An enzymatic spectrophotometric method for the determination of homogentisic acid in plasma and urine.

Previous methods for the determination of homogentisic acid have been based upon its ability to reduce silver (I), phosphomolybdic acid (a), or iodine (3-5). The error introduced by other biological substances which have similar reducing properties is inconsequential in the determination of the relatively large amounts of homogentisic acid present in urine from a patient with alcaptonuria. Such errors, however, are substantial in the determination of the relatively low concentrations of homogentisic acid in the plasma of alcaptonuric patients. Some attempts were made to gain greater specificity by preliminary extraction of homogentisic acid into ether, and by the use of an iodometric method which took advantage of the increase in oxidation potential of the quinone form of homogentisic acid upon lowering the pH (6). Neuberger (6) also utilized the rapid reduction of silver by homogentisic acid at pH 4.4 in the presence of colloidal gold to estimate levels of homogentisic acid in plasma. Nevertheless, these methods admittedly lacked specificity and precision when applied to alcaptonuric plasma. We have developed a simple and rapid enzymatic spectrophotometric method for the determination of homogentisic acid which uses partially purified homogentisic acid oxidase and detects as little as 1 pg of homogentisic acid. This method has been used to determine the plasma levels of homogentisic acid in patients with alcaptonuria, the diurnal variation in this level, and the changes produced by feeding phenylalanine.