Application of Glass Capillary Gas Chromatography to the Study of Urinary Steroid Excretion in Normal Children and in Patients With Various Endocrinopathies* ** By W. M. Teller and J. Homoki
Department of Pediatrics, University of Ulm, Federal Republic of Germany
With 7 Figures
Summary
A method of gas chromatography on glass capillary columns (g. c. c. c.) is presented which allows the determination of 26 urinary C19 and C21 steroid meta- bolites in one procedure.
Hundredthirtyseven normal individuals of both sexes from 6 months through 32 years of age were studied regarding their urinary steroid patterns. These were compared to the excretion patterns of patients with congenital adrenal hyperplasia before and during treatment and of a child with virilizing adrenal carcinoma. From the results it is concluded that g. c.c.c. may be considered a valuable tool in the study of steroid production and metabolism.
Considerable information regarding steroid metabolism and/or produc- tion may be gained by the analysis of urinary steroid patterns. Formerly the separation of steroids was achieved by paper and/or column chromatography.
In recent years two new methods were developed for the determination of steroids: The radioimmune assay of individual steroids, particularly in
* Supported by Deutsche Forschungsgemeinschaft, SFB 87, Project C3.
** Abbreviations used in the text: Oe = estratetrienol (= internal standard), Andro = androsterone, Et = etiocholanolone, DHA = dehydroepiandrosterone, 11-O-Andro = 11-ketoandrosterone, 11-O-ET = 11-ketoetiocholanolone, 11-OH- Andro = 11-hydroandrosterone, 11-OH-ET = 11-hydroxyetiocholanolone, PD = pregnanediol, TH-DOC = tetrahydrodesoxycorticosterone, PT = pregnanetriol, 45-PT = pregnenetriol, THA = tetrahydrodehydrocorticosterone, THB = tetra- hydrocorticosterone, THS = tetrahydro-21-desoxycortisol, IS = cholesterylbutyrate (= internal standard), THE = tetrahydrocortisone, THF = tetrahydrocortisol, allo-THF = allo-tetrahydrocortisol, Ch = cholesterol, MO-TMS = Methyloxim- trimethysilyl.
W. Swoboda et al. (eds.), Aktuelle Probleme der pädiatrischen Endokrinologie
@ Springer-Verlag/Wien 1977
plasma, and the gas chromatography of steroid metabolite patterns, parti- cularly in urine. The latter method proved to be unsuitable for the simul- taneous fractionation of C19 and C21 metabolites. Usually the penta- and hexahydroxylated C21 metabolites were destroyed during the chromato- graphic procedure with programming of the temperature.
In 1970 VÖLLMIN described a method of gas chromatographic separation of steroids on glass capillary columns. We modified his procedure for the application to the simultaneous fractionation of urinary C19 and C21 steroids in normal children and patients with various endocrinopathies.
Materials and Method
Subjects
Normal Control Group: The urinary steroid patterns were determined in 137 normal persons between 6 months and 32 years of age. They were 50 female and 78 male probands.
1/5 th_ 1/20th Volume of 24 hr
URINE
Adjust pH to 4.6
1
HYDROLYSIS 20 mg Helicase 48 hrs. at 37℃
Adjust pH to 5.2
Add Internal Standard Adjust pH to 12.5
EXTRACTION
2x 20ml ethylacetate : ether ( 2:1) wash 5 ml 8% NaHCO3 & 5 ml. water
!
Dry
FORMATION OF METHYLOXIME TRIMETHYLSILYL ETHERS
Add 0.1ml 0.8% Methyloxime - hydrochloride in methanol + 0.1 ml. 1% Potassium acetate in methanol. Heat 12 hrs. at 42°C. Dry. Add 01 ml BSTFA. Heat 2hrs. at 60°C.
1 Remove 1-2 ul
G.L.C.
Glass capillary column 20 or 50 m . x 0 32 mm. OV 101 Splitless injection. Temp. Progr 50℃ for 2 min , 170℃ for 20 min -1 2ºC/min - 245℃
Patients With Endocrinopathies: As examples of endocrine disturbances with involvement of the steroid producing organs we examined the urinary steroid pat- terns in 9 patients of various ages and both sexes with congenital adrenal hyper- plasia due to 21-hydroxylase deficiency before and during suppressive steroid therapy, and in one 76/12 year-old girl with adrenal carcinoma.