Measurement of 3 p-hydroxysteroid A 7-reductase activity in cultured skin fibroblasts utilizing ergosterol as a substrate : a new method for the diagnosis of the Smith

3p-hydroxysteroid A7-reductase activity in cultured skin fibroblasts utilizing ergosterol as a substrate: a new method for the diagnosis of the Smith-Lem I i-0pi tz sy nd rome Abstract A new sensitive and specific method for the evaluation of Sphydroxysteroid A'-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-5,22dien-3bl) produced by the incubation of ergosterol (ergosta-5,7,22-trien-3bl) with cultured human skin fibroblasts. Although the conversion of ergosterol to brassicasterol was slower than the transformation of ['HI 7dehydrocholesterol to [3H]cholesterol, cells from control subjects produced brassicasterol efficiently. In contrast, cells from SLO patients produced very little brassi-casterol (P < 0.0001, patients vs. parents or vs. controls). These results indicate that the reduction of ergosterol can be used as an assay for Jkhydroxysteroid A7-reductase in human skin fibroblasts, which avoids the many problems caused by the instability and lack of availability of radiolabeled 7dehy-drocholestero1.l The present method made it possible to diagnose the SLO syndrome with high sensitivity and reliability using a commercially available compound. Measurement of 3phydroxysteroid A7-reductase activity in cultured skin fibroblasts utilizing ergosterol as a sub-strate: a new method for the diagnosis of the Smith-Lemli-Opitz syndrome. Supplementary key words cholesterol gas chromatography-mass spectrometry brassicasterol cholesterol 7dehydro-failure to thrive, and multiple congenital anomalies (1, 4-7), and biochemically by abnormally low plasma cholesterol concentrations and the accumulation of the final cholesterol precursor, 7-dehydrocholesterol (8, 9). Plasma sterol analysis is the most convenient and useful way to screen for the syndrome. However, it is important to develop more sensitive and accurate methods because occasional atypical cases exhibit normal plasma cholesterol levels with concentrations of 7dehydro-cholesterol that are considerably below that reported for most individuals with the syndrome (10, 11). In addition , increasing numbers of new metabolic and genetic studies of the syndrome require the measurement of 7-dehydrocholesterol A'-reductase activity. Procedures used up until now made use of the conversion of 13H] lathosterol (the precursor of 7dehydrocholes-terol) or of [3H] 7dehydrocholesterol to cholesterol. Although these substrates have been used successfully to demonstrate the enzyme defect (2, 3, 12), neither of these radiolabeled compounds is available commercially and r3H] 7-dehydrocholesterol is especially unstable and difficult to work with. This report describes a new method for the evaluation of 3phydroxysteroid A'-reductase activity in cultured human skin fibroblasts by gas chromatography-The Smith-Lemli-Opitz syndrome …

[1]  X. Chenivesse,et al.  Cloning by Metabolic Interference in Yeast and Enzymatic Characterization of Arabidopsis thaliana Sterol 7-Reductase (*) , 1996, The Journal of Biological Chemistry.

[2]  N. Venizelos,et al.  Detection of defective 3β-hydroxysterol Δ7-reductase activity in cultured human fibroblasts: a method for the diagnosis of Smith-Lemli-Opitz syndrome , 1996, Journal of Inherited Metabolic Disease.

[3]  G. Ness,et al.  Treatment of the cholesterol biosynthetic defect in Smith-Lemli-Opitz syndrome reproduced in rats by BM 15.766. , 1995, Gastroenterology.

[4]  A. Honda,et al.  Markedly inhibited 7-dehydrocholesterol-delta 7-reductase activity in liver microsomes from Smith-Lemli-Opitz homozygotes. , 1995, The Journal of clinical investigation.

[5]  V. P. Johnson,et al.  Correlation of severity and outcome with plasma sterol levels in variants of the Smith-Lemli-Opitz syndrome. , 1995, The Journal of pediatrics.

[6]  A. Honda,et al.  Defective conversion of 7-dehydrocholesterol to cholesterol in cultured skin fibroblasts from Smith-Lemli-Opitz syndrome homozygotes. , 1995, Journal of lipid research.

[7]  S. Scherer,et al.  Identification of a yeast artificial chromosome clone spanning a translocation breakpoint at 7q32.1 in a Smith-Lemli-Opitz syndrome patient. , 1995, American journal of human genetics.

[8]  R. Kelley.  Diagnosis of Smith-Lemli-Opitz syndrome by gas chromatography/mass spectrometry of 7-dehydrocholesterol in plasma, amniotic fluid and cultured skin fibroblasts. , 1995, Clinica chimica acta; international journal of clinical chemistry.

[9]  T. Buie,et al.  Abnormal cholesterol metabolism in the Smith-Lemli-Opitz syndrome: report of clinical and biochemical findings in four patients and treatment in one patient. , 1994, American journal of medical genetics.

[10]  G Salen,et al.  Defective cholesterol biosynthesis associated with the Smith-Lemli-Opitz syndrome. , 1994, The New England journal of medicine.

[11]  G. Tint,et al.  Defective cholesterol biosynthesis in Smith-Lemli-Opitz syndrome , 1993, The Lancet.

[12]  J. Clayton-Smith.  Syndromes of the Head and Neck , 1993 .

[13]  M. Axelson.  Occurrence of isomeric dehydrocholesterols in human plasma. , 1991, Journal of lipid research.

[14]  K. Taysi,et al.  Possible abnormalities of steroid secretion in children with Smith-Lemli-Opitz syndrome and their parents , 1985, Steroids.

[15]  R. Bittman,et al.  Distribution and movement of sterols with different side chain structures between the two leaflets of the membrane bilayer of mycoplasma cells. , 1984, The Journal of biological chemistry.

[16]  G. Tint,et al.  Biosynthesis of cholesterol, lanosterol, and delta 7-cholestenol, but not cholestanol, in cultured fibroblasts from normal individuals and patients with cerebrotendinous xanthomatosis. , 1982, Journal of lipid research.

[17]  M. Holick,et al.  Regulation of cutaneous previtamin D3 photosynthesis in man: skin pigment is not an essential regulator. , 1981, Science.

[18]  R. Anderson,et al.  Photosynthesis of previtamin D3 in human skin and the physiologic consequences. , 1980, Science.

[19]  J. Opitz,et al.  A NEWLY RECOGNIZED SYNDROME OF MULTIPLE CONGENITAL ANOMALIES. , 1964, The Journal of pediatrics.

[20]  J. Folch,et al.  A simple method for the isolation and purification of total lipides from animal tissues. , 1957, The Journal of biological chemistry.

[21]  G. Ness,et al.  Reproducing abnormal cholesterol biosynthesis as seen in the Smith-Lemli-Opitz syndrome by inhibiting the conversion of 7-dehydrocholesterol to cholesterol in rats. , 1995, The Journal of clinical investigation.

[22]  J. Opitz,et al.  Smith-Lemli-Opitz syndrome-type II: multiple congenital anomalies with male pseudohermaphroditism and frequent early lethality. , 1987, American journal of medical genetics.

[23]  M Buyse,et al.  Center for Birth Defects Information Services. , 1980, Birth defects original article series.

[24]  A. Truswell,et al.  SEPARATION OF CHOLESTEROL FROM ITS COMPANIONS, CHOLESTANOL AND DELTA-7-CHOLESTENOL, BY THIN-LAYER CHROMATOGRAPHY. , 1965, Journal of Lipid Research.