Source: UNIV OF MINNESOTA submitted to
TOXICOMETABOLOMICS OF HEATED VEGETABLE OILS-INDUCED ADVERSE EFFECTS
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
TERMINATED
Funding Source
Reporting Frequency
Annual
Accession No.
0222753
Grant No.
(N/A)
Project No.
MIN-18-082
Proposal No.
(N/A)
Multistate No.
(N/A)
Program Code
(N/A)
Project Start Date
Oct 1, 2010
Project End Date
Sep 30, 2013
Grant Year
(N/A)
Project Director
Chen, C.
Recipient Organization
UNIV OF MINNESOTA
(N/A)
ST PAUL,MN 55108
Performing Department
Food Science & Nutrition
Non Technical Summary
In recent years, consumption of food and feed containing heated and processed dietary fats such as heated vegetable oils has markedly increased in humans and animals. Under thermal stress, highly unsaturated oils are prone to be oxidized and degraded to various reactive lipid oxidation products (LOPs). Since the interaction between LOPs and biomolecules (protein, DNA and lipids) can potentially disrupt normal biochemical and signaling pathways regulating energy metabolism, inflammation, cell death and proliferation, the causative roles of consuming food prepared using highly unsaturated oils have been implicated in the pathogenesis of chronic diseases, including atherosclerosis, cancer and Alzheimer's disease, even though the underlying mechanisms are still poorly understood. Our current proposal intends to focus our efforts on understanding the influence of heated oils on the metabolic system and identifying biomarkers that can function as the indicators of prior exposures of heated vegetable oils and related toxic events, in the biological samples obtained from exposed humans and animals.
Animal Health Component
(N/A)
Research Effort Categories
Basic
(N/A)
Applied
(N/A)
Developmental
(N/A)
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
7231820115025%
7235010115025%
7111820115025%
7115010115025%
Goals / Objectives
Goal: The proposed work aims to examine the metabolic events elicited by consumption of heated highly-unsaturated vegetable oils and to identify small-molecule biomarkers associated with the toxicities using metabolomic approaches. Objective 1: To identify endogenous small-molecule biomarkers of heated soybean oil-induced toxicities through long-term feeding and metabolomic analysis of changes in urine, serum and tissue samples, and to establish the correlation between biomarkers and toxicological parameters. Objective 2: To identify fractions of heated soybean oil that are responsible for the toxicities and metabolic events associated with identified biomarkers, and to investigate the metabolic fates of hydroxyalkenals. Output: The successful completion of this project will not only provide novel information on the pathogenesis of LOPs-related toxicities, but also set the knowledge base for translating the identified biomarkers into clinical practice and developing new prevention strategies against the risks from the exposure of heated unsaturated oils.
Project Methods
In this study, we will combine advanced mass spectrometry (both LC-MS and GC-MS) with multivariate analysis and animal models to identify and characterize endogenous metabolites that can function as the biomarkers of adverse effects induced by heated soybean oils (HSO). In order to distinguish the endogenous metabolites related to the toxicities from the exogenous metabolites introduced by HSO exposure, the bolus dosing experiment will be conducted to define the chromatographic and spectral properties of LOPs and their metabolites, while the feeding experiment will be performed to identify the biomarkers through analyzing the kinetic profile of metabolome during the initiation and progression of HSO-induced toxicities. Identified biomarkers will be characterized by examining their correlations with general toxicological parameters, and the mechanisms of HSO-induced toxicities will be implied by the biological functions of identified biomarkers. In addition, fractions of HSO that are responsible for the toxicities and metabolic events associated with identified biomarkers will be determined, and metabolic fates of hydroxyalkenals will also be investigated. We expect the application of mass spectrometry (MS)-based metabolomics in this project will provide us a good opportunity to observe previously unnoticed effects of heated oils and obtain novel information for further mechanistic investigation.

Progress 10/01/10 to 09/30/13

Outputs
Target Audience: 1. General public: Information from this project could help define the risk of consuming large quantities of oxidized fats and related degradation products in humans and animals. 2. Biomedical research professionals: Novel information on the metabolic effects of thermally-oxidized oils and their impacts on health will become a scientific foundation for further mechanistic investigation on diet and disease. 3. Pig farmers: Results from this project could potentially provide useful information on the pros and cons of using oxidized oils and fats as a source of energy for farm animals Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided? The project has provided a great opportunity for training students who major in nutrition or animal nutrition. Overall, 4 graduate students in PI's lab (Ms. Xiaolei Shi, Mr. Lei Wang, Ms. Yuwei Lu, and Ms. Yiwei Ma), and one undergraduate (Mr. Darin Schwinkendorf) were trained in this project. Among them, two graduate students have graduated after completing the experiments related to this project. In addtion, 3 graduate student in Dr. Gerald Shurson's group (Mr. Pai Liu, Ms. Ran Rong, and Ms. Andrea Hanson) were trained by collaborating with my research group. How have the results been disseminated to communities of interest? We have published the results from this project in peer-reviewed journals (see Products), and also presented them in multiple scientific meetings in nutrition, biochemistry, and animal science. What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? Using liquid chromatography-mass spectrometry as an platform for untargeted and comprehensive metabolite analysis, we examined three aspects of chemical and metabolic changes associated with the consumption of heated vegetable oils: 1) heating-induced chemical changes in soyben oils; 2) the metabolites of lipid peroxidation products in mouse urine after consuming heated soyben oil; 3) changes in endogenous metabolism (mainly from nutrient metabolism) after consuming heated soybean oil. Our results have shown substantial amounts of reactive aldehydes were produced in heating process. The kinetics of their production was revealed in a multivariate statistical model. To effectively examine the bioactive and potentially toxic species in heated oils and in the aqueous matrix of biological samples (such as urine, serum, interstitial fluid), we have developed a chemical derivatization method (using 2-hydrazinoquinoline as the derivatization agent) to facilitate the detection of these compounds in LC-MS analysis. To test the efficacy of this method, we have applied the method to investigate the development of ketoacidosis in diabetic mice, a pathological condition that induces great increase of aldehyde and ketone metabolites in biofluids (published in 2013). In addition, we also identified that tryptophan metabolism was greatly affected by feeding heated soybean oil. Analysis of tryptophan and its metabolites revealed that tryptophan level in serum decreases after subchronic feeding of heated soybean oil in mouse chow, while tryptophan metabolites formed through tryptophan-kynurenine pathway increase in urine. Preliminary gene expression analysis on tryptophan metabolism pathway supports that heated soybean oil can selectively induce specific genes in tryptophan metabolism pathway. Using the methods and knowledge we obtained from our mouse studies, we further examined the metabolic changes in pigs consuming peroxidized lipids (In collaboration with Dr. Gerald Shurson in the Dept. of Animal Science). In a feeding experiment using thermally-oxidized vegetable oils and animal fats in pig diet,including corn oil, canola oil, poultry fat, and tallow, we observed the influence of consuming oxidized oils on lipids and amino acid metabolism (4 manuscripts in revision). In another feeding experiment using highly-oxidized dried distillers grains with solubles (DDGS) in pig diet, we oberved the protective effects of sulfur-containing amino acids against oxidized lipid-induced vitaminE depletion (2 manuscripts published in 2013). Besides being published in peer-reviewed journals, the results from these studies have been presented inseveral scientific meetings (total 17 abstracts in the last three years).

Publications

  • Type: Journal Articles Status: Published Year Published: 2013 Citation: 1. Lu Y, Yao D, Chen C*. 2-Hydrazinoquinoline as a derivatization agent for LC-MS-based metabolomic investigation of diabetic ketoacidosis. Metabolites. 3: 993-1010 (2013) (*corresponding author) 2. Song R, Chen C, Johnston LJ, Kerr BJ, Weber TE, Shurson GC. Effects of feeding diets containing highly oxidized dried distillers grains with solubles and increasing vitamin E levels to wean-finish pigs on growth performance, carcass characteristics, and pork fat composition. J Anim Sci. Accepted. 3. Wang L, Chen C*. Emerging Applications of Metabolomics in Studying Chemopreventive Phytochemicals. AAPS J. Epublished (* corresponding author) 4. Chen C*, Kim S. LC-MS-based metabolomics of xenobiotic-induced toxicities. Comput Struct Biotech J. 4: e201301008 (2013) (* corresponding author) 5. Song R, Chen C, Wang L, Johnston LJ, Kerr BJ, Weber TE, Shurson GC. High sulfur content in corn dried distillers grains with solubles (DDGS) protects against oxidized lipids in DDGS by increasing sulfur-containing antioxidants in nursery pigs. J Anim Sci. 91: 2715-28 (2013)


Progress 01/01/12 to 12/31/12

Outputs
OUTPUTS: In the past one year, we continued the metabolomic investigation of metabolic effects of thermally-oxidized vegetable oil in three separate efforts: 1. We examined the biofluids and tissue samples collected from a feeding experiments on C57BL/6 mice using 4 different customized diets, soybean oil-low fat (FSO-LF) diet (contain 7% fresh oil), fresh soybean oil-high fat (FSO-HF) diet(contain 16.7% fresh oil), heated soybean oil-low fat (HSO-LF) diet (contain 7% heated oil), and heated soybean oil-high fat (HSO-HF) diet (contain 16.7% heated oil). LC-MS analysis has been conducted to define the different effects of feeding these diets on amino acids, lipids, and organic acids. 2. We examined the biofluids and tissue samples collected from a feeding experiments using oxidized vegetable oils and animal fats in pig diet,including corn oil, canola oil, poultry fat, and tallow. Metabolomic and metabolite analyses have been performed to examine the changes in lipids and amino acids. 3. We examined serum samples collected from a feeding experiments using dried distillers grains with solubles (DDGS) in pig diet. Since DDGS contains oxidized lipids generated from drying process, we analyzed the influences of DDGS diet on lipids and amino acids. Dissemination: We have given a poster presentation on the results from study 1 (heated oil feeding experiment in mouse) in the Experimental Biology meeting 2012, San Diego, April 2012 and two poster presentations on the results from study 2 & 3 in the 2012 ASAS Midwestern Annual Meeting, Des Moines, Iowa. PARTICIPANTS: 1. Dr. Chi Chen is the principal investigator of this project, who is responsible for designing and performing both animal experiments and chemical analysis. 2. Dan Yao, a Research Fellow, is responsible for conducting LC-MS and chemical analysis of biological samples from this project. 3. Lei Wang, a PhD graduate student, is responsible for conducting animal experiments and performing metabolomic analysis. 4. Yuwei Lu, a MS graduate student, assists metabolite analysis. Collaborators: Dr. Gerald C. Shurson, Professor in Swine Nutrition and Management,Department of Animal Science, 335D An Sci/Vet Med,1988 Fitch Avenue, St. Paul, MN 55108-6012, Telephone: 612-624-2764, Fax: 612-625-1210 Email: shurs001@umn.edu TARGET AUDIENCES: 1. General public: Information from this project could help define the risk of consuming large quantity of oxidized fats and related degradation products in human and animals. 2. Biomedical research professionals: Novel information on the metabolic effects of thermally-oxidized oils and their impacts on health will become a scientific foundation for further mechanistic investigation on diet and disease. 3. Pig farmers: Results from this project could potentially provide useful information on the pros and cons of using oxidized oils and fats as a source of energy for farm animals. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.

Impacts
1. Metabolomic analysis of heated oil-induced metabolic changes. The focus of our experiments has been moved from general metabolite profiling to detailed analysis of tryptophan metabolism based on our finding from the study conducted in 2011. Overall, tryptophan level in serum decreases, while its metabolites formed through tryptophan-kynurenine pathway increase in urine, suggesting the increase of tryptophan catabolism in this pathway. The gene expression analysis supports this conclusion. All these observations warrant further investigation on the effects of thermally-stressed oils on amino acid metabolism. In addition, the metabolomics method established in this study has been applied to another project on alcohol, leading to the identification of a novel ethanol metabolite. 2. Pig feeding experiment: In addition to the observed changes in growth performance and feeding efficiency , activation of peroxisome proliferator-activated receptor alpha (PPARα)pathway was identified as a major metabolic events in pigs fed with oxidized oils and fats. A manuscript based on this result has been submitted to J. Animal Science. 3. In DDGS feeding experiment, even though the original hypothesis was that DDGS containing oxidized lipids may produce oxidative stress and reactive species, the results showed that oxidative stress level in DDGS-fed pigs didn't increase. Through amino acid analysis, we revealed that sulfur-containing amino acids significantly increase in DDGS-fed pigs, leading to higher levels of antioxidants (glutathione and vitamin E). The cause behind the protective effect of DDGS has been attributed to high sulfur content in DDGS diet through sulfur analysis. Overall, we have enhanced our capacity to use LC-MS-based metabolomics to conduct nutritional and biochemical analyses. New information provides mechanistic insight on the effects of oxidized lipids on health and nutrition of humans and animals.

Publications

  • JOURNAL ARTICLES: Shi X, Yao D, Chen C*. Identification of N-acetyltaurine as a novel metabolite of ethanol through metabolomics-guided biochemical analysis. J Biol Chem. 287: 6336-49 (2012) (* corresponding author)
  • Charves J, Chen C, Hegeman AD, Reineccius GA. Evaluation of instrumental methods for the untargeted analysis of chemical stimuli of orange juice flavor. Flavour and Fragrance J. 26: 429-440 (2011)
  • ABSTRACTS: Liu P., Kerr BJ, Weber TE, Chen C., Johnston LJ., and Shurson GC, Influence of thermally-oxidized oils and fats on growth performance and lipid profile of young pigs. 2012 ASAS Midwestern Annual Meeting, Des Moines, Iowa.
  • Song R., Chen C., Johnston LJ., Kerr BJ, Weber TE, and Shurson GC, High sulfur content in dried distillers grains with solubles (DDGS) protects against oxidized lipids in DDGS by increasing sulfur-containing antioxidants in nursery pigs. 2012 ASAS Midwestern Annual Meeting, Des Moines, Iowa.
  • PAPERS IN PROCEEDINGS: Wang L., Yao D., Chen C. Effects of consuming oxidized vegetable oils on amino acid metabolism. The FASEB Journal. 2012;26:637.3


Progress 01/01/11 to 12/31/11

Outputs
OUTPUTS: In the past one year, we examined the health effects of consuming thermally-oxidized vegetable oil in three different studies: Study 1. we have conducted feeding experiments on C57BL/6 mice using 4 different customized diets, including fresh soybean oil-low fat (FSO-LF) diet (contain 7% fresh oil), fresh soybean oil-high fat (FSO-HF) diet (contain 16.7% fresh oil), heated soybean oil-low fat (HSO-LF) diet (contain 7% heated oil), and heated soybean oil-high fat (HSO-HF) diet (contain 16.7% heated oil). After 4-week treatment, urine, serum and tissue (liver, heart, kidney and others) samples have been collected. The influences of consuming heated vegetable oils on the metabolic system have been examined through the metabolomics-guided biochemical analysis. Study 2. Commonly used vegetable oils (corn and canola oils) and animal fats (poultry fat, and tallow) in pig diets were thermally stressed by rapid oxidation or slow oxidation. The effects of these oxidized dietary lipids on the growth and lipid metabolism of young pigs was compared with the effects of corresponding control lipids after a 38-day feeding experiment that comprised of 28 days in the nursery and 10 days in the metabolic room. During the first 28 days, all the pigs were allowed free access water and consume feed freely, while during last 10 days, all pigs were fed about 4% of individual body weight of day 28. Study 3. we established a LC-MS method to analyze the 4-Hydroxynonenal(HNE) and other toxic aldehydes contents in French fries from fast food restaurants. We have given a poster presentation on the results from study 1 (heated oil feeding experiment in mouse) in the Experimental Biology meeting 2011, Washington DC. PARTICIPANTS: Individuals: 1. Dr. Chi Chen is the principal investigator of this project, who is responsible for designing and performing both animal experiments and chemical analysis. 2. Dan Yao, a Research Fellow, is responsible for conducting LC-MS and chemical analysis of biological samples from this project. 3. Lei Wang, a PhD graduate student, is responsible for conducting animal experiments and performing metabolomic analysis. Collaborators: 1. Dr. Gerald C. Shurson, Professor in Swine Nutrition and Management,Department of Animal Science, 335D An Sci/Vet Med,1988 Fitch Avenue, St. Paul, MN 55108-6012, Telephone: 612-624-2764, Fax: 612-625-1210 Email: shurs001@umn.edu 2. Dr. Agnes S. Csallany,Professor,Department of Food Science and Nutrition,148 FScN, Phone: (612) 624-3683, Fax: (612) 625-5272 ascsalla@umn.edu TARGET AUDIENCES: 1. General public: Information from this project could help define the risk of consuming large quantity of oxidized fats and related degradation products in human and animals. 2. Biomedical research professionals: Novel information on chemical identities of reactive species in thermally-oxidized oils and their impacts on the metabolic system will become a scientific foundation for further mechanistic investigation on diet and disease. 3. Food industry: Results from this project could potentially provide useful information for adjusting current practices in preparing fried food. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.

Impacts
1. Mouse feeding experiment: The results from targeted metabolite analysis indicated that, in addition to the significant changes in the cholesterol and triglyceride levels, HSO treatment also altered the homeostasis of amino acids, such as the decrease of tryptophan level and the increase of proline in the serum. Metabolomic analysis of urine samples further identified the metabolites of tryptophan as the prominent biomarkers that contributing to the separation of urine samples from FSO and HSO treatment. Nicotinamide N-oxide, a metabolite from tryptophan-NAD+ pathway, was increased by HSO, while xanthurenic acid, another metabolite of tryptophan, was reduced by HSO. All these observations warrant further investigation on the effects of thermally-stressed oils on amino acid metabolism. 2. Pig feeding experiment: During the first 28 days of feeding experiment, lipid oxidation level significantly affected pigs' body weights. Pigs fed rapidly-oxidized lipids had lower body weight, lower average daily gain (ADG), and tended to have a lower average daily feed intake (ADFI)than those fed unheated controls. Pigs fed canola oil had a lower feed to gain ratio (F/G) compared to those fed other lipids. Relative liver organ weight from pigs fed rapid oxidation lipids tended to be higher than those fed the original oil. Different levels of lipid oxidation affected the expression of peroxisome proliferator-activated receptor alpha (PPARα)-targeted genes in the liver. The activation of PPARα and its target genes in pigs fed oxidized lipids is consistent with the lower triacylglycerols (TAG)levels in the livers of these pigs. Furthermore, pigs fed either corn or canola oil tended to have higher hepatic TAG concentration than those fed tallow supplemented diets. In conclusion, feeding oxidized fats and oil negatively affects the growth performance and health status of pigs, as indicated by the decreased ADFI and ADG, relatively higher liver weight, and lower hepatic TAG content than those fed original fats and oils. 3. French fries experiment: HNE in French fries was identified and quantified by LC-MS. Results suggested that French fries from fast food restaurants contains HNE (ranging from 70-320 ppm) and other unsaturated aldehydes.

Publications

  • Chen C., Schwinkendorf D., Yao D., and Csallany A.S. 2011. Toxicometabolomics of Heated Vegetable Oils-induced Adverse Effects, FASEB J 2011 25:624.4


Progress 10/01/10 to 12/31/10

Outputs
OUTPUTS: In order to define the metabolic effects and toxicities of dietary exposure of thermally-oxidized soybean oil, we have conducted a feeding experiment (1-12 weeks) using C57Bl/6 mice. Urine, serum and tissue (liver, heart, kidney and others with pathological changes) samples have been collected at multiple time points. General biochemical and histological parameters of susceptible tissues, such as liver, have been examined through liver function assays and H-E staining. Preliminary metabolomic analysis of urine samples has been performed using a high-resolution liquid chromatography-mass spectrometry (LC-MS) platform. In addition, we conducted a bolus dosing experiment by feeding animals heated soybean oil through oral gavage. The goal of this experiment was to identify the metabolites that directly originate from the chemicals produced in the heating process. PARTICIPANTS: 1. Dr. Chi Chen is the principal investigator of this project, who is responsible for designing and performing both animal experiments and chemical analysis. 2. Dan Yao, a Research Fellow, is responsible for conducting LC-MS and chemical analysis of biological samples from this project. 3. Lei Wang, a PhD graduate student, is responsible for conducting animal experiments and performing metabolomic analysis. TARGET AUDIENCES: 1. General public: Information from this study could help define the risk of consuming large quantity of fried food, which is the staple of Western diet. 2. Biomedical research professionals: Novel information on chemical identities of reactive species in thermally-oxidized oils and their impacts on the metabolic system will become a scientific foundation for further mechanistic investigation on diet and disease. 3. Food industry: Results from this project could potentially provide useful information for adjusting current practices in preparing fried food. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.

Impacts
Our feeding experiment indicated that the exposure of thermally-stressed soybean oil, which is high in linoleic acid, an omega-6 essential fatty acid, could significantly alter the metabolic system in treated animal. Some physiological indicators of these adverse effects were liver enlargement and mild weight loss. Through preliminary metabolomic analysis of urine samples from control and heated oil-treated mice, it was clear that various new exogenous chemicals (xenobiotics) formed by the heating process were extensively absorbed and metabolized in vivo since high-resolution LC-MS analysis of those urine samples had led to distinctive classification of animal groups in a multivariate model. Currently, we are conducting structural analysis of major chemical species contributing to this group classification. We are especially interested in identifying endogenous metabolites that are affected by the exposure of heated edible oils

Publications

  • No publications reported this period