In 1937 Brunel (1) used the sensitive Rimini-Schryver reaction to identify glyoxylic acid as the product of the all,antoicase reaction. Young and Conway used this reaction for the determination of allantoin and allantoic acid (2). In their method, allantoin or allantoic acid is first hydrolyzed to glyoxylic acid, which in turn reacts with phenylhydrazine in dilute hydrochloric <acid at 190Β°C. The phenylhydrazone formed in this reaction is then oxidized with ferricyanide in the presence of concentrated hydrochloric ,acid. Although the reagent is very sensitive, the method has certain disadvantages : since spectrophotometers were not generally available in 1937 and Young and Conway still used a colorimeter to measure the color, the spectrum of the chromophore was not known. Moreover, under the drastic reaction condition used for the reaction of glyoxylic acid with phenylhydrazine, allantoic acid, ,a precursor of glyoxylic acid in the pathway of purine catabolism, hydroylzes spontaneously to glyoxylic acid (2), making it impossible to differentiate between glyoxylic acid and allantoic acid. Most 'biological fluids or reaction media contain ingredients which change either the pH or the ionic strength, or both, of the reaction medium; this, in turn, may affect the color yield.
The present paper reports the spectrum of the chromophore and the effect of pH, buffer concentration, timing, ionic strength, and reagent concentrations on the color development.
A rapid, quantitative, and sensitive method for the determination of glyoxylic acid formed in the allantoicase reaction is described.
EXPERIMENTAL METHODS
Reagents