Effects of a Nutritional Supplement Containing Omega 3 Fatty Acids, Plant Sterols, Folate, and B Vitamins on Cardiovascular Risk Factors
1-3 Ernst J. Schaefer, 1Joi A. Gleason, 3,4Michael L. Dansinger, 3Katalin Horvath, 2,3Masumi Ai, 2,3Seiko Otokozawa,2,3Katsuyuki Nakajima, 5Akira Tanaka, and 2,3Bela F. Asztalos
1Cardiovascular Research Clinic, 2Cardiovascular Research Laboratory, Friedman School of Nutrition Science and Policy at Tufts University and Tufts University School of Medicine, 3Lipid Metabolism Laboratory, Jean Mayer USDA Human Nutrition Research Center on Aging at Tufts University, 4Division of Endocrinology, Metabolism, and Molecular Medicine, Tufts-New England Medical Center, Boston, MA, and 5Kagawa Nutrition University, Sakado, Japan.
Supported by a grant from PBM Pharmaceuticals, Inc., Gordonsville, VA to carry out the clinical protocol, grants R01 HL-60935, HL 74753 and PO50HL083813 from the National Institutes of Health and contract 53-3K – 06 from the United Department of Agriculture Research Service to support the biochemical assays
Address correspondence to: EJ Schaefer, MD, Tufts University, 711 Washington Street, Boston, MA 02111 phone 617 556 3100 fax 617 556 3103 email: email@example.com Abstract Word Count: 211, Total Manuscript Word Count including Abstract, but not references and tables: 3,912
We assessed the effects of a daily nutritional supplement (Animi 3) rich in omega 3 fatty acids (mainly docosahexaenoic acid or DHA), plant sterols, folic acid, vitamin B6, and vitamin B12 versus corn oil placebo on measures of coronary heart disease (CHD) risk. Subjects 55-80 years of age with plasma triglyceride levels over 100 mg/dl were randomized in a blinded, placebo controlled 2:1 fashion, with 67 of 79 subjects completing the study (42 in the active and 25 in the placebo arm). We noted significant decreases (p<0.001) in plasma homocysteine levels (-23.6%) and significant increases in folate levels (+287%) and red blood cell phospholipid DHA content (+274.1%) in the active group versus placebo. We also noted significant percentage decreases versus placebo (p<0.05) in plasma levels of total cholesterol (-9.1%), non-high density lipoprotein (HDL) cholesterol (-14.4%), HDL cholesterol (+10.2%), the total cholesterol/HDL cholesterol ratio (-14.9%), triglycerides (-31.2%), and remnant lipoprotein cholesterol (-34.7%). In addition we noted significant increases (p<0.01) in HDL cholesterol (+10.2%), and in apoA-I in the large protective alpha 1 HDL subfraction (+37.0%). In conclusion the alterations induced by this nutritional supplement, especially on the total cholesterol/HDL cholesterol ratio, non-HDL cholesterol, triglycerides, remnant lipoprotein cholesterol, HDL cholesterol, and large HDL particles would be predicted to significantly reduce CHD disease risk.
Coronary heart disease (CHD) remains the leading cause of death and disability in our society, and is highly prevalent in the elderly population. Significant CHD risk factors as assessed in the Framingham Heart Study as well as other studies include increased age, male gender, cigarette smoking, increased levels of systolic blood pressure, diabetes (fasting blood glucose > 125 mg/dl), elevated levels of total cholesterol, non- high density lipoprotein (HDL), and low density lipoprotein (LDL) cholesterol, and decreased levels of HDL cholesterol (1,2). In addition elevated values of the total cholesterol/HDL cholesterol ratio and remnant lipoprotein cholesterol are very potent predictor of future CHD events, as are decreased levels of large alpha 1 migrating HDL in this same study and other studies (2-5). Increasing this latter parameter with the combination of simvastatin and niacin has been associated with significant benefit in preventing progression of coronary artery disease stenosis in CHD patients (6). Omega 3 fatty acid use has been associated with a significant reduction in the risk of recurrent CHD events and sudden death in CHD patients (7,8). A preparation containing 1800 mg of eicosapentaenoic acid given daily without DHA reduced CHD events in in a large Japanese intervention trial in hypercholesterolemic patients on statins, but had no significant benefit on mortality (9). At higher doses omega-3 fatty acid supplements also significantly lower plasma triglyceride levels (10), and we have documented that this also occurs with preparations containing DHA only (11,12). Moreover products enriched in plant sterols have been reported to decrease levels of total cholesterol and LDL cholesterol (13).
Our purpose in this study was to assess the effects of daily nutritional supplementation with omega 3 fatty acids, plant sterols, B vitamins and folate versus corn oil placebo on measures of coronary heart disease (CHD) risk in 75 subjects with moderate elevations in plasma triglyceride levels (> 100 mg/dl) over a 6 month period.
The objectives of the study were to carry out a double blind, randomized, placebo-controlled prospective trial in 75 subjects between 55-80 years of age with no prior history of dementia in a 2:1 randomization design over a 6 month period of time. We compared the effects of 4 capsules/day of a supplement (Animi 3) rich in omega 3 fatty acids. folic acid, vitamin B6, vitamin B12 and plant sterols versus corn oil placebo capsules on measures of cardiovascular (CVD) risk and measures of cognitive function. The CVD risk factors assessed at baseline and at 6 months included fasting plasma cholesterol, triglycerides, high density lipoprotein (HDL) cholesterol, non-HDL cholesterol, the total cholesterol/HDL cholesterol ratio, low density lipoprotein (LDL) cholesterol, small dense LDL cholesterol, HDL particles, red blood cell fatty acids, C reactive protein (CRP), homocysteine, docosahexaenoic acid (DHA), and folate. The duration of the study for each subject was 6-months after screening and enrollment. Our primary hypothesis was that the active supplement would have significantly better effects on markers of CHD risk than placebo.
Study Subjects, Protocol, and Safety Measurements
Subjects were recruited by direct mailing and newspaper advertising for subject recruitment. The following criteria were required for enrollment: 1) Age 55-80 with at least some high school education (defined as completion of grade 9); 2) Not carrying the diagnosis of stroke, dementia, Alzheimer’s disease, Parkinson’s disease, schizophrenia, cognitive dysfunction, significant memory loss, or other significant mental illness; 3) Not taking medications for cognitive dysfunction or lipid-lowering medication (atorvastatin, fluvastatin, lovastatin, pravastatin, rosuvastatin, simvastatin, ezetimibe, fenofibrate, gemfibrozil, niacin, colesevelam, colestipol, or cholestyramine); 3) No history of stroke, known malabsorption, current heavy alcohol intake, defined as more than 14 drinks per week in men or more than 7 drinks per week in women; 5) Not consuming fish more than once per week, on average, or using fish oil capsules; 6) Not allergic to fish, or fish products or any of the ingredients in the capsule, and 7) Not consuming high-dose vitamins other than a once-a-day multivitamin.
Eligible subjects gave informed consent for the screening and the study. The protocol was approved by the human investigation review board. Subjects were asked to fast overnight, to undergo a finger stick lipid profile to determine if their triglycerides were over 100 mg/dl, but less than 1000 mg/dl. Qualifying subjects then had a complete blood count including hemoglobin and white blood cell count, blood chemistries (liver transaminases, alkaline phosphatase, bilirubin, albumin, total protein, blood urea nitrogen, creatinine, calcium, glucose, sodium, potassium, chloride, and bicarbonate), insulin, and a lipid profile consisting of a total cholesterol, triglyceride, HDL cholesterol, and a calculated LDL cholesterol. All these tests were performed on fresh samples at baseline and fasting at the end of the study by Quest Laboratories (Cambridge, MA). All subjects were required to have a fasting serum triglyceride values > 100 mg/dl, normal liver enzymes, defined as less than 3 times the upper limits of normal, and a serum creatinine level less than 2.5 mg/dl. At the baseline examination a standard medical history and physical examination were carried out. Subjects were also excluded if they had any evidence of significant cognitive impairment or unstable medical problems. At that time the following measurements were carried out: blood pressure, height, weight, waist measurement, and subjects had fasting blood work done. All subjects received a stipend for participation.
A total of 79 qualifying subjects were randomized into the trial in a 2:1 fashion (active:placebo) and received either active nutritional supplement (Animi 3, a prescription item) prepared by PBM Pharmaceuticals Inc., of Gordonsville, Virginia USA, or placebo in a double-blinded, randomized fashion. Both the active supplements and the placebo consisted of two capsules to be taken twice daily for a total of four capsules per day. Four active capsules contained 1400 mg of docosahexaenoic acid (DHA), 140 mg of eicosapentaenoic acid (EPA), 800 mg of plant sterols, 4 mg of folic acid, 50 mg of vitamin B6, and 2000 mcg of vitamin B12, while the placebo capsules contained corn oil. Subjects were contacted at 1 month and 3 months by telephone to assure compliance. Two subjects withdrew from the placebo group due to individual complaints of hair loss and that the number and size of the capsules were too large. Ten subjects withdrew from the active group for the following individual reasons: 2 with complaints of hair loss, 2 with complaints of heartburn, and individual complaints of nausea, blood in the stools related to hemorrhoids, worsening of diabetes, too many capsules, acne, and recurrence of prostate cancer requiring radiation therapy. All adverse events were reported to the institutional review board. All subjects were given calendars to chart their use of capsules and were asked to bring unused capsules. A total of 67 subjects completed the study, of whom 42 subjects received active compound, and 25 received placebo. Those subjects completing the study all had the same vital signs, blood tests and cognitive function tests assessed at the 6 month time point as at randomization.
In the 42 subjects (20 men, 22 women) completing the active phase, their mean (standard deviation) age, body mass index, blood pressure and glucose values were 67.8 (12.7) years, 29.6 (4.6) kg/m2, 128.4 (17.2)/81.1 (9.5) mmHg, and 96.1 (12.2) mg/dl, respectively, while for the 25 subjects (12 men, 13 women) completing the placebo phase these values were 68.3 (11.0) years, 29.2 (4.6) kg/m2, 128.8 (15.6)/80.4 (10.1) mmHg, and 92.8 (12.2) mg/dl, with both groups having identical mean creatinine values of 0.9 (0.2) mg/dl). No significant differences between treatment groups for any of these parameters was noted. With regard to ethnicity 88% of the active group and 96% of the placebo group were Caucasian, 10% and 4% were African American, and 2% and 0% were Asian, respectively. In addition equal or similar percentages of subjects placed on placebo or active supplement were smokers at 12% and 12%, were taking blood pressure lowering medications at 24%% and 16%%, and were taking medications for diabetes at 0% and 4%. At baseline no statistically significant differences were observed for subjects placed on placebo or active supplement with regard to hemoglobin, white blood cell count, liver transaminases, alkaline phosphatase, bilirubin, albumin, total protein, blood urea nitrogen, calcium, glucose, sodium, potassium, chloride, and bicarbonate, insulin, sodium, potassium, and bicarbonate (data not shown). Moreover there were no differences in effects of placebo versus active supplement on any of these parameters except for the liver transaminases. Mean aspartate transaminase (AST) increased from 20.6 by 23.8% to 25.0 (P<0.001), and mean alanine transaminase (ALT) increased from 20.4 by 46.8% to 29.5 (p <0.001). Such increases have been previously observed and are consistent with increased uptake of omega 3 fatty acids by the liver with supplementation. These values however are still within the range of normality. No such effects on bilirubin or alkaline phosphatase were noted.
Specific Biochemical Measurements
Using frozen plasma and red blood cells obtained at baseline and after 6 months of treatment from samples which had never been previously thawed and had been frozen at -80 degrees C to carry out a variety of biochemical assays in our laboratory at Tufts University. High sensitivity C-reactive protein (CRP), apolipoprotein (apo) A-I, A-II, B, E, and lipoprotein (a) or Lp(a) were measured using immunoassay kits obtained from Wako Diagnostics (Richmond, VA) as previously described (14-18). Total cholesterol, triglyceride, and HDL cholesterol were measured as previously described Direct LDL cholesterol and small dense LDL cholesterol levels were measured using kits obtained from Denka-Seiken Corporation, Tokyo, Japan), and remnant lipoprotein cholesterol (using kits obtained from Kyowa-Medex Corporation, Tokyo, Japan) as previously described (19-21). ). All these assays were carried out on a Hitachi Model 911 automated analyzer, and had within and between run coefficients of variation of less than 5%. Plasma apoB-48 was measured by an enzyme linked immunosorbent assay obtained from the Shibayagi Company, Gunma, Japan (22). All these assays were carried out on a Hitachi Model 911 automated analyzer, and had within and between run coefficients of variation of less than 5%. ApoA-I in HDL subpopulations was determined by immunoblotting with specific antibody after two dimensional gel electrophoresis of plasma as previously described (4-6). Homocysteine and folate were measured by high-performance liquid chromatography as previously described (24). Our laboratories maintains lipid standardization with the Centers for Disease Control, Atlanta, GA. Red blood cell fatty acids were measured by gas liquid chromatography as previously described (11,12,25). All laboratory personnel were blinded from treatment information until all analyses were completed.
The results for various parameters obtained for study subjects at 6 months were compared to baseline as a function of absolute values, changes from baseline, and percent changes from baseline, and the active group and the placebo group were compared using analysis of variance, as well as paired t test analysis with SYSTAT software. P values of < 0.05 were considered statistically significant.
Plasma Lipid and Lipoprotein Cholesterol Levels and Changes
Baseline values and percentage changes in plasma lipid and lipoprotein cholesterol levels are shown in table 1. Use of the active supplement resulted in significant mean percentage changes versus placebo in total cholesterol (-9.1%), triglyceride (-31.2%), HDL-cholesterol (+10.2%), non-HDL cholesterol (-14.4%), total cholesterol/HDL cholesterol ratio (-14.9%), and remnant lipoprotein cholesterol (-34.7%). Similar percentage changes were observed versus baseline for the active supplement, but not for the placebo group.
Plasma Apolipoprotein Levels and Changes
Baseline values and percentage changes in plasma apolipoprotein levels are shown in table 2. Use of the active supplement resulted in non-significant mean percentage changes versus placebo in total apoB (-9.3%) and apoB-48 (-16.0%), but significant changes in apoA-I in large alpha 1 HDL of 32.2%, apoA-I in large alpha 2 HDL of 9.2%, and apoA-I in small alpha 3 HDL (-17.5%). Similar percentage changes were observed versus baseline for the active supplement, but not for the placebo group.
Other Biochemical Variable Levels and Changes
Baseline values and percentage changes in other biochemical variable levels are shown in table 3. Use of the active supplement resulted in significant mean percentage changes versus placebo in plasma homocysteine (-23.6%), plasma folate (+287.0%), and red blood cell docosahexaenoic acid (+268.8%), with no significant effects on changes in white blood cell count, C reactive protein, glucose, insulin, or adiponectin levels.
We sought to test whether supplementation with omega 3 fatty acids (especially DHA), plant sterols, vitamins B6, B12 and folate would have a favorable effect on cardiovascular risk factors. In the present study, as one would expect the active supplement increased levels of red blood cell DHA and plasma folate levels about 3 fold, and decreased plasma homocysteine by about 24% as compared to placebo. These data indicate that subjects in the active arm of the protocol had good compliance. The supplement tested in the current study was reasonably well tolerated, but some subjects did experience adverse side effects, with the most common complaints being heartburn and hair loss. Moreover we had 10 drop-outs in the active group and 2 in the placebo group.
Intake of fish or fish oil capsules (1-2 capsules/day) has been reported in a number of intervention trials to decrease death from coronary heart disease in patients with established CHD in a British study as well as in a large Italian study, and CHD events in a large Japanese primary prevention trial with 1800 mg of EPA in hypercholesterolemic patients all on statins (7-9). In the Italian trial no significant effects on triglycerides and other lipid measures versus placebo were noted (8). Many investigators have documented that high doses of oral omega 3 fatty acids decrease plasma triglyceride levels (25). We have previously documented that 1200 mg of DHA given orally per day was well tolerated, and lowered serum triglyceride levels by 24% and raise plasma DHA by 311% and HDL cholesterol by 6%, versus placebo (26).
Consistent with many previous studies we noted a very significant 31% reductions in plasma triglyceride levels with the active supplement versus placebo. In addition we documented 35% reductions in remnant lipoprotein cholesterol levels versus placebo. We have documented that elevated remnant lipoprotein cholesterol is a better marker of CHD risk than triglyceride levels in the Framingham Study, especially in women (3). The active supplement also significantly reduced non-HDL cholesterol, and the total cholesterol/HDL cholesterol ratio by 15% versus placebo, and increased HDL cholesterol by 10% versus placebo. These parameters are substantially more powerful predictors of CHD risk in the Framingham Offspring Study than is LDL cholesterol, as we have recently documented (2). We did observe a trend towards greater reduction in calculated LDL cholesterol (-9.2%) and direct LDL cholesterol (-4.6%) with the active supplement as compared to placebo, but these differences were not statistically significant. Consistent with other studies we saw no significant effects of the active supplement on levels of C reactive protein or white blood cell count (26).
We have reported that high levels of apoA-I in large alpha 1 and alpha 2 migrating HDL particles are better predictors of protection from CHD than is elevated HDL cholesterol (4,5). Moreover we have linked increases in these large HDL particles with the niacin/simvastatin combination with significantly less progression and more regression of coronary atherosclerosis as assessed by quantitative coronary angiography (6). The active supplement not only raised HDL cholesterol significantly relative to placebo, but dramatically and significantly increased apoA-I in large HDL particles by 37% as compared to placebo. The overall data indicates that the nutritional supplement used in this study was quite effective in reducing triglycerides, non-HDL cholesterol, and remnant lipoprotein cholesterol, as well as the total cholesterol/HDL cholesterol ratio, all associated with increased CHD risk, and increasing HDL cholesterol and large HDL particles associated with decreased CHD risk.
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Table 1 Plasma Lipid and Lipoprotein Test Values at Baseline and % Change at 6 Months+
|Supplement Group (n=42)||Placebo Group (n=25)|
|Variables (mg/dl)||Baseline||6-Month Change (%)||Baseline||6-Month Change (%)||P-Value for Difference++|
|Total Cholesterol (C)||232.5 ± 41.7||-6.2 ± 12.3*||225.5 ± 36.5||+2.9 ± 10.4||0.050|
|Triglyceride||169.1 ± 78.4||-25.3 ± 24.6*||147.2 ± 56.0||+5.9 ± 32.7||0.001|
|HDL C||52.9 ± 16.5||+14.2 ± 21.0**||58.7 ± 15.8||4.0 ± 14.1||0.006|
|Non HDL C||179.4 ± 42.0||-11.0 ± 14.3*||168.6 ± 33.4||+3.4 ± 15.4||0.001|
|TC/HDL C Ratio||4.7 ± 1.1||-13.1 ± 13.2**||4.1± 1.1||+1.8 ± 14.6||0.001|
|Calculated LDL C||146.0 ± 37.5||-5.5 ± 23.0*||139.2 ± 33.5||+3.7 ± 17.0||0.688|
|Direct LDL C||141.4 ± 38.5||-4.8 ± 24.4||139.1 ± 28.9||-0.2 ± 79.0||0.798|
|Small Dense LDL C||61.8 ± 34.3||-2.8 ± 28.3||54.6 ± 18.7||-8.8 ± 28.3||0.319|
|Remnant Lipoprotein C||11.2 ± 7.0||-32.1 ± 29.4*||9.9 ± 5.2||+2.6 ± 45.1||0.001|
|Lipoprotein (a)||21.4 ± 23.5||+4.0 ± 49.7||18.9 ± 24.8||+5.1 ± 27.9||0.931|
+Mean values and percentage changes at 6 months, with standard deviations; * p<0.05 versus baseline, ** p<0.01 versus baseline, ++p value for treatment differences as % change.
Table 2 Apolipoprotein Test Values in Plasma and HDL Particles at Baseline and % Changes at 6 Months+
|Supplement Group (n=42)||Placebo Group (n=25)|
|Variables||Baseline||6-Month Change (%)||Baseline||6-Month Change (%)||P-value++|
|Homocysteine (uM/L)||9.3 + 4.1||-15.6 + 15.2*||8.5 + 2.0||+8.0 + 18.7||0.001|
|Folate (ng/ml)||20.2+ 6.5||+298.8 + 398.3*||25.2 + 10.4||+11.8 + 57.8||0.001|
|RBC DHA (% of total)||4.01 + 0.41||+274.1 + 131.8**||4.05 + 0.35||+5.3 + 22.22||0.001|
|White Blood Cell Count 106/ml||6.2 + 1.1||-3.0 + 11.1||6.0 + 1.1||+1.5 + 15.8||0.930|
|C Reactive Protein (mg/L)||2.7 + 2.9||-2.8+ 34.8||4.0 + 4.2||-3.3 + 37.4||0.947|
|Glucose (mg/dl)||92.8 + 12.2||-1.3 + 8.5||96.1 + 12.2||-0.3 + 9.1||0.646|
|Insulin (uU/ml)||10.1 + 7.8||+14.2 + 39.7||10.0 + 7.7||+0.5 + 32.1||0.627|
|Adiponectin||15.1 + 8.0||+10.8 + 23.3*||12.1 + 6.1||0.5 + 20.5||0.083|
+Mean values and percentage changes at 6 months, with standard deviations; * p<0.05 versus baseline, ** p<0.01 versus baseline, ++p value for treatment differences as % change
Table 3 Other Biochemical Variable Test Values at Baseline and % Changes at 6 Months+
|Supplement Group (n=42)||Placebo Group (n=25)|
|Variables (mg/dl)||Baseline||6-Month Change (%)||Baseline||6-Month Change (%)||P-value++|
|Apolipoprotein (Apo) B||113.2 ± 25.0||-7.1 ± 14.8||108.9 ± 19.0||+1.2 ± 11.8||0.072|
|Apo B-48||0.6 ± 0.3||-20.7 ± 38.7||0.7 ± 0.4||-4.7 ± 42.0||0.156|
|Apo A-II||32.5 ± 3.9||-3.8 ± 9.5||36.3 ± 6.3||-0.7 ± 9.5||0.068|
|Apo E||4.9 ± 1.2||-0.8 ± 16.4||4.9 ± 1.0||+2.1 ± 14.3||0.965|
|Apo A-I||142.5 ± 26.4||+1.7 ± 3.4||156.5 ± 22.8||+0.4 ± 3.6||0.901|
|Apo A-I in small pre-beta 1 HDL||18.4 ± 6.9||-3.3± 21.7||19.3± 5.9||-3.1 ± 25.2||0.230|
|Apo A-I in small alpha 4 HDL||11.7 ± 3.1||+7.2± 19.8||13.0 ± 4.3||+3.8 ± 25.3||0.663|
|Apo A-I in intermediate alpha 3 HDL||27.3 ± 7.8||-12.8 ± 21.6*||26.6 ± 6.5||+4.7 ± 15.4||0.001|
|Apo A-I in large alpha 2 HDL||42.3 ± 14.0||+2.3 ± 23.7||49.1 ± 11.1||-6.9 ± 14.0||0.003|
|Apo A-I in large alpha 1 HDL||20.0 ± 38.5||+34.6 ± 39.9*||22.4 ± 9.7||+2.4 ± 25.9||0.001|
+Mean values and percentage changes at 6 months, with standard deviations; * p<0.05 versus baseline, ** p<0.01 versus baseline, ++p value for treatment differences as % change.