Source: UNIVERSITY OF CALIFORNIA, DAVIS submitted to NRP
METABOLIC SIGNATURES OF HUMAN DISEASES AND THEIR MODULATION USING NATURAL COMPOUNDS
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
COMPLETE
Funding Source
HATCH
Reporting Frequency
Annual
Accession No.
1005946
Grant No.
(N/A)
Cumulative Award Amt.
(N/A)
Proposal No.
(N/A)
Multistate No.
(N/A)
Project Start Date
Mar 16, 2015
Project End Date
Feb 1, 2018
Grant Year
(N/A)
Program Code
[(N/A)]- (N/A)
Recipient Organization
UNIVERSITY OF CALIFORNIA, DAVIS
410 MRAK HALL
DAVIS,CA 95616-8671
Performing Department
Interdepartmental
Non Technical Summary
Fruits and natural products produced in California are rich source of antioxidants, polyphenols, flavones and phytochemicals. We plan to use these xenobiotics to alter the course of toxic metabolic pathway(s). Biological processes are controlled by small molecules. These small molecules are the reactants / products of metabolism and include chemically diverse class of compounds, such as carbohydrates, lipids, amino acids, steroids, neurotransmitters etc. Comprehensive measurement of the small molecules in the cells/tissue/body fluids is called metabolomics . Using metabolomic technique hundreds of small molecules can be simultaneously identified and quantified in a biological sample, resulting in metabolic profile or signature. The metabolic profile of a healthy individual is a result from the interaction of its genetic make-up with the environment. However, in case of disease the metabolic profile may get altered due to cause or effect of disorder. We plan to explore the metabolic pathways in humans/rats/cells and get the metabolic signatures for healthy as well as pathologic (cancer/neurological disease/etc.) conditions using tandem mass spectrometry. We hypothesize that statistical comparison of the metabolic profiles of cases with diseases will lead to simple biomarker for the disease. Further, we plan to use fruit/plant based xenobiotics to alter the metabolic profiles that are a cause/result of pathological condition.
Animal Health Component
50%
Research Effort Categories
Basic
50%
Applied
50%
Developmental
0%
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
70274101010100%
Knowledge Area
702 - Requirements and Function of Nutrients and Other Food Components;

Subject Of Investigation
7410 - General technology;

Field Of Science
1010 - Nutrition and metabolism;
Goals / Objectives
Our overall long term goal is to use nutrients/diet/phytochemicals to alter the course of metabolic pathways that are responsible for diseases, mainly cancer and neurological disorders. To accomplish this, we will use metabolomics methods to explore the metabolic pathways in humans/rats/cells and get the metabolic signatures for healthy as well as pathologic conditions using tandem mass spectrometry. Further, we plan to use fruit/plant based xenobiotics to alter the metabolic profiles that are a cause of pathological condition. We will meet some of our goals by accomplishing the following objectives. First, develop UPLC-MS/MS analytical platforms to isolate, identify and measure small molecule metabolites from human/rat tissues and bio-fluids. Here the purpose is to develop methods to enrich the small molecule metabolites in the human/rat bio-specimen samples and carry out their comprehensive analysis by UPLC-MS/MS. This will allow us to profile the metabolites from both control and cases groups. Second, measure and using statistical multivariate methods compare the metabolic profiles of patients with disease and matched control samples. This objective is to create metabolic profile of a healthy individual and compare it with metabolic profile of cases. We hypothesize that statistical comparison of the metabolic profiles of cases and controls will lead to simple biomarker for disease. Third, modulate metabolic profile using nutrients/diet/phytochemicals. Here we will use external factors such as diet, nutrients or phytochemicals to change the metabolic signature so that it is similar to that obtained from healthy specimen.
Project Methods
Objective 1: Develop UPLC-MS/MS analytical platforms.1.1 Urine, serum and tissue samples. Human urine, serum and tissue samples from cases with neurological disease or cancer and age matched healthy controls will be obtained from UC Davis's Alzheimer's disease research centre and Cancer Centre. We will also obtain samples from experimental animals. 1.2 Processing of samples: This will be done by the method as previously developed in our lab [8,40]. Briefly, 1ml milliliter aliquots of urine/serum will be adjusted with 1 M NaOH or 1M HCl to pH 7.0 and concentrated. The concentrate will be extracted with 1ml of methanol and centrifuged. The methanol fraction will be removed and concentrated using a Speed-Vac and lyophilizer, and subjected to ultraperformance liquid chromatography-tandem mass spectrometry (UPLC/MS-MS) analysis. For tissue samples Approximately 100 - 500mg of tissue will be ground in liquid N2. The resulting tissue powder will be extracted with buffer and organic solvent and will be analyzed as above.1.3 UPLC/MS-MS analysis. UPLC analyses of small molecule metabolites in SPE extracts will be carried out with a Waters Acquity UPLC system connected with the high performance Xevo-TQ mass spectrometer. Analytical separations on the UPLC system will be conducted using an Acquity UPLC BEH C18 or phenyl 1.7 µ columns (1 X 150 mm) at a flow rate of 0.15 ml/min. All MS experiments will be performed by using electrospray ionization (ESI) in positive ion (PI) and negative ion (NI) mode. MS system parameters: capillary voltage, extractor cone voltage, detector voltage, desolvation gas and cone gas flow, desolvation and source temperature, etc. will be optimized for minimum limit of detection. Resulting data will be processed by using TargetLynx 4.1 software (Waters).1.4 Statistical analysisStatistical analysis will be carried out for UPLC-MS/MS method validation. Coefficient of variation, standard deviation, standard error etc. parameters will be determined after triplicate analysis. Objective 2: Measure and using statistical multivariate methods compare the metabolic profiles.2.1 Measure the metabolites in bio-specimens from controls and casesSmall molecule metabolites present in the urine/serum/tissue will be extracted as previously described and quantified by UPLC-ESI-MS/MS in either positive and negative ionization mode. The metabolite peak, area under curve (AUC), will be integrated to get their concentrations using MassLynx 4.1 software. 2.2 Statistical Analysis and Sample SizeCharacteristics of cases and controls will be descriptively compared using percents, means, medians, standard deviations and ranges. One-way analysis of variance (ANOVA) will be used to assess whether significant mean differences in metabolites exist in cases compared to controls. Significance will be assessed using the Bonferroni method. It is anticipated that those who have disease will have a pattern that is not seen in controls. Using multivariate logistic regression analysis we will determine which of the combination of biomarkers best predict case-control status adjusting for important confounders. Finally partial least-squares discriminant analysis (PLS-DA) of centred and pareto scaled data will be done to give a balance of group separation and predictive power. The primary measure of interest is the Area under curve because we are interested in whether the metabolites can be different between cases and controls.Objective 3: Modulate metabolic profile using nutrients/diet/phytochemicals.3.1 Cell Culture and treatmentCells will be obtained from American Type Culture Collection (ATCC) and will be maintained in appropriate medium with 10% fetal bovine serum at 5% CO2 and 37°C. Confluent cells will be split and seeded into 6-well plates and incubated for 18 -24h. Media will be removed and replaced with serum-free media and cells will be treated with test compounds. Each treatment and time point will be repeated 3-5 times for reproducibility. At the end of incubation cells will be removed and will be frozen at -80ºC until the extraction. 3.2 Animal TreatmentAppropriate male/female rats weighing 180- 200 g will be used in these studies. Test compounds will be orally administered to rats once daily for 30 days. Control and experimental rats will be housed separately in stainless steel metabolism cages with free access to food and water. Urine and blood serum will be collected daily until 7 more days after stopping test compound dose and will be frozen at -80ºC until the extraction.3.3 Analysis We will statistically compare the normal metabolic profiles to those treated with various doses of test compounds, using t-tests on metabolites that can be transformed to produce a normal distribution and the Wilcoxon two-sample non-parametric test for those that cannot be made normal by the Wilks-Shapiro test.

Progress 03/16/15 to 02/01/18

Outputs
Target Audience: Nothing Reported Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided? Nothing Reported 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? Nothing Reported

Impacts
What was accomplished under these goals? Nilesh Gaikwad was on leave majority of federal fiscal year, so no effort has been reported. He seperated from the University of California, Davis, as of October 31, 2017, and was unavailble to submit a progress or final repport.

Publications


    Progress 10/01/15 to 09/30/16

    Outputs
    Target Audience:Undergraduate students Graduate students Industry Disease Support Groups/Organizations Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?Jiajun Han, Graduate Student, was mentored and trained to perform biological assays and mass spectrometry analysis. Stephanie Fernandez, Graduate Student, was mentored and trained to perform biological assays and mass spectrometry analysis. Huang Sun, Graduate Student, was mentored and trained to perform biological assays and mass spectrometry analysis. More than 5 undergraduate students were mentored as a lab interns during 2016 How have the results been disseminated to communities of interest?Results that were obtained were disseminated through presentations in national and international seminars/conferences and through publications. Abstracts/Presentations 1. 2016 Pharmacometabolomics Signatures in Women with Premenstrual Dysphoric Disorder Undergoing GnRH Agonist-induced Ovarian Suppression with and without Physiologic Estradiol and Progesterone Replacement. Daniel M. Rotroff, Nilesh W. Gaikwad, John M. Reuter, Chris Smith, Heidi R. Kucera, Tuong Vi Nguyen, David R. Rubinow, Alison A. Motsinger-Reif, Peter J. Schmidt, Rima Kaddurah-Daouk. Am. Soc. Clin. Pharmacol. Toxicol. conference in San Diego on March 9, 2016. (Selected to receive the ASCPT 2016 Presidential Trainee Award) 2. 2016 Steroidomics: Essential Tool for Endocrinologists Nilesh W. Gaikwad. Seoul International Congress of Endocrinology andMetabolism. Seoul, Korea April 28, 2016. (Selected for Travel Award) 3. The Steroid Metabolome in Women with Premenstrual Dysphoric Disorder (PMDD) duringGnRH Agonist-induced Ovarian Suppression: Effects of Estradiol and Progesterone Addback Tuong Vi Nguyen, John M. Reuter, Nilesh W. Gaikwad, Daniel Rotroff, Heidi R. Kucera, Alison Motsinger-Reif, Chris P. Smith, Lynnette K. Nieman, David R. Rubinow, Rima Kaddurah-Daouk, and Peter J. Schmidt. Society of Biological Psychiatry Conference, Atlanta, May, 2016 What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

    Impacts
    What was accomplished under these goals? During 2016 we have improved upon our global steroid metabolic profiling method based on liquid−liquid extraction followed by LCMS for simultaneous measurement of over 200 indigenous as well as exogenous steroids in about 12 min, without derivatization. During review period, our studies were focused on effect of various tea's, viz. green, black, oolong and white, on steroid metabolism. Recently, green tea and other types of tea have received much attention because of bioactive polyphenols. However, very little information is available on comparative assessment of health effects of different tea's on health. We hypothesize that bioactive compound(s) from tea could modulate estrogen metabolism towards non carcinogenic pathways resulting in a significant preventive effect. To test our hypothesis, we treated rats with extracts of white, black, green and oolong tea and using recently developed mass spectrometry based metabolomic platform, we investigated the molecular mechanisms which leads to alteration of estrogen and overall steroid metabolic pathways. Preliminary analysis of the data shows several interesting trends and physiological effects, which will result in several publications.

    Publications

    • Type: Journal Articles Status: Accepted Year Published: 2016 Citation: Acute toxicity and cytotoxicity of Pereskia aculeata, a highly nutritious cactaceae plant. D�bora O. Silva, Maur�cio Seifert, Fabiana R. Nora, Vera L. Bobrowski, Rog�rio A. Freitag, H. Kucera, Leonardo Nora, Nilesh W. Gaikwad. Journal of Medicinal Food, In Press, 2016. Impact Factor: 1.844
    • Type: Journal Articles Status: Accepted Year Published: 2016 Citation: Combined Effects of Gestational Phthalate Exposure and Zinc Deficiency on Steroid Metabolism and Growth. Nuttall, J., Kucera, H. R., Supasai S., Gaikwad, N. W., and Oteiza, P.I. Toxicological Sciences, In Press, 2016 Impact Factor: 3.880
    • Type: Journal Articles Status: Published Year Published: 2016 Citation: Nutritional marginal zinc deficiency disrupts placental 11�-hydroxysteroid dehydrogenase type 2 modulation Huang, Y.L., Supasai S., Kucera, H., Gaikwad, N. W., Adamo, A.M, Mathieu, P., Oteiza, P.I. Food and Function, 20:84-92, 2016. Impact Factor: 2.68
    • Type: Journal Articles Status: Submitted Year Published: 2016 Citation: The Steroid Metabolome in Women with Premenstrual Dysphoric Disorder during GnRH Agonist-induced Ovarian Suppression: Effects of Estradiol and Progesterone Addback Tuong Vi Nguyen*, John M. Reuter*, Nilesh W. Gaikwad*, Daniel Rotroff, HR. Kucera, Alison Motsinger-Reif, Chris P. Smith, Lynnette K. Nieman, David R. Rubinow, Rima Kaddurah-Daouk, and Peter J. Schmidt. Submitted, 2017


    Progress 03/16/15 to 09/30/15

    Outputs
    Target Audience:Undergraduate students Graduate students Industry Disease Support Groups/Organizations Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?Heidi Kucera, Graduate Student, was mentored and trained to perform biological assays and mass spectrometry analysis. Jiajun Han, Graduate Student, was mentored and trained to perform biological assays and mass spectrometry analysis. Stephanie Fernandez, Graduate Student, was mentored and trained to perform biological assays and mass spectrometry analysis. Huang Sun, Graduate Student, was mentored and trained to perform biological assays and mass spectrometry analysis. More than 10 undergraduate students were mentored as a lab interns during 2015 How have the results been disseminated to communities of interest?Results that were obtained were disseminated through presentations in national and international seminars/conferences and through publications. Abstracts/Presentations 1.Pharmacometabolomics of Estradiol and Progesterone Treatment in Women withPremenstrual Dysphoric Disorder. Daniel M. Rotroff, Nilesh W. Gaikwad, John M.Reuter, Chris Smith, Heidi R. Kucera, Tuong Vi Nguen, David R. Rubinow, Alison A. Motsinger-Reif, Peter J. Schmidt, Rima Kaddurah-DaoukMetabolomics Society Conference. San Francisco, CA., 2015 2.Identification of Active Components of Genistein Combined Polysaccharide through Fractionation, Bioassay, and Mass Spec Analysis. Neelu Batra, Heidi Kucera, Nilesh W. Gaikwad, Ralph W. deVere White, Paramita Ghosh, Ruth VinallCancer Center Conference. Sacramento, CA., 2015 3.Ultra-Performance Liquid Chromatography-Tandem Mass Spectrometry Profiling of Steroid Metabolome in Human Saliva. Stephanie G. Fernandez, Heidi R. Kucera, Nilesh W. Gaikwad. Metabolomics Society Conference. San Francisco, CA., 2015 4.Podocyte-specific protein tyrosine phosphatase 1B deficiency in mice improves glucose homeostasis and proteinuria under diabetic mellitus condition. Yoshihiro Ito, Heidi R. Kucera, Ahmed Bettaieb, Mingfu Hsu, Nilesh W. Gaikwad and Fawaz G. Haj. Metabolomics Society Conference. San Francisco, CA., 2015 5.Metabolomic investigation of tamoxifen induced endometrial cancer. Heidi R.Kucera, Juliana O. Martins, Nilesh W. Gaikwad. Metabolomics Society Conference. San Francisco, CA. What do you plan to do during the next reporting period to accomplish the goals?N/A

    Impacts
    What was accomplished under these goals? During 2015 we have improved upon our global steroid metabolic profiling method based on liquid−liquid extraction followed by LCMS for simultaneous measurement of over 200 indigenous as well as exogenous steroids in about 12 min, without derivatization. In humans many physiological processes are controlled by steroids, but they are also implicated in the development and/or progression of many diseases. Using above methods we are currently analyzing several human/animal tissues to explore how the steroid metabolism is altered in various diseases.

    Publications

    • Type: Journal Articles Status: Published Year Published: 2015 Citation: Nutritional marginal zinc deficiency disrupts placental 11�-hydroxysteroid dehydrogenase type 2 modulation Huang, Y.L., Supasai S., Kucera, H., Gaikwad, N. W., Adamo, A.M, Mathieu, P., Oteiza, P.I. Food and Function. In Press, 2015.
    • Type: Journal Articles Status: Published Year Published: 2015 Citation: Estrogen sulfotransferase ablation sensitizes mice to sepsis. Xie W, Chai X, Guo Y, Jiang M, Hu B, Li Z, Fan J, Deng M, Billiar T, Kucera H, Nilesh W. Gaikwad, Xu M, Lu P,� Yan J, Fu H, Liu Y, Yu L, Huang M, Zeng S. Nature Comm., 6:7979, 2015.
    • Type: Journal Articles Status: Published Year Published: 2016 Citation: Inflammatory Regulation of Steroid Sulfatase, A Novel Mechanism to Control Estrogen Homeostasis and Inflammation in Chronic Liver Disease. M. Jiang, M Klein, U M. Zanger, M K. Mohammad, M C. Cave, Nilesh W. Gaikwad, K W. Selcer, Y Guo, J He, X Zhang, Q Shen, W Qin, J Li, S Li, W Xie. Journal of Hepatology, S0168-8278, 2015.
    • Type: Journal Articles Status: Published Year Published: 2015 Citation: Intracrine androgens and AKR1C3 activation confer resistance to enzalutamide in prostate cancer Chengfei Liu, Wei Lou, Yezi Zhu, Joy C. Yang, Nagalakshmi Nadiminty, Nilesh W. Gaikwad, C. P. Evans, Allen C. Gao. Cancer Research, 75, 1413-1422, 2015.
    • Type: Journal Articles Status: Published Year Published: 2015 Citation: Metabolic disturbance in the ATP binding cassette transporter Abcb6 deficient mice results in suppression of hepatic Cytochrome P450 activity. H. Chavan, F. Li, R. Tessman, K. Mickey, K. Dorko, T. Schmitt, S. Kumer, Sumedha, Gunewardena, Nilesh Gaikwad, and Partha Krishnamurthy. Journal of Biological Chemistry, 290: 7871-7886, 2015.
    • Type: Journal Articles Status: Published Year Published: 2015 Citation: Estrogen sulfotransferase is an oxidative stress responsive gene that gender-specifically affects liver ischemia/reperfusion injury. Yan Guo, Bingfang Hu, Hai Huang, Allan Tsung, Nilesh Gaikwad, Meishu Xu, Mengxi Jiang, Songrong Ren, Jie Fan, Timothy R. Billiar, Min Huang, and Wen Xie. Journal of Biological Chemistry, 290:14754-64, 2015.
    • Type: Journal Articles Status: Published Year Published: 2015 Citation: Decreased Adiposity and Enhanced Glucose Tolerance in Shikonin-Treated Mice, Obesity. Ahmed Bettaieb, Ellen Hosein, Samah Chahed, Ahlam Abdulaziz, Heidi R. Kucera, Nilesh W. Gaikwad and Fawaz G. Haj. Obesity, 23: 2269-2277, 2015.