Dosage des pyridine nucléotides oxydés et réduits dans le sang et les tissus animaux

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Abstract
A fluorometric method is described for the estimation of di‐ and triphosphopyridine nucleotides (oxidized and reduced) in animal tissues.The continuous cycling reduction of DPN+ (or TPN+) in the presence of excess of ethanol and alcohol dehydrogenase (or glucose‐6‐phosphate and glucose‐6‐phosphate dehydrogenase) was coupled with non enzymatic reoxidation of pyridine nucleotides with phenazine methosulfate.Phenazine methosulfate reduced the non fluorescent compound, resazurine to the highly fluorescent compound, resorufine. The rate of production of resorufine is proportionnal to the concentration of pyridine nucleotides.The procedure is relatively simple and requires only two commercial enzyme preparations for complete determination. This method can be used for estimation of pyridine nucleotides in concentrations as low as 0.1 μM and quantities as small as 10 pmoles.The acid and alkaline extraction procedure was carried out essentially according to the method of Lowry. Conditions affecting the specificity, accuracy and reliability have been studied. During extraction, interfering material is formed, inhibiting the rate of reduction. A calibration procedure is described which overcomes this difficulty. Values for the concentrations of oxidized and reduced pyridine nucleotides in rat liver and in rat and human blood are presented and are in fairly good agreement with those of Lowry.