METABOLOMIC PROFILING OF PHYTOCHEMICALS IN CROPS FOR HUMAN HEALTH ENHANCEMENT

• Sponsoring Institution: National Institute of Food and Agriculture
• Project Status: COMPLETE
• Funding Source: HATCH
• Reporting Frequency: Annual
• Accession No.: 1020083
• Project Start Date: Jul 1, 2019
• Project End Date: Sep 30, 2023
• Program Code: [(N/A)]- (N/A)

Recipient Organization
OHIO STATE UNIVERSITY
1680 MADISON AVENUE
WOOSTER,OH 44691

Performing Department
Horticulture and Crop Science

Non Technical Summary
The mission of the Cooperstone laboratory is to better understand the ways by which fruits and vegetables, and their phytochemicals may be acting to alter risk for chronic diseases. If we can define scientifically-validated phytochemical targets that impact health, then we can develop strategies for their modification. By exploiting our understanding of plant genetics, we can alter or select for accessions/varieties that have the appropriate phytochemical profiles to test how single phytochemical changes affect biological outcomes. This can be done within the context of a plant-containing diet for maximum translatability. Our goal is to develop fruit and vegetable varieties that our purposefully designed for enhanced health, backed up by clinical trial data and worthy of government supported health claims. These varieties will provide value across the food chain, from farmers who can sell crops for a premium, to consumers who can enhance their health without substantially changing their diets.

Animal Health Component

40%

Research Effort Categories

Basic

20%

Applied

40%

Developmental

40%

Classification

Knowledge Area (KA)	Subject of Investigation (SOI)	Field of Science (FOS)	Percent
204	1460	1010	100%

Knowledge Area
204 - Plant Product Quality and Utility (Preharvest);

Subject Of Investigation
1460 - Tomato;

Field Of Science
1010 - Nutrition and metabolism;

Keywords

phytochemicals

metabolomics

nutrition

horticulture

food science

mass spectrometry

Goals / Objectives
A major challenge of the 21st century will be to adequately support the growing population. As the number of inhabitants of our planet is expected to reach 9.8 billion in 2050, and 11.2 billion by 2100 (1), new and innovative solutions are needed not only to feed, but to sufficiently nourish these individuals. This endeavor requires researchers in agriculture to think about ways to improve the productivity, resiliency and quality of our food system. In tandem with our growing population, rates of cardiovascular disease, obesity and diabetes are increasing in the United States (2). One half of Americans suffer from one or more diet-related chronic disease (3). But paradoxically, we still observe the double burden of malnutrition (4) at the individual level, with caloric excess existing in diets lacking in essential micronutrients and other health-beneficial compounds. Diets have become more homogenous (5) despite the Dietary Guidelines for Americans recommending a varied diet (3).Dozens of studies have examined the relationship between fruit and vegetable intake and cancers, cardiovascular disease and all-cause mortality (6). The Dietary Guidelines for Americans recommends that half of one's plates should be fruits and vegetables, and to consume a variety of different types. Despite this recommendation, 87% and 75% of Americans eat fewer vegetables and fruits, respectively, than recommended (3).At the same time, there is a push to adopt more sustainable food systems to improve the health of our planet. The EAT-Lancet Commission on Food, Planet and Health, which aims to develop a scientific consensus for what defines a healthy and sustainable diet, has estimated that we will need to produce 100% more vegetables by 2050 - a huge task (7). With an increased push towards more plant based diets to improve the health of people as well as the sustainability of our food system (7), in combination with our growing population, requires agricultural scientists to think about ways to produce more, high quality, healthful food.There is an enormous opportunity to improve the nutritional quality of our crops (8). Most of the efforts to breed crops for enhanced nutrition have focused on essential micronutrient deficiencies (like vitamin A and iron) in staple crops. Significant efforts have been exerted to produce products using both genetic modification (e.g., Golden Rice) and conventional breeding approaches (e.g., orange fleshed sweet potato, orange maize). Horticulture crops (i.e., fruits and vegetables) are rarely biofortified, despite being naturally rich in phytochemicals, and the recommendation that they make up half of the American plate. Consumption of many of these fruits and vegetables has been associated with decreased risk of disease, and more positive health outcomes in pre-clinical and clinical trials, although the exact compound(s) responsible for this effect are often unknown.There is currently a gap in our ability to improve the nutritional quality of our fruits and vegetables by a lack of understanding of the particular phytochemicals imparting health benefit. There are many phytochemicals associated with positive health outcomes via epidemiology (e.g., lycopene and prostate cancer), pre-clinical models (e.g., resveratrol and cardiovascular disease) and in vitro effects (e.g., anthocyanins and antioxidant properties), though none of these relationships have enough scientific evidence to infer causality instead of correlation.The mission of the Cooperstone laboratory is to better understand the ways by which fruits and vegetables, and their phytochemicals may be acting to alter risk for chronic diseases. If we can define scientifically-validated phytochemical targets that impact health, then we can develop strategies for their modification. By exploiting our understanding of plant genetics, we can alter or select for accessions/varieties that have the appropriate phytochemical profiles to test how single phytochemical changes affect biological outcomes. This can be done within the context of a plant-containing diet for maximum translatability. Our goal is to develop fruit and vegetable varieties that our purposefully designed for enhanced health, backed up by clinical trial data and worthy of government supported health claims. These varieties will provide value across the food chain, from farmers who can sell crops for a premium, to consumers who can enhance their health without substantially changing their diets.

Project Methods
Targeted phytochemical analyses: phytochemicals of interest (carotenoids, flavonoids, phenolic acids, steroidal glycoalkaloids) will be extracted from both plants and biological samples using appropriate techniques fit for each category of analyte, as has been done previously (9-12). Extracts will be run using HPLC-PDA on an Agilent 1260 and/or HPLC-MS/MS on a Waters Acquity interfaced with a TQD MS.Untargeted metabolomics:Metabolomics is an emerging technique capable of investigating the totality of small molecules within a biological system. Metabolomics is perhaps the newest of the 'omics fields, and is more sensitive to changes due to a short term intervention as compared to the genome, transcriptome or proteome. Metabolites, the end-products of biological pathways, are influenced by interactions of genotype, the microbiome and environmental factors, including one's diet. By employing untargeted metabolomics, one can profile thousands of small molecular weight metabolites present in plants, foods, or biological system, without a preconceived bias as to which may be different between groups. This allows the investigator, using big data analytical techniques, to analyze metabolite differences between groups and attempt to explain these biological differences to develop novel and testable hypotheses. Studies of this type have been conducted on plants (11,13), and with other nutritional interventions (14,15) and dietary patterns (16).We will use open-ended metabolomics to capture as broad a range of compounds as possible as ions in full scan MS experiments. A state-of-the-art Agilent 1290 UHPLC coupled with a QTOF 6545 provides us high resolution chromatography, high dynamic range MS detection (5 orders of magnitude), high mass resolution (45K) and mass accuracy (0.8 ppm) for reliable relative comparisons as well as the ability to resolve nominally isobaric species and generate tentative identities. We analyze plants, foods and biological samples in both positive and negative modes of ionization for comprehensive coverage, creating a highly powered dataset to examine statistically significant differences in compounds between groups of interest. The planned workflow is shown in Figure 1. We will identify specific compounds through accurate mass searches of public databases (e.g. METLIN Metabolite Database at Scripps Research Institute, the Human Metabolome Database at the University of Alberta and others). We will guard against artifactual results during the MS data acquisition through both machine and analytical replicates in addition to randomized injection order. To sift through the large amount of data produced in these experiments, our workflow includes m/z extraction, confirmation of detected ions through m/z orthogonal searches, and further data reduction by ion abundance and ranking.Pooled quality control (QC) samples will be evaluated to determine the variability in detected compounds over the course of the study. Analysis will be restricted to those metabolites having ion intensities with a coefficient of variation (CV) less than 30% across identical QC samples. Mass Profiler Professional Software (Agilent Technologies) and R will be used as visualization tools for MS-based metabolomics data. Unsupervised principal component analysis (PCA) and hierarchal clustering analysis (HCA) will be used to visualize natural clusters in the dataset, which can be further examined by fold change and P-value. SAS® 9.3 and R will be used for the statistical analyses. Analyses will be conducted to determine if metabolites in plasma and urine are significantly different from baseline to four weeks after each tomato juice treatment, and the metabolome differences between the two tomato juice treatments. A filtering method based on percentage of metabolites above a noise cutoff (<10th percentile) will be applied to filter out low expression metabolites. Appropriate normalization methods (e.g. quantile, percentile shift, external scalar) and baselining will be used to correct for non-experiment variability and remove bias across metabolites (17).

Progress 10/01/19 to 09/30/20

Outputs
Target Audience:Target audience is scientists, students, and the public. We have reached scientists through peer reviewed journals and book chapters. Students through teaching and advising, and the public through our outreach efforts. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?Under this project, 3 M.S. and 2 Ph.D. students have been progressing towards their graduate degrees.3 M.S. and 1 Ph.D. students completed their degrees in August 2020. Bilbrey EA. Seeding multi-omics improvement in apple [M.S. Thesis]. [Columbus, OH]: The Ohio State University; 2020. Dzakovich MP. Exploring metabolic and genetic diversity in tomato secondary metabolites [Ph.D. Dissertation]. [Columbus, OH]: The Ohio State University; 2020. Goggans M. Elucidating tomato steroidal glycoalkaloid metabolism and effects of consumption on the gut microbiome in a pig model [M.S. Thesis]. [Columbus, OH]: The Ohio State University; 2020. Miller JL. Discovering potential urinary biomarkers of tomato consumption using untargeted metabolomics [M.S. Thesis]. [Columbus, OH]: The Ohio State University; 2020. How have the results been disseminated to communities of interest?We have reached scientists through peer reviewed journals and book chapters, students through teaching and advising, and the public through our outreach efforts. What do you plan to do during the next reporting period to accomplish the goals? To comprehensively characterize small molecular weight metabolites present in crops Phytochemicals of interest from plants will be quantitated using targeted high performance liquid chromatography (HPLC) and HPLC-tandem mass spectrometry (MS). Global metabolic fingerprinting will be conducted using high resolution MS. To comprehensively characterize the in vivo response to plant or phytochemical intake. Phytochemicals of interest from blood plasma, urine, and/or tissues will be quantitated using targeted ultra-high performance liquid chromatography (UHPLC) with photodiode array detection (PDA) and UHPLC-tandem mass spectrometry (MS). Global metabolic fingerprinting will be conducted using high resolution MS and a metabolomics workflow.

Impacts
What was accomplished under these goals? The mission of the Cooperstone laboratory is to better understand the ways by which fruits and vegetables, and their phytochemicals may be acting to alter risk for chronic diseases. If we can define scientifically-validated phytochemical targets that impact health, then we can develop strategies for their modification. By exploiting our understanding of plant genetics, we can alter or select for accessions/varieties that have the appropriate phytochemical profiles to test how single phytochemical changes affect biological outcomes. This can be done within the context of a plant-containing diet for maximum translatability. Our goal is to develop fruit and vegetable varieties that our purposefully designed for enhanced health, backed up by clinical trial data and worthy of government supported health claims. These varieties will provide value across the food chain, from farmers who can sell crops for a premium, to consumers who can enhance their health without substantially changing their diets.? Our projects in tomato span: Developing methods to analyze steroidal glycoalkaloids in tomato fruit. Phenotyping steroidal glycoalkaloid diversity in cultivated and wild tomato accessions. Understanding the chemical basis of bitterness in high alpha-tomatine tomatoes. Understanding tissue distribution of steroidal glycoalkaloids in pigs after tomato consumption. The effects of tomato consumption on the microbiome in pigs via shotgun metagenomics. Discovery of urinary biomarkers of tomato consumption. Our projects in apple span: Linking the apple genome with the apple fruit metabolome. Understanding genomic regions associated with phenolic production Our projects in black raspberry span: Understanding the effects of black raspberries and their phytochemicals on atopic dermatitis. Our projects in mixed systems: The effect of tomato and soy on markers of inflammation in humans. We have focused specifically this year on the focus areas below: How do genetic factors affect biosynthesis of phytochemicals in crops? Tomatoes: Epidemiological studies show that people who consume tomato rich diets tend to have lower incidence of many chronic diseases. The majority of research in this area has focused on lycopene (the red tomato pigment) as the principal bioactive compound. However, pre-clinical studies have found an enhanced effect from consuming tomatoes vs. lycopene alone or a lack of enhanced effect with more bioavailable lycopene, suggesting other phytochemicals contribute to this bioactivity. We discovered an understudied class of metabolites called steroidal glycoalkaloids from tomato that may confer some of the benefit of tomato rich diets (Cooperstone et al., Sci Reports 2017). Studying these compounds are a major focus of research in my group. However, considerable barriers to their study existed, which we have worked to overcome. Because no quantitative methods for extraction and analysis of these compounds existed, we developed on allowing the determination of concentrations of ~20 steroidal glycoalkaloids in tomato (Dzakovich et al., Frontiers in Plant Science 2020). Little was known about the genetic structure governing their production, so in collaboration with David Francis (OSU HCS), we surveyed accessions across various tomato clades and found large glycoalkaloid diverse in wild cherry tomatoes. After conducting a metabolite-genome wide association study (mGWAS) and appropriate metabolite-quantitative trait loci (mQTL) validation, we learned production of these alkaloids is largely under genetic control, and discovered a unique genomic regions responsible for elevated content of different alkaloids. We have developed mapping populations for our continued, integrated, genomic and metabolomic analyses. These plant-based resources will allow us to continue to ask questions about what role these compounds play within the tomato plant (including in diseases resistance), and to select for specific phytochemical profiles that enable targeted nutritional testing. Apples: As one of the most commonly consumed fruits in America, apples present an opportunity to impact the health of consumers. Health benefits conferring the adage "an apple a day keeps the doctor away" have largely been attributed to the fruit's phytochemical composition. To this end, integrated quality and nutritional improvement of apple has been hamper by a long juvenile period, which delays fruit evaluation for quality traits, such as phytochemical composition. To enable marker-assisted selection in apple, in collaboration with Jonathan Fresnedo Ramirez, Diane Miller (both OSU HCS), and Emmanuel Hatzakis (OSU FST), we have developed an integrated genomic-metabolomic platform to identify marker-phytochemical associations in breeding-relevant, and wild apple germplasm. By integrating both mass spectrometry and NMR based metabolomics with apple SNP data, we were able to discover unique metabolite-gene relationships using both GWAS and a pedigree-based approach. Our platform determining marker-phytochemical relationships in apples will inform breeding decisions and facilitate future marker-assisted selection for improved nutrition, while provide value to the Rosaceae community to map traits attributes related to flavor, post-harvest quality, and disease resistance etiology.

Publications

• Type: Journal Articles Status: Accepted Year Published: 2020 Citation: Mukherjee D, DiVincenzo M, Torok M, Choueiry F, Kumar R, Deems A, Miller JL, Hinton A, Geraghty C, Maranon JA, Kulp SK, Coss C, Carson III WE, Hart PA, Cooperstone JL, Mace TA. Soy-tomato enriched diet reduces inflammation and disease severity in a pre-clinical model of chronic pancreatitis. Accepted. Scientific Reports.
• Type: Journal Articles Status: Published Year Published: 2020 Citation: Anderson K, Ryan N, Pero T, Siddiqui A, Volpedo G, Cooperstone JL, Oghumu S. Black raspberries and protocatechuic acid mitigate DNFB-induced contact hypersensitivity by down-regulating dendritic cell activation and inhibiting mediators of effector responses. Nutrients, 2020;12(6):1701. https://doi.org/10.3390/nu12061701
• Type: Journal Articles Status: Published Year Published: 2020 Citation: Dzakovich MP, Hartman JL, Cooperstone JL. A high-throughput extraction and analysis method for steroidal glycoalkaloids in tomato. Front Plant Sci, 2020;11:767. https://doi.org/10.3389/fpls.2020.00767
• Type: Journal Articles Status: Published Year Published: 2020 Citation: Shetge SA, Dzakovich MP, Cooperstone JL, Kleinmeier D, Redan BW. Concentrations of the opium alkaloids morphine, codeine, and thebaine in poppy seeds are reduced after thermal and washing treatments but are not affected when incorporated in a model baked product. J Agric Food Chem, 2020;68(18):5241-5248. https://doi.org/10.1021/acs.jafc.0c01681
• Type: Journal Articles Status: Accepted Year Published: 2020 Citation: Fenstemaker S, Miller J, Cooperstone JL, Francis DM. Estimating parental contributions to hybrid rootstocks in grafted tomato. Accepted, Acta Horticulturae, 2020.
• Type: Journal Articles Status: Published Year Published: 2020 Citation: Knobloch NA, Charoenmuang M, Cooperstone JL, Patil BS. Developing interdisciplinary thinking in a food and nutritional security, hunger and sustainability graduate course. Journal of Agricultural Education and Extension 2020;26(1):113-127. https://doi.org/10.1080/1389224X.2019.1690014
• Type: Book Chapters Status: Published Year Published: 2020 Citation: Cooperstone JL. Lycopene: Food Sources, Properties, and Effects on Human Health. In: The Handbook of Nutraceuticals and Functional Foods, 3rd edition. Eds. Wildman REC, Bruno RS. CRC Press, Boca Raton, FL, 2020. p.37-53. ISBN 9781498703727

Progress 07/01/19 to 09/30/19

Outputs
Target Audience:Target audience is scientists, students, and the public. We have reached scientists through peer reviewed journals and book chapters. Students through teaching and advising, and the public through our outreach efforts. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?Under this project, 3 M.S. and 2 Ph.D. students have been progressing towards their graduate degrees. How have the results been disseminated to communities of interest? Nothing Reported What do you plan to do during the next reporting period to accomplish the goals? To comprehensively characterize small molecular weight metabolites present in crops Phytochemicals of interest from plants will be quantitated using targeted high performance liquid chromatography (HPLC) and HPLC-tandem mass spectrometry (MS). Global metabolic fingerprinting will be conducted using high resolution MS. To comprehensively characterize the in vivo response to plant or phytochemical intake. Phytochemicals of interest from blood plasma, urine, and/or tissues will be quantitated using targeted ultra-high performance liquid chromatography (UHPLC) with photodiode array detection (PDA) and UHPLC-tandem mass spectrometry (MS). Global metabolic fingerprinting will be conducted using high resolution MS and a metabolomics workflow.

Impacts
What was accomplished under these goals? The mission of the Cooperstone laboratory is to better understand the ways by which fruits and vegetables, and their phytochemicals may be acting to alter risk for chronic diseases. If we can define scientifically-validated phytochemical targets that impact health, then we can develop strategies for their modification. By exploiting our understanding of plant genetics, we can alter or select for accessions/varieties that have the appropriate phytochemical profiles to test how single phytochemical changes affect biological outcomes. This can be done within the context of a plant-containing diet for maximum translatability. Our goal is to develop fruit and vegetable varieties that our purposefully designed for enhanced health, backed up by clinical trial data and worthy of government supported health claims. These varieties will provide value across the food chain, from farmers who can sell crops for a premium, to consumers who can enhance their health without substantially changing their diets. To meet these aims, in 2019 we have published 4 peer reviewed publications and 1 book chapter and 2 abstracts in proceedings. Knobloch TJ, Ryan NM, Bruschweiler-Li L, Wang C, Bernier MC, Somogyi A, Yan PS, Cooperstone JL, Mo X, Bruschweiler RP, Weghorst CM, Oghumu S. Metabolic regulation of glycolysis and AMP activated protein kinase pathways during black raspberry-mediated oral cancer chemoprevention. Metabolites 2019;9(7):140. https://doi.org/10.3390/metabo9070140 Knobloch NA, Charoenmuang M, Cooperstone JL, Patil BS. Developing interdisciplinary thinking in a food and nutritional security, hunger and sustainability graduate course. Epub ahead of print, Journal of Agricultural Education and Extension 2019. https://doi.org/10.1080/1389224X.2019.1690014 Dzakovich MPg, Gas-Pascual E, Orchard CJ, Sari EN, Riedl KM, Schwartz SJ, Francis DM, Cooperstone JL. Analysis of tomato carotenoids: comparison extraction and chromatographic methods. J AOAC Int. 2019;102(4):1069-1079. https://doi.org/10.5740/jaoacint.19-0017 Teegarden MDg, Schwartz SJ, Cooperstone JL. Profiling the impact of thermal processing on black raspberry phytochemicals using untargeted metabolomics. Food Chem, 2019;274:782-788. https://doi.org/10.1016/j.foodchem.2018.09.053 Cooperstone JL. Lycopene: Food Sources, Properties, and Effects on Human Health. In: The Handbook of Nutraceuticals and Functional Foods, 3rd edition. Eds. Wildman REC, Bruno RS. CRC Press, 201X. Forthcoming. DiMarco D, Bilbrey EAg, Cichon MJ, Luz-Fernandez M, Cooperstone JL. Impact of 3 eggs/day on the plasma lipidome of young, healthy adults. Curr Dev Nutrition 2019;3(1):OR19-4-19. Fenstemaker S, Miller Jg, Cooperstone JL, Francis DM. Estimating parental contributions to hybrid rootstocks in grafted tomato. Submitted, Acta Horticulturae. Our projects in tomato span: Developing methods to analyze steroidal glycoalkaloids in tomato fruit. Phenotyping steroidal glycoalkaloid diversity in cultivated and wild tomato accessions. Understanding the chemical basis of bitterness in high alpha-tomatine tomatoes. Understanding tissue distribution of steroidal glycoalkaloids in pigs after tomato consumption. The effects of tomato consumption on the microbiome in pigs via shotgun metagenomics. Discovery of urinary biomarkers of tomato consumption. Our projects in apple span: Linking the apple genome with the apple fruit metabolome. Our projects in black raspberry span: Understanding the effects of black raspberries and their phytocheicals on actopic dermatitis.

Publications

• Type: Conference Papers and Presentations Status: Published Year Published: 2019 Citation: DiMarco D, Bilbrey EAg, Cichon MJ, Luz-Fernandez M, Cooperstone JL. Impact of 3 eggs/day on the plasma lipidome of young, healthy adults. Curr Dev Nutrition 2019;3(1):OR19-4-19.
• Type: Book Chapters Status: Awaiting Publication Year Published: 2019 Citation: Cooperstone JL. Lycopene: Food Sources, Properties, and Effects on Human Health. In: The Handbook of Nutraceuticals and Functional Foods, 3rd edition. Eds. Wildman REC, Bruno RS. CRC Press, 201X. Forthcoming.
• Type: Journal Articles Status: Published Year Published: 2019 Citation: Knobloch TJ, Ryan NM, Bruschweiler-Li L, Wang C, Bernier MC, Somogyi A, Yan PS, Cooperstone JL, Mo X, Bruschweiler RP, Weghorst CM, Oghumu S. Metabolic regulation of glycolysis and AMP activated protein kinase pathways during black raspberry-mediated oral cancer chemoprevention. Metabolites 2019;9(7):140. https://doi.org/10.3390/metabo9070140
• Type: Journal Articles Status: Awaiting Publication Year Published: 2019 Citation: Knobloch NA, Charoenmuang M, Cooperstone JL, Patil BS. Developing interdisciplinary thinking in a food and nutritional security, hunger and sustainability graduate course. Epub ahead of print, Journal of Agricultural Education and Extension 2019. https://doi.org/10.1080/1389224X.2019.1690014
• Type: Journal Articles Status: Published Year Published: 2019 Citation: Dzakovich MP, Gas-Pascual E, Orchard CJ, Sari EN, Riedl KM, Schwartz SJ, Francis DM, Cooperstone JL. Analysis of tomato carotenoids: comparison extraction and chromatographic methods. J AOAC Int. 2019;102(4):1069-1079. https://doi.org/10.5740/jaoacint.19-0017
• Type: Journal Articles Status: Published Year Published: 2019 Citation: Teegarden MD, Schwartz SJ, Cooperstone JL. Profiling the impact of thermal processing on black raspberry phytochemicals using untargeted metabolomics. Food Chem, 2019;274:782-788. https://doi.org/10.1016/j.foodchem.2018.09.053
• Type: Conference Papers and Presentations Status: Submitted Year Published: 2020 Citation: Fenstemaker S, Miller J, Cooperstone JL, Francis DM. Estimating parental contributions to hybrid rootstocks in grafted tomato. Submitted, Acta Horticulturae.