Measurement of 7-dehydrocholesterol and cholesterol in hair can be used in the diagnosis of Smith-Lemli-Opitz syndrome

Abstract

7-dehydrocholesterol (7-DHC) and cholesterol (CHOL) are biomarkers of Smith-Lemli-Opitz Syndrome (SLOS), a congenital autosomal recessive disorder characterized by elevated 7-DHC level in patients. Hair samples have been shown to have great diagnostic and research value, which has long been neglected in the SLOS field. In this study, we sought to investigate the feasibility of using hair for SLOS diagnosis. In the presence of antioxidants (2,6-ditert-butyl-4-methylphenol and triphenylphosphine), hair samples were completely pulverized and extracted by micro-pulverized extraction in alkaline solution or in n-hexane. After microwave-assisted derivatization with N,O-Bis(trimethylsilyl)trifluoroacetamide, the analytes were measured by GC-MS. We found that the limits of determination for 7-DHC and CHOL were 10 ng/mg and 8 ng/mg, respectively. In addition, good linearity was obtained in the range of 50-4000 ng/mg and 30-6000 ng/mg for 7-DHC and CHOL, respectively, which fully meets the requirement for SLOS diagnosis and related research. Finally, by applying the proposed method to real hair samples collected from 14 healthy infants and two suspected SLOS patients, we confirmed the feasibility of hair analysis as a diagnostic tool for SLOS. In conclusion, we present an optimized and validated analytical method for the simultaneous determination of two SLOS biomarkers using human hair.

Keywords: diagnostic test; gas chromatography-mass spectrometry; hair analysis; micro-pulverized extraction; microwave-assisted derivatization.

Graphical abstract

Fig. 1

Syndactyly of the second and the third toes, clinical appearance of two suspected SLOS patients. SLOS, Smith-Lemli-Opitz syndrome.

Fig. 2

Representative GC-MS/MS chromatograms of 7-DHC (m/z 325, 351) and CHOL (m/z 129, 329, 368). Results of blank sample (A); spiked sample with 7-DHC (50 ng/mg, LLOQ) and CHOL (30 ng/mg, LLOQ) (B); sample from the one SLOS patient (C); arrow 1 refers to CHOL; arrow 2 refers to 7-DHC. 7-DHC, 7-dehydrocholesterol; CHOL, cholesterol; LLOQ, lower limit of quantification.

Fig. 3

Concentrations of hair 7-DHC and CHOL in seven female and seven male infants and two suspected patients. Statistical results: ns, P > 0.05; ∗P ≤ 0.05; ∗∗P ≤ 0.01. 7-DHC, 7-dehydrocholesterol; CHOL, cholesterol.

Fig. 4

Effects of different extraction solutions and methods on the extraction of (A) 7-DHC and (B) CHOL from hair and effect of different ethanol concentrations and the presence or absence of KOH on the extraction of (C) 7-DHC and (D) CHOL from hair (n = 4). Statistical results: ns, P > 0.05; ∗P ≤ 0.05; ∗∗P ≤ 0.01; ∗∗∗P ≤ 0.001; ∗∗∗∗P ≤ 0.0001. Range bar, standard deviation (sd). 7-DHC, 7-dehydrocholesterol; CHOL, cholesterol; KOH, potassium hydroxide; ACN, acetonitrile; TFA, trifluoroacetic acid; MeOH, Methanol; MPE, micro-pulverized extraction; aqu, aqueous solution.

Fig. 5

Effect of antioxidant concentration on recovery rates of 7-DHC and cholesterol (CHOL) (n = 4). Statistical results: ns, P > 0.05; ∗P ≤ 0.05; ∗∗P ≤ 0.01; ∗∗∗P ≤ 0.001; ∗∗∗∗P ≤ 0.0001. Range bar, standard deviation (sd). 7-DHC, 7-dehydrocholesterol.