Xotchil Medina NFSC 440 10/15/2013 AND Evidence Analysis Worksheet Citation Sanders TA, Hall WL, Maniou

Z, Lewis F, Seed PT, Chowienczyk PJ. Effects of low doses of long-chain n-3 PUFAS on endothelial function and arterial stiffness: a randomized control trial. Am. J Clin Nutr. 2013;94(4):973-80. doi: 10.3945/ajcn. 111.018036. Randomized Control Trial A Does consuming n-3 long-chain PUFAS-equivalent to 1, 2, or 4 portions of oily fish per week affect endothelial function and arterial stiffness? Men and women aged 4570 y attended a screening visit in the fasting state for measurement of height, weight, waist circumference, seated BP, liver function, glucose, lipids, and hematologic indicators. A spot urine sample was provided to check absence of albumin and to confirm nonsmoking status by measuring cotinine. Exclusion criteria for this study included a medical history of cardiovascular disease; overall risk of cardiovascular disease >20% over the next 10 years; cancer (excluding basal cell carcinoma) in the previous 5 years; type 1 diabetes mellitus; uncontrolled type 2 diabetes (fasting plasma glucose >7 mmol/L); chronic renal, liver, or inflammatory bowel disease; history of substance abuse or alcoholism; pregnancy; weight change of >3 kg in preceding 2 mo; and BMI (in kg/m2) <20 and >35. Dietary intervention involved supplementation with encapsulated n-3 longchain PUFAs at 3 different doses: 0.45, 0.9, 1.8 g/day. The placebo consisted of olive oil. Each treatment consisted of a run-in period(3placebo capsules/d for 1 mo) and a 12-mo intervention phase (3 capsules/d). Each capsule had peppermint added to disguise the fish taste from DHA and EPA. The blends of oil provided 1.8, 0.9, and 0.45 g/day and the ratio of EPA to DHA was 1.51:1. Participants would receive new batches periodically and the unused capsules were counted and recorded. Compliance was assessed by measuring the proportion of EPA and DHA in erythrocyte lipids at baseline, 6 months, and 12 months. Measurements of endothelial function and arterial stiffness were measured at baseline and after 12 months of treatment. The day before measurement participants were asked to abstain from extraneous physical activity, avoid caffeinated beverages or alcohol, and to consume a low-fat frozen meal in the evening before. They were also asked to fast overnight then check in in the morning and complete the dietary and lifestyle questionnaire. Blood pressure and a blood sample were taken upon arrival. After 15 minutes of rest in the supine position, arterial stiffness and supine blood pressure were measured. After further rest of 30 minutes, the brachial artery was measured by a professional using high resolution ultrasound.

Study Design Class Research Purpose Inclusion Criteria

Exclusion Criteria

Description of Study Protocol

Data Collection Summary

Summary of Results

The independent variables of the study are the amounts of n-3 long-chain PUFAs that are being administered to the participant. The dependent variables are blood pressure, endothelial function and arterial stiffness. Some confounding factors would be the gender, age, genetics, and lifestyle of the participants. These factors can affect how the body uses the administered n-3 long-chain PUFAs. The independent variable was measure by having a capsule made that contains specific amounts of EPA and DHA in them. These amounts varied from 0.45, 0.9, and 1.8 g/day. The dependent variables were measured periodically throughout the intervention. Blood pressure and a blood sample were taken upon arrival on test days. After 15 minutes of rest in the supine position, arterial stiffness and supine blood pressure were measured. After further rest of 30 minutes, the brachial artery was measured by a professional using high resolution ultrasound. To measure endothelial function, reactive hyperemia was induced by inflation of a pneumatic tourniquet placed around the forearm to a pressure of 250 mmHg for 5 minutes. Images were recorded continuously for 1 minute in baseline state, 5 minutes in cuff inflation, and 5 minutes post cuff release. After a 10 minute rest, another scan was taken. The diameter of the brachial artery was determined by using computer-assisted edge detection software. The result is expressed as a percentage increase in brachial artery diameter from baseline to maximal dilation, which is usually 30-90 seconds after release of the cuff. Arterial stiffness was measured 15 minutes after supine rest. It was estimated using SphygmoCor analysis software by measuring PWVc-f. This was computed from the time delay between the upstroke of arterial pressure wave at the carotid and femoral arteries and the anatomic carotid to femoral distance. Blood pressure was monitored 24 hours a day by wearing a device that took measurements every half an hour during the day and hourly during the night. This was done at the end of the run in period, 6 months, and 12 month intervention. Serum lipid and C-reactive protein concentrations and erythrocyte lipid composition were determine from the blood collected at the end of the intervention. The statistics used was STATA 11 software that adjusted for baseline values, age, sex, BMI, and ethnicity. When multiple measurements were available, the mean values were used for analysis. Standard distributional checks were made and the analyses were attempted after log transformation. After, geometric means and ratios were calculated. Significance tests for comparison between groups were done using means of trends tests. Spearmans rank correlation test was used to examine associations between variables. There were a total of 367 participants, but by the end there was complete data for 310. During the trial, body weight remained stable between the different treatments. There was a decrease in triacylglycerol concentrations with the 1.8g/day of the n-3 long-chain PUFAs. The p-value for this significance is 0.014. 45% of men and 33% of women started with FMD values that indicated impaired endothelial function. These properties did

Author Conclusion

Reviewer Comments

not change after intervention and no statistically significant differences were found between treatment groups. No significant changes were found in PWVc-f, peripheral or central augmentation indexes, central supine BP, or 24-h ambulatory BP. No other significant differences were noted. Evidence was insufficient to support firm recommendations. Their study was the first randomized controlled trial to be sufficiently statistically powered to test this hypothesis. They used a standardized protocol for measurement of FMD that requires participant to avoid foods high in fat, caffeine, or alcohol on the previous day and to avoid strenuous exercise. Measurements were made after an overnight fast at the time of day by an experienced vascular ultrasonographer. The findings of the current study indicate that, in healthy adults without clinically overt coronary heart disease, no effects of n3 long-chain PUFA intakes in the range of 0.45 to 1.8 g/d on endothelial function were found. They were unable to demonstrate any effects on arterial stiffness at the dose that they used in the intervention. They were also unable to show any changes in blood pressure. A limitation to their study was that it was conducted in adults at mild-tomoderate risk of cardiovascular disease rather than in those with clinically evident disease. In conclusion, the randomized controlled trial indicated that intakes of n3 long-chain PUFAs 1.8 g/d do not improve endothelial function in nonsmoking healthy adults with a mild-to-moderate increased risk of cardiovascular disease. Some strengths to the study are that they excluded people that were already at risk for developing heart disease and that they used different doses of omega 3s. This made it so that their results were not skewed by the affects of heart disease and helped determine what the lowest level of supplementation was effective. The fact that they had blood pressure monitors to monitor the participants 24 hours a day helped them monitor outside of the analysis day. Some limitations to the study are that they were not able to monitor lifestyle choices and other fats that they might have been eating. The participants could have already been consuming some omega 3s while some could be consuming fats that are hindering the effects the intervention. I have some concerns with the generalizability because the age group was broad and didnt include younger people that could have different effects to the intervention. I do think that the validity of the study was strong because it was double blind and they used professionals that didnt know what they were looking for to measure arterial pressure, endothelial function, and blood pressure. At the end, it says that none of the authors had a financial or commercial interest in any company or organization sponsoring the research. Some of the sponsors were CRODA Europe Ltd (oil) and St Thomas Hospital Clinical Research Centre.

11/3/13 AND Evidence Analysis Worksheet 2 Citation Din JN, Sarma J, Harding SA, Lyall K, Newby DE, Flapan AD. Effect of -3 fatty acid supplementation on endothelial function, endogenous fibrinolysis and platelet activation in patients with a previous myocardial infarction: a randomised controlled trial. BMJ Open. 2013;3(9):e003054. Randomized Control Trial A What is the effect of omega 3 fatty acid supplementation on endothelial vasomotor function, endogenous fibrinolysis, and platelet and monocyte activation in patients with coronary heart disease? Twenty patients with a myocardial infarction at least 3 months previously were recruited to participate in the study. Exclusion criteria included dietary fish allergy or intolerance, consumption of one or more than one fish meal per week, renal or hepatic failure, or any intercurrent illness likely to be associated with an inflammatory response. Intervention: Participants were randomized into two different groups based on what they would be receiving. One group would receive omega 3 fatty acid supplements and the other would have matching placebo capsules for 6 weeks. 6 weeks later there was a 4 week washout period. Participants then crossed over to the other treatment for another 6 weeks. The omega 3 fatty acid capsules contained 85-88% eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) as ethyl ester in a ratio of 1.2:1. The placebo treatment contained olive oil. Both treatments contained 4 mg alpha-tocopherol. Measurements: All 20 participants had peripheral blood taken for fasting lipid profile, plasma fatty acid analysis and flow cytometric analysis of platelet activation at baseline and at the end of each treatment period. A subset of 12 participants also underwent measurement of forearm blood flow and endogenous fibrinolysis at the end of each treatment period. Variables: Independent: The amount of omega 3 fatty acid being delivered in the supplementation. Dependent: Endothelial vasomotor function, endogenous fibrinolysis, and platelet and monocyte activation. Confounding/Control: Type of MI, medical history, age, gender, genetics, and lifestyle. Measurement Methods: Blood collection- Venous blood was drawn from a large antecubital vein and anticoagulated with EDTA and the direct thrombin inhibitor PPACK. - Blood anticoagulated with PPACK was immunolabelled within 5

Study Design Class Research Purpose

Inclusion Criteria Exclusion Criteria Description of Study Protocol

Data Collection Summary

minutes of being drawn for cytometric analysis. - Plasma was prepared from blood anticoagulated with sodium EDTA by centrifugation. Flow cytometry- Several reagents and their appropriate isotype control were used. Portions of blood anticoagulated with PPACK were incubated with appropriate antibodies and their isotype matched controls for 20 minutes at room temperature. - To evaluate platelet-monocyte aggregates and CD-40 on monocytes, samples were fixed and red cells lysed by the addition of FACS-Lyse solution - To evaluate platelet surface P-selectin and CD-40 ligand, samples were fixed with 1% paraformaldehyde. - Samples were analyzed using a Coulter EPICS XL flow cytometer equipped with a 488nm wavelength laser within 6 hours of labeling. - Monocytes and platelets were identified by gating for CD 14 and CD 42a positive cells respectively. Plasma fatty acid analysis- fatty acid composition of plasma phospholipids was determined from blood anticoagulated with EDTA. - Total lipids recovered from plasma using dichloromethanemethanol containing 0.005% butyrated hydroxytoluene as an antioxidant. - Phospholipids isolated by solid phase extractionusing aminopropyl silica columns and fatty acids converted into methyl esters. - Fatty acid methyl esters analysis was performed with and HPINNOWAX capillary column and peaks identified with known fatty acid methyl ester standards. - Total phospholipid fatty acids were expressed as the individual fractions of fatty acids and fatty acid groups as relative values. Vascular studies- Participants fasted for 6 hours prior to the study and avoided caffeine and alcohol for the previous 24 hours. - Blood pressure and heart rate were recorded throughout the study using a semi-automated non-invasive oscillometric sphygmomanometer. - All participants underwent brachial artery cannulation under controlled conditions. o Starts with baseline saline infusion then acetylcholine at 5, 10 and 20 ug/min, substance P at 2, 4, and 8 pmol/min and sodium nitroprusside at 2, 4, and 8 ug/minute were infused for 6 minutes at each dose. o The solution sequence was randomized and each separated by a saline solution flush. - Forearm blood flow was measured in infused and non-infused arms by venous occlusion plethysmography with mercury-in-silicone elastomer strain gauges. o Blood was drawn before and during infusion and then collected into acidified buffered citrate and citrate.

Summary of Results

Author Conclusion

Reviewer Comments

o Plasma t-PA antigen and activity and PAI-1 antigen and activity concentrations were determined by ELISAs. - Haematocrit was determined by centrifusion at baseline. Statistics: Variables were reported as a mean SE of the mean. Statistical analyses were performed using one way and two way ANOVA with Bonferronis post tests for multiple comparisons All calculation were performed using GraphPad Prism Plasma phospholipid fatty acid composition: There was an increase in EPA percentage compared to both baselines (p<0.0001)- Table 2 o From 3.7 0.4% vs 2.0 0.2% and 3.7 0.4% vs 1.7 0.1% There was an increase in DHA compared to both baselines (table 2) o Supplement: 5.6 0.2% to 4.8 0.3% (p<0.01) o Placebo: 5.60.2% to 4.40.3% (p<0.0001) The levels dropped back to the baseline after supplementation Reduction in plasma phospholipid percentage of arachadonic acid, but no effect on -linolenic acid, linoleic acid, palmitic acid, stearic acid, or oleic acid with omega 3 or placebo. Lipid Profile: No effect on total cholesterol, LDL-C, HDL-C or triglycerides. Stimulated t-PA activity: Substance P infusion caused a dose-dependent increase in plasma tPA activity concentrations after omega 3 supplementation and the placebo (p<0.0001) Did not affect plasma TPA activity, TPA antigen, or PAI-1 concentrations compared to placebo No difference in net release of t-PA activity after omega 3 supplementation compared to placebo Platelet-monocyte aggregation and CD40/CD40 ligand No affect on platelet-monocyte aggregation, platelet-neutrophil aggregation, platelet surface expression of P-selectin or CD40L, or monocyte of CD 40 The finding suggest that any cardiac benefits conferred by omega 3 fatty acids in coronary heart disease are unlikely to be mediated through effects on endothelial function, endogenous fibrinolysis or platelet activation. It is not likely that these results are due to noncompliance because blood levels indicated increase in EPA and DHA. This is a going against previous studies and it could be because the others dont use a placebo and they are not double blinded. It is conceivable that endothelial function cannot be improved with omega 3s in coronary heart disease patients already treated with modern medical therapy. They believe they lacked power measure for small changes, but they believe the study had sufficient power to detect any clinically relevant effects of omega 3 fatty acids. Some strengths not identified in the article are that it is randomized and double blind. It also crosses over without the participants and analysts

knowing so that they can look at the numbers alone to take observations. Some weaknesses to the study are that the degree of the myocardial infarction could be affecting the outcome of supplementation. I dont really have any concerns about the validity of the study. The funding source of the study was a British Heart Foundation Project Grant and it states that there were no competing interests.