Acid or alkaline extraction provides better results than acetonitrile extraction or boiling, which cause oxidation of NADH or breakdown of NAD. The activities of several enzymes are measured in crude lysates made from fresh or, when suitable, stored erythrocytes: nicotinate phosphoribosyltransferase (NA–PRT), nicotinamide phosphoribosyltransferase (NAm–PRT), nicotinamide-mononucleotide and nicotinate-mononucleotide adenylyltransferase (NMN–AT and NAMN–AT), and NAD synthetase (NAD-s). All assay reactions are started by lysate addition. Hemoglobin content is measured in lysates by the cyanmethemoglobin method and used to quantify enzyme specific activity.