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Assessment of adrenocortical activity by chemical or biological assay of corticosteroids and their metabolites in urine is of limited value: these compounds are in part excreted as water-soluble conjugates, and the existing methods for their hydrolysis are not entirely satisfactory (Marrian, 1951; Bayliss, 1952). The decrease in circulating eosinophils following administration of adrenocorticotrophic hormone or cortisone has provided only an indirect and non-quantitative index of increased amounts of circulating corticosteroids. Various methods have been used for direct estimation of adrenal steroids in blood. Vogt (1943) and Paschkis, Cantarow, Walking & Boyle (1950) used bioassay procedures involving the cold-protection and the glycogen-deposition tests, expressing the results in terms of cortisone and 11-dehydrocorticosterone (Kendall's compound A) respectively. In dogs, quantities ranging from 1 to 5 mg./100 ml. plasma were found. These methods are not suitable for clinical use. Corcoran & Page (1948) determined the quantity of formaldehydogenic material in plasma and found in human peripheral blood the equivalent of 100-400 jpg. 11-deoxycorticosterone/100 ml. plasma. The exact nature of the compounds liberating formaldehyde is unknown, but Pincus (1952) suggests that some are glycols, which may or may not be steroids. Paper-partition chromatography shows that peripheral blood contains several hormones, of which 17-hydroxycorticosterone (Kendall's compound F) and corticosterone (Kendall's compound B) are present in the largest amount by weight and may prove to be clinically the most important (Hechter, 1952; Bush, 1952, 1953). Such methods are only semi-quantitative and require large volumes of plasma. Recently, Nelson & Samuels (1952) have described a chemical method for estimating in blood and plasma steroids that give a colour reaction with phenylhydrazine in sulphuric acid. According to Porter & Silber (1950), who first described it, this colour reaction is specific for 17:21-dihydroxy-20ketosteroids, and Nelson, Samuels, Willardson & Tyler (1951) state that by their technique the chief

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1. Results of tests for the diagnosis of Cushings syndrome of varoius aetiologies are discussed for twenty-five patients in whom the pathology was established by operation or autopsy. 2. Control values for the urinary excretion of free cortisol, 17-OHCS, Porter-Silber chromogens (P-SC) and 17-OS and plasma levels of P-SC are compared with those for normal subjects. 3. The results indicated that urinary values are within the normal range for some patients with Cushing's syndrome. 4. Plasma levels of P-SC in the morning were within the normal range for the majority and elevated for the rest. 5. Some patients showed day-night variation of plasma P-SC but evening values were above the normal range. 6. The expected response for low dosage dexamethasone was found in all patients tested but unexpected responses followed high dosage in some. 7. Plasma 11-OHCS in the five patients tested failed to respond to insulin induced hypoglycaemia. 8. Metyrapone administration and corticotrophin infusion tests had limited usefulness in establishing the aetiology of the disease. The 17-OHCS excretion became raised in the response to corticotrophin and the evaluation was prolonged beyond normal responsiveness.

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