acetylglucosamine; HQC, high quality control; LC/MS/MS, liquid chromatography/tandem mass spectrometry; LQC, low quality control; MQC, middle quality control; NPC, Niemann-Pick disease type C; NPC1, NPC intracellular cholesterol transporter 1; NPC2, NPC intracellular cholesterol transporter 2; Niemann-Pick disease type C; S7B-Δ 5-CA, 3β-sulfooxy-7β-hydroxy-5-cholen-24-oic acid; S7O-Δ 5-CA, 3β-sulfooxy-7-oxo-5-cholen-24-oic acid; SNAG-Δ 5-CA, nonamidated 3βsulfooxy-7β-N-acetylglucosaminyl-5-cholen-24-oic acid; SNAG-Δ 5-CG, glycineamidated 3β-sulfooxy-7β-N-acetylglucosaminyl-5-cholen-24-oic acid; SNAG-Δ 5-CT, taurine-amidated 3β-sulfooxy-7β-N-acetylglucosaminyl-5-cholen-24-oic acid; SRM, selected reaction monitoring; ROC, receiver operating characteristic.

Background: Niemann-Pick disease type C (NPC) is an autosomal recessive inherited disorder with progressive neuronal degeneration. Because conventional diagnostic methods are complicated and invasive, biomarker tests have drawn attention. We aimed to evaluate three urinary conjugated cholesterol metabolites as diagnostic biomarkers for NPC. Methods: Urine samples from 23 patients with NPC, 28 healthy controls, and 7 patients with inherited metabolic disorders were analyzed. 3β-Sulfooxy-7β-N-acetylglucosaminyl-5-cholen-24-oic acid and its glycine and taurine conjugates in urine were quantified by liquid chromatography-tandem mass spectrometry. The diagnostic performance of the three metabolites and their total concentration was evaluated. Result: Creatinine-corrected concentrations of three metabolites and their total concentration were all significantly higher in NPC patients (0.0098 < P < 0.0448). The area under the receiver operating curve for all metabolites exceeded 0.95, the clinical specificity was 92-100%, and the clinical sensitivity was ~95%. In the urine of patients with other inherited metabolic diseases, the concentrations of the metabolites were lower than those in the urine of patients with NPC. Conclusion: These conjugated cholesterol metabolites in urine can serve as useful diagnostic markers for noninvasive screening of NPC.

Niemann–Pick disease type C (NPC) is an autosomal recessive disorder caused by the mutation of cholesterol-transporting proteins. In addition, early treatment is important for good prognosis of this disease because of the progressive neurodegeneration. However, the diagnosis of this disease is difficult due to a variety of clinical spectrum. Lysosphingomyelin-509, which is one of the most useful biomarkers for NPC, was applied for the rapid and easy detection of NPC. The fact that its chemical structure was unknown until recently implicates the unrevealed pathophysiology and molecular mechanisms of NPC. In this study, we aimed to elucidate the structure of lysosphingomyelin-509 by various mass spectrometric techniques. As our identification strategy, we adopted analytical and organic chemistry approaches to the serum of patients with NPC. Chemical derivatization and hydrogen abstraction dissociation–tandem mass spectrometry were used for the determination of function groups and partial structure, respectively. As a result, we revealed the exact structure of lysosphingomyelin-509 as N-acylated and O-phosphocholine adducted serine. Additionally, we found that a group of metabolites with N-acyl groups were increased considerably in the serum/plasma of patients with NPC as compared to that of other groups using targeted lipidomics analysis. Our techniques were useful for the identification of lysosphingomyelin-509.