Figures and data
Study design and workflow. A 5-phase study (discovery phase [discovery set 1, the discovery set 2], validation phase 1, validation phase 2, supplemental phase, and cohort phase) design. A: The workflow of the discovery phase, validation phase 1, supplemental phase, and cohort phase were analyzed by liquid chromatography-mass spectrometry (LC-MS) for untargeted metabolomics. B: The workflow of validation phase 2 was analyzed chemiluminescence immunoassay for targeted detection. C: A total of 440 patients and controls were recruited and assigned to discovery set 1 (n = 140), the discovery set 2 (n = 180), and validation set 1 (n = 120). The biomarker signature was identified on the metabolomic data from the discovery phase, comparing primary angle closure glaucoma (PACG) with control patients. These data were used as a discovery set for the algorithm. D: Validation phase 2 (n = 176) was included as the second validation cohort. E: Three measurements were performed in the supplemental phase. F: Cohort phase were performed to validate the predictive value of biomarker (n = 97).
Metabolic profiles discriminate participants with primary angle closure glaucoma (PACG) from normal controls (NC). A: Orthogonal projection to latent structure-discriminant analysis (OPLS-DA) score plot of the comparison between the PACG and NC groups in the discovery phase (discovery set 1 and discovery set 2). Samples in the encircled areas are within the 95% confidence interval. B: Volcano plot of differential metabolites. Metabolites with a fold change of <0.85 and a false discovery rate (FDR) of <0.1 were considered significantly down-regulated. Metabolites with a fold change of >1.15 and an FDR of <0.1 were considered significantly up-regulated. Changes in other metabolites were not significant (insignificant). C: Venn diagram displaying the 32 differential metabolites that were altered as biomarker candidates from the two comparisons in the discovery phase. D: Heatmap of differential metabolites in the discovery set 1 (Data were normalized to min-max). E: Heatmap of differential metabolites in the discovery set 2 (Data were normalized to min-max). FA: fatty acid.
The clinical and demographic characteristics of all subjects in the discovery and validation phases
Identification of a unique PACG-associated blood metabolite fingerprint and its behavior in the discovery phase. A: Heatmap of the area under the receiver-operating-characteristic curve assessing the discriminating accuracy of each of the 32 metabolites in differentiating PACG from normal control in the discovery set 1 and discovery set 2. B: The eigenmetabolite of the 32-metabolite cluster between primary angle closure glaucoma (PACG) and control patients in the discovery set 1. C: The eigenmetabolite of the 32-metabolite cluster between primary angle closure glaucoma (PACG) and control patients in the discovery set 2. Wilcox test was used.
Biomarker discovery discriminates primary angle closure glaucoma (PACG) from normal in the discovery phase. A: A heatmap of correlation analysis between ocular clinical characteristics and 32 potential biomarkers in the discovery phase in PACG subjects. B: The serum level of androstenedione between PACG and normal group in the discovery set 1 (Unit for y-axis is peak areas). C: The serum level of androstenedione between PACG and normal group in the discovery set 2 (Unit for y-axis is peak areas). D: Receiver operating characteristic curves of androstenedione to discriminate PACG from normal in the discovery set 1. E: Receiver operating characteristic curves of androstenedione to discriminate PACG from normal in the discovery set 2. Independent student’s t-test was used.
Biomarker validation in two independent validation phases to discriminate primary angle closure glaucoma (PACG) from normal. A: The serum level of androstenedione between PACG and normal group in validation phase 1 (Unit for y-axis is peak areas). B: The serum level of androstenedione between PACG and normal group in validation phase 2 (Unit for y-axis is peak areas). C: Receiver operating characteristic curves of androstenedione to discriminate PACG from normal in validation phase 1. D: Receiver operating characteristic curves of androstenedione to discriminate PACG from normal in validation phase 2. Independent student’s t-test was used.
Specificity of circulating androstenedione in patients with primary angle closure glaucoma (PACG) in supplemental phase. A: Sampling scheme and workflow to investigate the temporal changes in androstenedione levels. B: Differential level of serum androstenedione between patients with PACG before and three months after treatment (Unit for y-axis is peak areas). C: Sampling scheme and workflow to determine whether aqueous humor levels of androstenedione were high in patients with PACG. D: The aqueous humor level of androstenedione between PACG and cataract (Unit for y-axis is peak areas). E: Heatmap of correlation analysis between ocular clinical characteristics and aqueous humor level of androstenedione. F: Comparison means aqueous humor levels of androstenedione between mild, moderate, and severe PACG (Unit for y-axis is peak areas). G: 7 paired serum-aqueous humor samples from the same PACG patients were included (Unit for y and x-axis is peak areas). A significant correlation between serum and aqueous humor levels of androstenedione was observed. Kruskal-Wallis test and one-way ANOVA was used. *P<0.05; **: P<0.001.
Kaplan-Meier curves stratified by the men value in terms of androstenedione. A: male+female; B: Female; C: Male. We categorized study participants into 2 groups based on their mean levels of androstenedione.
