A method for determination of pyridine nucleotides using a single extract

Intracellular redox levels play an important role in physiology and pathoph ysiology. The principal intracellular reductant is NADPH, which is required for both the proper activity of the entire antioxidant system and importan t prooxidant enzymes such as nitric oxide synthase and NADPH oxidase. Thus an easy and accurate measurement of NADPH is very desirable. The method des cribed in this paper is based on the fact that NADH and NADPH (not NAD(+) a nd NADP(+)) affect absorbance at 340 nm. A single cell extract is separated into three aliquots (A(1), A(2), and A(3)). A(1) is untreated and the abso rbance at 340 nm is measured. A(2) is treated with an enzyme that converts all of the NADP(+) to NADPH and then the absorbance at 340 nm is measured. A(2) is treated with an enzyme that converts all of the NADPH to NADP(+) an d then the absorbance at 340 nm is measured. A(1) - A(3) is the NADPH conte nt and A(2) - A(1) is the NADP(+) content of the extract. Using this method , we have obtained full recovery of all added nucleotides from cell extract s, thus making the method suitable for the quick determination of NADP(+) a nd NADPH in living cells. We conclude that this method for the measurement of NADP(+) and NADPH is rapid, simple, accurate, and reliable. (C) 2000 Aca demic Press.