Differentiation of soluble aqueous humor metabolites in primary open angle glaucoma and controls
Graphical abstract
Introduction
Glaucoma refers to a group of irreversible blinding optic neuropathies that affect approximately 70 million people worldwide (Quigley and Broman, 2006). Primary open angle glaucoma (POAG) is the most common form of the disease, which is frequently associated with elevated intraocular pressure (IOP). Elevated IOP in glaucomatous eyes is consistent with findings of impeded aqueous humor (AH) outflow due to pathologic changes in the anterior chamber angle. The trabecular meshwork (TM), a filter-like structure in the anterior chamber angle, is thought to be a site of resistance to aqueous humor outflow (Morrison and Acott, 2003). However, resistance to AH outflow may arise distal to the TM, further downstream (Carreon et al., 2017).
Our understanding of the dynamic nature of AH, and its generation and egress from the anterior eye chamber has advanced through several decades (Mark, 2010). AH is actively secreted by the ciliary body (CB) and/or its non-pigmented epithelium (Civan and Macknight, 2004). AH outflow occurs through the conventional pathway in the anterior chamber angle structures that involve TM or through the uveoscleral pathway that involves the CB. The TM has been subjected to extensive investigation to understand how its properties undergo pathologic changes in glaucoma. This includes multi-omics analysis: genomics, proteomics (Bhattacharya et al., 2005), lipidomics, (Aribindi et al., 2013a; Aribindi et al., 2013b) and metabolomics. Comprehensive analysis of the AH proteome, lipidome and metabolome are expected to provide complementary insight into pathologic changes associated with outflow resistance. Proteomics, lipidomics, (Edwards et al., 2014a; Edwards et al., 2014b) and metabolomics (Buisset et al., 2019; Mayordomo-Febrer et al., 2015) studies of AH have been conducted. Due to the large number of isomeric lipids and metabolites, multiple studies of AH lipidomics and metabolomics are desirable. A comparison of CB secretions (Margolis et al., 2018) with AH has suggested that the metabolite composition of AH may likely change as AH passes through the anterior segment as ocular tissues absorb and release metabolites into the AH. This is consistent with emerging localized studies of lipids and metabolites using imaging mass spectrometry (Guerra et al., 2015). As noted above, the possible involvement of distal outflow regions and all tissues of the anterior eye segment towards AH level balance and IOP homeostasis is increasingly being realized.
Non-destructive nuclear magnetic resonance (NMR) analysis of AH followed by destructive analysis using mass spectrometry to yield a greater confidence quantification of metabolites and possible wider identification, remains yet to be done. The metabolome is thought to be the most transient repertoire of all molecular omics, therefore several independent approaches to compare identifications is desirable. This was the rationale behind our use of NMR and mass spectrometry. The isotopic ratio outlier analysis combined with mass spectrometry is a recent method that enables a higher confidence in metabolite identification compared to other mass spectrometric methods, which was the rationale behind using this method. The experimental metabolome can be compared with a previously identified proteome hub for AH and TM to provide insights into our understanding, which is also lacking in the current literature, motivating us to undertake the studies presented here.
Section snippets
Human subjects/donors/chemicals
All materials were collected from human donors without identifiers under institutional review board exemption/approval. We acquired 58 aqueous humor (AH) samples from participants at the Veterans Administration (VA) Medical Center (Miami, FL). Patient samples used for 1H-Nuclear Magnetic Resonance (NMR) and Isotopic Ratio Outlier Analysis (IROA) consisted of individuals with POAG (n = 23) and non-POAG controls (n = 35). Patient information is presented in Table 1. All the chemicals utilized in
Metabolomic profiling of POAG aqueous humor
We performed high performance liquid chromatography (HPLC) tandem mass spectrometry (LC-MS/MS) and nuclear magnetic resonance (NMR) spectroscopy analysis on aqueous humor from 23 POAG and 35 non-POAG control patients. Table 1 shows the characteristics of the patients.
A total of 77 metabolites including unknowns were identified in NMR based on the characteristics of the chemical shifts. These metabolites were common to both control and POAG AH (Fig. 1). There were 206 metabolites identified in
Discussion
We present the analysis of aqueous humor (AH) metabolites from non-glaucoma patients undergoing cataract surgery with otherwise normal healthy eyes (control) and from POAG patient eyes here. POAG patients also underwent cataract surgery, thus AH samples are not different with respect to cataract surgeries. Aqueous humor composition is dependent on metabolites produced during its generation plus the metabolic interchanges with other intraocular tissues during its passage (De Berardinis et al.,
Conclusion
Current analyses of metabolites identified the presence of L-arginine, L-lysine, cysteine, threonine, glycine, anthranilate, ascorbate, 4-hydroxybenzoate, myo-inositol, acetate, propylene glycol, 2-hydroxy-butyrate, creatine, choline, L-phenylalanine, glutamine, 4-aminobutanoate and isopropanol total from human control and POAG AH respectively. Our results show a significant increase and decrease in metabolites that have been previously implicated in specific biological roles in AH production
Funding
The funding support for this study was partly provided by a DOD grant W81XWH-15-1-0079, NIH grant EY14801, an unrestricted grant from Research to Prevent Blindness and Glaucoma Foundation of New York. NMR study was partly performed at Southeast Center for Integrated Metabolomics, supported by NIH award U24DK097209 and partly at the National High Magnetic Field Laboratory, supported by National Science Foundation Cooperative Agreement No. DMR-1644779 and the State of Florida. Metabolomics
Acknowledgements
We thank Drs. Santanu Banerjee and Matthew E. Merritt for their assistance with IROA and NMR analysis respectively. We thank Dr. Manik Goel for his critical comments on the manuscript.
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