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Journal of the American College of Nutrition, Vol. 24, No. 3, 189-199 (2005)
Published by the American College of Nutrition

Lipid Responses to a Dietary Docosahexaenoic Acid Supplement in Men and Women with Below Average Levels of High Density Lipoprotein Cholesterol

Kevin C. Maki, PhD, Mary E. Van Elswyk, PhD, RD, Deanna McCarthy, RD, Serena P. Hess, MA, Patricia E. Veith, MPH, RD, Marjorie Bell, Papasani Subbaiah, PhD and Michael H. Davidson, MD

Radiant Development (K.C.M., S.P.H., P.E.V., M.B., M.H.D.), Chicago, Illinois
University of Illinois (P.S.), Chicago, Illinois
Martek Biosciences Boulder Corporation, Boulder, Colorado (M.E.V.E., D.M.)

Address reprint requests to: Michael H. Davidson, M.D., Radiant Development, 515 N. State St., 27th Floor, Chicago, IL 60610. E-mail: MichaelDavidson{at}RadiantResearch.com


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 CONCLUSIONS
 ACKNOWLEDGMENTS
 REFERENCES
 
Objective: To assess fasting lipid responses to a docosahexaenoic acid (DHA) supplement in men and women with below-average levels of high-density lipoprotein (HDL) cholesterol.

Methods: This randomized, double-blind, controlled clinical trial included 57 subjects, 21–80 years of age, with fasting HDL cholesterol concentrations ≤44 mg/dL (men) and ≤54 mg/dL (women), but ≥35 mg/dL. Subjects were randomly assigned to receive either 1.52 g/day DHA from capsules containing DHA-rich algal triglycerides or olive oil (control) for six weeks.

Results: There were no significant differences between groups in baseline lipid values. The DHA supplemented group showed significant changes [–43 (DHA) vs. –14 (controls) mg/dL, p = 0.015] and percent changes [–21% (DHA) vs. –7% (controls), p = 0.009] in triglycerides, total (12 vs. 3 mg/dL; p = 0.021 and 6% vs. 2%; p = 0.018) and low-density lipoprotein (17 vs. 3 mg/dL; p = 0.001 and 12% vs. 3%; p = 0.001) cholesterol concentrations, and in the triglyceride to HDL cholesterol ratio (–1.33 vs. –0.50, p = 0.010), compared with controls. In addition, there was a significant reduction in the percentage of LDL cholesterol carried by small, dense particles in the DHA supplemented group (changes = –10% vs. –3%, p = 0.025).

Conclusions: Supplementation with 1.52 g/d of DHA in men and women with below-average HDL cholesterol concentrations raised the LDL cholesterol level, but had favorable effects on triglycerides, the triglyceride/HDL cholesterol ratio and the fraction of LDL cholesterol carried by small, dense particles. Further research is warranted to evaluate the net impact of these alterations on cardiovascular risk.

Key words: docosahexaenoic acid (DHA), fatty acids, high-density lipoprotein (HDL), low-density lipoprotein (LDL), hypertriglyceridemia


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 CONCLUSIONS
 ACKNOWLEDGMENTS
 REFERENCES
 
Evidence from epidemiologic studies [1–6], as well as randomized, controlled clinical trials [5,7,8] suggests that consumption of omega-3 (n-3) fatty acids reduces cardiovascular event risk. Prospective secondary prevention studies indicate that intake of 0.5 to 1.8 g/d of long-chain n-3 fatty acids (eicosapenatenoic acid, EPA; docosahexaenoic acid, DHA) from fish or dietary supplements significantly reduces all-cause and cardiac mortality [5]. A recent meta-analysis of data from randomized clinical trials showed that consumption of long-chain n-3 fatty acids from dietary and supplemental sources was associated with a 20–30% reduction in risk for various cardiovascular outcomes, including fatal and non-fatal myocardial infarction and sudden death [9].

The American Heart Association has recently issued recommendations for long-chain n-3 fatty acid intake [5]. The recommendations state that for people without documented coronary heart disease, consumption of at least two fish meals per week is recommended. For those with documented coronary heart disease, use of oily fish and supplemental sources, if needed, are recommended to achieve intakes of DHA and EPA of ~1 g/d. For patients in need of triglyceride lowering, consideration should be given to using 2 to 4 g/d of EPA+DHA as capsules under a physician’s care.

A variety of mechanisms have been proposed to account for the cardioprotective effects of long-chain n-3 fatty acids, including their influence on serum lipids and lipoproteins, blood pressure, platelet activity and hemostasis, inflammation, susceptibility to cardiac arrhythmias, and effects on vascular structure and function [5]. Effects of long-chain n-3 fatty acids on serum lipids and lipoproteins have received a great deal of attention in the scientific literature. An analysis of results from 65 randomized, controlled trials concluded that intake of roughly 4 g/d of long-chain n-3 fatty acids from marine oil (EPA+DHA) reduced serum triglycerides by 25–34%, increased LDL cholesterol by 5–11% and raised HDL cholesterol by 0–3% [10]. Both the degree of triglyceride reduction and the increase in LDL cholesterol were more pronounced in subjects with elevated baseline triglyceride concentrations [10].

The individual effects of EPA and DHA on the lipid profile are less well understood. The limited data published to date suggest that both EPA and DHA lower triglycerides [11–13]. Results from some [11,12], but not all [13], studies have suggested that DHA may have a slightly greater hypotriglyceridemic effect and ability to raise HDL cholesterol levels, particularly cholesterol carried by larger HDL2 particles.

Depressed HDL cholesterol concentration is a strong predictor of cardiovascular disease risk, the influence of which is statistically independent of the level of LDL cholesterol and other cardiovascular risk factors [14–16]. Epidemiologic data indicate that this relationship is continuous with no threshold [14]. Each 1% reduction in the level of HDL cholesterol level is associated with a 2–3% increase in coronary heart disease event risk [15]. However, the degree to which this increased risk is attributable to lower levels of HDL cholesterol concentration per se is uncertain, since depressed HDL cholesterol is often accompanied by other lipid disturbances that have been linked to increased cardiovascular risk, including elevated levels of triglycerides and triglyceride-rich lipoproteins and a greater proportion of cholesterol carried by small, dense LDL particles [17–19]. In addition, low HDL cholesterol is a component of the metabolic syndrome, a constellation of risk factors that may confer increased risk beyond that captured by evaluation of traditional risk markers [20]. Because of the high degree of covariation and metabolic links between the HDL cholesterol level and other metabolic disturbances, it is difficult to statistically separate their individual contributions to cardiovascular risk [21].

Although the effects of long-chain n-3 fatty acid supplements on HDL cholesterol have generally been modest and often not statistically significant [10], potentially beneficial responses have been reported on the other lipid abnormalities associated with depressed HDL cholesterol concentration, including lowered levels of fasting and postprandial triglycerides, reduced concentrations of triglyceride-rich lipoproteins and their remnants, and enhancement of LDL particle size [12,22–25].

Given that some reports have suggested a greater ability of DHA than EPA to raise HDL cholesterol concentration [11,12,23] and the potentially favorable effects of DHA on other lipid abnormalities associated with depressed HDL cholesterol, the authors felt that it would be of interest to evaluate the impact of DHA supplements on the serum lipid profile in a sample selected on the basis of having below-average levels of HDL cholesterol. The present study was designed to assess fasting lipid and lipoprotein responses to consumption of a DHA supplement (1.52 g/d DHA) in men and women with HDL cholesterol concentrations below the sex-specific median levels for the United States population.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 CONCLUSIONS
 ACKNOWLEDGMENTS
 REFERENCES
 
This was a randomized, double-blind, controlled clinical trial. The study was performed according to Good Clinical Practice Guidelines, the Declaration of Helsinki (1996), and US 21 CFR Part 50—Protection of Human Subjects, and Part 56—Institutional Review Boards. An institutional review board (Schulman Associates IRB, Inc., Cincinnati, OH) approved the protocol prior to the initiation of the study. Study procedures were reviewed with subjects and each participant provided written informed consent before protocol-specific procedures were carried out.

Subjects
Potential participants (21 to 80 years of age) were recruited from the Chicago metropolitan area and pre-screened by telephone. Eligibility was further assessed at screening and baseline visits (weeks –2, –1, and 0). At the week –2 visit, eligible subjects were required to have HDL cholesterol levels below the sex-specific median for the U.S. population (≤44 mg/dL for men and ≤54 mg/dL for women), but ≥35 mg/dL. Participants also needed to be in apparent good health, to be willing to abstain from alcohol for 48 hours prior to each clinic visit, and to maintain body weight and usual level of physical activity. Women of childbearing potential were required to have a negative urine pregnancy test at the week –1 visit and to use an approved method of contraception throughout the study.

Subjects with body mass index (BMI) > 40 kg/m2 were excluded, as were those with a serum total cholesterol concentration > 300 mg/dL or a serum triglyceride level > 350 mg/dL at week –2. Subjects had to abstain from all foods (e.g., seafood and flax-based foods) and supplements with significant omega-3 fatty acid content for two weeks prior to the week –2 screening visit and throughout the trial. Use of hypolipidemic medication within four weeks of week –2 was also exclusionary, as was the use of dietary fiber supplements, and products containing phytosterols/stanols within two weeks of week –2.

Poorly controlled hypertension (systolic blood pressure ≥160 mm Hg and/or diastolic blood pressure ≥100 mm Hg) was an exclusion criterion, although subjects with adequately controlled hypertension were allowed to participate provided that their dose of anti-hypertensive therapy had remained constant for two months prior to the week –2 visit.

Clinic Visits
Subjects visited the clinic at weeks –2 and –1 (screening), week 0 (baseline), and weeks 3, 5, and 6 (treatment) for assessments of vital signs, height (week –2 only), weight, and a fasting serum lipid profile (total, LDL, HDL, and non-HDL cholesterol, and triglycerides). A telephone visit was performed between weeks –1 and 0, to ensure that subjects were not consuming exclusionary foods, medications, or supplements and to confirm each subject’s visit schedule. A brief physical examination was performed at baseline (week 0). Serum chemistry and hematology panels and a urine pregnancy test (for women of childbearing potential) were completed at screening (week –1) and at the end of treatment (week 6). Waist circumference was measured, the Stanford 7-Day Physical Activity Recall questionnaire [26] was completed, and serum EPA and DHA concentrations were assessed at weeks 0 and 6. Three-day diet records were dispensed at weeks –1 and 5, and collected and analyzed at weeks 0 and 6, respectively. A study product questionnaire was also completed at the end of treatment (week 6).

Subjects were randomized at baseline (week 0) to one of two double-blind treatments: DHA capsules that provided 1.52 g DHA per day (DHA treatment) or olive oil capsules (control). Assessments of concomitant medication use and adverse events were performed at each treatment visit (weeks 3, 5, and 6).

Study Products
Study products were supplied by Martek Biosciences Inc. They included DHA (derived from Schizochytrium sp.) and control (olive oil) capsules (Table 1), which were indistinguishable from one another in appearance and odor. The primary fatty acid in the control capsules was oleic acid (0.65 g/capsule), and the primary fatty acid in the DHA capsules was DHA (0.38 g/capsule). Subjects were instructed to take four capsules per day.


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  		Table 1. Major Fatty Acid Compositions of the DHA and Control Capsules1,2

 
To verify the fatty acid profile of the capsules, aliquots of oil were transesterified using anhydrous HCl in methanol in order to produce the study products. Resultant fatty acid methyl esters were identified and quantified using NuChek Prep analytical reference standard 502 using an internal standard (19:0) to determine empirical response factors. Gas-liquid chromatography of methyl esters was performed using a Hewlett-Packard Model 6890 Series II gas-liquid chromatograph equipped with a Hewlett-Packard autosampler, ChemStation software, a 30 m x 0.32 mm SP-2380 capillary column (Supelco), and a flame-ionization detector. The oven temperature was held at 120°C for 3 min, programmed to 190°C at 5°C/min, held at 190°C for 1 min, programmed to 260°C at 20°C/min, then held for 3 minutes at 260°C. The injector temperature was set at 295°C and the detector temperature was set at 280°C. Helium was used as a carrier gas and a split injection technique was employed.

Laboratory Measurements
Medical Research Laboratories (Highland Heights, KY) performed laboratory measurements including the serum chemistry, hematology, lipid profiles, and fibrinogen. Serum chemistry analysis was conducted on the Hitachi 747 (Roche Diagnostics, Indianapolis, IN) and serum hematology testing utilized the Coulter STKS (Coulter Corporation, Miami, FL). Fibrinogen was measured using the Behring Nephelometer (Dade Behring Diagnostics, Auckland, New Zealand).

Serum lipids were analyzed with Centers for Disease Control and Prevention lipid standardization program methods [27]. Cholesterol and triglycerides were measured enzymatically using the Hitachi 747 (Roche Diagnostics, Indianapolis, IN). Heparin and manganese chlorides were used to isolate HDL cholesterol. Low-density lipoprotein cholesterol in mg/dL was calculated using the Friedewald equation (LDL cholesterol = total cholesterol – HDL cholesterol – triglycerides/5) [28].

Lipoprotein subfractions were analyzed by the Vertical Auto Profile II (VAP-II). This method assessed the concentration of cholesterol carried in large, buoyant (LDL1 and LDL2) and small, dense (LDL3 and LDL4) LDL particles and in large (HDL2) and small (HDL3) HDL particles, as explained in detail elsewhere [29]. Briefly, blood samples were drawn and serum was diluted with saline/EDTA to meet VAP-II sensitivity. Serum lipoproteins were separated by single vertical spin density-gradient ultracentrifugation and the cholesterol content was assessed at close intervals throughout the density spectrum with the VAP II analyzer (Atherotech, Inc., Birmingham, AL). A deconvolution algorithm was applied to define cholesterol carried by various subfractions [29,30].

Serum fatty acids [DHA, EPA, linolenic acid (LNA), docosapentaenoic acids (DPA n-3 and DPA n-6), and arachidonic acid (AA)] were measured from samples collected at weeks 0 and 6. The total lipids were extracted from an aliquot of plasma with chloroform-methanol by the procedure of Bligh and Dyer [31]. Fatty acid methyl esters were prepared from the total lipids using BF3-methanol reagent (Supelco Inc, Bellafone, PA). The dried lipid sample was heated at 90°C under nitrogen with 1 ml of BF3-methanol, and the methyl esters were extracted twice with 2 ml hexane. The methyl esters were analyzed by capillary GC (Shimadzu GC-9A) equipped with a flame ionization detector [32]. Peaks were identified by comparing with retention times of methyl ester standards. The concentrations of various fatty acids were calculated with reference to a 17:0 internal standard. The temperature was initially 172°C for 8 minutes, and was raised at a rate of 6°C per minute to a final temperature of 220°C, which was maintained for 20 minutes.

Diet Assessment
Dietary counseling was reinforced at each clinic visit. Subjects completed diet records for three consecutive days (including one weekend day) during the weeks prior to baseline (week 0) and the last treatment visit (week 6). Three-day diet records were analyzed using the University of Minnesota Nutrition Data System for Research, version 4.03_31 (2000).

Physical Activity Assessment
At the initial screening clinic visit (week –2), subjects were instructed to maintain their usual levels of physical activity throughout the trial. At each clinic visit, they were specifically asked about any unusual or vigorous activity that they had participated in over the past 48 hours. Subjects completed the Stanford 7-day Physical Activity Recall questionnaire at the baseline (week 0) and final (week 6) clinic visits. This questionnaire was used to calculate an activity score, based on the amount of time spent engaged in various moderate, hard, or very hard physical activities over the previous seven days [26].

Study Product Questionnaire
At the end of treatment (week 6), subjects completed a questionnaire that asked whether they experienced any gastrointestinal disturbances after consumption of the capsules and whether they thought the capsules had a fishy taste or marine odor.

Safety
Safety and tolerability were assessed by monitoring treatment-emergent adverse events at each post-randomization clinic visit and by serum chemistry and fibrinogen assessments (taken at weeks –1 and 6). Subjects were questioned about adverse signs or symptoms at each post-randomization clinic visit. A study physician evaluated each adverse event and made a determination as to severity and possible relation to test product, recorded the outcome and any action taken. All assessments were completed prior to breaking the treatment code, thus without knowledge of treatment assignment.

Statistical Analyses
Statistical analyses were conducted using the SAS version 8.0 statistical analysis package (SAS Institute, Cary, NC) for the personal computer. All tests for significance were performed at {alpha} = 0.05, two-tailed. In some cases, values were not normally distributed, so rank transformations were employed prior to calculating inferential statistics.

Baseline comparability of treatments for demographic, anthropometric and lipid values was assessed by analysis of variance (ANOVA), chi-square tests, or Fisher’s exact test as appropriate. Possible differences in the incidence of treatment-emergent adverse events were assessed with Fisher’s exact test. Spearman correlation analyses were performed to assess relationships between changes in lipid, lipoprotein and lipoprotein subclass cholesterol concentrations during treatment.

Differences between groups in serum lipid responses, dietary intake and physical activity were also assessed by ANOVA. For fasting lipid variables, models were run assessing the changes from baseline to the end of treatment with the baseline value included in each model as a covariate. For the components of the fasting lipid profile (triglycerides and total, HDL and LDL cholesterol), baseline was defined as the average of values collected at weeks –2, –1 and 0 and end of treatment was defined as the average of values collected at weeks 5 and 6. For lipoprotein subfraction and serum fatty acid analyses, baseline and end or treatment values represent those collected at weeks 0 and 6. For normally distributed variables, least squares means and standard errors (SEMs) are reported. For variables that were not normally distributed, median and minimum (min) and maximum (max) values are reported.

Sample size calculations were based on the number of subjects needed to detect a statistically significant (alpha = 0.05, two sided) difference between treatments in HDL cholesterol response. The number of evaluable subjects required in order to have 80% power to detect a 2.8 mg/dL difference in HDL cholesterol response was estimated to be 25 per group. These calculations assumed a pooled standard deviation of 3.5 mg/dL for the HDL cholesterol response.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
CONCLUSIONS
 ACKNOWLEDGMENTS
 REFERENCES
 
Subjects and Demographics
One hundred-three people were screened to identify the 61 subjects randomized. Two of the randomized subjects dropped out of the study prior to completing the treatment period. One was lost to follow-up and the other had an abnormal laboratory value at week –1, but was inadvertently randomized and then removed by the Principal Investigator.

Results are presented for a per protocol sample (n = 57) which excluded data from the two subjects who did not complete the treatment period, as well as data from two who had clinically important weight changes during the treatment period: one subject gained ~5 kilograms and another lost ~5 kilograms. The decision to exclude these subjects was made prior to breaking the treatment code.

Baseline characteristics of subjects are shown in Table 2. There were no significant differences between groups in sex, race, age, height, weight, BMI, blood pressure, waist circumference, or activity score. Two DHA-supplemented and six control subjects were taking hypertension medication during the trial (1 subject on a calcium channel blocker and 2 subjects each either on an angiotensin II receptor antagonist, a ß-adrenergic receptor blocker, or on an angiotensin-converting enzyme inhibitor). Participants had a mean age of ~54 years and a mean BMI of ~30 kg/m2. Forty-nine percent of the sample qualified as having the metabolic syndrome as defined by the National Cholesterol Education Program Adult Treatment Panel III [33].


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  		Table 2. Demographic and Baseline Characteristics of Subjects1,2

 
Mean compliance (evaluated by counting unused capsules returned to the clinic at each treatment visit) with study product consumption was 98.2% in the DHA supplemented group and 99.9% among subjects assigned to the control group. The study product questionnaire results showed that there were no differences between treatments in study product odor or difficulty of consumption (e.g., reports of gastrointestinal cramping, flatulence, or diarrhea). A significantly greater percentage of subjects in the DHA supplemented group reported that the capsules had a fishy taste (14.8% vs. 0% for control; p = 0.044). Both groups reported a low incidence of burping/indigestion (14.3% in the DHA supplemented group vs. 9.7% in the control group; p = 0.697).

Plasma Fatty Acids
Median changes from baseline to the end of treatment in plasma fatty acid concentrations are shown in Table 3. Levels of both DHA and EPA were significantly (p < 0.001) increased in the DHA supplemented group. The elevation in EPA likely reflects some degree of in vivo retro-conversion of DHA to EPA. Concentrations of DPA n-6 were elevated (p = 0.052), while DPA n-3 declined (p = 0.029) in the DHA supplemented group. Concentrations of linolenic and arachidonic acids were not altered significantly.


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  		Table 3. Plasma Fatty Acid Levels According to Treatment1

 
Changes in Body Weight and Physical Activity Score
Body weight did not change significantly during the treatment period in either group (mean change = 0.8 kg for both groups). The DHA supplemented group showed a median decrease of 3.2 metabolic equivalent-hours/week, compared to a median increase of 2.3 metabolic equivalent-hours/week in the control group. This difference approached statistical significance (p = 0.083).

Dietary Analyses
Results of the three-day diet record analyses completed at baseline and at the end of treatment are reported in Table 4. Intakes of study products were included in the analyses where applicable. At baseline (week 0), the only significant difference between groups was a slightly higher intake of polyunsaturated fat in the DHA supplemented group (7.7% vs. 6.4% of energy, p = 0.057). At week 6, the DHA supplemented group had higher intakes (% of energy) of n-3 fatty acids (1.66 vs. 0.66, p < 0.001), DHA (0.78 vs. 0.02, p < 0.001), EPA (0.05 vs. 0.00, p < 0.001), and DPA n-6 (0.31 vs. 0.01, p < 0.001) than the control group, which is consistent with expectations based on the composition of the supplement.


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  		Table 4. Diet Record Analyses at Baseline and the End-of-Study According to Treatment1,2

 
Predicted changes in LDL cholesterol based on changes in dietary intake estimated from diet records were calculated according to the equation of Yu et al. [34]. The DHA supplemented group had a mean predicted increase in LDL cholesterol of 3.9% and the control group a predicted change of –5.3% (p = 0.0012, p < 0.0001 for the difference between groups). This difference resulted from alterations in the ratio of cholesterol-raising saturated (12:0 to 16:0) to unsaturated fatty acids between weeks 0 and 6. Saturated fatty acid intake was unchanged in the DHA supplemented group and reduced slightly among controls. Unsaturated fatty acid intake increased somewhat among controls and showed a small decline in the DHA supplemented group.

Serum Lipids and Lipoproteins
No significant differences were present between the control and DHA supplemented groups with regard to baseline triglyceride, total cholesterol or lipoprotein cholesterol values (Table 5). During treatment, serum triglycerides were reduced to a greater degree in the DHA supplemented group (–42.9 mg/dL, –21.4%) than controls (–14.1 mg/dL, –7.0%; p = 0.015). Total cholesterol was increased [12.4 (5.8%) vs. 2.7 mg/dL (1.5%), p = 0.021] and there was also a trend toward an increase in HDL cholesterol [3.9 (9.0%) vs. 2.2 mg/dL (5.3%), p = 0.080] in the DHA supplemented group compared with controls. The concentrations of cholesterol carried by HDL subfractions were not significantly altered.


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  		Table 5. Lipid Levels According to Treatment123

 
The LDL cholesterol concentration increased in the DHA supplemented group [16.6 (12.0%) vs. 3.4 mg/dL (2.6%), p = 0.001]. This elevation appears to have been attributable to an increase in cholesterol carried by larger, less dense particles. Subjects in the DHA group showed a mean elevation of 20.6 mg/dL in LDL1 + LDL2 cholesterol vs. 2.7 mg/dL among controls, p = 0.006. There was also a near significant mean reduction in cholesterol carried by small, dense LDL3 + LDL4 particles in the DHA supplemented group (–13.5 and –4.3 mg/dL in the DHA supplemented and control groups, respectively, p = 0.074). Together, these changes resulted in a significant net reduction in the DHA supplemented group, relative to controls, in the percentage of LDL cholesterol carried by small, dense particles (–9.7 vs. –3.0%, p = 0.025).

The total to HDL cholesterol ratio response did not differ between DHA vs. control (–0.1 vs. –0.2, respectively; p = 0.517), although, the ratio of triglycerides to HDL cholesterol declined significantly more in the DHA supplemented group than in controls (–1.3 vs. –0.5, p = 0.010).

Correlation Analyses
Spearman correlation coefficients for relationships between changes in lipid, lipoprotein and lipoprotein subclass cholesterol concentrations are shown in Table 6. In the control group, the only significant correlations were between the changes in triglycerides with HDL cholesterol (r = –0.486, p = 0.01) and LDL3 + LDL4 cholesterol with HDL2 cholesterol (r = –0.542, p = 0.05).


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  		Table 6. Spearman Correlation Coefficients for Changes from Baseline to End of Treatment in Lipid, Lipoprotein and Lipoprotein Subfraction Cholesterol Concentrations Presented by Treatment Group

 
Within the DHA supplemented group, changes in triglyceride concentration were inversely associated with changes in HDL cholesterol (r = –0.385, p = 0.05) and cholesterol carried by large, buoyant LDL1 + LDL2 (r = –0.591, p = 0.05) and correlated directly with changes in cholesterol carried by small, dense LDL3 + LDL4 particles (r = 0.700, p = 0.01). Changes in LDL cholesterol concentration were associated with those in cholesterol carried by LDL1 + LDL2 (r = 0.705, p = 0.01) and HDL2 particles (r = 0.626, p = 0.05). The association between the change in HDL cholesterol level was stronger with the change in HDL3 (r = 0.780, p = 0.01) than HDL2 cholesterol (r = 0.539, p = 0.058). In addition, there was a strong inverse relationship between changes in cholesterol carried by small, dense LDL3 + LDL4 and larger, buoyant LDL1 + LDL2 particles (r = –0.779, p = 0.01).

Safety
There were no statistically or clinically important differences in the incidence of abnormal clinical chemistry values or vital signs from baseline to the end of treatment (data not shown). In addition, there were no differences between groups in the incidence of treatment-emergent adverse events overall or for any body system.


   DISCUSSION
 
The present trial investigated the effects of a moderate dose of supplemental DHA (1.52 g/d) on serum lipids, lipoproteins and lipoprotein subfractions in subjects with below-average levels of HDL cholesterol. As expected, the sample was enriched with subjects displaying obesity, mildly elevated triglyceride concentrations and the metabolic syndrome, all of which are associated with depressed HDL cholesterol concentration.

DHA supplementation produced significant elevations in both plasma DHA and EPA concentrations and in DPA n-6, while the DPA n-3 concentration declined. This suggests some degree of conversion of DHA to EPA in vivo. Decreased plasma DPA n-3 likely reflects feedback inhibition of dietary {alpha}-linolenic conversion to EPA and DPA due to accumulation of plasma DHA [35,36].

The supplement also resulted in significant reductions in serum triglycerides, the triglyceride to HDL cholesterol ratio and the percentage of LDL cholesterol carried by small, dense particles. A trend (p = 0.08) was present toward a larger increase in HDL cholesterol than observed in controls. However, these potentially beneficial effects were accompanied by elevations in total and LDL cholesterol concentrations.

Previous studies providing DHA supplements as ethyl esters [11–13] or algal oil [22,23,37,38] ranging in dose from 0.7 to 4.0 g/d, have consistently shown a hypotriglyceridemic effect, with net (control-adjusted) reductions of 14 to 29%. These results are in line with the 14% net decline observed in the present trial.

HDL cholesterol and/or HDL2 cholesterol increased relative to control in most of the previous DHA supplementation trials [11–13,22,23,37]. The net increase in HDL cholesterol in the present investigation was 1.7 mg/dL (3.7%), which did not reach statistical significance (p = 0.08), but is in general agreement with results from prior studies (median of 4.7%, range 0.3 to 18.0%). However, the change in HDL2 cholesterol was not significantly different from that of controls. Thus, the results were equivocal with regard to the effects of DHA on HDL metabolism.

Most previous studies of DHA supplementation have shown increases in LDL cholesterol concentrations [11,12,22,23,37,38] with net LDL cholesterol responses ranging from –2.8% to 16.0% (median = 7.2%). Studies of lipid responses following fish oil supplementation have also reported LDL increases between 5% and 11% [10]. In the present trial, the control-adjusted increase in LDL cholesterol was 13.2 mg/dL (9.4%). Examination of LDL subclass distributions in a subset of subjects revealed that the increase in LDL cholesterol was apparently attributable to an increase in cholesterol carried by larger LDL1 + LDL2 particles, which was accompanied by a near-significant (p = 0.074) reduction in cholesterol carried by smaller, more dense LDL3 + LDL4 particles. In combination, these changes resulted in a significant decline in the percentage of cholesterol carried by small, dense particles, which is in accordance with the findings of other investigations that have assessed the influence of DHA supplementation on LDL particle size [12,22].

In large cohorts, concentrations of cholesterol carried by the three major lipoprotein classes, HDL, LDL and VLDL (reflected by the triglyceride concentration), contribute independently to the prediction of cardiovascular event rates [33,39–41]. Since the HDL cholesterol level is inversely associated with cardiovascular event risk, and concentrations of cholesterol carried by apolipoprotein B100-containing particles (VLDL and LDL) are associated directly with risk, the total/HDL cholesterol ratio is often used as a composite measure to account for the net impact of interventions that alter multiple aspects of the fasting lipid profile [42–44]. In the present study, no significant differences were observed between groups in the response of this ratio, with small declines observed in both treatment arms.

The mechanism(s) responsible for the changes in the lipid profile associated with DHA supplementation are not fully understood. Kinetic studies have shown that the hypotriglyceridemic effects of fish oil containing EPA and DHA are attributable to reduced hepatic VLDL production, due principally to reduced hepatic triglyceride synthesis and enhanced fatty acid beta-oxidation [45,46]. To date, no kinetic data have been published evaluating the effects of DHA and EPA individually. During supplementation, VLDL particles secreted by the liver tend to be smaller and less triglyceride-rich, which allows more rapid conversion through the delipidation process to particles in the LDL density range [46–49]. Without a commensurate increase in the fractional catabolic rate of LDL, this results in an elevation of LDL cholesterol, consistent with the observations in the present study. However, changes in fatty acid intake other than DHA, particularly palmitate, may have also contributed to the elevation in LDL cholesterol as indicated by the predicted changes based on the Yu equation [34].

With less circulating triglyceride substrate for cholesteryl ester transfer protein, exchange of VLDL triglyceride for HDL and LDL cholesterol would be reduced, which favors the maintenance of larger, more buoyant LDL and HDL particles [21,50]. While this was evident in the distribution of cholesterol across LDL subfractions, there was no clear indication of a shift in the distribution of cholesterol toward larger HDL2 particles. This may have been due to a lack of power, since lipoprotein subfraction analyses were only conducted for a subset of the participants and previous trials have generally shown increased HDL2 levels after DHA supplementation with doses of 1.68 to 4.0 g/d [12,13,23].

Despite the potential for net increases in LDL cholesterol, current guidelines [5] recommend fish oil supplements for patients with coronary artery disease and/or hypertriglyceridemia who do not wish to, or cannot, obtain the recommended doses of EPA + DHA from fish. Data from clinical trials [5] and epidemiologic studies [2] suggest that the cardioprotective effects of n-3 fatty acids may occur at intakes below that studied in the present trial. Elevated triglycerides, depressed HDL, and small dense LDL are hallmarks of the metabolic syndrome indicating that DHA supplementation may be an alternative to pharmaceutical interventions in this population.

A review of 11 studies investigating the relationship between n-3 consumption and coronary heart disease risk concluded that the risk reduction was stronger for fatal coronary events than non-fatal myocardial infarction, suggesting that mechanisms other than lipid changes (e.g., anti-arrhythmic effects) may contribute to the cardiovascular benefits [9].

Although it is not possible to conclude that the observed increase in LDL cholesterol is benign, the lack of increase in the total/HDL cholesterol ratio, the decline in the triglyceride/HDL cholesterol ratio and the reduction in the proportion of cholesterol carried by small, dense LDL particles render the changes in LDL cholesterol level less worrisome.


   CONCLUSIONS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 CONCLUSIONS
ACKNOWLEDGMENTS
 REFERENCES
 
Supplementation with 1.52 g/d of DHA in men and women with below-average HDL cholesterol concentrations raised the LDL cholesterol level, but had potentially favorable effects on triglycerides, the triglyceride/HDL cholesterol ratio and the fraction of LDL cholesterol carried by small, dense particles. Further research is warranted to evaluate the net impact of these alterations on cardiovascular risk.


   ACKNOWLEDGMENTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 CONCLUSIONS
 ACKNOWLEDGMENTS
REFERENCES
 
This research was funded by Martek Biosciences Boulder Corporation. The authors wish to thank Nkengyal Barber, Denise Umporowicz and Mary Sue Witchger for their assistance with the conduct of this study.

Received April 23, 2004. Accepted January 19, 2005.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 CONCLUSIONS
 ACKNOWLEDGMENTS
 REFERENCES
 

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