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Metabolomics analysis of human vitreous in diabetic retinopathy and rhegmatogenous retinal detachment Nathan R Haines, Niranjan Manoharan, Jeffrey L Olson, Angelo D'Alessandro, and Julie A. Reisz J. Proteome Res., Just Accepted Manuscript • DOI: 10.1021/acs.jproteome.8b00169 • Publication Date (Web): 07 Jun 2018 Downloaded from http://pubs.acs.org on June 8, 2018
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Journal of Proteome Research
Metabolomics analysis of human vitreous in diabetic retinopathy and rhegmatogenous retinal detachment
Nathan R. Haines,1,3 Niranjan Manoharan,1 Jeffrey L. Olson,1 Angelo D’Alessandro,2 and Julie A. Reisz,2,* 1
Department of Ophthalmology, University of Colorado - Anschutz Medical Campus, Aurora, CO USA 2 Department of Biochemistry and Molecular Genetics, University of Colorado - Anschutz Medical Campus, Aurora, CO USA 3 Current address: Piedmont Retina Specialists, Greensboro, NC USA
*Corresponding author: Julie A. Reisz Department of Biochemistry and Molecular Genetics University of Colorado - Anschutz Medical Campus 12801 E. 17th Ave Aurora, CO USA Phone 303-724-3339
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Abstract The vitreous humor is a highly aqueous eye fluid interfacing with the retina and lens and providing shape. Its molecular composition provides a readout for the eye’s physiological status. Changes in cellular metabolism underlie vitreoretinal pathologies, but despite routine surgical collection of vitreous, only limited reports of metabolism in the vitreous of human patients have been described. Here, vitreous samples from patients with rhegmatogenous retinal detachment (n=25) and proliferative diabetic retinopathy (n=9) were profiled along with control human vitreous samples (n=8) by untargeted mass spectrometry-based metabolomics. Profound changes were observed in diabetic retinopathy vitreous, including altered glucose metabolism and activation of the pentose phosphate pathway, which provides reducing equivalents to counter oxidative stress. In addition, purine metabolism was altered in diabetic retinopathy, with decreased xanthine and elevated levels of related purines (inosine, hypoxanthine, urate, allantoate) generated in oxidant-producing reactions. In contrast, the vitreous metabolite profiles of retinal detachment patients were similar to controls. In total, our results suggest a rewiring of vitreous metabolism in diabetic retinopathy that underlies disease features such as oxidative stress and furthermore illustrate how the vitreous metabolic profile may be impacted by disease.
Keywords (10): untargeted metabolomics, absolute quantification, vitreous, vitreoretinal disease, diabetic retinopathy, mass spectrometry, retinal detachment
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Journal of Proteome Research
Introduction The vitreoretinal interface plays a significant role in surgical retinal diseases ranging from symptomatic vitreous floaters to more serious problems of macular holes, rhegmatogenous retinal detachments (RD), ocular traumas, and diabetic eye disease. Severity of disease and other factors, such as age and ethnicity, affect but do not fully explain disparities that exist in surgical outcomes. Underlying these pathologies are cellular changes that may be causative or correlative factors. For example, an epithelial to mesenchymal transition (EMT) of retinal pigmented epithelial (RPE) cells following rhegmatogenous RD allows for their migration into the vitreous and is associated with extracellular matrix remodeling leading to proliferative vitreoretinopathy (PVR)1. Though the development of PVR affects a minority of RD patients, a subsequent surgery is required and ultimate visual outcomes are often poor. In diabetic patients, there is significant variability in the severity of diabetic retinopathy which is not fully explained by the patient’s glucose control 2. Diabetic retinopathy (DR), the leading cause of blindness among working age adults in the U.S. and developed world (Centers for Disease Control), requires intravitreal injection, laser therapy and/or surgical intervention simply to halt disease progression. Certain patients have refractory DR that fails to improve even despite aggressive medical and surgical therapy3. Early and minimally invasive methods for observing systemic changes in the eye may provide robust and facile tools to inform management of higher risk patients and further develop the understanding of how cellular metabolism is altered in vitreoretinal diseases. Retinal and vitreous metabolism vary depending on underlying retinal pathology, and to date, a lack of full metabolic profiling of the vitreous in healthy and disease states hinders the use of this powerful analytical approach to distinguish characteristics of disease states and patient outcomes. Obtaining vitreous fluid for analysis is fairly straightforward in patients undergoing vitrectomy surgery, and has significantly lower risk than obtaining retinal tissue. To date, however, there exist only limited descriptions of vitreous metabolism in human patients focusing on a small subset of metabolites4. Here, we examined the metabolic profiles of human vitreous fluid from patients with retinal pathologies (RD or DR) and patients without significant retinal disease. High-throughput5 generation of a comprehensive profile of the vitreous metabolome in the context of healthy and disease states provides the means to identify key metabolic axes that are disrupted in retinal damage or disease. Moving forward, robust and rapid global profiling may aid in stratification of disease states and/or predicting adverse postoperative outcomes such as PVR.
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Experimental Vitreous sample collection Colorado Multiple Institutional Review Board (COMIRB) approval was obtained for retrieval of human vitreous at the time of routinely scheduled vitrectomy on patients at the University of Colorado Health Eye Center (Aurora, CO). After anesthesia, the eye was prepped in usual sterile ophthalmic fashion. Trocars were inserted in standard fashion for 3-port pars plana vitrectomy. Prior to any surgical maneuvers and with infusion off, the vitrector was used to manually aspirate samples into a tuberculin syringe (approximately 0.3 mL) while cutting at 7500 cuts per minute. Samples were de-identified and patient age, gender, diagnosis, ocular history, medical history, preoperative visual acuity, and date of sample collection were documented. Vitreous samples were classified as control (epiretinal membrane), retinal detachment, and diabetic retinopathy, and were immediately cooled and stored at -80 oC. UHPLC-MS metabolomics All solvents were Optima grade (Fisher Scientific). Vitreous samples were thawed on ice then 25 µL of vitreous was mixed with 50 µL of ice cold extraction buffer (5:3:2 MeOH:ACN:H2O). For absolute quantification, all stable isotope labeled standards were purchased from Cambridge Isotope Laboratories. Where applicable, the extraction buffer contained 3.75 µM of an amino acid mixture (MSK-A2-1.2), an acylcarnitine mixture (NSK-B) diluted 1:200 according to the manufacturer’s instructions (final concentrations: free carnitine D9 – 0.76 nM, acetylcarnitine D3 – 0.19 nM, propanoyl D3 – 0.038 nM, butyryl D3 – 0.038 nM, isovaleryl D9 – 0.038 nM, octanoyl D3 – 0.038 nM, myristoyl D9 – 0.038 nM, palmitoyl D3 – 0.076 nM), [2,2,4,4-D4]citrate (1.5 µM), [U-13C]α-ketoglutarate (1.5 µM), [U-13C]succinate (1.5 µM), [1,4-13C2]fumarate (1.5 µM), and [1-13C]pyruvate (1.5 µM). Samples were vortexed at 4 oC for 30 min then spun 10 min at 10,000 g and 4 oC. Protein and lipid pellets were discarded and supernatants were analyzed by ultra high pressure liquid chromatography-mass spectrometry (UHPLC-MS) on a Thermo Vanquish UHPLC (San Jose, CA) coupled to a Thermo Q Exactive mass spectrometer (Bremen, Germany) in positive and negative ion modes (separate runs). Solvents were water and acetonitrile supplemented with formic acid (0.1% - positive mode) or ammonium acetate (5 mM - negative mode). Initial analysis utilized a 3 min isocratic run as previously described5, 6; subsequent extensive analysis (including absolute quantification) was performed using a 9 min gradient from 5-95% acetonitrile organic phase, as described7. Samples were randomized and a quality control sample was injected every 10 runs. Data analysis was performed using Maven (Princeton University) following file conversion by
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Journal of Proteome Research
MassMatrix (Case Western Reserve University). Absolute concentrations were obtained using the following equation: [light] = (peakarealight/peakareaheavy)[heavy]*DF where DF = dilution factor, in this case, 3 (i.e. 25 µL of vitreous in a total 75 µL volume).
Absolute concentrations for additional acylcarnitines (Table S3) were estimated using the labeled acylcarnitine with closest structural similarity (i.e. similar fatty acyl moiety carbon backbone length). Relative quantification data was normalized to median and auto-scaled within the MetaboAnalyst 3.0 platform prior to visualization and statistical analysis. Hierarchical clustering analysis was performed using GENE-E (Broad Institute); bar graphs prepared using GraphPad Prism 5.03; receiver operating characteristic curves, partial least squaresdiscriminant analysis, and statistical analysis (ANOVA) for heat maps prepared using MetaboAnalyst 3.0.
Results & Discussion Patient vitreous samples were obtained during the course of standard vitrectomy. As an aqueous medium interfacing with the retina, lens, and numerous cell types, the biomolecular composition of the vitreous humor provides a systemic overview of eye physiology. To assess the global metabolism of vitreous samples from patients with detached retina or DR vs control, batched analysis was performed on a preliminary set of 34 patient samples (epiretinal membrane: n=9, age 68 ± 6 yrs; rhegmatogenous RD: n=17, age 62 ± 10 yrs; proliferative DR: n=8, age 41 ± 10 yrs) via metabolite extraction followed by analysis by untargeted UHPLC-MS metabolomics (Figure 1A). This analysis utilized only 25 µL of vitreous per patient and resulted in the identification of >100 named metabolites (KEGG, Table S1), with 17 metabolites demonstrating statistically significant differences among cohorts (p
