Serum 3-hydroxysteroids (3-OHS) were estimated fluorometrically with hydroxysteroid dehydrogenase (HSD) coupled with diaphorase-resazurin system. The serum sample (0.5ml) was deproteinized with 1.5M HCIO₄ and the supernatant was heated at 100°C for 10 minutes. The hydrolyzed steroids were extracted with methylene chloride and evaporate to dryness. The residue was incubated with NAD, HSD, diaphorase and resazurin, and resorufin, the reaction product, was measured by fluorometry.(λ₁=57 Onm, λ₂=580nm). The sensitivity of this method increased about 20 times compared with photometry of NADH (340nm), and 6 times compared with fluorometry of NADH.
Two hydrolysis method, solvolysis and acid hydrolysis, were studied and the latter was more suitable for the application of laboratory routine work.
Normal levels of 3-OHS were 1.69±0.49μg/ml (mean±2 S. D., n=14) for male and 1.41±0.64μg/ml (n=8) for female.
Correlation between urinary steroids (17-KS and 17-OHCS) and serum steroids (3-OHS and 11-OHCS) were studied and high correlation was observed between urinary 17-OHCS and serum 11-0HCS (r=0.698), and between urinary 17-KS and serum 3-OHS (r=0.600). Serum 3-OHS could be an indicator of adrenal androgenic function as a substitute of urinary 17-KS.

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The trypsin activity from trypsin-α₁-antitrypsin complex was restored about 50% on the treatment with acetone. The restoration ratio of trypsin activity was almost constant in various sera. These findings show that the absolute amount of serum trypsin was not obtained, but the method described in the present paper can be utillized for the diagnosis if the relationship between serum trypsin activity and diseases is clarified.

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Recently isoenymes of various enzymes have been investigated and each clinical significance has been elucidated.
Author has studied isoenzymes of NADP dependent isocitrate dehydrogenase (EC 1.1. 1.42) in human tissue using starch gel electrophoresis.
Three isoenzymes move towards the anode and one isoenzyme moves toward th cathod. Main isoenzyme band is IDH, in heart muscle and IDH2 in other tissues.
Manganese as a metal ion has been used in measurement of IDH activity and in the identification of NADP-IDH isoenzymes by tetrazolium method. However, manganese shows inhibition to the step of non-enzymatic reaction and decreases the production of formazan in tetrazolium method. Magnesium has not demonstrated this inhibition.

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As previously reported1) human serum cortisol was assayed using high pressure liquid chromatography. After an intravenous single injection of 250μg of synthetic α_1-24 ACTH (Cortrosyn), serum cortisol level was followed until 180min passed, with endocrinologically healthy patients. The time-course curve of serum cortisol obtained by the pressent method, was almost consistent with the curve of a rapid ACTH test measured by the conventional fluorometric method2), except the values before ACTH and at 180min pased after ACTH were lower in our results. From these results the synthetic rate of cortisol was calculated, it showed that ACTH stimulates about 10 times the synthesis of cortisol. Also, the curves of abnormal responses against ACTH administration, such as non-reactive or delayed-type, were shown.

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