The estimation of 4-hydroxy-3-methoxymandelic acid in urine.

Armstrong & McMillan (1957) identified 4hydroxy-3-methoxymandelic acid (vanilmandelic acid) in normal human urine and demonstrated that increased quantities were excreted after noradrenaline infusion and by patients with the catecholamine-secreting tumour, phaeochromocytoma. Subsequent work has established that 3methoxylation followed by oxidative deamination to 4-hydroxy-3-methoxymandelic acid is a major metabolic pathway of both noradrenaline and adrenaline (see Axelrod, 1959). Some 4-hydroxy-3methoxymandelic acid is also formned by an alternative pathway in which oxidative deamination precedes 3-methoxylation (Kopin, 1960). About 45% of the total noradrenaline and adrenaline production in man is excreted as 4hydroxy-3-methoxymandelic acid (Kopin, 1960) compared with a free-amine excretion of only 1-4% (Euler & Luft, 1951; Goldenberg, Serlin, Edwards & Rapport, 1954). Estimation of small concentrations of free catecholamines in urine demands specialized skills and equipment for the biological or fluorimetric procedures involved; results are often conflicting and depend on the method of assay (Goldenberg et al. 1954). Catechol compounds generally are inherently unstable. It is thus likely that 4-hydroxy-3-methoxymandelic acid, by virtue of its stability and relatively large output, will provide a better guide to noradrenaline and adrenaline turnover, if a simple technique for its assay were available. A number of quantitative techniques have been described (Armstrong, Shaw & Wall, 1956; Robinson, Ratcliffe & Smith, 1959; Smith, Schweitzer & Wortis, 1959; Weise, Mc. Donald & LaBrosse, 1960; Studnitz & Hanson 1959) but none of these methods is well suited to the clinical laboratory. We have given preliminary reports of a colorimetric method for 4-hydroxy-3-methoxymandelic acid assay (Sandler & Ruthven, 1959a, b). The modification described here is both simpler and more specific. It involves the absorption of urinary 4-hydroxy-3-methoxymandelic acid on an anion-exchange resin, and its elution, extraction and subsequent conversion into vanillin, which is measured spectrophotometrically. Apart from its value in the diagnosis of phaeochromocytoma, it is suitable for the study of small changes in 4hydroxy-3-methoxymandelic acid output at normal excretion levels.