Simultaneous assay for individual sulphated 3 alpha- and beta-hydroxysteroids in serum using high-performance liquid chromatography combined with 3 alpha- and beta-hydroxysteroid dehydrogenases immobilized on one column.
A high-performance liquid chromatographic method for the simultaneous determination of individual sulphated 3 alpha- and beta-hydroxysteroids in serum using 3 alpha- and beta-hydroxysteroid dehydrogenases (3 alpha-HSD and beta-HSD, respectively) immobilized on one column and a fluorimeter to detect the resulting NAD+ to NADH transformation is described. Individual sulphated 3 alpha- and beta-hydroxysteroids in serum are extracted with ethanol, solvolysed with sulphuric acid in ethyl acetate and then separated by high-performance liquid chromatography. The hydroxysteroids thus separated are subsequently mixed with NAD+ and then passed through the column in which the following catalytic reaction occurs: (formula, see text) The detection limits are as low as 0.5-1.0 microgram/dl for sulphated 3 alpha- or beta-hydroxysteroids in serum. The present assay method is highly specific, reliable and reproducible and is thus applicable to a clinical study on the metabolism of sulphated 3 alpha- and beta-hydroxysteroids in patients with adrenal or gonadal diseases.
Takagi
K
K
Nagoya University College of Medicine Technology, Japan.
Okumura
K
K
Morikawa
N
N
Okuyama
S
S
Wu
M C
MC
Narita
O
O
D015096:3-Hydroxysteroid Dehydrogenases; D045002:3-alpha-Hydroxysteroid Dehydrogenase (B-Specific); D000328:Adult; D002851:Chromatography, High Pressure Liquid; D004800:Enzymes, Immobilized; D005260:Female; D006801:Humans; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D006914:Hydroxysteroids / Q000097:blood*; D007535:Isomerases; D008297:Male; D009097:Multienzyme Complexes; D009243:NAD / Q000032:analysis; D011375:Progesterone Reductase; D011863:Radioimmunoassay; D012997:Solvents; D013251:Steroid Isomerases
C021425:3 beta-hydroxysteroid oxidoreductase-delta(5) 3-ketosteroid isomerase; D004800:Enzymes, Immobilized; D006914:Hydroxysteroids; D009097:Multienzyme Complexes; D012997:Solvents; D009243:NAD; D015096:3-Hydroxysteroid Dehydrogenases; D011375:Progesterone Reductase; D045002:3-alpha-Hydroxysteroid Dehydrogenase (B-Specific); D007535:Isomerases; D013251:Steroid Isomerases