Abstract
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.
Highlights
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 [1]
We describe the results obtained for the determination of free cholesterol, total cholesterol and its derivatives in 39 amniotic fluids without genetic alterations, considered as control fluids and in 60 amniotic fluids withdrawn from pregnant women, showing a fetal growth retardation potentially related to Smith-Lemli-Opitz syndrome (SLOS), in order to confirm the correlation between this pathology and the sterol levels in amniotic fluid
In the screening phase the determinations were carried out by means of the GC/flame ionization detector (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
Summary
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 [1]. In particular the Smith-Lemli-Opitz syndrome (SLOS), is an autosomal recessive disorder affecting 1:20000 individuals, characterized by a recognizable pattern of minor facial anomalies, limb abnormalities and multiple congenital anomalies including pseudohermaphroditism in males, neonatal hypotonia, mental retardation and failure to thrive [9]-[12] This disease, caused by a 7-dehydrocholesterol reductase deficiency, is characterized by high blood levels of specific metabolites of postsqualene cholesterol biosynthesis [13]. 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. Standard Solutions Stock standard solutions (1 mg·mL−1) of cholesterol, 7-DHC and stigmasterol (internal standard, IS) were prepared in cyclohexane and diluted in volumetric flasks to concentrations of 50.0, 100.0, 150.0, 200.0, 300.0, 400.0, 600.0, 800.0 μg·mL−1 for cholesterol; 2.0, 5.0, 7.0, 10.0, 15.0, 20.0, 30.0, 50.0 μg·mL−1 for 7-DHC and 50.0 μg·mL−1 for IS and stored at 4 ̊C for up to a month
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