The Smith-Lemli-Opitz syndrome (SLOS), is an autosomal recessive disorder caused by a 7-dehydrocholesterol reductase deficiency, which is characterized by abnormally elevated amniotic fluid 7-DHC (7-dehydrocholesterol) concentrations. A GC/FID (gas-chromatography with a flame ionization detector) and GC/MS (gas-chromatography with a mass detector) method was optimized for the detection of cholesterol and 7-DHC in amniotic fluids. The quantitative determination of cholesterol in 39 control amniotic fluids evidenced that between the fourth and fifth month of pregnancy the levels of this analyte are quite constant, the concentration of total and free cholesterol being respectively 10.3 μg·mL ﹣1 (SD = ±3.6) and 1.7 μg·mL ﹣1 (SD = ±0.91), while the analysis of 60 amniotic fluids potentially related to SLOS, showed a higher variability of cholesterol levels. Moreover, in 13 samples an analyte which did not correspond either to cholesterol or to 7-DHC was detected. A GC/MS investigation allowed us to identify this compound as lathosterol, a precursor of cholesterol in the biosynthetic pathway.
Cholesterol is an essential lipid which is a precursor for many sterol-based compounds and is involved in the regulation of the precise pattering of embryonic structures [
Cholesterol is produced from lanosterol through a complex biosynthetic pathway involving several multienzymatic reactions, including demethylations, isomerization, desaturation, and double-bond reductions. Several polimalformative disorders [
The fetal growth retardation were divided into three classes: an overt intra uterine growth retardation (IUGR), a smaller fetus respect to the gestational age (SGA) and cases in which further biomedical studies were necessary (ACC).
In the case of the amniotic fluids related to fetal growth retardations only the quantitative determination of total cholesterol was carried out, in fact it was not possible to determine its free portion because of the small amount of amniotic fluid available after the routine cytogenetic investigations.
In the screening phase the determinations were carried out by means of the GC/FID technique, which allows a fast and accurate quali-quantitative detection of the analytes of interest; the fluids which presented altered concentration values of sterols were further investigated by GC/MS, a technique able to detect compounds structurally related to cholesterol, belonging to its biosynthetic pathway.
All reagents and solvents were of analytical-reagent grade. Cholesterol, 7-DHC, lathosterol and stigmasterol were purchased from Sigma (St. Louis, MO), cyclohexane was obtained from J.T. Baker (Phillipsburg, NJ), ethanol from Carlo Erba (Milano), KOH and pyridine from Merck (Darmstadt), 0.9% NaCl solution from Baxter (Trieste).
Derivatization reagents were N,O-bis(trimethylsilyl)trifluoroacetamide (BSTFA) with 1% trimethylchlorosilano (TMCS) (Supelco inc., Bellafonte).