Glaucoma is a leading cause of blindness worldwide, which affected approximately 95 million people in 2022. This number is predicted to rise to 112 million by 2040 (Tham et al., 2014). Primary angle-closure glaucoma (PACG), which is most prevalent in Asia (76.7 %) (Tham et al., 2014), is characterized by the peripheral iris obstructing or permanently adhering to the trabecular meshwork. This obstruction leads to the blockage of aqueous humor outflow and, subsequently, increases intraocular pressure (IOP). Patients with PACG often experience severe eye pain, vision loss, unilateral migraines, nausea, vomiting, and other symptoms. Without timely and appropriate treatment, PACG can result in a worse prognosis because of the higher prevalence of bilateral blindness. Despite numerous studies in recent years identifying mechanisms, such as extracellular matrix remodeling (Liu et al., 2020), oxidative stress (Hondur et al., 2017; Adav et al., 2019; Ye et al., 2022), apoptosis, autophagy (Liu et al., 2020; Ye et al., 2022), and inflammatory factor leakage-associated with PACG (Kong et al., 2010; Chua et al., 2012), the key factors remain largely unknown. The provocation test, a technique used to artificially increase IOP to diagnose suspected glaucoma in advance, provides a rapid clinical assessment for patients; however, its results are not definitive. Indeed, a negative result does not rule out angle-closure glaucoma in the future, and a positive result does not always indicate acute angle-closure. Therefore, the development of a safe, effective, convenient, and rapid method for identifying the factors associated with PACG is needed.

Currently, the identification of differential ingredients is a prominent focus in glaucoma research. Over the years, researchers have attempted to identify ingredients for glaucoma in various body fluids, such as blood, tears, aqueous humor, and the vitreous body (Ussher et al., 2016; Buas et al., 2017; Wang et al., 2021). However, each of these fluid samples has its respective limitations. For example, blood and tears do not directly interact with intraocular tissues, such as the trabecular meshwork and optic nerves, making them potentially unsuitable for studying differential ingredients in patients with glaucoma. In contrast, the vitreous body, which is in direct contact with retinal ganglion cells, holds significant potential as a sample for investigating differential ingredients associated with glaucoma. However, obtaining vitreous body samples is invasive and generally performed during vitrectomy, a procedure not commonly performed in patients with glaucoma. Therefore, the collection of vitreous samples from these patients is challenging. Moreover, an increase in IOP owing to anterior chamber angle closure is a principal risk factor for PACG. The balance between the secretion and drainage of the aqueous humor is crucial for maintaining a stable IOP. The evaluation of small molecules in the aqueous humor may provide insights into the pathogenesis of PACG. Therefore, aqueous humor specimens can be used to detect differential ingredients in glaucoma research.

In recent years, proteomics and metabolomics have been introduced to detect differential ingredients associated with glaucoma, which have significant potential to improve the diagnosis, prognosis, and treatment response of this condition. However, the protein content in human aqueous humor is relatively low, ranging from 120 to 500 ng/mL (Browne et al., 2011; McNally and O'Brien, 2014). Moreover, proteins are easily degraded during the collection process, which complicates their analysis. Conversely, metabolomics involves the study of metabolic fingerprints. Compared with other omics approaches, metabolomics offers several advantages, including the easy detection of changes at the metabolite level, direct evidence of the number of metabolites, and the ability to analyze comprehensive biological information. Additionally, proteins are less stable than metabolites and require more stringent sample processing conditions. Metabolomics can systematically reveal the pathophysiological status of the body by observing the changes in metabolites. It has recently become a broad and sensitive method for evaluating differences in metabolic states, making it possible to analyze ocular clinical samples, such as tears, aqueous humor, and vitreous body, in eye diseases. However, a few studies on the role of metabolomics in glaucoma have been conducted, with several focusing on primary open-angle and pseudo-exfoliation glaucoma (Burgess et al., 2015; Cabrerizo et al., 2017; Nzoughet et al., 2020; Myer et al., 2020a, Myer et al., 2020b). These studies primarily employed non-targeted metabolomics to identify differential metabolites using libraries of small-molecule metabolites. However, further absolute quantitative analysis of these differential metabolites is lacking. Therefore, although metabolomics presents a promising avenue for glaucoma research, especially for detecting molecular changes in ocular samples, more targeted and quantitative studies are needed to fully realize its potential in the field of glaucoma, particularly for conditions including PACG.

In this study, we aimed to identify differential metabolites in the aqueous humor of patients with PACG. First, we compared the metabolomic characteristics of the aqueous humor between patients with PACG and those with age-related cataracts (ARC), using non-targeted metabolomics analysis to identify differences in metabolites. Second, targeted metabolomics analysis was employed to validate the differential molecules identified in the initial analysis (Fig. 1). Furthermore, we explored the potential mechanisms underlying PACG by conducting functional annotations of differential metabolites detected in the aqueous humor.