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.

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.

Early diagnosis of Niemann-Pick diseases (NPDs) is important for better prognosis of such diseases. N-Palmitoyl-O-phosphocholine-serine (PPCS) is a new NPD biomarker possessing high sensitivity, and with its combination with sphingosylphosphocholine (SPC) it may be possible to distinguish NPD-C from NPD-A/B. In this study, a rapid liquid chromatography-tandem mass spectrometry (LC-MS/MS) method (method 1) and a validated LC-MS/MS analysis (method 2) of PPCS and SPC were developed, and we have proposed a diagnostic screening strategy for NPDs using a combination of serum PPCS and SPC concentrations. Nexera and API 5000 were used as LC-MS/MS systems. C18 columns with lengths of 10 and 50 mm were used for method 1 and 2, respectively. 2 H 3 -Labeled PPCS and nor-SPC were used as internal standards. Selective reaction monitoring in positive-ion mode was used for MS/MS. Run times of 1.2 and 8 min were set for methods 1 and 2, respectively. In both methods 1 and 2, two analytes showed high linearity in the range of 1-4000 ng/mL. Method 2 provided high accuracy and precision in method validation. Serum concentrations of both analytes were significantly higher in NPD-C patients than those of healthy subjects in both methods. Serum PPCS correlated between methods 1 and 2; however, it was different in the case of SPC. The serum PPCS/SPC ratio was different in healthy subjects, NPD-C, and NPD-A/B. These results suggest that using a combination of the two LC-MS/MS analytical methods for PPCS and SPC is useful for diagnostic screening of NPDs.

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