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Volcano plot relating significance levels and fold change in data from glaucoma and control samples. Compounds for which differences have a p < 0.0001 score are shown in red.
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The composition of the aqueous humor of patients with glaucoma is relevant to understand the underlying causes of the pathology. Information on the concentration of metabolites and small molecules in the aqueous humor of healthy subjects is limited. Among the causes of the limitations is the lack of healthy controls since, until recently, they were...
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... Arginine levels have been reported higher in the plasma or aqueous humor of individuals with primary openangle glaucoma (POAG) than in controls [17][18] . Similarly, the concentrations of spermidine and various acyl-carnitines and lysophosphatidylcholines [19] , as well as L-carnitine and L-acetylcarnitine [20] , have been reported higher in the aqueous humor of patients with than without open angle glaucoma. The present study also found that the levels of L-arginine, L-carnitine and acetyl-L-carnitine were higher in the retinas of mice with NMDA-induced RGC injury glaucoma mice, and that the levels of these metabolites were increased by eicosane treatment. ...
AIM: To investigate the protective effects, antioxidant potential, and anti-inflammatory mechanisms of eicosane on glutamate-induced cell damage and on N-methyl-D-aspartate (NMDA)-induced retinal ganglion cell (RGC) injury in a mouse model of glaucoma. METHODS: The protective effects of eicosane on the rat R28 retinal precursor cell line were assessed using cell counting kit-8 assays and Hoechst-propidium iodide staining. Intracellular reactive oxygen species (ROS) production was measured using the fluorescent probe 2'-7'-dichlorofluorescin diacetate and flow cytometry. The protective role of eicosane on NMDA-induced RGC injury in a mouse glaucoma model was determined by immunostaining of frozen sections of retina. The effects of eicosane on the metabolome of the retina in mice with NMDA-induced RGC damage were evaluated by liquid chromatography-mass spectroscopy (LC-MS) and untargeted metabolomics analyses. RESULTS: Eicosane treatment significantly attenuated glutamate-induced damage to R28 cells in vitro. Eicosane also protected RGCs against NMDA-induced injury in a mouse glaucoma model. Untargeted metabolomics analyses showed that eicosane increased multiple metabolites, including L-arginine and L-carnitine, in the retina. CONCLUSION: Eicosane has protective effects, antioxidant potential, and anti-inflammatory properties in an in vitro model of glutamate-induced cell damage and in an in vivo model of NMDA-induced RGC injury in mouse glaucoma through modulation of L-arginine and/or L-carnitine metabolism.
... Data (concentration of every metabolite in each sample) used in the present study are available in previously published papers [5,14]. We used R 4.3.0 ...
... The altered concentration of some metabolites found in the AH of patients in comparison to healthy individuals is providing valuable insights into the pathophysiological mechanisms of multiple diseases, including, but not limited to, open-angle glaucoma and type 2 diabetes [5,14], but a diagnostic tool is missing. Our laboratory has recently provided metabolomics-based evidence of altered arginine metabolism as a principal factor in Parkinson's disease [20]. ...
... In our previous publications concerning the composition of AH, we were interested in looking for differentially concentrated metabolites and why they could give insight into disease mechanism. We discovered that glutamine, kynurenine, acyl-carnitine and lysophosphatidylcholine levels are altered in the AH of glaucoma patients [14], that biogenic amines are differentially concentrated in the AH of type 2 diabetes patients [5] and that the metabolism of arginine is altered as deduced from the composition of the AH of Parkinson's disease patients [20]. In the present study, we use the data from previous studies to develop a novel methodology for disease diagnosis and management considering the perspective of personalized medicine. ...
Background: This paper aimed at devising an intelligence-based method to select compounds that can distinguish between open-angle glaucoma patients, type 2 diabetes patients, and healthy controls. Methods: Taking the concentration of 188 compounds measured in the aqueous humour (AH) of patients and controls, linear discriminant analysis (LDA) was used to identify the right combination of compounds that could lead to accurate diagnosis. All possibilities, using the leave-one-out approach, were considered through ad hoc programming and in silico massive data production and statistical analysis. Results: Our proof of concept led to the selection of four molecules: acetyl-ornithine (Ac-Orn), C3 acyl-carnitine (C3), diacyl C42:6 phosphatidylcholine (PC aa C42:6), and C3-DC (C4-OH) acyl-carnitine (C3-DC (C4-OH)) that, taken in combination, would lead to a 95% discriminative success. 100% success was obtained with a non-linear combination of the concentration of three of these four compounds. By discarding younger controls to adjust by age, results were similar although one control was misclassified as a diabetes patient. Conclusions: Methods based on the consideration of individual clinical chemical parameters have limitations in the ability to make a reliable diagnosis, stratify patients, and assess disease progression. Leveraging human AH metabolomic data, we developed a procedure that selects a minimal number of metabolites (3–5) and designs algorithms that maximize the overall accuracy evaluating both positive predictive (PPV) and negative predictive (NPV) values. Our approach of simultaneously considering the levels of a few metabolites can be extended to any other body fluid and has potential to advance precision medicine. Artificial intelligence is expected to use algorithms that use the concentration of three to five molecules to correctly diagnose diseases, also allowing stratification of patients and evaluation of disease progression. In addition, this significant advance shifts focus from a single-molecule biomarker approach to that of an appropriate combination of metabolites.
... Most of the research on the human AH has till now focused on the non-targeted analysis performed via nuclear magnetic resonance spectroscopy [5,9,14] and mass spectrometry, coupled with liquid chromatography [4][5][6]10], gas chromatography [7,8], or capillary electrophoresis [4]. So far targeted analysis was focused mainly on liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) with a focus on lipid metabolism-related molecules [15][16][17], diabetes [18], or glaucoma [19,20]. ...
... This assay has been optimized for various matrices, such as human milk [22] or murine tissues [23]. Although it was also used for AH analysis, the samples were prepared according to a standard protocol for plasma (10 µl) [19] or using 30 µl of AH sample [18,20], without prior validation. Therefore, the purpose of this study was, for the first time, to compare various volumes of AH for the analysis of metabolites using the Biocrates AbsoluteIDQ p180 kit, as well as, by this process, to indicate the optimal one. ...
... This was achieved using the standard protocol (10 µl), and 54 metabolites were detected [19]. Meanwhile, Lillo et al. compared AH samples from glaucoma or diabetes patients to control patients (myopic patients without additional ocular pathology undergoing refractive surgery) using 30 µl of AH, and thus detected 80 metabolites [18,20]. In our recent publication, in which we applied the methodology presented in this short communication, we compared the AH samples from patients undergoing simultaneous bilateral cataract surgery. ...
The aim of this study was to use the commercial kit AbsoluteIDQ p180 (Biocrates) for the quantification of metabolites in aqueous humor (AH), as well as to determine the optimal volume of AH that is necessary to obtain reliable and reproducible results. Different volumes of AH (10 µl, 20 µl, and 30 µl) were tested. Of the 188 metabolites measurable with the Biocrates kit, 69 were detected in AH. Depending on the volume used, 41, 51, and 63 metabolites were measured using 10 µl, 20 µl, and 30 µl of AH, respectively. The repeatability of the measurements improved with increasing AH volume. Considering only those metabolites that were obtained with a CV < 15%, 34 metabolites at 10 µl, 41 at 20 µl, and 44 at 30 µl AH were received. On this basis, it can be concluded that the tested method can be successfully applied to analyze metabolites in the human AH. To achieve the most comprehensive detection range and highest repeatability of measurements, it is recommended to use 30 µl AH.
... Other studies have equally reported reduced spermidine levels in glaucomatous eyes [66]. Lillo et al. reported, however, elevated levels of spermidine in the aqueous humor of open-angle glaucoma patients [68]. Wang et al. also reported reduced levels of spermidine to be implicated in glaucomatous damage [69]. ...
Glaucoma is a chronic optic neuropathy that can lead to irreversible functional and morphological damage if left untreated. The gold standard therapeutic approaches in managing patients with glaucoma and limiting progression include local drops, laser, and/or surgery, which are all geared at reducing intraocular pressure (IOP). Nutrients, antioxidants, vitamins, organic compounds, and micronutrients have been gaining increasing interest in the past decade as integrative IOP-independent strategies to delay or halt glaucomatous retinal ganglion cell degeneration. In our minireview, we examine the various nutrients and compounds proposed in the current literature for the management of ophthalmology diseases, especially for glaucoma. With respect to each substance considered, this minireview reports the molecular and biological characteristics, neuroprotective activities, antioxidant properties, beneficial mechanisms, and clinical studies published in the past decade in the field of general medicine. This study highlights the potential benefits of these substances in glaucoma and other ophthalmologic pathologies. Nutritional supplementation can thus be useful as integrative IOP-independent strategies in the management of glaucoma and in other ophthalmologic pathologies. Large multicenter clinical trials based on functional and morphologic data collected over long follow-up periods in patients with IOP-independent treatments can pave the way for alternative and/or coadjutant therapeutic options in the management of glaucoma and other ocular pathologies.
... For example, taurine has been found to exhibit a protective effect against mitochondria-related metabolic impairments in the retinal pigment epithelium [9], whereas nicotinamide has shown to have neuroprotection on glaucoma [10]. Carnitines are a class of metabolites that have been recently shown to be related to ocular diseases [11][12][13][14][15][16][17][18][19][20][21][22][23][24][25][26][27]. Nevertheless, most clinical studies involving metabolomics studies are still preliminary and show important limitations. ...
... Additionally, Lillo et al. investigated the metabolomic composition of the aqueous humor of patients with POAG, comparing them with healthy controls [23]. This study confirmed the study of Buisset et al. regarding the changes in the levels of C0, C2, C3, and C4 that increased in glaucoma patients. ...
... These alterations in the levels of acylcarnitines seem to be of mitochondrial origin, and the role of C10 carnitine, which was the one that decreased, should be evaluated further regarding its role in the functionality of the mitochondria. Indeed, in the past, the exogenous administration of this metabolite interestingly led to impairment in mitochondrial handling, as well as fatty acid oxidation and the inhibition of ketogenesis [23]. ...
Several common ocular diseases are leading causes of irreversible visual impairment. Over the last decade, various mainly untargeted metabolic studies have been performed to show that metabolic dysfunction plays an important role in the pathogenesis of ocular diseases. A number of metabolites in plasma/serum, aqueous or vitreous humor, or in tears have been found to differ between patients and controls; among them are L-carnitine and acylcarnitines, which are essential for mitochondrial fatty acid oxidation. The metabolic profile of carnitines regarding a variety of diseases has attracted researchers’ interest. In this review, we present and discuss recent advances that have been made in the identification of carnitines as potential metabolic biomarkers in common ocular diseases, such as age-related macular degeneration, diabetic retinopathy, retinopathy of prematurity, central retinal vein occlusion, primary open-angle glaucoma, rhegmatogenous retinal detachment, and dry eye syndrome.
... The median value of all molecules whose concentration can be reliably determined is similar. Adding more controls does not significantly change the findings that were reported in a previous study related to altered AH composition in glaucoma [18]. As in glaucoma and with respect to metabolites whose concentration is significantly altered, the AH of diabetic patients shows much more increase than decrease in the concentration of metabolites. ...
... Increased alanine concentration in AH also occurs in glaucoma, but to a lesser extent [18]. The increase when the general trend of amino acids is to decrease in diabetes may reflect impaired glucose disposition and the underlying metabolic syndrome. ...
... In glaucoma, glutamate buffering occurs through the formation of glutamine, the level of which is not significantly altered in diabetes. Indeed, the pattern of variation in glaucoma, where Gln, various acyl-carnitines, and lysophosphatidylcholines are increased [18], is different from that found in diabetes. ...
The composition of the aqueous humor of patients with type 2 diabetes is relevant to understanding the underlying causes of eye-related comorbidities. Information on the composition of aqueous humor in healthy subjects is limited due to the lack of adequate controls. To carry out a metabolomics study, 31 samples of aqueous humor from healthy subjects without ocular pathology, submitted to refractive surgery and seven samples from patients with type 2 diabetes without signs of ocular pathology related to diabetes were used. The level of 25 compounds was significantly (p < 0.001) altered in the aqueous humor of the patient group. The concentration of a single compound, N-acetylornithine, makes it possible to discriminate between control and diabetes (sensitivity and specificity equal to 1). In addition, receptor operating characteristic curve and principal component analysis for the above-mentioned six compounds yielded significantly (p < 0.001) altered in the aqueous humor of the patient group. In addition, receptor operating characteristic curve and principal component analysis for six compounds yielded cut-off values and remarkable sensitivity, specificity, and segregation ability. The altered level of N-acetylornithine may be due to an increased amount of acetate in diabetes. It is of interest to further investigate whether this alteration is related to the pathogenesis of the disease. The increase in the amino form of pyruvate, alanine, in diabetes is also relevant because it could be a means of reducing the formation of lactate from pyruvate.
Various substances within the aqueous humor (AH) can directly or indirectly impact intraocular tissues associated with intraocular pressure (IOP), a critical factor in glaucoma development. This study aims to investigate individual changes in these AH substances and the interactions among altered components through a multi‐omics approach. LC/MS analysis was conducted on AH samples from patients with exfoliation syndrome (XFS, n = 5), exfoliation glaucoma (XFG, n = 4), primary open‐angle glaucoma (POAG, n = 11), and cataracts (control group, n = 7). Subsequently, differentially expressed proteins and metabolites among groups, alterations in their network interactions, and their biological functions were examined. Both data‐independent acquisition and data‐dependent acquisition methods were employed to analyze the AH proteome and metabolome, and the results were integrated for a comprehensive analysis. In the proteomics analysis, proteins upregulated in both the XFG and POAG groups were associated with lipid metabolism, complement activation, and extracellular matrix regulation. Metabolomic analysis highlighted significant changes in amino acids related to antioxidant processes in the glaucoma groups. Notably, VTN, APOA1, C6, and L‐phenylalanine exhibited significant alterations in the glaucoma groups. Integration of individual omics analyses demonstrated that substances associated with inflammation and lipid metabolism, altered in the glaucoma groups, showed robust interactions within a complex network involving PLG, APOA1, and L‐phenylalanine or C3, APOD, and L‐valine. These findings offer valuable insights into the molecular mechanisms governing IOP regulation and may contribute to the development of new biomarkers for managing glaucoma.
Glaucoma is one of the leading causes of visual impairment and blindness worldwide, and is characterized by the progressive damage of retinal ganglion cells (RGCs) and the atrophy of the optic nerve head (ONH). The exact cause of RGC loss and optic nerve damage in glaucoma is not fully understood. The high energy demands of these cells imply a higher sensitivity to mitochondrial defects. Moreover, it has been postulated that the optic nerve is vulnerable towards damage from oxidative stress and mitochondrial dysfunction. To investigate this further, we conducted a pooled analysis of mitochondrial variants related to energy production, specifically focusing on oxidative phosphorylation (OXPHOS) and fatty acid β-oxidation (FAO). Our findings revealed that patients carrying non-synonymous (NS) mitochondrial DNA (mtDNA) variants within the OXPHOS complexes had an almost twofold increased risk of developing glaucoma. Regarding FAO, our results demonstrated that longer-chain acylcarnitines (AC) tended to decrease, while shorter-chain AC tended to increase in patients with glau-coma. Furthermore, we observed that the knocking down cpt1a (a key rate-limiting enzyme involved in FAO) in zebrafish induced a degenerative process in the optic nerve and RGC, which resembled the characteristics observed in glaucoma. In conclusion, our study provides evidence that genes encoding mitochondrial proteins involved in energy metabolisms, such as OXPHOS and FAO, are associated with glaucoma. These findings contribute to a better understanding of the molecular mechanisms underlying glaucoma pathogenesis and may offer potential targets for therapeutic interventions in the future.
Lipids are biological molecules that play vital roles in all living organisms. They perform many cellular functions, such as 1) forming cellular and subcellular membranes, 2) storing and using energy, and 3) serving as chemical messengers during intra‐ and inter‐cellular signal transduction. The large‐scale study of the pathways and networks of cellular lipids in biological systems is called “lipidomics” and is one of the fastest‐growing omics technologies of the last two decades. With state‐of‐the‐art mass spectrometry instrumentation and sophisticated data handling, clinical studies show how human lipid composition changes in health and disease, thereby making it a valuable medium to collect for clinical applications, such as disease diagnostics, therapeutic decision‐making, and drug development. This review gives a comprehensive overview of current workflows used in clinical research, from sample collection and preparation to data and clinical interpretations. This is followed by an appraisal of applications in 2022 and a perspective on the exciting future of clinical lipidomics.
