Measurement of 3 beta-hydroxysteroid delta 7-reductase activity in cultured skin fibroblasts utilizing ergosterol as a substrate: a new method for the diagnosis of the Smith-Lemli-Opitz syndrome.
A new sensitive and specific method for the evaluation of 3 beta-hydroxysteroid delta 7-reductase activity, the defective enzyme in the Smith-Lemli-Opitz (SLO) syndrome, is described. The assay is based on the use of gas chromatography-mass spectrometry with selected-ion monitoring to measure the mass of brassicasterol (ergosta-5,22-dien-3 beta-ol) produced by the incubation of ergosterol (ergosta-5,7,22-trien-3 beta-ol) with cultured human skin fibroblasts. Although the conversion of ergosterol to brassicasterol was slower than the transformation of [3H]7-dehydrocholesterol to [3H] cholesterol, cells from control subjects produced brassicasterol efficiently. In contrast, cells form SLO patients produced very little brassicasterol (P < 0.0001, patients vs. parents or vs. controls). These results indicate that the reduction of ergosterol can be used as an assay for 3 beta-hydroxysteroid delta 7-reductase in human skin fibroblasts, which avoids the many problems caused by the instability and lack of availability of radiolabeled 7-dehydrocholesterol. The present method made it possible to diagnose the SLO syndrome with high sensitivity and reliability using a commercially available compound.
Honda
M
M
Department of Medicine, Veterans Affairs Medical Center, East Orange, NJ 07018, USA.
Tint
G S
GS
Honda
A
A
Batta
A K
AK
Chen
T S
TS
Shefer
S
S
Salen
G
G
D002775:Cholestadienols / Q000032:analysis; D004875:Ergosterol / Q000378:metabolism*; D005347:Fibroblasts / Q000201:enzymology; D008401:Gas Chromatography-Mass Spectrometry; D006801:Humans; D010088:Oxidoreductases / Q000032:analysis*; D044925:Oxidoreductases Acting on CH-CH Group Donors; D010840:Phytosterols; D012867:Skin / Q000201:enzymology*; D019082:Smith-Lemli-Opitz Syndrome / Q000175:diagnosis*; D013265:Stigmasterol / Q000032:analysis
D002775:Cholestadienols; D010840:Phytosterols; C003532:brassicasterol; D013265:Stigmasterol; D010088:Oxidoreductases; D044925:Oxidoreductases Acting on CH-CH Group Donors; C019995:7-dehydrocholesterol reductase; D004875:Ergosterol