A fluorophotometric method for the determination of ~0x0 acids has been developed based on their interaction with pyridoxamine and Zn(I1) ion in pyridine-methanoI solution to yield highly fluorescent chelates. This fluorophotometric procedure is simple and highly sensitive to o-0x0 acids. No detectable interference for the determination of pyruvate occurred with any of the amino acids, amines, sugars, organic acids, and inorganic salts. Recoveries for pyruvate added to bloods and serum ranged from 90.1 to 103.2%. As little as 5 x IO-ii mol of ~0x0 acid may be measured, ~0x0 acids in biological materials have been determined by chemical and biological methods. Among them a calorimetric method based on formation of 2,4-dinitrophenylhydrazone
(1) and an uv method using enzymic reaction (2) are most widely used in clinical chemistry. Although sensitivity of the spectrophotometric method is sufficient for many purposes, more sensitive assays for ~0x0 acids are often needed in biochemical studies. As fluorophotometry is known to increase the sensitivity of assays by several orders of magnitude, some investigators tried to develop a fluorophotometric assay for CX-0x0 acids. Spinker ef af.
(3) developed a fluorophotometric method for the determination of a-0x0 acids based on its condensation with o-phenylenediamine in a strong acidic medium. The resulting quinoxaline derivatives are stable and highly fluorescent. Mizutani et al. ( 4) introduced the use of 4'-hydrazino-2stilbazole to assay ~0x0 acids in the form of resulting hydrazones.
Our previous papers reported a new fluorophotometric method for the determination of amino sugars (5) and amino acids (6), based on the formation of a highly fluorescent chelate (7), using pyridoxal and zinc acetate in pyridine-methanol media. The transamination of ~0x0 acid with pyridoxamine catalyzed by some metal ions, such as aluminum(II1) and zinc(I1) ions, is well known as the model reaction for the transaminase-enzyme reaction (8). Based on this reaction, a new fluorophotometric method for the determination of ~0x0 acids has been developed and applied to the determination of total ~0x0 acids in biological materials.