Differential metabolic markers associated with primary open-angle glaucoma and cataract in human aqueous humor

Primary open-angle glaucoma (POAG) is the most common subtype of glaucoma and the major cause of irreversible blindness throughout the world [1]. Although numerous studies have identified several important ocular risk factors for POAG such as increased intraocular pressure (IOP) [2, 3], myopic refractive errors [4], larger optic disc size [5, 6] and thinner central corneal thickness [7, 8], these findings are limited in understanding the pathophysiology of POAG. Further knowledge regarding the pathophysiology might help to create new drug development research lines and expand current therapeutic targets for POAG. In current clinical practice, the treatment strategy of POAG mainly relies on IOP-lowering medications or surgeries. Although increased IOP is widely accepted to be the primary predictor for POAG, glaucomatous neuropathy is still observed in some patients with normal or even lower-than-normal IOPs, suggesting that other mechanisms exist in the pathophysiology of POAG.

Metabolomics is a widely used technology to assess biomarkers for diseases and provide molecular information regarding disease phenotype since metabolites are the ultimate product of gene, mRNA and protein activities [9]. Variations in the metabolome represent the interplay of genetic and environmental factors and are in relation to disease states, which may shed some lights in mechanism and pathophysiology of the disease [10]. With regard to eye diseases, metabolomics has been successfully used in identifying the metabolic signatures of diabetic retinopathy [11]. However, there were less studies focusing on POAG, especially in human participants. A previous analysis comparing plasma metabolic signatures as measured by mass spectrometry observed significant differences in some specific metabolic processes such as palmitoylcarnitine, sphingolipids, vitamin D-related compounds, and steroid precursors between POAG patients and healthy controls [12]. These differences observed in metabolome might be linked to mitochondrial dysfunction and energy metabolism changes [12]. However, we believe that aqueous humor samples are more sensitive to detect biomarkers in glaucomatous eyes, which may provide novel insights into more new pathogenic pathways for this ocular condition. To the best of our knowledge, few studies have focused on the aqueous humor metabolite markers of POAG in human beings. To address this gap, we performed a clinical-based case-control study and aimed to identify novel metabolite markers of POAG in human aqueous humor samples.

In clinical practice, POAG remains a poorly understood condition with limited therapeutic options. Therefore, there is a pressing need to develop personalized approach to guide clinical management. In this study, we systematically explored the metabolic differences in aqueous humor samples between patients with POAG and controls of patients with cataract. We identified fourteen metabolites as possible biomarkers in aqueous humor samples that had the potentials to distinguish POAG patients from controls. The identification of novel metabolite markers for POAG in human aqueous humor provides insights into potential new pathogenic pathways for this vision-threatening condition and could potentially lead to new drug development research lines.

Previous animal and human studies have provided some preliminary results on metabolites associated with glaucoma. For example, an experimental study found that metabolites in relation to osmotic stress, oxidative stress and glucose metabolism were related to the retinal ganglion cell death in rats, which is regarded as the common mechanism for glaucoma [19]. Another mouse model of chronic glaucoma identified unique profiles of sphingolipid and ceramide species between normotensive and hypertensive aqueous humor and found that sphingosine- and sphinganine-1-phosphates greatly increased in hypertensive mice [20]. With regard to the studies on humans, an untargeted plasma metabolomic study observed increased levels of palmitoylcarnitine; putatively-identified sphingosine, sphinganine, and decreased levels of sphingosine-1-phosphate in patients with POAG [12]. To the best of our knowledge, few studies have explored the POAG-associated metabolites in human aqueous humor samples. The analysis of metabolites in human aqueous humor may reveal novel potential biomarkers of POAG with greater sensitivity and specificity which might be detected in blood samples. We identified 14 metabolite markers in human aqueous humor which might potentially distinguish POAG from cataract. We also searched public databases such as KEGG [17] and MetaboAnalyst [18] and other published literatures, trying to find possible metabolic pathways for POAG observed in this study.

We found that Biotin metabolism was highly impacted in the pathway analysis, implying that Biotin may play major roles in POAG pathophysiology. Biotin was reported be oxidized into the retina [21]. It was also indicated that in vivo administration of biotin to early embryonic chick eyes at moderately elevated levels induced malformations, mainly affecting lens structures [22]. As the controls in this study were patients undergoing cataract surgeries whose lens structures might have been malformed compared with cases free of cataract, it is reasonable that the levels of Biotin is lowers in POAG patients observed in this study.

The phenomenon that Methylmalonic acid was reduced in POAG patients compared with controls probably suggested increased oxidative damage may play a major pathophysiological role in POAG [23].. Methylmalonic acid was reported to be able to provoke oxidative damage and compromise antioxidant defenses in nerve terminal and striatum [24]. According to the results of a recent meta-analysis including 22 case-control studies with 1614 with glaucoma and 1319 healthy controls, some biomarkers increased in glaucomatous aqueous humor such as superoxide dismutase and glutathione peroxidase. In addition, high levels of Methylmalonic acid were also found to increase the risk of optic neuropathy [25‐27].

The level of Ribitol in aqueous humor was found to be higher in POAG compared with cataract in our study. Increased level of Ribitol was reported to play a role in prevention of cataract formation and act as a cofactor for glutathione reductase, which is linked to cataract formation by decreased glutathione levels in the lenses. On the other hand, Ribitol deficiency is implicated in the formation of cataracts due to the concentration of reduced glutathione in the lens and its ability to protect the tissue from oxidative damage [28]. Thus, our finding is consistent with previous studies.

The level of Sorbitol was expected to be lower in POAG patients as compared with those with cataract. It is well established that diabetes is a major risk factor cataract [29]. Sorbitol is a key marker in osmotic stress. Lens fiber cell resulting from excessive accumulation of sorbitol has been proposed as a possible mechanism in cataract formation in diabetic patients. A previous study reported a strong relationship between the abundance of polyol pathway metabolites sorbitol and blood glucose levels in lenses extracted from diabetic patients [30].

Mannose was found to be increased in POAG patients compared with cataracts. Mannose is a simple sugar but have a complex health effect on different parts of the body including the eyes. A previous study have indicated that an upregulation of the lectin pathway-associated mannose-serine-protease-2 was observed in the optic nerves of the optic nerve homogenate antigen group [31], which supports the finding in the current study.

We have to acknowledge some limitations of this study. One major limitation was the small sample size, which may have prevented changes in certain metabolites from being apparent. In addition, some sources of bias such as differences in the time of day of sample collection may have distorted the findings. Particularly, intake of drugs such as IOP-lowering medications in patients with POAG may have potentially altered the metabolome distributions. However, it is unlikely to control the effect of IOP-lowering medications between cases and controls in this study. Furthermore, the control group was patients undergoing cataract surgery rather than “healthy” individuals, and the selection of control group might have distorted the findings as cataract should have its own distinct metabolic profiling which should be different from POAG. It is ideal if we could obtain aqueous humor samples in healthy people without eye diseases. Finally, metabolic pathways of some metabolites identified in this study could not be founded in public databases or published literatures. Whether they are true biomarkers for POAG warrants further clarifications.

This study investigated the metabolic markers associated with POAG in human aqueous humor samples. As a result, 14 metabolites were identified as potential biomarkers that could discriminate between patients with POAG and controls. Biotin metabolism was highly impacted in pathway analysis and may play important roles in the regulation of this pathway. Overall, this study provided new clues in the disease pathogenesis of POAG. Validation of the results is warranted in other studies.