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Life sciences · Journal article
Diagnostics · September 12, 2026
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Background/Objectives: Cholesterol sulfate (CS) is a multifunctional signaling molecule implicated in diverse physiological and pathological processes. Its clinical translation as a biomarker is hindered by the lack of established reference intervals, unclear disease-specific alterations, and undefined relationships with routine laboratory parameters. This study aimed to establish a serum CS reference range for Chinese populations, evaluate its associations with various diseases and clinical laboratory parameters, and define its clinical diagnostic utility. Methods: A total of 642 subjects were enrolled, comprising 372 healthy controls and 270 patients with one of six diseases: Alzheimer’s disease, osteoporosis, obesity, type 2 diabetes mellitus (T2DM), non-alcoholic fatty liver disease (NAFLD), or Crohn’s disease. Serum CS concentrations were quantified using LC-MS/MS. Reference intervals were established using non-parametric methods. Univariate analyses included Spearman’s rank correlation, Mann–Whitney U test, and receiver operating characteristic (ROC) curve analysis; multivariate analysis employed a gamma-family generalized linear model (GLM) with a log-link function, incorporating age, sex, clinical laboratory parameters, and disease status as covariates. The age-by-sex interaction was examined to assess synergistic effects. Incremental diagnostic value of CS was evaluated using the DeLong test and likelihood ratio test. Results: The serum CS reference interval for healthy individuals was 0.55–2.24 mg/L (males: 0.60–2.25 mg/L; females: 0.54–2.20 mg/L), with a significant sex difference. The age-by-sex interaction effect was significant (two-way ANOVA interaction p = 0.005; GLM interaction β = 0.102, p = 0.002). Stratified analysis revealed a significant positive correlation between age and CS in males, but no such association in females. In univariate analysis, CS correlated significantly with total cholesterol (TC), non-HDL-C, hemoglobin A1c (HbA1c), and other biomarkers; CS levels were elevated in Alzheimer’s disease and reduced in Crohn’s disease. In the multivariate GLM, only TC and male sex emerged as independent significant determinants of CS, whereas HbA1c and NAFLD showed borderline effects; no disease group retained independent significance. After adjustment for TC and sex, the residual area under the curves (AUCs) of CS for all diseases were close to 0.50, and DeLong’s test indicated no incremental diagnostic value beyond the baseline model (all p > 0.70). Conclusions: This study is the first to establish a serum CS reference range for the Chinese population. Serum CS levels are independently regulated by TC and sex, with a significant age-by-sex interaction. The associations between CS and disease status were largely mediated by confounding from lipid profiles and age. CS is not suitable as an independent disease biomarker but may serve as a supplementary indicator for assessing lipid metabolism—particularly in male populations, where it may indirectly reflect age-related changes in cholesterol metabolism.