A sterol panel for rare lipid disorders: sitosterolemia, cerebrotendinous xanthomatosis and Smith-Lemli-Opitz syndrome

Abstract

Disease-specific sterols accumulate in the blood of patients with several rare lipid disorders. Biochemical measurement of these sterols is important for correct diagnosis and sometimes monitoring of treatment. Existing methods to measure sterols in blood, particularly plant sterols, are often laborious and time consuming. Partly as a result, clinical access to sterol measurements is limited in many parts of the world. A simple and rapid method to extract free sterols from human serum and quantitate their concentration using isotope-dilution liquid chromatography-tandem mass spectrometry (LC-MS/MS) without derivatization was developed. The method was designed to be compatible with routine workflows (e.g., 96-well format) in a clinical lab and extensively validated. Serum from at least 125 controls were analyzed and used to estimate the upper reference limits for sitosterol, campesterol, stigmasterol, desmosterol, 7-dehydrocholesterol (7DHC), lathosterol, and cholestanol. Serum from patients with the rare lipid disorders sitosterolemia (n = 7), Smith-Lemli-Opitz syndrome (SLOS; n = 1), and cerebrotendinous xanthomatosis (CTX; n = 1) were analyzed. All seven sitosterolemia patients had greatly elevated levels of free plant sterols (sitosterol, campesterol, and stigmasterol) compared to the controls. The SLOS serum contained massively increased concentrations of 7DHC. CTX serum contained greatly increased concentrations of cholestanol, as well as 7DHC and lathosterol. Spiking experiments indicated that the method is likely also useful for the diagnosis of desmosterolosis and lathosterolosis. The reported method is a relatively simple and fast LC-MS/MS method capable of quantitating diagnostically important sterols and differentiated patients with three rare lipid disorders from controls.

Keywords: 7-dehydrocholesterol; LC-MS/MS; campesterol; cholestanol; sitosterol; stigmasterol.

Quantitation of diagnostic sterols (free, nonesterified) in human serum using a simple and fast LC-MS/MS method. Sitosterolemia, Smith-Lemli-Opitz syndrome (SLOS), and cerebrotendinous xanthomatosis (CTX) patients had greatly increased concentrations compared to controls.

Fig. 1

Measurement of free sterols in human serum. Overview of method (A) and qualitative visualization of sterol profiles (extracted ion chromatograms; XICs) (B to G). A: Serum is combined with water, isopropanol (IPA), and internal standard (ISTD) stock in a 96-well plate. Calibrators are prepared using 2-fold concentrated calibrator stocks (2xCal; in IPA) added to lipoprotein-depleted serum. After equilibration, the mixture is transferred to a supported liquid extraction plate and extracted with ethyl acetate (EtOAc). Extract is evaporated, reconstituted, and injected on LC-MS/MS employing electrospray ionization to measure free (non-acyl conjugated) sterols. B: Standards in lipoprotein-depleted serum (Calibration standard 6). C: Control serum pool from presumed nonaffected patients. D: Control pool spiked with 10 μM of each sterol (excluding cholesterol). E: Sitosterolemia patient serum (pool). F: Smith-Lemli-Opitz Syndrome (SLOS) patient serum. G: Cerebrotendinous Xanthomatosis (CTX) patient serum. Numbers indicate analyte (parenthesis indicate absence or the cholesterol-signal present in the lathosterol XIC), and either all analytes (B to D) or analytes elevated compared to control (E to G) are indicated. Magenta arrowhead indicate presence of elevated unidentified sterol (presumed to be 8DHC). Solid XICs are from the quantitation-transitions and dashed-lines the ISTDs. For increased visual clarity, the XICs were trimmed around the known retention time and were normalized to the max. peak height of the 7DHC-ISTD. Created in BioRender (https://BioRender.com/h87p026).

Fig. 2

Method comparisons. Comparison of measured sitosterol (A), campesterol (B), 7DHC (C), and cholestanol (D) concentrations. A and B: Passing-Bablok comparison of results measured by reported method (OUS) to free plant sterol concentrations measured by Karolinska Institutet (KI). C and D: Comparison to reported consensus concentrations (median) for ERNDIM external quality assurance samples, arranged based on the consensus concentration. For D, filled circles indicate concentration values above the upper reference limit of our method (see Table 1). The ERNDIM consensus values are confidential and therefore not reported.

Fig. 3

Greatly increased free plant sterol concentrations in sitosterolemia. Measured concentrations of free plant sterols in seven sitosterolemia patients (six on treatment to lower plant sterols) and 126 controls. A: Sitosterol. B: Campesterol. C: Stigmasterol. Points show individual values, and black bars indicate group median. The dashed gray lines indicate the method lower limit of quantitation (mLLOQ) and the gray text the no. of samples below the mLLOQ. Asterisk (∗) indicates statistically significant Mann-Whitney U test results, P-value < 0.001.