Figures and data
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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).
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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.
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The clinical and demographic characteristics of all subjects in the discovery and validation phases
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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.
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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.
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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.
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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.
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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.
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