Development of a simultaneous analytical method for five conjugated cholesterol 2 metabolites in urine and investigation of their performance as diagnostic markers for 3 Niemann–Pick disease type C

47 Niemann–Pick disease type C (NPC) is an autosomal recessive disorder characterized 48 by progressive nervous degeneration. Because of the diversity of clinical symptoms and 49 onset age, the diagnosis of this disease is difficult. Therefore, biomarker tests have 50 attracted significant attention for earlier diagnostics. In this study, we developed a 51 simultaneous analysis method for five urinary conjugated cholesterol metabolites, 52 which are potential diagnostic biomarkers for a rapid, convenient, and noninvasive 53 chemical diagnosis, using liquid chromatography/tandem mass spectrometry 54 (LC/MS/MS). By the method, their urinary concentrations were quantified and the NPC 55 diagnostic performances were evaluated. The developed LC/MS/MS method showed 56 high accuracy and and satisfied all analytical method validation criteria. Analyzing the 57 urine of healthy controls and patients with NPC, three of five urinary conjugated 58 cholesterol metabolites concentrations corrected by urinary creatinine were significantly 59 higher in the patients with NPC. As a result of receiver operating characteristics 60 analysis, the urinary metabolites might have excellent diagnostic marker performance. particularly

poor, it is important to diagnose NPC early and apply the treatment to maintain the 79 quality of life of the patient (5). However, few trained specialists are available and the 80 process leading to the discovery and diagnosis of NPC is complex. As conventional 7 20 urine samples and preliminarily investigated their diagnostic performance, assuming 98 that they may be useful for NPC screening (11). However, several patients with NPC 99 had extremely low concentrations of the relevant metabolites and false-negatives. Thus, 100 a comprehensive analysis method was used to search for other biomarker candidates 101 (12), which yielded two strongly detected metabolite peaks in urine of patients with 102 NPC, 3β-sulfooxy-7β-hydroxy-5-cholenoic acid (S7B-Δ 5 -CA) and 3β-sulfooxy-7-oxo-   A total of 50 μL of water was used as a surrogate matrix and 50 μL of IS curves were prepared using the least squares method with 1/x 2 weighting.   diluted 20-fold with water (Dilute 2) and Dilute 1 and 2 were analyzed as described 199 above. Dilution factor (%) was calculated as follows. it is necessary to use a surrogate matrix. Therefore, we investigated the matrix effects 226 for quantification of analytes. Procedure of sample preparation for calibration curves,

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QC samples and urine samples were summarized in Supplementary Table 2,   228 respectively. We prepared calibration curves using water as a surrogate matrix, and the 229 all calibration curves showed high linearity over wide range from 0.3 to 1000 ng/mL 230 (Supplemental Table 3A). Next, the matrix effects were investigated. The matrix effect 231 is usually calculated by the ratio of peak intensity of the standard solution spiked in a 232 pretreated matrix to that of the neat standard solution (17). However, the analytical 233 system used herein features an online solid phase extraction, so we could not evaluate 234 the typical method (17). Therefore, it was evaluated using MF which is the parameter 235 combining the pretreatment extraction efficiency and matrix effects from biological 236 contaminants (7). As a result, the MFs of all analytes and IS was 101-105% 237 (Supplemental Table 3B). The IS normalized MFs of all analytes were nearly 100% and 238 it was found that the analytes could be quantified without considering the matrix effect.

Reproducibility test
The method reproducibility was investigated using QC samples. Accuracy was 242 evaluated by subtracting the concentration in the healthy control urine as Blank. The 243 accuracy of the inter-and intra-day assays were within 100% ± 10% for all QC samples 244 and their precision (%) were within 10% (Table 1). urine, water was used as a surrogate matrix. The influence on the quantitative value was investigated and it was found that 20-fold dilution of the urine sample by water did not 258 affect the quantitative results (Table 1B).  Table 4.

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The age of each groups did not differ between healthy controls (0.33-47 years) and  Table 6). All metabolites were significantly higher in patients with NPC

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Because the concentrations of urinary cholesterol metabolites were generally higher 301 than plasma oxysterols (Fig. 3, Supplemental Table 7 and (6) Finally, the NPC diagnostic performance of each urinary cholesterol 307 metabolites was evaluated using ROC analysis (Fig. 4). This study investigated the 308 biomarkers for a rare lysosomal disease NPC, and we experienced difficulty collecting    100% and the specificities were 81.1% to 100%. The cut-off concentrations ranged from