N-Acetyl- l-cysteine: An alternative to 2-mercaptoethanol in the reduction of Udenfriend's chromophore used for the determination of urinary 5-hydroxyindoleacetic acid

Abstract

The routine laboratory technique for the determination of 5-hydroxyindoleacetic acid (5-HIAA) in urine relies on the original observation of Udenfriend et al [ 11, which is based on measurement of the violet chromophore formed by the reaction of I-nitroso-2-naphthol and 5-HIAA in the presence of nitrous acid. Later studies by Udenfriend et al [2] and by others [3,4] confirmed both the lack of sensitivity and specificity of the technique. Goldenberg noted a marked increase in the specificity of the nitrosonaphthol reaction for 5-HIAA when 2-mercaptoethanol (2ME) is added to the reaction mixture [5]. The colours produced by other phenols and phenol carboxylic acids, some of which are of biological interest, are discharged; at the same time the violet SHIAA chromophore suffers a bathochromic shift with a two-fold increase in extinction at the new absorption maximum (645 nm). The new method, although simpler and more specific than the previous methods, is still plagued with disadvantages viz. the notorious odour of 2-mercaptoethanol, the relatively low sensitivity because colour measurement is done at 590 nm instead of 650 nm where Beer’s Law is not obeyed, and the need for an incubator at 37’C to complete the determination which takes about 2 h. In view of the above mentioned shortcomings of the Goldenburg technique, we proceeded to investigate the use of other reducing agents of comparable efficiency, but with no untoward effects. Our results show that N-acetyl-L-cysteine is a better alternative to 2-mercaptoethanol. The reduction is faster and complete in 4 min at room temperature. Furthermore, inclusion of dimethyl sulphoxide (DMSO) in the reaction mixture, makes it possible for the first time to read the extinction at 650 nm where Beer’s Law is obeyed at high concentrations of SHIAA.