Basic study on urinary 17-hydroxycorticosteroids in male newborn infants

Abstract

The urine was collected daily from 10 male newborns, beginning shortly after birth until the 7th day of life. Daily urinary samples from these infants were pooled to be used for quantitative analysis of steroids. The urine was first treated with limpet β-glucuronidase. After extraction of the steroids with ethyl acetate, steroid sulfate present in aqueous phase was hydrolysed by the method of Burstein and Lieberman. The steroids thus obtained were separated by Girard's reaction and the ketonic fractions were used for analysis. Polar corticoids, present in free state, were extracted with ethyl acetate from urine saturated with sodium sulfate. These fractions were separated by the elution chromatography on partially esterified Amberlite IRC-50 using a mixture of ethanol, methanol and water (9 : 3 : 8, by vol.) as eluent. In this chromatographic system 6β-hydroxycortisol, cortisone, allo-tetrahydrocortisol and tetrahydro-11-desoxycortisol were separated from each another satisfactorily, but tetrahydrocortisol, tetrahydrocortisone and cortisol were eluted together into the same fraction. These three steroids were separated by a partition chromatography on Dowex 50 w×4 using a mixture of ethanol, benzene, n-hexane and water (20 : 100 : 40 : 1, by vol.) as a moving phase. 17-hydroxycorticosteroids present in the effluent fractions were determined photometrically by the Porter-Silber reation.The daily total amounts of Porter-Silber chromogens of which dominant components were polar corticoids containing 6β-hydroxycortisol were 102 μg/24 hrs. in the 1st day and almost 200 μg/24 hrs. in the 2nd to the 7th day of life. The most specific feature was the dominance of tetrahydrocortisone and cortisone over tetrahydrocortisol and cortisol in the urine of male newborns. The ratio of tetrahydrocortisone to tetrahydrocortisol was more than twenty, whereas the ratios obtained from the urine of their mothers and adults ranged from 1.3 to 2.3.Tetrahydrocortisone fraction was tentatively identified by : elution volume in the chromatographies on Amberlite IRC-50 and Dowex 50w×4, typical Porter-Silber chromogen, absence of absorption at 240 mμ, absence of absorption at 405 mμ with Oertel-Eik-Nes reagent, positive reaction with blue tetrazolium and the formation of 11-ketoetiocholanolone alone by the oxydation with sodium bismuthate. The recoveries of tetrahydrocortisol and cortisol were recognized to be 40% and 56%, respectively, by the method used in this experiment after the addition of standard tetrahydrocortisol and cortisol before hydrolysis.It is suggested from this result that cortisol might be metabolised to cortisone at a much higher rate in newborns than in adults.