Specific photometric determination of 5-hydroxyindoleacetic acid in urine.

Udenfriend ‘s nitrosonaphthol test for 5-hydroxyindoleacetic acid (5-HIAA) is subject to interference by urinary phenolic acids and drug metabolites. Studies in our laboratory have shown that the specificity and sensitivity of the nitrosonaphthol reaction with 5-hydroxyindoles can be greatly enhanced by the addition of 2-mercaptoethanol. This observation serves as the basis of a simple, improved method for the photometric determination of urinary 5-HIAA. The 5-HIAA is extracted from urine and transferred to a colorimeter tube. Treatment with nitrosonaphthol and nitrous acid results in formation of Udenfriend’s violet chromophore, which is converted to an intense blue chromophore upon addition of mercaptoethanol. Extraneous colors caused by reactive phenols and indoleacetic acid in urine are destroyed at this point by the mercaptoethanol. The intensity of the remaining blue color is proportional to the 5-HIAA concentration. Recoveries of added 5-HIAA by the proposed method are quantitative. The normal 24-hour excretion values obtained for human subjects fall in the range of 0.8 to 6.0 mg (95% confidence limits), with a mean of 3.9 mg. Drugs that produce a false positive test for intestinal carcinoid with earlier photometric methods have no effect on the new method.