Progress 10/01/08 to 09/30/13
Outputs
Target Audience: Scientific community, industry and general public through publications in scientific peer reviewed journals, conference presentations and seminars. Changes/Problems: Due to the lack of adequate funds for the measurements of the health effects of dietary HNE was not established. Measurements of the health effects of heated soybean oil in mice has been started but the final results are not established yet. The other in vivo experiments are summarized in the Accomplishments previously. What opportunities for training and professional development has the project provided? Training graduate students, both MS and Ph.D. Training postdoctoral fellow. How have the results been disseminated to communities of interest? Journal articles in peer reviewed journals. Seminars. Conference presentations. What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
Some but not all the objectives were completed due to the broad and complex nature of objectives and the lack of adequate funds. For Project A, the accomplishments were the following: 1) Established the temperature dependence of HNE formation in vegetable oils and butter oil; 2) Identified alpha, beta-unsaturated 4-hydroxy aldehydes such as HHE, HOE, HNE and HDE in thermally oxidized methyl esters; 3) Determined that 2, 4 decadienal is not a precursor in the formation of HNE; 4) Determined that the linoleic acid-containing imitation Mozzarella cheeses contain much higher levels of the toxic HNE then natural Mozzarella cheeses on heating; and 5) Established the concentration of HNE in French fries from 6 different fast food chain restaurants. Experiments are in progress to measure the relationships between linoleic acid concentration, temperature and time of heating on the formation of HHE, HOE, HNE and HDE in the following oils and fats: corn, soybean, peanut, canola oils, lard and beef tallow. Experiments also are continuing on determining the supressing effect of HNE formation in the presence of synthetic and natural antioxidants and their mixtures in corn oil at frying temperature (185 C) and for various times of heat treatments. In relation to the goals of Project B) the objectives of the experiments were changed based on availability of funds. Experiments were completed on the comparison between high polyunsaturated fatty acid and low polyunsaturated fatty acid containing fats on lipid peroxidation and low density lipoprotein oxidation in vivo. The effects of conjugated linoleic acids on 15-1 lipoxigenase-1 in vitro and in vivo were studied. Experiments were completed on the in vivo effect of dietary omega-3-fatty acids in human subjects. A significant increase of urinary HHE was found in vivo lipid peroxidation due to the higher linoleic acid containing diet. HDE was isolated for the first time and identified by LC/MS from human urine due to a high level w-3 fatty acid in the diet.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2013
Citation:
Kadiiska, Maria B., Basu, Samar, Brot, Nathan, Cooper, Christopher, Csallany, A. Saari, Davies, Michael J., George, Magdalene M., Murray, Dennis M., Roberts II, L. Jackson, Shigenaga, Mark K., Sohal, Rajindar S., Stocker, Roland, Van Thiel, David H., Wiswedel, Ingrid, Hatch, Gary E., Mason, Ronald P. Biomarkers of oxidative stress study. V. Ozone exposure of rats and its effect on lipids, proteins, and DNA in plasma and urine. Free Radical Biology and Medicine 61:408-415.
- Type:
Journal Articles
Status:
Published
Year Published:
2012
Citation:
Youn, Moonyeong, Csallany, A. Saari and Gallaher, D. Daniel. Whole grain consumption has a modest effect on the development of diabetes in Goto-Kikisakirat. Brit. J. Nutr. 10:192-201.
- Type:
Conference Papers and Presentations
Status:
Other
Year Published:
2013
Citation:
Seminar Presentation, Department of Nutrition, University of Capetown, south Africa, August 1, 2013.
- Type:
Journal Articles
Status:
Published
Year Published:
2011
Citation:
Youn, M., Csallany, A. Saari and Gallaher, D.D. The processing of wheat bran to release bound phenolic compounds improves insulin resistance and reduces visceral adiposity and liver cholesterol in ZDF rat. FASEB J. 25:329-4.
- Type:
Journal Articles
Status:
Published
Year Published:
2010
Citation:
Reckmeyer, Nicole M., Vickers, Zata M. and Csallany, A.S. Effect of free fatty acids on sweet, salty, sour and umani tastes. J. Sensory Studies 25:751-760.
- Type:
Journal Articles
Status:
Published
Year Published:
2010
Citation:
The antioxidant functions of tocopherol and tocotrienol homologues in oils, fats and food systems. J. Am. Oil Chem. Soc. 87:469-481.
- Type:
Journal Articles
Status:
Published
Year Published:
2009
Citation:
Han, I.H. and Csallany, A. Saari. Formation of toxic alpha, beta-unsaturated 4-hydroxy-aldehydes in thermally oxidized fatty acid methyl esters J. Am. Oil Chem. Soc. 86:253-260.
- Type:
Journal Articles
Status:
Published
Year Published:
2008
Citation:
Han, I.H. and Csallany, A. Saari. Temperature dependence of HNE formation in vegetable oils and butter oil. J. Am. Oil Chem. Soc. 85:777-782.
- Type:
Theses/Dissertations
Status:
Other
Year Published:
2013
Citation:
Ainslie-Waldman, Cheryl Elaine. Novel studies of fat and nutrient intakes and the risk of human cancer. Ph.D. Dissertation, University of Minnesota, pp. 338.
- Type:
Theses/Dissertations
Status:
Other
Year Published:
2008
Citation:
Youn, Moon Yeong. Effect of whole grain or whole grain components on Type 2 Diabetes in rats. Ph.D. Dissertation, University of Minnesota, pp. 198.
- Type:
Theses/Dissertations
Status:
Other
Year Published:
2008
Citation:
Cho, H. Effects of conjugated linoleic acids on 15-1 lipoxygenase-1 in vitro and in vivo. Ph.D. Dissertation, University of Minnesota, pp. 60.
- Type:
Conference Papers and Presentations
Status:
Other
Year Published:
2013
Citation:
Invited seminar speaker, Budapest University of Technology, Department of Biochemistry and Food Technology, Budapest, Hungary, July 23, 2013.
- Type:
Conference Papers and Presentations
Status:
Other
Year Published:
2012
Citation:
Poster Presentation at the HNE Symposium of the 16th Bi-Annual Conference of the Free Radical Society and HNE Symposium, London UK, 09/01/12 to 09/09/12.
- Type:
Conference Papers and Presentations
Status:
Other
Year Published:
2012
Citation:
Invited seminar speaker at the Department of Applied Biotechnology and Food Science, Budapest University of Technology, Budapest, Hungary, January 11, 2012 and July 17, 2012.
- Type:
Conference Papers and Presentations
Status:
Other
Year Published:
2011
Citation:
Youn, M., Csallany, A. Saari and Gallaher, D.D. The processing of wheat bran to release bound phenolic compounds improves insulin resistance and reduces visceral adiposity and liver cholesterol in ZDF rat. FASEB Meeting, March 17, 2011.
- Type:
Conference Papers and Presentations
Status:
Other
Year Published:
2010
Citation:
Poster presentation at the 101th Annual Meeting of the American Oil Chemists Society, 2010. A. Saary Csallany, In Hwa Han, D.W. Shoeman, C. Chen. 4-Hydroxynonenal (HNE), a Toxic Aldehyde, in French Fries from Fast Food Restaurants.
- Type:
Conference Papers and Presentations
Status:
Other
Year Published:
2010
Citation:
Invited seminar speaker (by Professor A. Salgo); Budapest University of Technology, Department of Biochemistry and Food Technology, Budapest, Hungary, June 18, 2010.
- Type:
Conference Papers and Presentations
Status:
Other
Year Published:
2009
Citation:
Poster presentation at the 100th Annual Meeting of the American Oil Chemists Society. 2009. Han, InHwa and Csallany, A. Saari. Impact of heat on toxic HNE formation in imitation and natural cheeses.
- Type:
Conference Papers and Presentations
Status:
Other
Year Published:
2009
Citation:
Poster presentation at the Annual FASEB Meeting. 2009. Youn, Moonyeon, Csallany, A. Saari and Gallaher, D.D. Whole grain does not slow diabetic progression in GK rats.
- Type:
Conference Papers and Presentations
Status:
Other
Year Published:
2009
Citation:
Invited seminar speaker; Budapest University of Technology, Department of Biochemistry and Food Technology, Budapest, Ungary. May 26, 2009.
- Type:
Conference Papers and Presentations
Status:
Other
Year Published:
2009
Citation:
Invited seminar speaker; Agricultural University of Athens, Department of Food Science and Technology, Athens, Greece. June 4, 2009.
- Type:
Conference Papers and Presentations
Status:
Other
Year Published:
2009
Citation:
Invited seminar speaker; NOVUS International, St. Louis, MO. May 6, 2009.
- Type:
Conference Papers and Presentations
Status:
Other
Year Published:
2008
Citation:
Budapest University of Technology, Budapest, Hungary, August 19, 2008, Formation of alpha, beta-hydroxy alkenals from four fatty acid methyl ester model systems.
- Type:
Conference Papers and Presentations
Status:
Other
Year Published:
2008
Citation:
99th AOCS Annual Meeting, Seattle, WA, Han, In Hwa and A. Saari Csallany, (2008) Temperature dependence of HNE formation in vegetable oils and butter oil.
- Type:
Conference Papers and Presentations
Status:
Other
Year Published:
2008
Citation:
2008 FESEB Meeting, San Francisco, LA, Youn, Moonyeon, A. Saari Csallany, Daniel D. Gallaher, (2008) Whole grain consumption does not slow development of non-insulin-dependent diabetes mellitus in GK rats.
- Type:
Journal Articles
Status:
Published
Year Published:
2013
Citation:
Seppanen, Christine M., Hyejung Cho, Csallany, A. Saari. Comparison between high-PUFA and low-PUFA fats on llipid peroxidation and LDL oxidation. Food and Nutrition Sciences 4:572-579.
- Type:
Conference Papers and Presentations
Status:
Other
Year Published:
2008
Citation:
Budapest University of Technology, Budapest, Hungary, January 22, 2008, Temperature dependence of HNE in vegetable oils and butter oil.
- Type:
Conference Papers and Presentations
Status:
Other
Year Published:
2008
Citation:
Otago University, Duned, New Zealand, July 31, 2008, Temperature dependence of HNE in vegetable oils and butter oil.
|
Progress 01/01/12 to 12/31/12
Outputs
OUTPUTS: Experiments were continued to determine the in vivo pro-oxidant effect of dietary highly unsaturated omega-3 fatty acids (n-3 PUFA) compared to dietary low n-3 PUFA in human subjects. Fifteen postmenstrual women were fed a low-fat diet (LF), 20% of energy from fat, or a low-fat diet supplemented with 3% of energy from n-3 PUFA faty acids (LFn-3 PUFA) for 8 weeks. Secondary lipid peroxidation products such as nonpolar (NP) and polar (P) aldehydes were measured from the urine as 2,4-dinitrophenyl hydrazones by high performance liquid chromatography in each subject. Most of the urinary nonpolar aldehydes had a tendency to increase due to dietary omega-3 fatty acids but did not reach significance. The concentration of the urinary polar aldehydes increased nearly two times due to omega-3 fatty acids administration to the human subjects. Identification of the polar aldehydes are under investigation. Urinary 4-hydroxy-2-trans-hexenal (HHE) concentration significantly increased due to dietary omega-3 fatty acids ingestion, these fatty acids are precursor for HHE formation. Experiments were started to investigate the concentration of 4-hydroxy-2-transnonenal (HNE), a toxic aldehyde, formation in potato chips because preliminary data indicate that potato chips contain somewhat similar levels of HNE as French fries. Experiments were started to determine the formation of HNE in various oils containing differing levels of linoleic acid, a precursor for HNE, at various temperatures and various times of heat treatments. Experiments were also started to determine the suppressing effects of various synthetic and natural antioxidants and their mixtures on HNE formation in frying oils at 185C frying temperature. Results from these experiments are not available yet. PARTICIPANTS: Graduate students: In Hwa Han and Moonyeong Youn. Dan Gallaher (University of Minnesota) TARGET AUDIENCES: Present research findings are intended for the scientific community, the food industry and for the public. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
The results of the experiment indicate that a diet supplemented with 3% omega-3 fatty acids increases in vivo lipid peroxidation in post-menopausal women. The concentration of polar lipophilic aldehydes excreted in the urine increased by nearly two-fold, while there was not a significant change in non-polar lipophilic aldehydes. Although these results suggest that the process of lipid peroxidation, which produces several reactive, toxic metabolites, is enhanced in a diet moderately supplemented with omega-3 fatty acids, more research is needed to elucidate what effect this increased lipid peroxidation would have on health outcomes, such as cancer, cardiovascular, and neurodegenerative diseases.
Publications
- Han, In Hwa and A. Saari Csallany. 2012. The toxic aldehyde, 4-hydroxy-2-trans-nonenal (HNE) formation in natural and imitation Mozzarella cheeses: heat treatment effects. J. Am. Oil Chem. Soc. 89:1801-1805.
- Youn, Moonyeong, A. Saari Csallany and D. Daniel Gallaher. 2012. whole grain consumption has a modest effect on the development of diabetes in Goto-Kikisakirat. Brit. J. Nutr. 10:192-201.
|
Progress 01/01/11 to 12/31/11
Outputs
OUTPUTS: Lipid peroxidation of polyunsaturated fatty acids yields several electrophilic, reactive carbonyl metabolites. We hypothesized that an increased intake of the highly unsaturated omega-3 fatty acids (n-PUFA) would lead to increased lipid peroxidation metabolites compared to a diet low in n-3 PUFA. As part of a randomized crossover dietary intervention trial, urinary markers of whole body lipid peroxidation were analyzed. Study subjects (n=15) included healthy, postmenopausal women who were fed a low-fat diet (LF), 20% of energy from fat, or low-fat diet supplemented with 3% of energy from n-3 PUFA fatty acids (LFn-3 PUFA) for 8 weeks. Urine was collected during two consecutive 24-hour collections in the last two days of each diet. Whole body lipid peroxidation within each subject was measured. Analysis via urinary TBARS was followed by a sensitive HPLC separation and quantification of 2,4-dinitrophenylhydrazine derivatives of lipophilic aldehydes, including 4-hydroxynonenal, 4-hydroxyhexenal, and other polar and nonpolar aldehydes. The results of the HPLC analysis showed that LFn-3 PUFA resulted in increased levels of lipid peroxidation products by two-fold relative to control LF. The identity of the urinary secondary oxidation products such as the polar and nonpolar aldehydes are under further investigation. Poster presentation at the FASEB annual meeting and publication of a paper in the B.J.N. PARTICIPANTS: Nothing significant to report during this reporting period. TARGET AUDIENCES: Present research findings are intended for the scientific community and for the public. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
These results show that increased n-3 intake leads to increased production of in vivo lipid peroxidation and the excretion of urinary secondary lipid peroxidation products. Present results also indicate that even if n-3 PUFAs have been described to be beneficial in several diseases, our findings suggest remaining cations with n-3 PUFAs supplementation because of the simultaneous increased in vivo lipid peroxidation, reactive oxygen species (RSO) production, secondary peroxidation products and the in vivo formation of toxic aldehydes.
Publications
- Youn, Moonyeon, Csallany, A. Saari, Gallaher, D. Daniel. 2011. Whole grain consumption has a modest effect on the development of diabetes in the Goto-Kakisakirat. British Journal of Nutrition June 3, p. 1-10 (E-Pub. ahead of print).
- Youn, M., Csallany, A. Saari and Gallaher D.D. 2011. The processing of wheat bran to release bound phenolic compounds improves insulin resistance and reduces visceral adiposity and liver cholesterol in ZDF rat. FASEB J. March 17, 2011, 25:329-4.
|
Progress 01/01/10 to 12/31/10
Outputs
OUTPUTS: Experiments were started to measure the concentration of 4-hydroxy-2-trans nonenal(HNE)in commercial potato chips. Six different samples (2 store brands and 4 name brands) were purchased and the nonpolar aldehydes including HNE were isolated from the oils extracted from the potato chips. Evaluation of lipid peroxidation levels in corn dried distillers grams with solubles (DDGS) were conducted. DDGS were obtained from 31 ethanol plants. The oils extracted from the DDGS and unaltered corn samples were analyzed for thiobarbituric acid reactive substances (TBARS) and peroxide value (PV). Animal experiments have been started to feed a total of 48 nursery pigs with various levels of oxidized DDGS and vitamin E to measure the nutrient digestibility, metabolic oxidation balance and growth performance. Animal experiments have also been started to measure the above mentioned parameters on the dietary effects of heat treated oils. Thirteen groups of 8 nursery pigs (104 total) were fed 10% unheated and heated (185 C for 6 hours and 95 C for 3 days) corn, canola oils, poultry fat and beef tallow. Results of the above animal experiments are not available yet. An animal study using obese diabetic rats has been conducted to determine the effect of feeding wheat bran that has been processed to liberate ferulic acid on several health-related metabolic parameters. A poster presentation was made at the 101st meeting of the American Oil Chemists Society, May 2010. Two seminars have been presented; Dept. of Food Chemistry and Biotechnology, Budapest University of Technology, Budapest, Hungary and Medical School of University of Pecs, Pecs, Hungary, June 2010. PARTICIPANTS: PI - Professor A. Saari Csallany, Research Associate Don Shoeman, Professor Dan Gallaher and from the Department of Animal Science Professor Gerald Shurson Graduate Assistants Pai Liu and Ran Song. TARGET AUDIENCES: Consumers, medical and nutrition practioners, food industry PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
Average oil concentration of potato chips was approximately 30% on dry basis. Preliminary experiments indicate the total HNE concentration is similar to the previously reported french fries, about 20 microgram HNE/100 g potato chips, however the oil content of potato chips was 2 to 3 times higher than the french fries, therefore HNE in the extracted oil was lower/g oil. Final data is not available yet. Partial results indicate that the toxic HNE similarly incorporated into the potato chips from the frying oil as we previously reported for french fries. It also shows that HNE remains in the commercially produced products after distribution to the public. Since potato chip consumption is very frequent by the public, it may be a health concern since HNE toxicity is related to a number of common pathological conditions. Average oil concentration for DDGS were 10.4%, TBARS ranged between 1.0 to 5.2 ng MDA equivalents/mg oil, PV ranged from 4.2 to 84.1 meq/kg oil. Reference corn sample contained 0.2 ng MDA equivalents/mg oil, and PV was 3.1 meq/kg oil. The level of oxidation of DDGS was highly correlated with color of the samples. The fact that oxidized DDGS, a byproduct of ethanol production, and heated used fats are commonly fed to various farm animals, it remains to be seen from the ongoing animals experiments that what is the dietary effect of heated oils and oxidized DDGS on health and physiological changes in the animals. Our results of the wheat bran feeding indicate that the processed wheat bran lowers liver cholesterol, lowers fasting plasma glucose, improves insulin sensitivity, increases plasma adiponectin, and decreases fat pad weight. Thus, in this animal model of metabolic disease, wheat bran processed to release bound ferulic acid produces a number of positive metabolic changes.
Publications
- Seppanen, C.M., Song, Q. and Csallany, A.S. 2010. The antioxidant functions of tocopherol and tocotrienol homologues in oils, fats and food systems. J. Am. Oil Chem. Soc. 87:469-481.
- Reckmeyer, N.M., Vickers, Z.M. and Csallany, A.S. 2010. Effect of free fatty acids on sweet, salty, sour and umani tastes. J. Sensory Studies 25:751-760.
|
Progress 01/01/09 to 12/31/09
Outputs
OUTPUTS: Experiments were conducted to access the concentration of the toxic lipid peroxidation product 4-hydroxy-2-trans nonenal (HNE) in French fries obtained from six US commercial fast food restaurants. Duplicate samples of French fries were purchased the same day between 2 and 3 pm from all of the six restaurants and 2 duplicate samples were purchased from one of the restaurants at 12 to 1, 2 to 3, 4 to 5 and 6 to 7 pm. Samples were extracted for their oil content and analyzed for HNE concentrations by HPLC with our previously published method. HNE was identified by co-chromatography with pure HNE standard in different polarity solvent systems and by liquid chromatography mass-spectrometry (LC-MS). The extracted oils from French fries were measured for their fatty acid distributions using gas chromatography(GC). HNE concentrations in French fries from the six fast food restaurants purchased between 2-3 pm were contained between 32.15 and 7.83 microgram HNE/100 g French fries and 4.50 to 0.90 microgram HNE/g of fat. HNE concentrations in French fries purchased from the above mentioned two restaurants between 12 and 7 pm fluctuated between 32.15 and 19.07 and 10.21 and 7.47 microgram NHE/100 g French fries. Collaborative experiments were conducted on the effect of whole grain consumption on the development of diabetes in Goto-Kakisai (GK)rats. Collaboratiave experiments are underway to evaluate the physiological, biochemical and metabolic changes in mice due to dietary HNE feeding heat treated soybean oil (6 h, 190 C). Results are not yet available. Additional collaborative experiments also started to measure the effect of dietary heat treated soybean oil (high linoleic acid) and compare to heat treated beef tallow (low linoleic acid) on human arterial construction. Linoleic acid is a precursor for HNE. Results are not yet available. Partial findings of the projects have been reported in a poster presentation at the Annual Meeting of the American Oil Chemists Society (2009). Two seminars have been presented; one at the University of Athens, Athens, Greece and the other at the Budapest University of Technology, Budapest, Hungary in June 2009. PARTICIPANTS: Dr. Han, In Hwa, Research Scientist; Dr. Shoeman, D., Research Scientist; Collaborations: Dr. Gallaher, D.D., Dr. Chen, C., Dr. Dengel, D. TARGET AUDIENCES: Scientific community, industry, general public PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
Results show that high PUFA oils (containing high levels of linoleic acid) due to continuous heat at frying temperature produced considerable amounts of HNE, a toxic degradation product of linoleic acid, which incorporated into the fried food from the oil. Frequently consumed food containing considerable amounts of HNE, a toxic aldehyde, may be a public health concern since its toxicity is related to a number of common pathological conditions. Collaborative experiments on the effects of whole grain consumption of diabetes in GK rats showed that whole grains, barley and oats, may have a modest beneficial effect early in the course of type 2 diabetes.
Publications
- Han, I.H. and Csallany, A.S. 2009. Formation of toxic alpha, beta-unsaturated 4-hydroxy-aldehydes in thermally oxidized fatty acid methyl esters. Journal of the American Oil Chemists' Society, 86: 253-260.
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Progress 01/01/08 to 12/31/08
Outputs
OUTPUTS: Some of the findings of the project have been demonstrated at the Annual Meeting of the American Oil Chemists Society (2008) Two seminars have been presented at the Budapest University of Technology, Budapest, Hungary and one at the University of Otago, New Zealand. Some results of the above experiments have been discussed in a course 8636 Lipid Chemistry. PARTICIPANTS: Dr. Han, In Hwa. Post Doctoral Research Associate. Collaboration with Dr. Gallaher, D.D. and Dr. Vickers, Z. M. TARGET AUDIENCES: Scientific community, industry, general public PROJECT MODIFICATIONS: None
Impacts
Experiments were conducted to demonstrate that heat treated natural low moisture part skim mozzarella cheeses (NC) produce much less 4-hydroxy-2-trans nonenal (HNE), a toxic compound, than imitation mozzarella cheeses (IC). The fat source in IC is partially hydrogenated vegetable oil which contains much higher levels of linoleic acid (LA) than NC. Due to heat treatment, such as baking, LA degrades to HNE and this toxic compound is absorbed from the food into the body after consumption. Lowering the consumption of toxic compounds from food is an objective in maintaining good health. A new method was developed to isolate and quantify the HNE formation in heat treated cheeses. A new procedure had to be developed because of the difficulty of HNE isolation from baked cheeses compared to the, previously published, method used for the isolation of HNE from vegetable oils and fats. One hundred gram batches of cheese samples were heated at 204 C for 0, 30 and 60 min, and at 232 C for 0 and 30 min to mimic baking conditions. Unheated cheeses were used as controls. After solvent extractions, the extracted aldehydes were converted to 2,4-dinitrophenylhydrazones, subjected to prepurification by thin layer chromatography (TLC) and the HNE-hydrazone was separated from other aldehyde hydrazones by high performance liquid chromatography (HPLC)and quantified at 380 nm by UV absorption. Results showed that HNE formations were higher in IC than in NC at both temperatures and no HNE was found in unheated cheeses. HNE concentrations after 30 min of heat treatment at 204 degree C were 8.5 times higher in IC than in NC and at 232 degree C 4.7 times higher in IC than in NC. When heating time was continued at 204 degree C for 1 hour in both cheeses the HNE continued to increase. HNE concentration was 7.5 times higher in IC and 13.6 times higher in NC at 232 degree C compared to 204 degree C for 30 min of heating periods. Since IC are made with vegetable oils which contain much higher levels of LA than dairy fat it is not surprising that in heat treated IC the HNE formation is higher than in NC. Since HNE formation is temperature and time dependent lower heating temperature and shorter heating time is recommended to reduce the formation of toxic HNE in cheeses. Experiments have also been conducted to find the optimum temperature and time to produce maximum levels of fatty acid hydroperoxides in methyl esters of beef tallow (BF), olive oil (OO) and oleic acid (OA) for possible use for acetal formation in production of biodiesel. The samples were peroxidized in open beakers in the presence of bubbling air at several temperatures; 78, 85, 95 degree C and at room temperature. Hydroperoxide productions were repeatedly measured during the peroxidation process. Optimum temperatures were found to be 85 degree C for BF, and 78 degree C for OO and OA and maximum peroxide levels were 875 meq/kg at 33hours for BF, 850 meq/kg at 28 hours for OO and 1275 meq/kg at 51 hours for OA.
Publications
- Han, I.H. and Csallany, A.S. 2008. Temperature dependence of HNE formation in vegetable oils and butter oil. J. Am. Oil Chem. Soc. 85:777-782.
- Cho, H. 2008. Effects of conjugated linoleic acids on 15-1 lipoxygenase-1 in vitro and in vivo. Ph.D. Dissertation. University of Minnesota. pp. 187.
- Reckmeyer, N. 2008. Effects of fatty acids on taste. MS. Plan A Thesis. University of Minnesota. pp. 60.
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Progress 01/01/07 to 12/31/07
Outputs
OUTPUTS: Some of the findings of the project have been demonstrated at the Annual Meeting of the American Oil Chemists Society (2007). Seminar has been presented at the Budapest University of Technology, Budapest, Hungary. Some results of the above experiments have been discussed in a course: Topics in Nutrition, Nutr 8620.
PARTICIPANTS: Dr. In Hwa Han, Associate Scientist
Impacts
Model systems of methyl linoleate (MLO) and methyl linolenate (MLN) were used to determine the thermal formation of toxic alpha, beta unsaturated aldehydes in high PUFA vegetable oils. MLO and MLN were heated at 190 C (frying temperature) for 0 to 6 hours. Isolation by HPLC and identification in three different polarity systems showed that MLO is a precursor for the toxic 4-hydroxynonenal (HNE) with the highest concentration, followed by 4-hydroxy octenal (HOE) and 4-hydroxy hexenal (HHE). MLN is a precursor for HOE with highest concentration followed by HHE. MLN is not a precursor for HNE, the most toxic of the four aldehydes. 2,4, decadienal was not converted to HNE due to thermal treatment up to six hours, therefore it is not an intermediate in HNE formation from linoleic acid (LA) oxidation. Temperature dependence of HNE formation was measured in high and low LA containing oils; corn, soybean and butter oils respectively at 190 C and 218 C from 0 to 30 min. HNE
concentrations were much lower at both temperatures for butter oil than in the high PUFA oils. Increase of HNE formation at 218 C versus 190 C after 30 min heating were 770, 390, and 270% for butter, corn and soybean oils respectively. The formation of toxic HNE in heated vegetable oils is dependent on the concentration of LA, the temperature and the length of heating time. The MLO and MLN model systems show the genesis of toxic HNE, HOE and HHE in heated edible oils. Due to the present findings, oils in the future can be better selected for low LA content, consequently for lower toxic compound (HHE, HOE, HHE) formations due to heat treatment (frying). It is also demonstrated that lower temperature for frying is healthier than frying at high temperature, because of lower toxic compounds formation at lower temperatures. Butter oil, because of its low LA content, produces lower toxic compounds due to heat treatment, therefore it is a healthier source of fat than high LA containing
vegetable oils used with elevated temperatures.
Publications
- No publications reported this period
|
Progress 01/01/06 to 12/31/06
Outputs
Experiments were continued to investigate the formation of four toxic alpha, beta-unsaturated-hydroxy aldehydes; 4-hydroxy-2-trans-hexenal (HHE), 4-hydroxy-2-trans-octenal (HOE), 4-hydroxy-2-transnonenal nonenal (HNE), and 4-hydroxy-2-trans-decenal (HDE) in model systems such as methyl-stearate, methyl-oleate, methyl-linoleate and methyl-linoleate, heated at 185 C (frying temperature) at 0, 1, 3 and 6 hour (using the method developed in this laboratory) to determine their precursors and the rate of formation of these four compounds. Isolation and identification of the secondary peroxidation compounds revealed that methyl stearate (no double bond) and methyl oleate (one double bond) due to heat treatment, at frying temperature, do not degrade to any of the four aldehydes (HHE, HOE, HNE and HDE). Present results are indicating that saturated and monounsaturated fatty acids are not precursors of the above mentioned toxic alpha, beta-unsaturated-hydroxy aldehdyes.
Experiments are continuing to determine the formation of HHE, HOE, HNE and HDE from methyl linoleate (two double bonds) and methyl linolenate (three double bonds) at frying temperature using the above mentioned heating methods. The formation of 2, 4-decadienal will be also determined in these experiments; a possible road to the alpha, beta-unsaturated aldehyde formation. Experiments have been started to measure the health effects of butter compared to low trans hydrogenated fats. Butter oil and soybean oil samples were heated at 185 C for 0, 1, 2, 3 and 6 hours. The secondary lipid peroxide products, the nonpolar and polar aldehydes and related carbonyl compounds, as 2, 4 dinitrophenyl hydrazones were isolated and quantitated by HPLC using two different concentrations of methanol:water gradient elution profiles. Preliminary results indicate that heated soybean oil contains higher levels of oxidative degradation products including the toxic alpha, beta unsaturated aldehydes compared to
heated butter oil under the same conditions. An animal feeding trial (a collaborative study with Dr. Gallaher) is also in progress investigating the effect of whole grain containing diets on diabetes, using the GK rat, a commonly used model of type 2 diabetes. After two months of feedings, blood samples were collected and the glycosylated hemoglobin values, a long-term measure of glucose control, and fasting serum insulin concentrations are determined. Plasma glucose concentration and insulin values are being determined which will allow to calculate insulin resistance.
Impacts
Using pure fatty acid acid methyl ester model systems will reveal the genesis of toxic alpha, beta-unsaturated hydroxy aldehydes, HHE, HOE, HNE and HDE, from the individual fatty acids which will be useful in determining their existence in various oils depending on their fatty acid composition. The butter oil project will show the beneficial effect of the use of butter and butter oil in baked goods and in frying because of its low levels of heat induced toxic compound (HNE) formation, compared to high linoleic acid containing vegetable oils.
Publications
- Cho, H. and Csallany, A.S. 2006. Effects of esterified linoleic acid and conjugated linoleic acid on pulmonary 15 lipoxygenase-1 activity. FASEB J. LB495.
- Fritz, K. 2006. Modification of the production of secondary lipid peroxidation products in vivo in humans and rats by dietary fat and soy isoflavones. Ph.D. Thesis. University of Minnesota. p. 147.
- Matter, B., Malejka-Giganti, D., Csallany, A.S. and Tretyakova, N. 2006. Quantitative analysis of the oxidative DNA lesion, 2, 2-diamino-4-(2deoxy-(beta)-D-erythro-pentofuranosyl) amino] -5 (2H)-oxazolone (oxazolone), in vitro and in vivo by isotope dilution-capillary HPLC-ESI-MS/MS. J. Nucleic Acid Res. Oct. 4, (EPub. ahead of print).
- Seppanen, C.M. and Csallany, A.S. 2006. The effect of intermittent and continuous heating of soybean oil at frying temperature on the formation of HNE and other alpha-, beta-unsaturated hydroxyaldehydes. J. Am. Oil Chem. Soc. 83(2):121-127.
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Progress 01/01/05 to 12/31/05
Outputs
Experiments were continued to determine the protective effect of stearic acid containing diet compared to high polyunsaturated fatty acids (PUFA) diet on in vivo lipid peroxidation. Rats were fed 10% beef tallow or 10% soybean oil as a fat source for 19 weeks. Urinary excretion of secondary oxidation products such as nonpolar and polar aldehydes and related carbonyl compounds were measured after 24 hours of fasting by reverse phase high performance liquid chromatography (HPLC). Both the sum of the individually measured nonpolar and polar aldehydes and related carbonyl compounds were significantly increased due to the high PUFA diet compared to the high stearic acid diet. Four of the individual polar secondary oxidation products increased signficantly and eight compounds showed near significant increases due to the high PUFA diet compared to the high stearic acid diet. The livers of rats, from both dietary groups, were analyzed for the level of lipid peroxidation
products. In the livers the thiobarbituric acid reactive substances (TBARS), measuring secondary peroxidation products, was found to increase significantly due to the high PUFA diet when compared to the high stearic acid diet. Experiments are continued to investigate the differences in the formation of the toxic 4-hydroxy-2-trans-nonenal (HNE) in the livers of rats fed the high PUFA and high stearic acid diets using HPLC and fluorescent detection. Experiments were started to investigate the formation of the four toxic alpha, beta-unsaturated-hydroxy aldehydes, such as 4-hydroxy-2-trans-hexenal (HHE), 4-hydroxy-2-trans-nonenal (HNE), 4-hydroxy-2-trans-octenal (HOE) and 4-hydroxy-2-trans-decenal (HDE) in model systems such as methyl-stearate, methyl-oleate, methyl-linoleate and methyl-linolenate heated at 185 C (frying temperature) using the method developed in this laboratory to determine their precursors and the rate of formation of the individual products.
Impacts
The urinary excretions of aldehydes and related carbonyl compounds and liver TBARS both measure the secondary products of in vivo lipid peroxidation. Both of these experiments demonstrate that a diet high in stearic acid generates lower level of lipid peroxidation in vivo than a high PUFA diet. This is in good agreement with our previous findings that high stearic acid diet increased the oxidative resistance of VLDL and LDL measured by diene conjugation. Using pure fatty acid methyl ester model systems will reveal the genesis of toxic alpha, beta-unsaturated hydroxy aldehydes, HHE, HOE, HNE and HDE, from the individual fatty acids which will be useful in determining their existence in various oils depending on the fatty acid composition of their triglycerides.
Publications
- Cho, H., Gallaher, D. and Csallany, A.S. 2005. Conjugated linoleic acid, cis-9, trans-11, is a substrate for pulmonary 15-lipoxygenase-1 in rat. J. Agr. Food. Chem. 53:7262-7266.
- Csallany, A.S., Seppanen, C.M. and Fritz, K.L. 2005. Effect of high stearic acid containing fat on markers for in vivo lipid peroxidation. Intl. J. Food Sci. Nutr. 56(8):1-13.
- Seppanen, C.M. and Csallany, A.S. 2005. The effect of intermittent and continuous heating of soybean oil at frying temperature on the formation of 4-hydroxynonenal and other polar lipophilic aldehydes. 96th AOCS Annual Meeting Abstracts, AOCS Press, p. 77.
- Cho, H., Gallaher, D.D. and Csallany, A.S. 2005. Trans-10, cis-12 conjugated linoleic acid isomer exhibits stronger inhibition of 15-lipoxygenase-1 than cis-9, trans-11 conjugated linoleic acid isomer in rat. FASEB J. (19) A994.
- Christine M. Seppanen. 2005. Ph.D. Thesis. Isolation and identification of polar lipophilic aldehydes in oxidized vegetable oils. Univ. of Minnesota. p. 147.
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Progress 01/01/04 to 12/31/04
Outputs
Experiments were continued in conjugated linoleic acid (CLA) which has been shown to relate to the protection of breast carcinogens. The inhibition of rat pulmonary 15-LOX-1 enzyme using linoleic acid (LA) as a substrate by two conjugated linoleic acid (CLA) isomers, cis 9, trans 11 and trans 10, cis 12 was investigated. Activity was measured by the production of 13(S)-hydroxyoctadecadienoic acid (13(S)-HODE), an LA metabolite. Both CLA isomers c9, t11 and t10, c12 were found to inhibit 15-LOX-1 in a dose-dependent manner, resulting in a decrease in 13(S)-HODE production from 8 mM CLA by 33% and 57% respectively. Inhibition of 15-LOX-1 was also inhibited by nordihydroguaiaretic acid (NDGA), a known LOX inhibitor, and was used as a control for enzyme activity. Results indicate that both the above CLA isomers inhibit 15-LOX-1 activity, with t10, c12 being a more effective inhibitor of LA oxidation than the c9, t11 isomer. The above CLA isomers were also investigated as
possible substrates for 15-LOX-1. It was found that c9, t11 CLA was 25% as active as LA as a substrate for 15-LOX-1, however, no 13(S)-HODE was produced from t10, c12 CLA. The authenticity of the production of 13(S)-HODE from c9, t11 as a substrate was established by isolation and co-chromatography with pure standard on HPLC using nonradioactive and 14c9, t11 CLA. The formation of toxic a-b unsaturated hydroxy aldehydes was investigated in thermally oxidized vegetable (soybean) oil at frying temperature. Besides 4-hydroxy-2-trans-nonenal (HNE), three more of the very reactive a-b unsaturated hydroxy aldehydes were isolated and identified in the heated oil. These are 4-hydroxy-trans-hexenal, 4-hydroxy-trans-octenal and 4-hydroxy-trans-decenal, however the concentrations of these compounds were much smaller than HNE, the major compound found due to thermal oxidation of LA in vegetable oils. Experiments were continued to measure the in vivo effects of 10% beef tallow (BT), 10% soybean
oil (SO) or 5% BT and 5% SO mixture. BT diet increased the oxidative resistance of VLDL and LDL measured by dienne conjugation compared to SO and BT and SO without increasing total serum triglyceride levels. Whole body oxidation measured by urinary excretion of secondary oxidation products (nonpolar and polar aldehydes) are under investigation.
Impacts
CLA is an anticarcinogen and can be found in butter and other dairy products. Present experiments investigate the inhibition of CLA on lipoxygenase enzyme induced in vivo LA oxidation and the formation of its metabolite which induce cell proliferation. The formation of toxic compounds in heated vegetable oil was investigated. Three more very reactive toxic lipid aldehydes were found besides the previously reported toxic HNE at frying temperature. Experiments were continued to determine the in vivo effect of high stearic acid diet compared to high PUFA diet on LDL and whole body oxidation measured by various parameters.
Publications
- Seppanen, C.M. and Csallany, A.S. 2004. Incorporation of the toxic aldehyde 4-hydroxy-2-trans-nonenal into food fried in thermally oxidized soybean oil. JAOCS 81(12):1137-1141.
- Cho, H., Gallaher, C.M., Gallaher, D.D. and Csallany, A.S. 2004. Dietary synthetic and natural linoleic acid inhibits 15-lipoxygenase and reduces cancer risk in rats. FASEB J. 18(4):LB264.
- Seppanen, C.M. and Csallany, A.S. 2004. The effect of intermittent heating of soybean oil on formation of 4-hydroxynonenal and other polar lipophilic aldehydes at frying temperature. Annual Meeting Abstracts, AOCS Press, p. 93.
- Csallany, A.S. and Seppanen, C.M. 2004. Formation of nonpolar lipophilic aldehydes and related carbonyl compounds in saturated and unsaturated oils at frying temperature. Annual Meeting Abstracts, AOCS Press, p. 94.
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Progress 01/01/03 to 12/31/03
Outputs
Conjugated linoleic acids (CLA), especially the cis9, trans11 (c9, t11) isomer, has been shown to relate to the protection of breast cancer. However, the mode of action has not been elucidated. This laboratory is examining the relation of c9, t11-CLA to the function of 15-1 lipoxygenase (15-1 LOX) activity which may elucidate its relation to its anticarcinogenic activity. Experiments were continued to determine the effect of c9, t11-CLA and trans10, cis12 (t10, c12) CLA on the activity of 15-1 LOX enzyme on the formation of its oxidation product, 13(s) hydroxyoctadecadienoic acid (13(s) HODE) in vitro and in vivo. When linoleic acid methyl ester (LAME) was used as a substrate for 15-1 LOX, in the presence of c9, t11-CLA the production of 13(s) HODE was greatly increased. In the presence of t10, c12-CLA, however 13(s) HODE was only slightly increased and in the presence of NDGA (enzyme inhibitor) almost no 13(s) HODE was produced. Verification of 13(s) HODE production
was achieved by cochromatography with standard cold and C14-13(s) HODE [isolated from the reaction of C14- c9, t11-CLA with rat lung 15-1 LOX] c9, t11-CLA was also found to be a substrate for 15-1 LOX. Synergism seems to exist between LAME and c9, t11-CLA in the production of 13(s) HODE. Experiments were also conducted to measure the possible in vivo effect of c9, t11-CLA in isolated rat liver 15-1 LOX enzyme and measured by 13(s) HODE production using linoleic acid (LA) as a substrate. Diets containing 0, 6 and 30 mg c9, t11-CLA/g fat (20% total fat) were fed to rats for 9 weeks. The activity of liver 15-1 LOX was depressed significantly due to dietary CLA administration measured by 13(s) HODE production. Lower in vivo 13(s) HODE production may relate to CLA anticarcinogenic activity through the depressed 15-1 LOX enzyme activity. The uptake of lipophilic aldehydes, especially the toxic 4-hydroxynonenal (HNE) into foods fried in thermally oxidized soybean oil (5 hrs, 185 C), was
investigated. Oil extracted from fried potato had the same concentrations of aldehydes including the toxic HNE as the frying oil (57 to 59 mg HNE/100 g oil). Intermittent heating of oil (1 hr/day for 5 days) contained the same quantity of lipophilic aldehydes as continuous heating, indicating the accumulation of the toxic HNE in reheated oils and the importance of heating time in the process. Experiments have been started to determine the presursor of HNE using methyl esters of stearic, oleic, linoleic and linolenic acids, which are the main components of edible oils and fats. Investigations started to determine the effects of dietary beef tallow vs. soybean oil on low-density lipoprotein (LDL) oxidation in rats. Rats were fed for 19 weeks, 10% beef tallow, 10% soybean oil or 5% beef tallow and 5% soybean oil containing diets. In vivo oxidation of LDL + VLDL was assessed by baseline diene conjugation, copper induced oxidation, TBARS measurements, cholesterol and tricylgeride content.
Final data is not available yet.
Impacts
The present experiments investigate the mode of action of CLA, an anticarcinogen, on its effect on the formation of enzyme induced lipid oxidation products. Dietary CLA repressed the formation of enzyme induced lipid oxidation in rat liver tissues, indicating that CLA anticarcinogenic action maybe related to 15-1 LOX enzyme activity. The incorporation of toxic HNE into food fried in heated oil has been determined. HNE concentration increased at the same rate both in continuously or intermittently heated oils. Experiments started to determine the fatty acid precursor of HNE and to measure LDL oxidation in vivo in the presence of dietary beef tallow and soybean oil.
Publications
- Fritz, K.L., Seppanen, C.M., Kurzer, M.S. and Csallany, A.Saari. 2003 The in vivo antioxidant activity of soybean isoflavones in human subjects. Nutr. Res. 23(4): 479-487.
- Cho, H., Seppanen, C., and Csallany, A. Saari. 2003. Effect of low and high stearic acid diets on diabetes-induced in vivo lipid oxidation in rats. FASEB J. 17(4):749.
- Seppanen, C.M., and Csallany, A. Saari. 2003. 4-Hydroxynonenal in foods fried in thermally oxidized oil. Annual Meeting Abstracts, AOCS Press, p.41.
- Cho, H., Gallaher, D.D., and Csallany, A. Saari. 2003. 13(S)-Hydroxyoctadecadienoic acid production from 15-lipoxygenase in the presence and the absence of CLA using radioisotope. Annual Meeting Abstracts, AOCS Press, p. 49.
- Song, Q. 2003. The antioxidant functions of tocopherol and tocotrienol homologues in oils, fats and food systems. M.S. Thesis, University of Minnesota, pp. 55.
- Cho, H., Gallaher, D.D., and Csallany, A. Saari. 2003. Non-radiometric HPLC measurement of 13(S)-hydroxyoctadecadienoic acid from rat tissues. Anal. Biochem. 318(1):47-51.
- Seppanen, C.M., Rahmani, M., Csallany, A. Saari. 2003. Simultaneous determination of chlorophylls, pheophytins, beta-carotene, tocopherols, and tocotrienols in olive and soybean oils by high-performance liquid chromatography. J. Food Sci. 68(5):1644-1647.
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Progress 01/01/02 to 12/31/02
Outputs
The effects of beef tallow (BT)(high stearic acid)(SA) and soybean oil (SO)(high PUFA) diets were investigated on in vivo lipid peroxidation in diabetic rats by copper-induced serum oxidation and urinary TBARS measurements. Positive correlation was found between the above measured parameters. Lipid oxidation was so strongly elevated in diabetes for both diets that the protective effect of SA in BT was not measurable in diabetes. The control BT group had the highest resistance to serum oxidation. Investigation of the dietary effects of BT and SO on low-density lipoprotein (LDL) oxidation has started with 30 experimental rats using 10% BT or 10% SO or 5% BT + 5% SO containing diets. Identification of lipophilic aldehyde formation in SO at 185C continued by identifying the nonpolar aldehydes and ketones by cochromatography in various polarity solvent systems. Experiments started to measure the incorporation of toxic 4-hydroxynonenal into fried potato from heated SO. A
new HPLC method was developed for the determination of 13(S)-hydroxyoctadecadienoic acid (13S-HODE) from rat liver and lung tissues using silica column, 13-S-HODE was isolated in the presence and absence of conjugated linoleic acid (CLA) c9t11 and t10c12, from 15-lipoxygenase-1 oxidized linoleic acid (LA). The formation of 13-S-HODE was verified by the use of radio isotopes indicating a synergism between LA and CLA. The effect of CLA is being measured for 13S-HODE levels in liver and lung rat tissues.
Impacts
Lipid peroxidation in vivo was determined to be so strongly elevated in diabetes that the protective effect from stearic acid in BT was not measurable. From heated SO a toxic compound, 4-hydroxynonenal, was isolated and its incorporation into fried food is being investigated. CLA is an anticarcinogen and can be found in butter and other dairy products. Present experiments investigate the mode of action of this anticarcinogenic agent. In the presence of CLA, LA oxidation was increased by lipoxygenase enzyme, the product was measured by a newly developed analytical procedure.
Publications
- Csallany, A.S., Lee, J.H. and Shoeman D.S. 2002 Protection of superoxide-induced cholesterol oxidation by antioxidants in protic conditions. Int. J. Food Sci. & Nutr. 53:403-409.
- Seppanen, C.M. and Csallany, A.S. 2002. Formation of 4-hydroxynonenal, a toxic aldehyde, in soybean oil at frying temperature. J. Am. Oil Chem. Soc. 79(7):1033-1038.
- Seppanen, C.M. and Csallany, A.S. 2002. The effect of paprika carotenoids on in vivo lipid peroxidation measured by urinary excretion of secondary oxidation products. Nutr. Res. 22(9):1055-1065.
- Seppanen, C.M., Rahmani, M., Csallany, A.S. 2002. Simultaneous determination of chlorophylls, pheophytins, B-carotene, tocopherols, and tocotrienols in vegetable oils and fats by HPLC. 2002 Annual Meeting Abstracts. 93rd AOCS Annual Meeting & Expo., Montreal, Quebec, Canada, AOCS Press: p. S18.
- Cho, H., Gallaher, D.D. and Csallany, A.S. 2002. A new HPLC method for the determination of 13(S)-hydroxyoctadecadienoic acid in rat liver and lung tissues. FASEB J. 16(5):A633.
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Progress 01/01/01 to 12/31/01
Outputs
Investigation began to measure the protective effect of dietary beef tallow, a high stearic acid containing fat, on in vivo lipid peroxidation in diabetic rats. Experiments were conducted on the formation and decomposition of toxic polar lipophilic aldehydes in soybean oil at frying temperature, 0 to 10 hours, in the presence of alpha-, beta-, gamma- and delta-tocopherols and beta-carotene. Gamma-tocopherol showed the fastest loss, alpha-tocopherol and beta-carotene were completely consumed, but delta-tocopherol decreased the least at the end of the heating period. Since delta tocopherol is the least effective antioxidant results suggest the relative heat stability of the chromanol ring of these compounds and the disappearance was due to its antioxidant function at high temperature and not the decomposition of the antioxidant. An HPLC system was developed for simultaneous measurement of tocopherol, tocotrienol, chlorophyll and pheophytin homologs and beta-carotene in
vegetable oils using in series a UV and a fluorescent detector. Investigations were continued to measure the effect of conjugated linoleic acid (CLA) (cis 9, trans 11, and trans 10, cis 12) on 15-lipoxygenase-1, activity and on the formation of 13-hydroxy octadecadienoic acid (13 HODE) formation in vivo. It was found that in the presence of CLA, 15-lipoxygenase-1 increase significantly 13-HODE production from linoleic acid. The increase of 13-HODE may relate to down regulation of cyclooxygenase activity, suppressing prostaglandin synthesis, which relate to carcinogenesis, therefore CLA would act as an anticarcinogen. Verification of results continue with radioactive isotope studies.
Impacts
Lipid oxidation is high in diabetes which relates to increased heart disease. Dietary beef tallow is being tested for lowering lipid peroxidation in vivo in diabetes. Vegetable oils contain toxic compounds at frying temperatures which have been shown to relate to certain pathological conditions. Antixodants are used to test the retardation of their formation at frying temperature. Conjugated linoleic acid is an anticarcinogenic agent and can be found in butter and other dairy products. Present experiments investigate the mode of action of this anticarcinogenic agent.
Publications
- Seppanen, C.M. and Csallany, A. Saari. 2001. Formation of the toxic lipophilic aldehydes 4-hydroxynonenal and 4-hydroxyhexenal in heated vegetable oil. Inform 12: 584.
- Seppanen, C.M. and Csallany, A. Saari. 2001. Simultaneous determination of lipophilic aldehydes by high-performance liquid chromatography in vegetable oil. J. Am Oil Chem Soc. 78: 1253-1260.
- Seppanan, C.M., Fritz, K.L. and Csallany, A. Saari. 2001. Comparison of the effects of dietary saturated fat and polyunsaturated fat on the in vivo lipid peroxidation. FASEB J. 15:A 609.
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Progress 01/01/00 to 12/31/00
Outputs
Studies on the beneficial effect of high stearic acid diet on in vivo lipid peroxidation were completed. The following analyses were conducted in the third year of this study; vitamin E isomers, b-carotene, fatty acid distribution of the dietary beef tallow, soybean oil, cocoa butter and manhaden oil used in feeding experiment of rats. a-Tocopherol, oxygen-radical absorbance capacity, triglycerides, cholesterol and fatty distribution of serum were also analyzed. Beef tallow diet for lowered serum oxidation, whole body oxidation and did not raise cholesterol levels compared to high PUFA diet in the experimental animals. Beef tallow diet lowered lipid peroxidation in vivo vs. PUFA diet. Experiments were continued on the isolation and identification of toxic lipophilic aldehydes in soybean oil at frying temperature. Soybean oil was heated at 185 deg for 2, 4, 6, 8 and 10 hours. 4-Hydroxyhexenal (HHE) and 4-hydroxynonenal (HNE) were significantly increased at 2 hours and
reached a maximum at 6 hours of heating. The existence of HNE was confirmed by co-chromatography by GC/MS. The unheated oil did not contain HHE or HNE. Studies continued to investigate the anticarcinogenic effect of conjugated linoleic acid (CLA). The dietary effect of CLA and butter on 15-lipoxygenase (LOX) was examined. Rats fed 0.8% CLA and 20% CLA butter containing diets had lower levels of hepatic LOX activity than the control groups. There was moderate correlation between hepatic 13S-HODE concentration and colonic aberrant crypt number. The inhibitory effects of trans-vaccinic acid, NDGA and synthetic CLA isomers on LOX activity are under investigation.
Impacts
Dietary stearic acid compared to other highly oxidizable fats seem to lead to less lipid oxidation in vivo which has been associated with various diseases. Understanding of the formation of toxic secondary lipid oxidation products in frying oils will lead to protection against these compounds and safer public use. Study of the beneficial effect of consumption of high CLA containing butter against colon cancer is important for public health.
Publications
- Csallany, A. Saari, Kim Song-Suk and Gallaher, D.D. 2000. Response of urinary aldehydes and related carbonyl compounds to factors that stimulate lipid peroxidation in vivo. Lipids 35:855-862.
- Kim, Song-Suk, Gallaher, D.D. and Csallany, A. Saari. 2000. The antioxidants vitamin E and probucol reduce renal enlargement and in vivo peroxidation in streptozotocin-treated diabetic rats. Lipids 35:1225-1237.
- Rahmani, M. and Csallany, A. Saari. 2000. Stability studies of Moroccan olive oils. J. Olivae. 82-June:37-40.
- Draper, H.H., Csallany, A. Saari and Hadley, Mary. 2000. Urinary aldehydes as indicators of lipid peroxidation in vivo. Free Radical Biology and Medicine 29:1071-1077.
- Gallaher, C.M., Cho, H.-J., Baer, R.J., Csallany, A.S. and Gallaher, D.D. 2000. Development of colonic pre-cancerous lesions in rats fed synthetic and natural sources of conjugated linoleic acid and nordihydroguaiaretic acid. The FASEB Journal 14:A472.
- Seppanen, C.M. and Csallany, A. Saari. 2000. Hydroxyalkenal identification in heated vegetable oil by HPLC and GC-MS. Inform 11:S96.
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Progress 01/01/99 to 12/31/99
Outputs
Experiments were continued to investigate the anticarcinogenic effect of conjugated linoleic acid (CLA) (cis-9,trans-11,18:2) in butterfat against colon cancer in experimental rats. Normal and high CLA butter samples were analyzed by capillary gas chromatography. Eight of the CLA isomers were well separated. In normal butter CLA was about 4 mg/g butter and in high CLA it was about 24 mg/g butter. Experiments are also being conducted to measure the lipoxygenase activity in the liver and lungs of rats fed normal and high CLA containing butter in order to measure the possible inhibition of the formation of 13-hydroxyoctadecadienoic acid (13-HOD) by dietary CLA. 13-HOD has been detected by HPLC. Data on tissue enzyme activity is not yet available. Experiments were also conducted to reevaluate the concentration of three toxic hydroxyaldehydes (4-hydroxy-2-trans-hexenal (HHE), 4-hydroxy-2-trans-octenal (HOE), and 4-hydroxy-2-trans-nonenal (HNE)) in soybean oil heated at
frying temperature for 8 hours. Toxic aldehyde formation in heated soybean oil was found to be much higher than originally estimated. The presence of HHE, HOE, and HNE was confirmed by co-chromatography with pure standards in three different polarity solvent systems by HPLC analysis. HHE, HOE, and HNE were found to be 1.8 ng/g oil, 4.9 ng/g oil, and 36.8 ng/g oil, respectively. These lipophilic alpha,beta-unsaturated aldehydes have been found to be mutagenic and cytotoxic and are easily absorbed from food. Studies on the beneficial effect of high stearic acid diets on in vivo lipid peroxidation were continued. Four urinary nonpolar and four polar lipophilic aldehydes, measured by HPLC as 2,4-dinitrophenylhydrazones, were found to be significantly lower in experimental rats fed beef tallow diets as compared to soybean oil diets (8% dietary fat). Lipophilic aldehydes are secondary products of lipid peroxidation in vivo. Results indicate less whole body oxidation as measured by urinary
lipophilic aldehyde excretion. A method was also developed to measure protein oxidation in liver, as protein carbonyl content, from rats fed previously for 19 weeks four different diets containing 8% beef tallow, cocoa butter, menhaden oil, or soybean oil. After precipitation of proteins from tissue homogenates, protein carbonyl groups were measured. Liver proteins were not found to be significantly different among the diet groups indicating that urinary measurement of secondary oxidation products are better indicators for in vivo lipid peroxidation. Preliminary serum total cholesterol and triglyceride measurements indicated that there was no significant difference between the beef tallow or soybean oil diet groups.
Impacts
Study of the beneficial effect of consumption of high CLA-containing butter against colon cancer is important for public health. Understanding of the formation of toxic secondary lipid oxidation products in frying oils will lead to protection against these compounds and safer public use. Investigation of the effects of dietary stearic acid compared to other highly oxidizable fats may lead to less lipid oxidation in vivo which has been associated with various diseases.
Publications
- Kim, S.-S., Gallaher, D.D., and Csallany, A. Saari. 1999. Lipophilic aldehydes and related carbonyl compounds in rat and human urine. Lipids. 34:489-496.
- Fritz, K.L., Seppanen, C.M., Cho, H., and Csallany, A. Saari. 1999. Reduction of urinary lipid peroxidation products in rats by dietary beef tallow when compared to soybean oil. FASEB Journal 13:A904.
- Seppanen, C.M., Fritz, K.L., Cho, H., and Csallany, A. Saari. 1999. Effects of high stearic acid diets on copper-induced oxidation of serum lipids. FASEB Journal 13:A903.
- Seppanen, C.M. and Csallany. A. Saari. 1999. Formation of secondary oxidation products in vegetable oils under various conditions. INFORM 10:S107.
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Progress 01/01/98 to 12/31/98
Outputs
Experiments were continued to determine the formation of secondary lipid peroxidation (SLP) products in commercial vegetable oils in the presence of air or nitrogen at room temperature or heat. Fifty gram oil samples in a closed system were: a) heated for 8 h at 185 C in the presence of air (97 ml/min), b) same as a) but in the presence of nitrogen, c) same as b) but at room temperature, d) room temperature in the presence of nitrogen. Volatile LP were directly collected into 20 ml of 2,4-dinitrophenylhydrazine (2,4-DNPH) reagent. The SLP products, such as lipophilic polar and nonpolar aldehydes (PA and NA) and related carbonyl compounds (RCC) which formed in the oil and vaporized were measured as 2,4-DNPH derivatives. After prepurification on TLC the samples were measured by HPLC in two different solvent systems. Separation of PA and NA were achieved using 50% or 75% MeOH in water respectively with a combination of isocratic and gradient elutions. Due to heat and air
exposure total SLP increased. The sum of the HPLC separate total NA + RCC were 21 times more, and total PA + RCC were 11 times more in soybean oil and vapor than at room temperature. Ratio of vaporized total NA to oil total NA was 21:1 due to heat, and 0.008:1 at room temperature. Butanal, butane-2-one, pentanal and pentane-2-one were the most prevalent as volatilized compounds but they were not present in heated oil in the presence of air. Vapor to oil ratio of the sum of the HPLC separated total PA + RCC at room temperature and after heat treatment at 185 C for 8 h in the presence of air remained unchanged 0.24:1.0. Concentration of PA increased both in oil and vapor about 10 times due to heat compared to room temperature. 4-hydroxy-hexenal (HHE) and 4-hydroxy-nonenal (HNE) were both identified in heated soybean oil in the presence of air by co-chromatography in three different polarity solvent systems by HPLC. In heated soybean oil (185 C, 8 h, air) 8 ng HNE/g of oil and 2 ng
HHE/g of oil was found. These results indicate that due to heat treatment not only the toxic PA such as HHE, HNE increase greatly but because these compounds are not very volatile their concentrations accumulate in the oil. The lipophilic alpha, beta-unsaturated aldehydes such as HHE and HNE have been found to be cytotoxic and mutagenic, form PUFA, and can be absorbed to the body from the diet. Factors that contribute to their formation in food is of importance relating to human health. One of the isomers of conjugated linoleic acid (CLA) cis-9, trans 11, 18:2 has been shown to be anticarcinogenic and is high in dairy products. Experiments have been started to investigate the anticarcinogenic effect of CLA in butterfat on the protection of colon cancer in rats. The analysis of 8 CLA and fatty acid standards and butterfat by gas chromatography was standardized. Conditions for CLA in butterfat by GC are: SP2560 capillary column 100m x 0.25mm x 0.20um; helium, carrier gas; flame
ionization detector; injector 250 C; detector 250 C; column 160 C for 100 min, then raised 5 C/min to 220 C and held for 33 min.
Impacts
(N/A)
Publications
- RAHMANI, M., LAMRINI, M. and CSALLANY, A.S. 1998. Development of a simple method for the determination of the optimum harvesting for olives. J Olivae. 69:48-51.
- RAHMANI, M. and CSALLANY, A.S. 1998. Role of minor constituents in Photooxidation of Virgin Olive Oil. J Am Oil Chem Soc. 75:837-843.
- SEPPANEN, C.M. and CSALLANY, A.S. 1998. Lipophilic aldehydes in vegetable oils measured by HPLC. Proc. 89th AOCS Meeting. A98.
- SEPPANEN, C.M. 1998. Lipophilic aldehydes and related carbonyl compounds in thermally oxidized vegetable fats and oils. M.S. Thesis. Pgs 110. (Advisor: Csallany, A.S.)
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Progress 01/01/97 to 12/31/97
Outputs
Experiments were conducted to determine the oxidative degradation and secondary carbonyl product formation of commercial oils at room temperature and after heating at 185 deg C for extended periods. The nonpolar and polar lipophilic aldehydes and related carbonyl compounds were measured, by a method developed in this laboratory, as 2-4 dinitrophenyl-hydrazones using HPLC and two different solvent systems using a combination of isocratic and linear gradient elutions. The rate of oxidation at 185 deg C was followed by measuring the increase of TBARS concentration for each oil. Maximum TBARS concentration of heated oils as malondialdehyde equivalents (ng) were for: coconut 20 (25 hours), corn 104 (13 hours), soybean 137 (8 hours) and cod liver oil 1755 (6 hours). The secondary oxidation products for each heated oil were measured at maximum TBARS concentration by the above mentioned HPLC method. Compounds identified were butanal, 2-butanone, pentanal, 2-pentanone,
hexanal, 2,4-heptadienal, 2-heptenal, octanal, 2-nonenal, 2,4-decadienal, 4-hydroxy-2-trans-hexenal (HHE), 4-hydroxy-2-trans-octenal (HOE) and 4-hydroxy-2-trans-nonenal (HNE). Secondary oxidation product formation due to thermal oxidation was affected by the fatty acid composition of an oil. Canola oil was more susceptible than soybean oil which was more susceptible than corn oil to thermal oxidation as measured by production of secondary oxidation products. The presence of lipophilic aldehydes and carbonyl compounds in coconut, corn, soybean, and cod liver oils generally increased due to heating. In general, the shortest chain nonpolar compounds, butanal, 2-butanone, pentanal, and 2-pentanone were most prevalent in the unheated oils. These compounds decreased with heating, while the longer chain compounds, hexanal, 2,4-heptadienal, 2-heptenal, octanal, 2-nonenal, 2,4-decadienal, decanal, and undecanal increased with heating. The amount of polar carbonyl compounds present increased
with heating. Some of the toxic, polar hydroxyaldehydes such as HHE, HOE and HNE were detected not only in heated oils but also in unheated oils.
Impacts
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Publications
- LEE, J.-H., SHOEMAN, D.W., KIM, S.-S. and CSALLANY, A. S. 1997. The effect of superoxide anion in the production of seven major cholesterol oxidation products in aprotic and protic conditions. Intl. J. Food Sci. Nutr. 48:151-159.
- RAHMANI, M. and CSALLANY, A. S. 1997. Stability studies of Moroccan virgin olive oils. J. Olives. In Press.
- RAHMANI, M., LAMRINI, M. and CSALLANY, A. S. 1997. Development of a simple method for the determination of the optimum harvesting for olives. J. Olives. In Press.
- SEPPANEN, C.M. and CSALLANY, A. S. 1997. Lipophilic aldehydes in vegetable oils measured by HPLC. 88th AOCS Annual Meeting, Addendum. Abstract.
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Progress 01/01/96 to 12/30/96
Outputs
Previously an HPLC method was developed in this laboratory for the measurement of lipophilic aldehydes as 2,4-dinitrophenylhydrazones in vegetable oils. In the present experiments, the formation of secondary lipid oxidation products in saturated, monounsaturated, and polyunsaturated vegetable oils (coconut, olive, corn, soybean, and fish oils) were investigated. Lipophilic aldehydes such as alkanals, alkenals, alkanones, and hydroxyaldehydes, were measured in the oils by HPLC at room temperature and after heating the oils for five hours at 60 deg C and 185 deg C. The lipophilic aldehydes formed and their concentrations are compared at the different temperatures and among the various oils. Identification of the individual aldehydes are made by co-chromatography with pure standards in three solvent systems of differing polarities. Some of the lipophilic aldehydes are toxic substances and have been implicated in the formation of plaque in heart disease, DNA damage, and
other cell injuries because of their high reactivities with proteins. Therefore, it is important to know the extent to which lipophilic aldehydes are formed in vegetable oils of varying degrees of unsaturated. The above experiments are currently in progress. Analysis for lipophilic aldehydes and hydroxy aldehydes are currently in progress for a soy project. The objective of the experiment is to see the in vivo antioxidant effect of soy components.
Impacts
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Publications
- LEE, J-H., SHOEMAN, D. W., KIM, S-S. and CSALLANY, A.S. 1996. The effect of superoxide anion in the production of seven major cholesterol oxidation productsin aprotic and protic conditions. Int. J. Food Sci. Nutr. 48:1-9.
- RAHMANI, M. and CSALLANY, A.S. 1996. The role of minor constituents in the photooxidation of virgin olive oil. J. Am. Chem. Soc. In Press.
- CSALLANY, A.S. and SEPPANEN, C.M. 1996. The formation of lipophilic aldehydes inheated oils measured by HPLC. 87th AOCS Annual Meeting, Indianapolis. Abstract.
- JACOB, R., KUTNINK, A., CSALLANY, A. S., DAROSZEWSKA, M. and BURTON, W.G. 1996. Vitamin C nutriture does not affect short-term vitamin E metabolism in healthy women. J. Nutr. 126:2268-2277.
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Progress 01/01/95 to 12/30/95
Outputs
Lipophilic aldehydes are secondary lipid peroxidation products. A method was dvlp. for measurement of lipophilic aldehydes in veget. oils. Method consists of oil extraction with 75% methanol, conversion of aldehydes to hydrazones, thin layer chromatography & high performance liquid chromatography. Heated soybean oil samples were monitored for oxidative deterioration at 185oC & 215oC for 12 hrs by measuring total thiobarbituric acid reactive substances (TBARS). Incre. of TBARS was faster at higher temps. but their decrease was also accelerated. The highest conc. of TBARS was found between 4-6 hrs of heating for both temps. The concentration of the indvl lipophilic aldehydes was measured, with the newly developed method, after 4 and 6 hrs heating at both temperatures. The following lipophilic aldehydes were identified: butanal, butanone, pentanal, pentan 2-one, hexanal, heptadienel, heptenal, octanal, octenal, decanal, decadienal, undecanal and 4-hydroxy nonenal. The
total concentrations of the individual aldehydes in commercial soybean oil were 33% less at 4 hours at 185oC, 42% less at 6 hours at 215oC, and 70% less at 4 hours at 215oC than at 6 hours of heating at 185oC. Some of the lipophilic aldehydes are toxic substances, and 4-hydroxy nonenal is implicated in plaque formation in heart disease, thus it is important to measure their formation in heated oils. Comparison of various veget. oils and fats for lipophilic aldehyde concentrations at elevated temps. are under investigation.
Impacts
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Progress 01/01/94 to 12/30/94
Outputs
Experiments were carried out to identify the previously isolated aldehydic degradation products which related to in vivo lipid peroxidation and to heat induced oxidation of beef tissue samples. Identification, of the individual aldehydes, was carried out by co-chromatography using high performance liquid chromatography (HPLC). The individual aldehyde fractions from samples and standards were separately converted to their hidrazone derivatives (2-4 dinitro phenylhydrazone), extensively purified and co-chromatographed in several solvent systems. The following lipid degradation related saturated and unsaturated aldehydes and ketones were identified: butanal, butanone, pentan-2-one, hexanal, trans, trans-2,4-heptadienal, octanal, 2-nonenal, decadienal. Four of these aldehydes significantly increased to oxidative conditions. The identification of the hydroxy aldehydes are under investigation. Results indicate that not only malonaldehyde but other toxic aldehydic degradation
products were formed during in vivo lipid oxidation.
Impacts
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Progress 01/01/93 to 12/30/93
Outputs
Experiments were carried out to develop a sensitive method to determine the antioxidant effect of heat treatment in meat and meat products. At the late stages of the Maillard browning reaction, various antioxidants are produced and their effect could be measured by the diminished formation of aldehydic degradation products which relate to fatty acid autoxidation. Beef heart samples were used for the method development which involves homogenization, derivatization pretreatment with thin layer chromatography (TLC) and then high performance liquid chromatography (HPLC) for separation and measurement of the aldehyde derivatives. Further experiments are needed to complete the method development. By the use of this method the antioxidant activity of heat induced Maillard browning reaction will be measured in meat and will be related to oxidative rancidity and stability. Experiments were continued to study the effects of colupulon in a variety of areas. Hairless mice were
exposed to UV light to stimulate psoriasis and colupulon was tested for anti-proliferative activity. Results are inconclusive. Usnic acid and vulpinic acid, which have antibiotic activity, were tested for their induction of the activity of in vivo P450 enzyme. It was found that both of these compounds are similar to colupulon and highly inducing P450 3A enzyme systems. Colupulon was tested for hemoglobin glycetion in normal and diabetic mice. Glycetion was not affected by feeding colupulon.
Impacts
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Progress 01/01/92 to 12/30/92
Outputs
Studies on cholesterol oxidation by superoxide anion have been completed. It wasfound that superoxide anion is not involved in the formation of the major free radical induced oxidation products of cholesterol: cholesterol (alpha) and (beta) epoxides, 7-ketocholesterol, 7(alpha) and (beta) hydroxycholesterol and 25 hydroxycholesterol. Experiments were conducted to measure the recoveries of a) hydroxytyrosol, b) tyrosol, c) p-hydroxyphenylacetic acid, d) homovanillic acid and e) caffeic acid from olive oil by our newly developed HPLC method using electro chemical detection. Recoveries were: a) 93%, b) 97%, c) 100%, d) 99%, respectively. Electro chemical detection provided selectivity and sensitivity. Minimum detection limit ranged from 0.5-8 nanogram per injection. Experiments have continued on the biological effects of the hop components, lupulones and humulones, with references to a) their inductive effects on hepatic cytochromes P-450, and b) their inhibitory effects
on cell proliferation, including chicken embryo angiogenesis, inflammation of the rat cornea.
Impacts
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Progress 01/01/91 to 12/30/91
Outputs
Studies of cholesterol oxidation by superoxide are being extended to include measurements of alpha and beta epoxides and cholestane triol formation under protic or aprotic conditions. A method for the measurement of phenolic compounds in olive oil was developed. RP-HPLC with electrochemical detection gave sensitivity and selectivity for the five most important antioxidants present. Four commercial oils were analyzed, and showed variation in both the amount and kinds of phenolic antioxidants present. The fatty acid distribution and tocopherol content of high or low oil soybeans as well as the parent strain were determined. Tocopherol content was found to correlate with unsaturated fatty acid content across the three strains. The oil content and fatty acids distribution in 10 strains of corn bread for herbicide resistance were measured. No difference between resistant and non-resistant strains was detected. The HPLC-florescence method developed in this laboratory
for tocopherol analysis was adapted to avocado. Alpha-Tocopherol content was found to be highest in peel (75 mu g/g), followed by meat (25 mu g/g) and seed (20 mu g/g). Seed was also found to contain a rather high level (300 mu g/g) of alpha-tocotrienol.
Impacts
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Progress 01/01/90 to 12/30/90
Outputs
Studies were continued on free radical and superoxide induced oxidation of fattyacids in vivo and in vitro. Degradation of polyunsaturated fatty acids (PUFA) were measured in rat urine by high performance liquid chromatography (HPLC). Significant increases in malonaldehyde-amino acids conjugates (MDA-AC) and three carbonyl containing thiobarbituric acid positive substances were found in Vitamin E deficiency compared to normal conditions. This finding indicates that oxidative degradation of biomembranes leads to the formation of toxic aldehydes in vivo. Analysis of the MDA-AC revealed that not only lysine but other amino acids are also involved in MDA-AC formation. Recent studies have suggested that lipid peroxidation is increased in diabetes mellitus. Diabetic and normal rat urine was measured for MDA-AC. It was found that MDA-AC was very significantly increased in the urine of diabetic rats compared to the control group. Present findings confirm the presence of
increased lipid peroxidation in diabetes mellitus.
Impacts
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Progress 01/01/89 to 12/30/89
Outputs
Studies were continued on free radical and superoxide induced oxidation of fattyacids in vivo. Degradation of polyunsaturated fatty acids (PUFA) were measured in infant urines using the thiobarbituric acid (TBA) method for the measurements of TBA-reactive substances. It was found that infants fed intravenously and kept under oxygen atmosphere excreted 350% more TBA-reactive substances than controls. This finding indicates a large increase in oxidative degradation in biomembranes and an inbalance of vitamin E in the oxygenated systems. Experiments have started to isolate and identify free and bound malondialdehyde and 3 unidentified carbonyl containing PUFA degradation products from urine by high performance liquid chromatography (HPLC). Experiments are continued in the development of HPLC methods for the measurements of various polyphenols in oils using HPLC and electrochemical detection. Tyrosol and caffeic acid were used for the standardization of the method on mu
Bondapeck C(18) HPLC column and 10% acetonitrile in water as a mobile phase with a 0.5 ml/min flow. Experiments have also started for the determination of vitamin-E and changes in fatty acid distribution in fish eggs from young and older fish. Results are not available yet.
Impacts
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Progress 01/01/88 to 12/30/88
Outputs
Studies were continued in chlorophyll sensitized singlet oxygen induced oxidation of oils and the effect of other active oxygen species on oil degradation with special reference to polyphenols such as tyrosol, hydroxy tyrosol, tocopherols and carotenoids. A new high performance liquid chromatographic (HPLC) method is being developed for the determination of tyrosol, hydroxy tyrosol, caffeic acid, homovallinic acid and 4-hydroxy phenyl acetic acid (polyphenols) from oils by the use of electrochemical detector which enables determining minute changes caused by free radicals and singlet oxygen in the oils. The high performance liquid chromatographic (HPLC) method developed by this laboratory for the determination of chlorophylls and pheophytins from oils was adopted for beta-carotene determination by using an ultraviolet/visible detector. Isolation of tyrosol standard is carried out by fermentation, and the isolation of hydroxy tyrosol standard is from olive tree
leaves.
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Progress 01/01/87 to 12/30/87
Outputs
Studies on superoxide (0(2)), free radicals and singlet oxygen (0(2)) induced oxidation of lipids were continued. High-performance liquid chromatographic (HPLC) method was developed on a normal-phase column, mobile phase 95:4.5:05 hexane;chloroform;isopropanol for the separation of alpha-tocopherol (alpha-T) and its oxidation products; alpha-tocopheroxide (TO), alpha-tocopheryl quinone (TQ), dimer (D), dihydroxy dimer (DHD), trimer (T), compound A and compound B. The alpha-chromanoxy radical was produced by the reaction of alpha-T with 0(2) in anhydrous condition. This radical was further oxidized to a secondary oxidation product, which was separated by normal-phase HPLC. The secondary oxidation product was sensitive to both water and nitrogen, and deomposed to the equimolar of compounds A and B. Compounds A and B were isolated by HPLC, and identified as 7,trans,hydroxy-8,8a,trans,epoxy-alpha-tocopherone and 7,cis,hydroxy-8,8a,cis, epoxy-alpha-tocopherone,
respectively, by ultravilet (UV), infra-red (IR), mass spectroscopy (MS), and nuclear magnetic resonance (NMR). The action of dismutated 0(2) produced four different oxidation products of alpha-T and these were identified as TO, TQ, D, DHD by UV, IR, MS and NMR. Selective scavengers used to identify the oxygen species derived from the 0(2) dismutation. 0(2) produced TQ epoxide, OH produced TQ TQ epoxide, D, DHD from alpha-T.
Impacts
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Progress 01/01/86 to 12/30/86
Outputs
Studies on superoxide (O-/2) induced oxidation of lipids were continued. The reactions of O-/2 and O-/2 derived active oxygen species with cholesterol were investigated. O-/2 was generated by the electrochemical reduction of molecular oxygen in anhydrous acetonitrile, containing tetrabutylammonium bromide as an electrolyte. Experiments were conducted in aprotic and protic conditions. When cholesterol was reacted with O-/2 in dry media no cholesterol oxidation products were detected. When O-/2 was dismutated in the presence of water oxidation products also did not appear. The effect of H(2)O(2) on the decomposition of O-/2 in the presence of water was also investigated. In the presence of added H(2)O(2), O-/2 decomposition was accelerated and oxidation of cholesterol occurred. The major oxidation products were 7-keto cholesterol, 5 alpha-hydroxy cholesterol and 5-beta-hydroxy cholesterol. Three unknown cholesterol oxidation products were also found and separated
by HPLC. (The method was developed in this laboratory.) The effect of ferrons ion on O/2 dismutation in water and H(2)O(2) was also investigated and an inhibitory rather than an accelerating effect was found in relation to cholesterol oxidation. Cholesterol oxidation was greatly retarded in the presence of water and H(2)O(2) by antioxidants such as alpha-tocopherol, BHA and BHT. Cholesterol was found to be a relatively stable compound in its relation to O-/2. 25-Hydroxycholesterol was not detected in any of these experiments.
Impacts
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Progress 01/01/85 to 12/30/85
Outputs
Experiments on alpha-tocopherol oxidation medicated by superoxide anion (O(2)). The reactions of O(2) and O(2) derived oxygen species with alpha-tocopherol were investigated. O(2) was generated by the electrochemical reduction of molecular oxygen in anhydrous acetonitrile, containing tetrabutylammonium bromide as an electrolyte. The basal reaction mixture contained 3.3 x 10M alpha-tocopherol and 2.0 x 10M O(2). The decomposition of O(2) was achieved by the addition of 10% water to the reaction mixture to produce mostly hydroxyl free radicals. The effect of H(2)O(2) on the decomposition of O(2) in the presence of water was also investigated. Disappearance of alpha-tocopherol and the appearance of the oxidation products in the above reactions were measured by a newly developed high performance liquid chromatographic method. Oxidation of alpha-tocopherol by O(2), in the absence of water, produced two unstable intermediates which decomposed into two stabile tocopherol
oxidation products. The first intermediate has a half life of 30 seconds and can fully be regenerated to be alpha-tocopherol by reducing agents. An attempt was made to characterize these compounds by UV, MS, IR and NMR spectroscopy. Oxidation of alpha-tocopherol with water and H(2)O(2) dismutated 0(2), resulting in the following oxidation products: alpha-tocopherol quinone, alpha-tocopheryl quinone oxide, dimer and dihydroxy dimer.
Impacts
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Progress 01/01/84 to 12/30/84
Outputs
Studies were continued on the oxidation of alpha-tocopherol by superoxide-induced free radicals. When alpha-tocopherol was reacted with superoxide in dry state the main oxidation product was alpha-tocopherol quinone. However, in the presence of water (superoxide decomposes to singlet oxygen or hydroxy free radicals) some oxidation products which are less polar than alpha-tocopherol were produced. Of these oxidation products, the dimer, dihydroxy-dimer and trimer were identified. An HPLC system was developed for the identification and quantification of cholesterol and some of its oxidation products in food and biological systems. Standard cholesterol oxidation products were separated on a mu-Porasil column using hexane: isopropanol mixtures as the mobile phase and UV and refractive index detection. Identification of cholesterol oxidation products in liver, brain and muscle has been demonstrated. Minimum detectable concentrations are in the ppm range. 7-keto
cholesterol is the only cholesterol oxidation product that has been found in any tissue assayed. 7-keto cholesterol concentration increases following UV irradiation of tissue samples in a time-dependent fashion.
Impacts
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Progress 01/01/83 to 12/30/83
Outputs
Superoxide induced oxidation and chlorophyll sensitized singlet oxygen induced oxidation are being investigated in olive oil and in model compounds such as methyl linoleate in the presence and absence of alpha-tocopherol. In connection with these studies, an HPLC method was developed for analysis of chlorophyll pigments and their degradation products, pheophytins, by direct injection of vegetable oils onto a C18 reversed-phase column in 98.5:1.5 hexane: isopropyl alcohol (mobile phase). Studies were performed on the oxidation of alpha-tocopherol by superoxide-induced free radicals Alpha-tocopherol was reacted with superoxide and four oxidation products were separated on a C18 reversed-phase column in 95:5 methanol:water. One of these products was alpha-tocopheryl quinone. The identity of the other compounds is being investigated. A new HPLC method is being developed for the determination of cholesterol oxidation products in food and biological systems.
Impacts
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Progress 01/01/82 to 12/30/82
Outputs
In connection with the development of rancidity which is potentially one of the major types of chemical changes in fatty foods and can lead to economic and nutritional losses, experiments were carried out to test the effect of superoxide (O -(2)) initiated oxidation on antioxidants such as alpha-tocopherol and polyunsaturated fatty acids systems: Superoxide was developed electrolitically and was reacted under various conditions with several concentrations of alpha-tocopherol containing systems, a high pressure liquid chromatographic method (HPLC) was developed to measure the formation of alpha-tocopherol oxidation products; the destruction of 0 -(2) was followed by spectrophotometry in moisture free condition and in the presence of water, (water initiates the dismutation of 0 -(2) to singlet oxygen or hydroxy free radicals and alpha-tocopherol acts as a free radical scavenger in these systems; by the newly developed HPLC method, 0 -(2) induced oxidation products such
as alpha-tocopherol quinone, alpha-tocopherol dimer and alpha-tocopherol-dihydroxydimer were identified, presently work is being carried out on the identification of the two unknown oxidation products induced by 0 -(2). In separate experiments the final development of an HPLC method for the determination of malondialdehyde (MDA), a major degradation product of oxidizing polyunsaturated fatty acids, was carried out.
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Progress 01/01/81 to 12/30/81
Outputs
A high performance liquid chromatography (HPLC) method for the quantitation of free MDA was developed. The HPLC separation was performed using a TSK G1000 PW column (7.5 mm I.D. x 30 cm) with a mobile phase of 0.1 M Na(3)PO(4), pH 8.0 buffer at a flow rate of 0.6 ml/min. The eluant was monitored at 267 nm. Free MDA in the samples were separated and identified in approimately 50 min. The lowest amount of MDA that can be determined by this HPLC technique is approximately one nanogram per injection. This method was successfully applied to rat liver, beef, pork and chicken muscle and was compared with the thiobarbituric acid (TBA) test. It was found to be more sensitive, accurate and specific for the determination of free MDA than the TBA method. It was found that free MDA was actually two to ten fold less than previously found by the use of the nonspecific TBA method. Superoxide was electrolytically produced and reacted with Alpha-tocopherol. Identification of the
tocopherol oxidation products are presently conducted.
Impacts
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Progress 01/01/80 to 12/30/80
Outputs
Experiments were carried out to further develop the high pressure liquid chromatographic method (HPLC) for the direct determination of malondialdehyde (MDA). After considerable problems, because of the instability of MDA and its reactions to HPLC column packing, it was found that MDA can be best measured on Zorbax ODS column (25 cm x 4.6 mm i.d. Dupont) and a mobil phase of phosphate buffer (0.01 M) pH 7.5 and 0.00005 M tetrabutyl ammonium phosphate (PIC A reagent). MDA is measured by UV absorption at 267 nm in its enol nonvolatile form. Sensitivity of the method in pure system is 5 x 10 - 8 M MDA, similar to the sensitivity of the thiobarbituric acid (TBA) method. Presently experiments are conducted to adopt the HPLC method from pure systems to actual foods, oils and biological materials. By the previously developed HPLC method of this laboratory, vitamin E was analyzed in turkey livers. Four different type fat-containing and three different levels of vitamin
E-containing diets were fed to the turkeys. Seven duplicate livers, altogether 168 tissue samples, were analyzed in the experiment. Results indicate that the vitamin E requirement in fat-supplemented turkey feed depend on the type, the source and the amount of fat used. Hepatic and tissue levels depend on their variations in the feed. The present data are being correlated with the storage stability of fresh and frozen turkey meat.
Impacts
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Progress 01/01/79 to 12/30/79
Outputs
Experiments were carried out to develop an improved method for malondialdehyde (MDA). Measurement of MDA by the use of the change in uv absorption wavelengths and in molecular extinction, due to changes of pH was successful, but sensitivity was not adequate (10 - 5 g) in very low concentrations. A new high pressure liquid chromatography (HPLC) method is under development. With HPLC by the use of a C-18 column and phosphate buffer as mobile phase, 10 - 7 g concentrations of MDA can be adequately measured. Since MDA is a very reactive substance, more experiments are needed for the standardization of the method before it can be used successfully. A new HPLC method was developed for the determination of vitamin E in turkey tissue. Samples are extracted by acetone, centrifuged, supernatant separated, washed, dried over N(2) gas and applied to HPLC, with methanol as a mobile phase and a uv detector set at 280 nm. Recovery is 98%. Eighty-six turkeys were fed (by others) 4
different dietary fats. Each fat group was divided into 3 groups of 7. Two groups of each dietary fat group were supplemented with low and high levels of vitamin E to test storage stability of the meat. Livers and muscles were analyzed for vitamin E by the HPLC method. Vitamin E will be correlated with storage stability TBA and dietary treatments. This experiment is not yet finished.
Impacts
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Progress 01/01/78 to 12/30/78
Outputs
Malondialdehyde is a carcinogenic breakdown product of peroxidizing polyunsaturated fatty acids in foods, oils and biological systems. Presently, malondialdehyde concentrations are determined only by the nonspecific TBA (thiobarbituric acid) method, which is reacting with various carbonyl groups and not only with malondialdehyde in a given system. Experiments were carried out to develop a specific and sensitive method for malondialdehyde determination; using the difference in ultraviolet absorption wavelengths and absorption maxima (E 1% 1 cm) between the acidified enol form and the neutralized keto form of malondialdehyde. The hydrolysis of 1,1,3,3-tetramethoxy propane by acids and resins were compared for producing total conversion to malondialdehyde. It seems that malondialdehyde has to be distilled by steam distillation from food or tissue homogenates or from oils before the spectrophotometric method can be successfully used. 0 graduate student years.
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Progress 01/01/77 to 12/30/77
Outputs
Methyl linoleate and distilled corn oil samples, containing 0, 1500, or 15,000 Mu g Alpha-tocopherol/g oil were exposed to N(2), air, 15 ppm NO(2) or 15 ppm O(3) at 60 degrees C. Oxidative stability was measured by: inducation time, maximum positive slope, maximum peroxide value, time required to attain maximum peroxide value and area beneath the peroxide curve. Results showed that 1) methyl linoleate, oxidized faster then corn oil, 2) the relative oxidative abilities of the gases were, O(3) greater than NO(2) greater than air greater than N(2) in both corn oil and methyl linoleate, 3) the antioxidant protection was not found to be linearly related to the concentration of Alpha-tocopherol. A study was conducted to determine the direct oxidative effects of gases on Alpha-tocopherol. Silicone oil samples containing 15,000 Mu g Alpha-tocopherol/g silicone oil were exposed to N(2), air, 15 ppm NO(2) 15 ppm O(3) at 60 degrees C. Oxidation of Alpha-tocopherol in oil
systems exposed to gaseous oxidants was found to result from the additive effects of two oxidative mechanisms: 1)direct oxidation of Alpha-tocopherol by gaseous oxidants, 2) oxidation of Alpha-tocopherol resulting from the antioxidant function.
Impacts
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Progress 01/01/76 to 12/30/76
Outputs
Whole, halves and ground samples of three varieties (low and high oil) of soybeans were continuously exposed to air, 15 ppm No(2) or 1.5 ppm 0(3) for 100 hours. Lipid oxidation was measured by changes in polyunsatured fatty acid (PUFA) composition, tocopherol (TOC) destruction, and formation of lipofuscin pigments (LP). No destruction of TOC or PUFA occurred in whole intact seeds. Seed halves showed limited TOC and PUFA oxidation. Ground samples showed substantial destruction of TOC and PUFA when exposed to NO(2) or 0(3). Lipofuscin-like (LP) compounds were isolated from soybean samples. Small quantities of the LP were found in whole soybean seeds; however, halves and ground samples showed increased levels. Three varieties of corn samples were also continuously exposed to air, NO(2) or O(3) for 100 hours. No destruction of PUFA, TOC or LP was found in whole kernels. Samples of TOC-free corn oil (CO) and pure methyl linoleate (ML) were supplemented with 0, 1500 or
15000 ppm alpha-TOC and were exposed to N(2), air, NO(2), or 0(3) for 100-300 hours. Lipid oxidation was measured by peroxide formation, PUFA and alpha-TOC destruction. ML was more oxidatively labile than CO. Exposure to 0(3) produced rapid oxidations of PUFA and alpha-TOC in both ML and CO samples. Exposures to NO(2), air and N(2) respectively caused less destructions of PUFA and alpha-TOC. ML and CO samples containing 15000 ppm alpha-TOC showed the least oxidation of PUFA and alpha-TOC.
Impacts
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Progress 01/01/75 to 12/30/75
Outputs
Methods have been developed for the extraction, purification and quantitation oflipofuscin pigments from plant tissues. These apply molecular exclusion column chromatography and spectrophotometric analysis of the pigments. Recent finding indicates the existence of lipofuscin pigments in soybean, a grain whose high linoleic acid content makes it susceptible to lipid peroxidation. The existenceof the pigments in banana, apple, and carrots was disproved by quantitative spectrophotofluorometric measurements. Methods for the extraction of lipids from corn and soybean grains, quantitative fatty acids ethyl ester transesterification of the seed oils, and gas chromatographic separation and quantitation of the fatty acid methyl esters, have been developed. Apparatus has been designed and made for the short-term, small-scale exposure of corn and soybean grain to ozone and nitrogen dioxide. Methods have been standarized for the extraction, purification, thin-layer
chromatographic separation and spectrophotometric quantitation of each of the tocopherol isomers found in corn and soybean grains. Efforts are being made to study the inducing effect of NOy and ODT on the peroxidative process of soybean oil, corn oil and methyl linoleateand the oxidative degradation of tocopherols.
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Progress 01/01/74 to 12/30/74
Outputs
Short term NO(2) Exposure of Soybean: Six groups of selected soybean samples have been exposed to 15 ppm of NO(2) for 1 1/2 hours a day for 3 months. Six groups of identical samples were exposed to ambient air for the same amounts of time. The samples were of the following soybean varities; "Wayne" higher protein (42%), lower oil (21%), and 13% moisture containing soybean, "Amsoy" lower protein (39%), higher oil (23%), and 13% moisture containing soybeans. Each variety has been separated into 3 groups; undamaged seeds, broken shelled seeds, and split seeds. After completion of the gas exposure, the samples have been subjected to chemical analysis. Data not available yet from this part of the experiment due to the short project period. Long term NO(2) and 0(3) Exposure: Three environmental chambers have been made ready for the long term exposure of selected varieties of soybean and corn samples. In the first chamber 0.15 ppm of 0(3) and 0.45 ppm of NO(2) are
simultaneously maintained andin the third chamber only ambient room air is supplied. Data not yet available from this experiment due to the relatively short project period and the long term required for the exposure.
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