1. Trans-Fat and Heart Disease Paul Hinton 10 December 2010

2. Problem Hydrogenated vegetable oils contain unhealthy trans-fat that when incorporated in the diet lead to serious health problems, how much of this fat can a human consume before it becomes unhealthy? Hypothesis It is unsafe for any levels of trans-fat, from hydrogenated vegetable oil, to be consistently consumed by humans.

3. Trans-Fat In Foods Since 2006 companies were allowed to report 0% trans-fat if there was less than.5 grams per serving In 2003, the estimated trans-fat consumption was 7 g/day for men and 5 g/day for women. 1 Foods with partially hydrogenated vegetable oils still contain trans-fats. 1 14 different isomers of octadecenoic acid and octadecadienoic acid are formed during hydrogenation of vegetable oils. 1 Oleic AcidElaidic Acid An octadecadienoic acid

4. Trans-Fat Links to Heart Disease Increases Low density lipoprotein levels and decreases High density lipoproteins levels in plasma which reduces flow mediated vasodilatation (widening of blood vessels). 1 Increased inflammation of the arteries and endothelial dysfunction (oxysterols) which increases risk of atherosclerosis. 1 Inhibition of the enzyme COX-2 which produces prostacyclin which regulates blood flow. 1 LDLOxLDLOxysterolsAtherosclerosis Blood Flow Inhibited Death Plasma Cell Change in Cell composition Incorporation of trans-fat Inhibition of EFAProstacyclin Blood Flow Inhibited Death

5. Proving the Hypothesis To test the hypothesis, prostacyclin and oxysterol levels in blood will be measured in test subjects on a controlled trans-fat diet. The levels of prostacyclin an oxysterol in the subjects’ blood will indicate whether or not that specific level of trans-fat was adverse to the subjects health. I will only be going into further detail about the analysis of prostacyclin. An oxysterol Prostacyclin

6. Test Subjects Since participating in the study could potentially be dangerous, mice will have to be used.* 123456 (mg trans fat/ g bodyweight) /day - mice 00.00140.00710.01430.07140.1428 g/day - human (70 kg.) 00.10.51510

7. Analytical Methods Analytical Technique Comments ProsCons Fluorescence Good sensitivity, good dynamic range Sample preparation (separation of an analyte of very low concentrations) CE Good sensitivity when coupled with a fluorescence or MS detector Sample preparation (analyte in low concentrations), complex matrix, not good enough resolution (ideal for proteins) HPLC-MS Good sensitivity, dynamic range, and separation. LOQ: 10 pg/mL 2 Expensive, long run times GC-MS Good sensitivity, dynamic range, and separation Expensive, long run times

8. Analytical Method of Choice ELISA PGI 2 Kit/Spectrophotometry Inexpensive: $864/$4695 Good sensitivity Can measure many samples quickly Very little sample preparation Good dynamic range: 31.2 – 2000 pg/mL 3 LOD: 11.6 pg/mL 3 Nondestructive

9. UNICO SQ-2802S SQ-2802S 4 Range: 190-1000 nm Bandwidth:.5, 1, 2, 5 nm Single Beam, 1200 lines/mm Accuracy: +/-.3 nm Repeatability: +/-.2 nm Stray light: <0.15% T

10. Sample Preparation Oxysterols 5 1 mL plasma sample is spiked with.001 mg 27-hydroxycholesterol. Sample undergoes alkaline hydrolysis under Ar atmosphere. Extraction with chloroform at pH 7. Extract dissolved in toluene; cholesterol and oxysterols are separated by solid-phase extraction. The samples were converted to trimethylsilyl ethers and dissolved in.1 mL Hexane. – GC-MS Prostacyclin 3 Collect plasma using EDTA as an anti-coagulant. Centriuge at 1000 g for 15 minutes at 5 o C. Apply samples to prostacyclin ELISA kit.

11. Data Analysis Plot (log) Absorbance vs. (log) Known Concentrations Prostacyclin, then use the regression line to find concentration of unknowns.

12. Bibliography 1. Kummerow, F. A. Atherosclerosis 2009, 205, 458-465. 2. Diteba Research Laboratories Case Studies page. http://www.diteba.com/case- studies/content/?Tag=UPLC%20Method%20Development (accessed December 7 th, 2010). 3. Antibodies Online Prostacyclin ELISA kit page. http://www.antibodies- online.com/kit/414685/Prostacyclin+PGI2+ELISA/ (accessed December 7 th, 2010). 4. Unico SQ-2802 page. http://www.unicosci.com/spectro/2802detail.htm# (accessed December 7 th, 2010). 5. Babiker, A. Scandinavia Journal of Clinical and Laboratory Investigation 2005, 65(5), 365-375. 6. Iuliano, L. Analytical Biochemistry 2003, 312(2), 217-223. 7. Mozzaffarian, D. New England Journal of Medicine 2006, 354(15), 1601- 1613.