Tandem mass spectrometry of serum cholestanoic (C) acids - Typical concentration ranges and application to the study of peroxisomal biogenesis disorders.

Background: Primary bile acid synthesis is impaired in peroxisomal disorders, leading to the accumulation of long-chain bile acids, specifically dihydroxycholestanoic and trihydroxycholestanoic acids. Quantification of the diastereoisomers of these C bile acids is essential for the differential diagnosis of these disorders.

Methods: High-performance liquid chromatography electrospray ionization-tandem mass spectrometry with stable-isotope dilution was used to quantify all eight diastereoisomers of cholestanoic acids in serum. Clinical ranges were established for patients with and without cholestatic liver disease, as well as for those with peroxisomal disorders.

Results: The assay was linear over the range of 20-2,500 ng/mL, and intra- and inter-assay imprecision was <20 % CV. The mean (±SEM) serum concentration of total C bile acids in 20 adult controls was low (0.007 ± 0.004 μmol/L). In non-cholestatic, moderately cholestatic, and severely cholestatic patients, total C bile acids measured 0.015 ± 0.011, 0.129 ± 0.034, and 0.986 ± 0.249 μmol/L, respectively. In contrast, patients with confirmed peroxisomal disorders (n = 49) exhibited concentrations >10-fold higher (14.06 ± 2.59 μmol/L). Patients with heterozygous mutations in PEX genes had low concentrations of serum C bile acids. In all groups, the (25S)- and (25R)-diastereomers were present in a ratio of 0.3. In cases of 2-methylacyl-CoA racemase deficiency, serum total C bile acids were markedly elevated (10.61 ± 0.92 μmol/L) and comprised exclusively the (25R)-diastereoisomer.

Conclusions: This tandem mass spectrometric assay quantifies all diastereoisomers of the C cholestanoic acids in serum and was used to establish typical clinical concentration ranges. The method is applicable to the diagnosis of peroxisomal disorders and differentiates 2-methylacyl-CoA racemase deficiency from other peroxisomal biogenesis disorders.