Correspondence

REPLY: We thank Liu and Chen for their correspondence and MS metabolomics on the Boston cohort because of the described interest in our recent article. They raised concern about the greater sensitivity and the complementary nature of NMR and MS.1 comorbidities in our study population, because only diabetes was an Given the interesting results from this project, we have proceeded exclusion criterion. We agree this could affect our results; however, with MS analysis on our entire study cohort, and hope to share we obtained a detailed medical history, using a standardized our results soon. questionnaire, for all participants. When comparing subjects with Finally, we were careful not to infer causality between the age-related macular degeneration (AMD) and the control group, changes observed in plasma metabolomic profiles of AMD patients there were no differences with respect to a diagnosis of and any anatomic change in the retina. We do highlight, however, hypertension (P ¼ 0.665), dyslipidemia (P ¼ 0.793), heart failure how metabolomic profiling may provide novel insights into these (P ¼ 0.728), coronary heart disease (P ¼ 0.243), stroke or relationships, and how our data support the previously described transient ischemia attack (P ¼ 0.251), kidney disease relevance of lipid-related metabolites in AMD. Our discussion (P ¼ 0.818), or liver disease (P ¼ 0.596). The same was presents potential biological explanations, to encourage the readers observed when comparing the 3 AMD severity stages to consider a range of potential implications. However, we reiterate (P 0.394). The assessment of total blood lipid concentrations the uncertainty by saying that, “Further investigation is needed to was not performed in our study, and this is duly acknowledged in fully understand and to validate the role of enzymes and metabolites our limitations section. However, we believe this does not change in AMD.” Our conclusion also clearly states that additional studies the relevance of our metabolomic findings. are needed to clarify the relevance and biologic significance of our Second, there was concern about the mass spectrometry (MS) findings. We do wish to emphasize the take-home message from platform used for metabolomics. As mentioned in our article, we this article: “MS plasma metabolomic profiling is a powerful tool to used a state-of-the art platform, where 4 different ionization modes identify participants with AMD and to distinguish the different were used to comprehensively cover a broad range of metabolites, stages of disease,” and that “Our findings also contribute to the and chemocentric approaches with standards for each identified current knowledge of AMD pathophysiology by highlighting the metabolite were considered. To our knowledge, using >1 platform role of lipid metabolism.” is not a common practice among metabolomics studies and, 1 importantly, there are currently no platforms able to measure every INÊS LAÍNS, MD, MSC 2 single metabolite across the whole metabolome, so this is an un- RACHEL S. KELLY,PHD 3 avoidable bias of metabolomics research. We would also like to JOHN B. MILLER,MD 3 clarify that MS and nuclear magnetic resonance spectroscopy DEMETRIOS G. VAVVAS,MD,PHD (NMR) are complementary methods, and therefore do not neces- 3 1 IVANA K. KIM,MD sarily assess the same metabolites. MS is more sensitive, and able 2 JESSICA LASKY-SU,PHD to detect much smaller metabolites than NMR; thus, there are 3 JOAN MILLER,MD several metabolites that are detectable using MS and not using 4 NMR, which may account for the differences observed in our 2 DEEBA HUSAIN,MD assessments.2 1Retina Service, Massachusetts Eye and Ear, Harvard Ophthalmology AMD Center of Excellence, Department of Ophthalmology, Harvard Regarding the question of the appropriate sample size for this Medical School, Boston, Massachusetts; Faculty of Medicine, University study, it is important to recognize that in metabolomics many of Coimbra, Coimbra, Portugal; Association for Innovation and factors complicate sample size determination, including its char- Biomedical Research on Light, Association for Innovation and acteristically untargeted approach. Indeed, there is currently no Biomedical Research on Light and Image (AIBILI), Coimbra, Portugal; standard method for sample size determination for metabolomics Centro Hospitalar e Universitário de Coimbra, Coimbra, Portugal; experiments.3 For clinical studies, 10 to 20 patients per group are 2Systems Genetics and Genomics Unit, Channing Division of Network considered appropriate to generate pilot data.2 Our study was the Medicine, Brigham and Women’s Hospital and Harvard Medical 3 first assessing plasma MS metabolomics of the entire spectrum of School, Boston, Massachusetts; Retina Service, Massachusetts Eye and AMD severity stages, and is in agreement with these standards. Ear, Harvard Ophthalmology AMD Center of Excellence, Department As mentioned in our Methods section, the subjects included in of Ophthalmology, Harvard Medical School, Boston, Massachusetts; 4Retina Service, Massachusetts Eye and Ear, Harvard Ophthalmology this study (n ¼ 120) were recruited as a subset of a larger project AMD Center of Excellence, Department of Ophthalmology, Harvard on AMD biomarkers, which is in progress in Boston, Medical School, Boston, Massachusetts Massachusetts, and in Coimbra, Portugal. As part of this initiative, we have recruited almost 500 subjects, following the protocol described in this article, including the collection of Financial Disclosures: The authors made the following disclosures: D.H.: fasting blood samples for all. A group of these samples from 396 Patent pending e Biomarkers for age-related macular degeneration. subjects were analyzed using NMR spectroscopy, as a screening J.L.-S.: Consultant e Metabolon, outside the submitted work; Patent technique and initial approach.4,5 We then proceeded to perform pending e Biomarkers of AMD pending.

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I.L.: Patent pending e Biomarkers for Age-Related Macular References Degeneration. R.S.K.: Patent pending e Biomarkers for Age-Related Macular Degeneration. 1. Marshall DD, Powers R. Beyond the paradigm: combining mass I.K.K.: Financial support e Genentech, Allergan, Iconic Therapeutics. spectrometry and nuclear magnetic resonance for metabolomics. J.B.M.: Personal fees e Allergan, outside the submitted work. Prog Nucl Magn Reson Spectrosc. 2017;100:1e16. J.W.M.: Consultant e Amgen, Inc., KalVista Pharmaceuticals, Ltd., 2. Barnes S, Benton HP, Casazza K, et al. Training in metab- Maculogix, Inc., Biogen Idec, Inc., Alcon Research Council; Financial olomics research. I. Designing the experiment, collecting and support e Lowy Medical Research Institute, Ltd.; Patents e Valeant extracting samples and generating metabolomics data. J Mass Pharmaceuticals, ONL Therapeutics, LLC; Patent pending e Bio- Spectrom. 2016;51:461e475. markers for Age-Related Macular Degeneration. 3. Billoir E, Navratil V, Blaise BJ. Sample size calculation in metabolic phenotyping studies. Brief Bioinform. 2015;16:813e819. Available online: ---. 4. Suhre K, Gieger C. Genetic variation in metabolic phenotypes: study designs and applications. Nat Rev Genet. 2012;13:759e769. Correspondence: 5. Laíns I, Duarte D, Barros AS, et al. Human plasma metabolomics Deeba Husain, MD, MBS, Mass Eye and Ear, 234 Charles Street, 12th in age-related macular degeneration (AMD) using nuclear mag- Floor, Boston, MA 02114. E-mail: [email protected]. netic resonance spectroscopy. PLoS One. 2017;12(5):e0177749.

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