The measurement of triphosphopyridine nucleotide and reduced triphosphopyridine nucleotide and the role of hemoglobin in producing erroneous triphosphopyridine nucleotide values.

A method is described for determining TPN+ and TPNH in a single tissue homogenate made in dilute NaOH at 0’. One portion, for TPN+, is acidified after adding ascorbic acid to prevent TPNH oxidation. A second portion, for TPNH, is heated (still alkaline) after adding cysteine to prevent oxidation. If desired, a third portion can be analyzed directly without heating for total TPN (TPN+ plus TPNH). The same homogenate can also be used for measuring DPNf and DPNH. In all cases the actual analyses are made with enzymatic cycling. Consequently, by reason of the high sensitivity, it is unnecessary to deproteinize. The procedure presented avoids the danger of TPNH oxidation to TPN+ by tissue hemoglobin which was responsible for our earlier erroneous conclusion that there exists an acid-labile tissue form of TPN+. The oxidation of TPNH by hemoglobin has been studied intensively. The oxidation by free hemoglobin occurs to the same degree in strong acids as in dilute. Therefore, the possibility of obtaining erroneously high TPN+ values is not eliminated by the preparation of homogenates in strong acid, although TPN+ formation is greatly decreased if red blood cells in the tissue are intact at the time of strong acid addition. Neither is the danger eliminated by preparation of extracts with heat at neutral pH. With the use of the revised procedure the levels of TPN+ and TPNH were measured in liver, kidney, heart, brain, and blood. The ratio of TPNH to TPNf was found to vary from 20 to 1 in heart to 3 to 1 in blood.