Metabolomics and the molecular approach to glaucoma diagnosis
Metabolomics reflects the physiological state of a tissue and provides information about interactions between genetics, environment and lifestyle
The numbers of individuals worldwide who are affected by glaucoma and who will be in the future are burgeoning. The current push is to identify biomarkers for earlier diagnosis, to determine those at higher risk and provide new therapeutic targets, according to Neeru Vallabh, PhD, Clinical Senior Lecturer in Eye and Vision Science, Institute of Life Course and Medical Sciences, University of Liverpool, and a Consultant Ophthalmologist for Glaucoma at St Paul’s Eye Unit, Liverpool University Hospitals NHS Foundation Trust, Liverpool, UK.
Figure 1. Spectroscopy instrumentation. Molecular samples can be evaluated through neutral magnetic resonance (NMR) spectroscopy, vibrational Raman spectroscopy and infrared spectometry.
“The current diagnostic challenges are the asymptomatic nature of glaucoma and the limitations of the traditional diagnostic methods. While genetic studies have been hugely successful in improving our understanding of the disease, the mechanisms through which genetic factors interact with non-genetic factors and lead to glaucoma are not well understood,” Ms Vallabh commented and pointed out the need for a transition from reactive medicine to predictive, preventative, and personalised medicine.1
This transition may be achieved through metabolomics.
Metabolomics defined
Metabolomics is the qualitative and quantitative analysis of low-molecular-weight small molecules,2 ie, metabolites with molecular weights under 1,500 Da, that include amino acids, carbohydrates, nucleosides/nucleotides, tricarboxylic acid, intermediates, and lipids.
The human metabolome, which is defined as the complete set of small-molecule substances, is now thought to contain in excess of 110,000 metabolites but many have not yet been identified.
Metabolomics, Ms Vallabh explained, reflects the physiological or pathological state of a cell or tissue and provides information about interactions between genetics, environment, and lifestyle and is more closely related to the phenotype in multifactorial disease.3
The metabolites in humans, which are the downstream end products of genes, RNA, and proteins and their interaction with the environment, are critical for growth and maintenance of cells and tissues in the body and they reflect the functional states of an individual.
Metabolomics studied
The route to study metabolomics can be via mass spectrometry and there are two choices within metabolomics studies, either gas chromatography mass spectrometry or liquid chromatography mass spectrometry.4,5 (Figure 1)
With this technique, a sample is vaporised (destroyed) and an electron beam bombards the vapours and converts them into charge particles that then are deflected by a magnetic field, Ms Vallabh explained. The result is that the particles with a lighter mass are deflected more. A detector measures the mass charge ratio and by so doing, the components of the sample can be evaluated.
Figure 2. Sample collection and analysis. The metabolomics workflow.7 (Figure data courtesy of Neeru Vallabh, MBBS, FRCOphth, PhD)
Another technique, nuclear magnetic resonance spectroscopy, can be undertaken during which the samples are not destroyed. The samples are placed within a strong magnetic field and the electromagnetic signals released by the atoms, usually hydrogen ions, are detected.6,7
In vibrational spectroscopy, another technique, the samples are bombarded with a specific wavelength of light. If the scattering of the light by the vibrating molecules is evaluated, this is termed Raman spectroscopy and allows detection of the functional groups in the sample, whereas if the absorption of the sample by infrared light is evaluated this is called infrared spectroscopy.6
Metabolomics workflow
This process involves a number of steps.7 The sample, which can be tissue or biofluids, is collected and prepared. Data then are collected via the spectroscopy method of choice. Analysis is conducted, and normalisation and metabolites are identified and statistical multivariate analyses are performed. Finally, further interpretation is done by enrichment and pathway identification to understand the significance of the changes that have been identified, she described. (Figure 2)
Metabolites in glaucoma
Ms Vallabh reported that in open-angle glaucoma, certain metabolites have a higher frequency including metabolites that are amino acids, such as arginine and glycine.8
Metabolomics studies of aqueous humour, plasma, serum and tear samples have been performed, but the results in the aqueous humour differ from the results in serum and tears.
She explained that in glaucoma the metabolic alterations include amino acid dysregulation, vitamin metabolism, fatty acid oxidation and glutaminolysis.
Table. High frequency metabolics related to open angle glaucoma9
A systemic review9 reported 24 significantly enriched metabolism pathways.
In glaucoma, the main pathways that are disturbed involve lipid metabolism and the carnitine shuttle pathway, with increased and decreased levels of metabolites as well as inconsistent changes.
“These altered metabolites can impact the mitochondrial fatty acid metabolism and the vulnerability of the retinal ganglion cells,” she stated. (Table)
She cited a study of a rat model in which intraocular pressure (IOP) elevations were induced.10 Twenty-five metabolites were found to be significantly altered after 3 days of induced IOP increases in the rat model. These changes were dominated by metabolites that implicate oxidative stress, increased metabolic need, and dysfunction of metabolite production.
Pretreament with oral nicotinamide for 7 days before the induced IOP increase showed that nicotinamide treatment prevented these changes. The impact of nicotinamide treatment in humans and metabolite regulation is going to be evaluated in ongoing research.
Ms Vallabh and colleagues performed a small pilot study using vibrational spectroscopy to evaluate differences between 10 glaucoma and 10 control aqueous humour samples from the Liverpool Research Eye Bank. The samples were assessed using infrared spectroscopy to measure the molecular vibrations in the sample.
The results showed that 77.7% of control samples were correctly identified as a control, whereas 78.1% of glaucoma samples were correctly assigned as glaucoma. The gas chromatography-mass spectroscopy results from some of these samples indicated that the signatures observed were mainly associated with amino acids, fatty acids, and some monosaccharides.
The largest scale metabolite study in glaucoma was a case control study11 that evaluated plasma metabolite profiles in primary open-angle glaucoma.
This was performed in three US groups that evaluated 369 metabolites using liquid chromatography-mass spectroscopy; the UK biobank study measured 168 metabolites using magnetic resonance spectroscopy. The study found that diglycerides and triglycerides were adversely associated with glaucoma and may play an important role in glaucoma pathogenesis.
Two in vivo assessments of metabolomics12,13 assessed the metabolomics changes. Sentosa and associates13 developed a custom-built combined infrared fundus imaging, swept-source optical coherence tomography, and Raman spectroscopy instrument for use in rat eyes. They found that Raman spectra could be observed.
Cakir and colleagues13 also developed a Raman spectroscope to image the retina and evaluate the oxidative stress status of mitochondria in haemoglobin in mice with induced high IOP.
Ms Vallabh pointed out that these are preliminary models and our understanding of what these spectra and peaks represent at this stage is limited, but this demonstrates the potential of harnessing our imaging techniques to evaluate metabolite changes in vivo.
Metabolomics limitations
Ongoing research has associated the different metabolites and their corresponding pathways that are altered in glaucoma. However, there are limitations of these approaches, including cost, sample sizes/types, limited number of metabolites, variable methodology/analysis, variable findings/standardisation, sample destruction, and effects of treatments/ surgery/processing.
Metabolomics future
“We hope to apply the findings of these changes in metabolites to clinical application. This is being helped by the development of metabolite databases to register these changes. The hope in the future is that these metabolite changes may provide novel therapeutic targets,” Ms Vallabh concluded.
References
1. Zhang Q, Wang N, Rui Y, et al. New insight of metabolomics in ocular diseases in the context of 3P medicine.EPMA J. 2023;14:53–71.
2. Metabolomics. Metabolomics Technologies, Inc. Accessed May 2024. https://www.mtidx.com/our-technology/metabolomics
3. Steuer AE, Brockbals L, Kraemer T. Metabolomic Strategies in Biomarker Research-New Approach for Indirect Identification of Drug Consumption and Sample Manipulation in Clinical and Forensic Toxicology?. Front Chem. 2019;7:319. Published 2019 May 10. doi:10.3389/fchem.2019.00319
4. Mass Spectrometry. Priyam Study Centre Learning Chemistry. Updated 28 August 2022. Accessed May 2024.https://www.priyamstudycentre.com/2022/02/mass-spectrometry.html
5. Jones A. Teaching Animations. Warwick School of Life Sciences. Updated 12 March, 2024. Accessed May 2024.https://warwick.ac.uk/fac/sci/lifesci/research/sigtraf/animations/
6. Onissiphorou K. A Level Chemistry Revision: Organic Chemistry – Nuclear Magnetic Resonance Spectroscopy, ReAgent Chemical Services. Updated 6 May, 2022. Accessed May 2024. https://www.chemicals.co.uk/blog/a-level-chemistry-revision-organic-chemistry-nuclear-magnetic-resonance-spectroscopy
7. Contreras A, Cocom-Chan B, Hernandez-Montes G, Portillo Bobadilla T. Host-microbiome interaction and cancer: potential application in precision medicine. Front Physiol. 2016; doi:10.3389/fphys.2016.00606.
8. Wang Y, Hou XW, Liang G, Pan CW. Metabolomics in Glaucoma: A Systematic Review. Invest Ophthalmol Vis Sci. 2021;62(6):9. doi:10.1167/iovs.62.6.9
9. Tang Y, Shah S, Cho KS, Sun X, Chen DF. Metabolomics in Primary Open Angle Glaucoma: A Systematic Review and Meta-Analysis. Front Neurosci. 2022;16:835736. Published 2022 May 12. doi:10.3389/fnins.2022.835736
10. Tribble JR, Otmani A, Sun S, et al. Nicotinamide provides neuroprotection in glaucoma by protecting against mitochondrial and metabolic dysfunction. Redox Biol. 2021;43:101988. doi:10.1016/j.redox.2021.101988
11. Zeleznik OA, Kang JH, Lasky-Su J, et al. Plasma metabolite profile for primary open-angle glaucoma in three US cohorts and the UK Biobank. Nat Commun. 2023;14(1):2860. Published 2023 May 19. doi:10.1038/s41467-023-38466-w
12. Sentosa R, Stiebing C, Eibl M, et al. Targeting the molecular information of the retina with Raman spectroscopy. Invest. Ophthalmol. Vis. Sci. Published June 2023. doi:64(9):PP0023.
13. Cakir B, Tomita Y, Yagi H, et al. In vivo noninvasive mitochondrial redox assessment of the optic nerve head to predict disease. PNAS Nexus. 2023;2(5):pgad148. Published 2023 May 2. doi:10.1093/pnasnexus/pgad148
Neeru Vallabh MBBS, PhD, FRCOphth
E: neeru.amrita.vallabh@liverpool.ac.uk
Vallabh is a glaucoma specialist at the Liverpool University Hospital NHS Foundation trust and a clinical senior lecturer at the University of Liverpool. She works as a committee member for the UK Eye Genetics group, a section editor for the Eye journal and is a next generation partner in the European Glaucoma Society.
