Quantitative analysis of five sterols in amniotic fluid by GC–MS: Application to the diagnosis of cholesterol biosynthesis defects

Cholesterol and its precursors, namely 7-dehydrocholesterol, desmosterol and lathosterol are important biochemical markers of cholesterol biosynthesis, and their quantification in body fluids is useful for the diagnosis of cholesterol biosynthesis pathway disorders. A rapid and sensitive gas chromatographic–mass spectrometric method was developed and validated for quantitative analysis of five sterols (cholesterol, 7-dehydrocholesterol, desmosterol, lathosterol and sitosterol) in amniotic fluid. The method was linear for all compounds (r2 > 0.99), and intra and inter-assay coefficients of variation were typically below 5%, and inaccuracy was within a ±12% interval. The method was applied to 330 amniotic fluid samples, grouped by gestational age between 13 and 22 weeks of pregnancy, in order to establish reference intervals for sterols in this specimen. The obtained concentrations (μmol/L) for each sterol was as follows: 22.1758 ± 4.2716 at 13 weeks and 78.5082 ± 12.9041 at 22 weeks for cholesterol; 0.0039 ± 0.0007 at 13 weeks and 0.1150 ± 0.0212 at 22 weeks for 7-dehydrocholesterol; 0.1562 ± 0.0406 at 13 weeks and 0.7691 ± 0.0821 at 22 weeks for desmosterol; 0.0272 ± 0.0035 at 13 weeks and 0.8551 ± 0.1791 at 22 weeks for lathosterol; and 0.0404 ± 0.0039 at 13 weeks and 0.2326 ± 0.0386 at 22 weeks for sitosterol. The method was also applied to one pathological sample that showed decreased levels of cholesterol, and higher concentration of 7-dehydrocholesterol, which is consistent with a 7-dehydrocholesterol-reductase deficiency. Our results showed that as long as pregnancy goes on, the concentrations of cholesterol and precursors increase in amniotic fluid, which is related to the increased need for cholesterol by the fetus. The reference range of each sterol in amniotic fluid was calculated at different gestational ages and will be useful for the interpretation and validation of biochemical prenatal diagnosis of inborn errors of sterol biosynthesis.