DLM: Elevated Triglycerides and Omega-3 Fatty Acids (2007)

Citation:
 
Study Design:
Class:
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Quality Rating:
Research Purpose:
To see whether a moderate dose of omega-3 fatty acids potentiates the beneficial effets of statins on the high risk for coronary heart disease in patients with combined hyperlipemia.
Inclusion Criteria:
  • Combined hyperlipemia
  • After dietary run in phase, achieve serum TG of 2-15 mmol/L and serum total cholesterol >5.3 mmol/L
Exclusion Criteria:
  • No lipid lowering medications, including fish oil concentrates, antioxidants and others known to affect lipid metabolism for three months prior to study
Description of Study Protocol:

Recruitment Referral to the Lipid Clinic at the Department of Medicine, University of Tromsø, Norway

  • Included individuals who had been on a cholesterol-lowering diet and previously treated with statins with or without omega-3 fatty acids

 

Design Randomized, parallel, double-blind, placebo-controlled trial

 

Blinding used Not described

 

Intervention

  • Dietary run in period for 12-16 weeks
    • Clinical RD advised individuals or group to adjust their macronutrient intake to comprise <30% of energy from fat, with < 10% from saturated fat, 55-60% from CHO (prefereably complex types), and 10-15% of energy from protein.
    • Encouraged to maintain stable body weight, low to moderate alcohol intake, and not to take fish oil supplement.
  • After dietary run in, provided with 10 mg/day atorvastatin for 5 weeks
  • After 5 weeks, randomized to one of the following for additional 5 weeks:
    • 10 mg/day atorvastatin + omega-3 fatty acids
    • 10 mg/day atorvastatin + placebo

Statistical Analysis

  • The number of subjects included in the study was based on the potential effects of omega-3 fatty acids in addition to atorvastatin on plasma triglyceride and HDL-C concentrations.
  • All data were checked with regard to frequency distribution and found normally distributed.
  • Changes in variables were defined as the mean value of two or three consecutive measurements obtained after minus the corresponding mean before intervention.
  • Paired t-test was applied for changes in normally distributed variables within a treatment group.
  • Unpaired t-test was used to compare changes between groups.
  • Repeated measure analysis of variance was performed to assess changes in serum and chylomicron TG during the fat tolerance test.
  • Spearman correlation coefficients were used to examine correlates between variables.
  • Linear trends for changes in EPA and DHA across quaratiles of bseline values were tested in multiple linear regression models.
  • Results were considered statistically significant when the two-sided p-values was <0.05.
  • Values are means±1SD.

 

Data Collection Summary:

Timing of Measurements

  3 months prior to baseline Baseline 5 Weeks 10 Weeks
Dietary education x      
FFQ   x   x
Physical exam   x x x
Blood sampling   x x x
Oral fat load   x   x

 

Dependent Variables

  • Dietary intake: food frequency questionnaire to monitor compliance with dietary advice  
  • Compliance: assessed by counting leftover capsules and measurement of serum total phospholipid fatty acid concentrations
  • Blood sampling
    • Liver and hematology tests, glucose, HbA1c, uric acid and fibrinogen were analyzed by routine tests
    • Serum total cholesterol, free cholesterol, total phospholipids and TG were all measured by enzymatic, colorimetric assays
    • Serum apolipoproteins A1 and B concentrations were measured immunochemically by rate nephelometry
    • Apolipoproteins CII and CIII were measured by turbidimetry
    • Apoliprotein E was measured by hephelometry and genotyped
    • Serum phospholipid fatty acid composition was measured by transmethylation and gas liquid chromatograhy
    • VLDL and chylomicron, LDL, intermediate density lipoproteins and HDL were separated by density gradient ultracentrifugation of plasma and LDL subfractions were isolated from plasma by density gradient ultracentrifugation. Single subfraction concentrations were determined by proportioning the mass.
    • Lipoprotein lipase and hepatic lipase
  • Post prandial lipema: assessed with oral fat load test with fat providing 1 gm/kg body weight

Independent Variables

  • Atorvastatin
  • Atorvastatin + omega-3 fatty acids
    • 1.68 gm/day omega-3 fatty acids as ethyl esters [45% EPA, 39% DHA; (Omacor)]
  • Atorvastatin + placebo
    • Placebo = 2 gm/day corn oil 

 

  • Characteristics of capsules
    • Placebo and omega-3 fatty acid capsules were indistinguishable from one another
    • Each capsule contained 1 gm of oil
    • Contained 4 mg alpha tocopherol according to manufacturer
    • Contents had a peroxide value <0.020 meq/gm and a p-Anicidine value <35.

 

 

Control Variables

 

Description of Actual Data Sample:

 

Initial N: N=42 (32 males/10 females)

  • Omega-3 fatty acid group: 16 males/6 females
  • Placebo: 16 males/4 females

Attrition (final N): N=42

Age: mean±SD

  • Omega-3 fatty acid group: 47.6±10.0
  • Placebo: 53.0±12.9

Ethnicity: not described

Other relevant demographics:

Anthropometrics

                     Omega-3 Fatty Acid Group            Control Group 
 Weight (kg)  86.6±11.6 86.6±14.5 
 BMI  28.25±3.73 27.89±4.10 
 Systolic blood  pressure (mm Hg)  128.8±18.5 125.3±13.7 
 Diastolic blood pressure (mm Hg)  77.5±11.2 77.2±12.4 
 Heart rate/min  68.8±9.7 65.5±10.3 
 Waist/Hip ratio  0.93±0.07 0.91±0.10 
 Glucose (mmol/L)  5.4±0.5* 6.0±1.2 
 P-HbA1c %  5.13±0.27 5.39±0.55 
 Uric acid (mmol/L)  368.6±68.3 377.3±81.3
 Fibrinogen (g/L)  3.22±0.62 3.28±0.97 
Thrombocytes (109/L) 240±57 221±57
Apo E genotypes    
E 2/2 1 2
E 2/3 5 3
E 2/4 0 2
E 3/4 10 5
E 4/4 2 1

*Between group difference at baseline: P<0.05

In addition to the above:

  • None of the subjects were on antihypertensive treatment.
  • None of the subjects were receiving postmenopausal hormone replacement therapy.
  • 2 with diabetes mellitus treated with diet only.
  • The others had no cardiovascular, liver or renal diseases; diabetes melltus; alcoholism or other diseases that might influence lipid metabolism.
  • Ten were regular smokers.

Location: Clinical Research Center, University Hospital, Tromsø, Norway

 

Summary of Results:

 Plasma Lipids, lipoprotein and liprotein subfractions   [means (SD)]

  Omega-3 FA                                             Placebo                Effects attributable to omega-3 FA
  Baseline Atorvastatin         Atorvastatin      + omega-3 FA Baseline Atorvastatin      Atorvastatin + Placebo            CO*
Total cholesterol (mmol/L) 7.68 (1.31) -2.63 (0.93) 3 -0.07 (0.42)  8.33 (2.76)  -2.34 (2.12) 3  -0.36 (0.74) 1  0.13
LDL cholesterol (mmol/L)  4.89 (1.67) -1.79 (1.16) 3   -0.12 (0.51) 5.31 (2.07)  -2.05 (1.59) 3  -0.27 (0.37) 2 0.28 
HDL cholesterol (mmol/L) 0.99 (0.19) +0.08 (0.09) 3   +0.07 (0.08) 3  1.04 (0.25) +0.12 (0.16) 2  +0.01 (0.09) 0.03 
TG (mmol/L)  3.84 (2.45) -1.39 (1.49) 3  -0.04 (0.60)  4.22 (2.46)  -0.08 (2.11) 1  -0.26 (1.71)  0.59 
Apo A 1 (g/L)  1.17 (0.18) +0.10 (0.13)  -0.05 (0.13)  1.32 (0.08)  +0.15 (0.16) 3  -0.08 (0.22) 1  0.57 
Apo B (g/L)  1.25 (0.26) -0.22 (0.20) 3  -0.06 (0.13) 1   1.28 (0.22) -0.27 (0.22) 3  -0.05 (0.12)  0.68 
Apo E (mg/L)  70.5 (18.2) -24.8 (15.6) 3  -3.1 (12.1)  75.6 (26.6) -20.5 (18.1) 3  -6.0 (15.4)  0.52 
Apo CII (mg/L)  73.0 (27.7) -18.5 (20.6) 3  -8.7 (14.3) 2  81.4 (53.8)  -20.6 (46.6) 1  -1.1 (12.9)  0.58 
Apo CIII (mg/L)  203.4 (89.2) -51.5 (56.5) 3  -11.1 (29.2)  224.5 (47.5)  -39.8 (113.0)  -7.7 (32.1)  0.60 

 Within group changes during intervention: 1 P<0.05; 2 P<0.01; 3 P<0.01

* Significance of change in the omega-3 FA group vs change in the placebo group (corn oil).

  1. Atorvastatin reduced total serum choelsterol by about 30%, LDL-C by 35%, TG by 25%, and Apo B and E by 20%.
  2. HDL-C and Apo A1 both increased by approximately 10%.
  3. Additional effect of omega-3 fatty acids was reflected in a significant further increase in HDL-C (P=0.03).

 

Plasma Concentrations of LDL and LDL subfraction before and after treatment with atorvastatin plus omega-3 fatty acids or placebo  [mean (SD)]

Plasma Concentration Omega-3 fatty acids (n=17)   Placebo (N=15)   Effects attributable to omega-3 fatty acids
mg/dl Baseline Change                 Baseline Change             P
LDL 294.6(142.1) -125.4 (149.8) 2 256.4 (96.0) -38.7 (128.9) 1 0.09
LDL-I 20.8 (46.0) -1.7 (53.3) 13.3 (30.5) -6.2 (28.3) 0.84
LDL-II 147.5 (140.5) -76.4 (126.5) 1 119.3 (106.8) -29.5 (141.4) 0.11
LDL-III 126.3 (113.2) -47.4 (115.5) 1 123.8 (106.9) -2.9 (101.9) 0.68

Significance of difference within groups: 1 P<0.05; 2 P<0.01 

  1. Total isolated LDL fraction was reduced in both groups, though more in the omega-3 fatty acid group (p=0.09).
  2. Significant redistribution of the LDL subfractions in the omega-3 fatty acid group with a significant reduction of both the small, dense LDL particles (LDL-III) and the LDL-II fraction, though these changes were not significantly different from the other group.

 

Effects of Atorvastatin plus omega-3 fatty acids or placebo on post-prandial triglyceridemia 8 hours after a standardized meal  [mean (SD)]

  Omega-3 fatty acids (n=22)   Placebo (n=20)   Effects attributable to omega-3 fatty acids
Variable Baseline      Change                       Baseline      Change                                      P
Area under the curve (AUC)                         
Plasma TG (mmol/L/hr 47.6 (30.1) -17.2 (16.5) 3 49.3 (21.7) -10.8 (14.9) 2 0.20
Chylomicron TG (µmol/L/hr 1084.3 (797.3) -357.9 (527.2) 3 1171.0(568.3) -238.8 (413.5) 1 0.42
Incremental AUC          
Plasma TG (mmol/L/hr 16.5 (12.9) -7.0 (12.6) 1 13.7 (7.8) -1.2 (15.4) 0.20
Chylomicron TG (µmol/L/hr) 878.5 (686.4) -347.0 (494.6) 2 871.8 (465.9) -186.4 (626.5) 1 0.37
Triglyceridemic response          
Plasma TG (mmol/L) 3.13 (2.28) -1.22 (2.03) 2 2.84 (1.34) -0.35 (2.34) 0.21
Chylomicron TG (µmol/L) 168.3 (124.0) -58.7 (95.5) 2 170.7 (80.7) -30.0 (110.7) 0.37

Significance of difference within group: 1 P<0.05; 2 P<0.01; 3 P<0.001

  1. Significant reductions were seen in plasma and chylomicron responses measured as total triglyceride AUC in both groups.
  2. Incremental AUCs were significantly reduced in both groups.
  3. Triglyceridemic response, measured as peaks, were only significantly reduced in the omega-3 fatty acid group.
  4. Compared with pre-treatment post-prandial lipid responses, the effects of atorvastatin plus omega-3 fatty acids were consistently, but not significantly more pronounced than in the placebo group.
  5. No significant relationship to apo E genotype was associated with post prandial triglyceride response to omega-3 fatty acids.

 

Effects of omega-3 fatty acids on pre- and post-heparin plasma lipoprotein lipase activity, hepatic triglyceride activity and apolipoprotein CII/CIII ratio

  1. No significant changes were induced in lipoprotein lipase (LPL) or hepatic triglyceride lipase before or after stimulation with heparin in the fasting state in either group.
  2. The apo CII and apo CIII levels were reduced by approximately 25% in both groups in parallel with apo E and apo B.
  3. The apo CII/CIII ratio was also reduced (P<0.05), but there was no difference compared with the placebo group.
  4. In the series as a whole, there were inverse correlations between the fasting triglyceride levels and stimulated LPL activity (P=0.001) and between LPL activity and apo CII (P=0.003) and of apo CIII (P=0.02) levels.
  5. No significant relationship was observed between the apo CII/CIII ratio and heparin-stimulated LPL activity.
  6. No significant changes in the relationship between LPL activiites and the lipid or lipoprotein factions were induced in either group.

Other Findings

  • Compliance was >94% in both groups
  • Subjects maintained their diet without significant differences between the groups.
  • No side effects were observed.
  • Body weight remained constant.
  • No significant changes were observed in serum ALT, gamma-glutamyltransferase or serum creatinine kinase.
  • Increase in EPA in serum total phospholipids was moderate and not significantly higher in subjects with low baseline levels, wherease those with a traditionally high intake (up to 5-6 fish meals a week) showed little or no increase. DHA concentrations displayed a similar pattern.
  • Systolic blood pressure was significantly decreased after supplementation with omega-3 fatty acids (-5.5±11.3 mm Hg, P<0.05)

 

Author Conclusion:
  • This study indicates that addition of a low dose of omega-3 fatty acids may further improve the risk profile for coronary heart disease patients with combined hyperlipemia treated with atorvastatin.
  • The effect is related to reduction of post-prandial hyperlipemia and redistribution of LDL subfractions.
Funding Source:
Reviewer Comments:
  • Unclear how many subjects entered the dietary run in phase but known that 42 achieved target serum TG and cholesterol levels.
  • Little description of inclusion/exclusion criteria.
  • Small sample size, unclear whether sample size calculation performed.
  • The N for plasma concentrations of LDL and LDL subfractions is different than the N in the study: omega-3 fatty acid group=17 while the placebo group=15. No explanation given.
Quality Criteria Checklist: Primary Research
Relevance Questions
  1. Would implementing the studied intervention or procedure (if found successful) result in improved outcomes for the patients/clients/population group? (Not Applicable for some epidemiological studies) Yes
  2. Did the authors study an outcome (dependent variable) or topic that the patients/clients/population group would care about? Yes
  3. Is the focus of the intervention or procedure (independent variable) or topic of study a common issue of concern to dieteticspractice? Yes
  4. Is the intervention or procedure feasible? (NA for some epidemiological studies) Yes
 
Validity Questions
1. Was the research question clearly stated? Yes
  1.1. Was (were) the specific intervention(s) or procedure(s) [independent variable(s)] identified? Yes
  1.2. Was (were) the outcome(s) [dependent variable(s)] clearly indicated? Yes
  1.3. Were the target population and setting specified? Yes
2. Was the selection of study subjects/patients free from bias? Yes
  2.1. Were inclusion/exclusion criteria specified (e.g., risk, point in disease progression, diagnostic or prognosis criteria), and with sufficient detail and without omitting criteria critical to the study? Yes
  2.2. Were criteria applied equally to all study groups? Yes
  2.3. Were health, demographics, and other characteristics of subjects described? Yes
  2.4. Were the subjects/patients a representative sample of the relevant population? Yes
3. Were study groups comparable? Yes
  3.1. Was the method of assigning subjects/patients to groups described and unbiased? (Method of randomization identified if RCT) No
  3.2. Were distribution of disease status, prognostic factors, and other factors (e.g., demographics) similar across study groups at baseline? Yes
  3.3. Were concurrent controls or comparisons used? (Concurrent preferred over historical control or comparison groups.) Yes
  3.4. If cohort study or cross-sectional study, were groups comparable on important confounding factors and/or were preexisting differences accounted for by using appropriate adjustments in statistical analysis? N/A
  3.5. If case control study, were potential confounding factors comparable for cases and controls? (If case series or trial with subjects serving as own control, this criterion is not applicable.) N/A
  3.6. If diagnostic test, was there an independent blind comparison with an appropriate reference standard (e.g., "gold standard")? N/A
4. Was method of handling withdrawals described? Yes
  4.1. Were follow-up methods described and the same for all groups? Yes
  4.2. Was the number, characteristics of withdrawals (i.e., dropouts, lost to follow up, attrition rate) and/or response rate (cross-sectional studies) described for each group? (Follow up goal for a strong study is 80%.) Yes
  4.3. Were all enrolled subjects/patients (in the original sample) accounted for? Yes
  4.4. Were reasons for withdrawals similar across groups? N/A
  4.5. If diagnostic test, was decision to perform reference test not dependent on results of test under study? N/A
5. Was blinding used to prevent introduction of bias? Yes
  5.1. In intervention study, were subjects, clinicians/practitioners, and investigators blinded to treatment group, as appropriate? ???
  5.2. Were data collectors blinded for outcomes assessment? (If outcome is measured using an objective test, such as a lab value, this criterion is assumed to be met.) Yes
  5.3. In cohort study or cross-sectional study, were measurements of outcomes and risk factors blinded? N/A
  5.4. In case control study, was case definition explicit and case ascertainment not influenced by exposure status? N/A
  5.5. In diagnostic study, were test results blinded to patient history and other test results? N/A
6. Were intervention/therapeutic regimens/exposure factor or procedure and any comparison(s) described in detail? Were interveningfactors described? Yes
  6.1. In RCT or other intervention trial, were protocols described for all regimens studied? Yes
  6.2. In observational study, were interventions, study settings, and clinicians/provider described? N/A
  6.3. Was the intensity and duration of the intervention or exposure factor sufficient to produce a meaningful effect? Yes
  6.4. Was the amount of exposure and, if relevant, subject/patient compliance measured? Yes
  6.5. Were co-interventions (e.g., ancillary treatments, other therapies) described? Yes
  6.6. Were extra or unplanned treatments described? N/A
  6.7. Was the information for 6.4, 6.5, and 6.6 assessed the same way for all groups? Yes
  6.8. In diagnostic study, were details of test administration and replication sufficient? N/A
7. Were outcomes clearly defined and the measurements valid and reliable? Yes
  7.1. Were primary and secondary endpoints described and relevant to the question? No
  7.2. Were nutrition measures appropriate to question and outcomes of concern? Yes
  7.3. Was the period of follow-up long enough for important outcome(s) to occur? Yes
  7.4. Were the observations and measurements based on standard, valid, and reliable data collection instruments/tests/procedures? Yes
  7.5. Was the measurement of effect at an appropriate level of precision? Yes
  7.6. Were other factors accounted for (measured) that could affect outcomes? Yes
  7.7. Were the measurements conducted consistently across groups? Yes
8. Was the statistical analysis appropriate for the study design and type of outcome indicators? Yes
  8.1. Were statistical analyses adequately described and the results reported appropriately? Yes
  8.2. Were correct statistical tests used and assumptions of test not violated? Yes
  8.3. Were statistics reported with levels of significance and/or confidence intervals? Yes
  8.4. Was "intent to treat" analysis of outcomes done (and as appropriate, was there an analysis of outcomes for those maximally exposed or a dose-response analysis)? Yes
  8.5. Were adequate adjustments made for effects of confounding factors that might have affected the outcomes (e.g., multivariate analyses)? N/A
  8.6. Was clinical significance as well as statistical significance reported? Yes
  8.7. If negative findings, was a power calculation reported to address type 2 error? ???
9. Are conclusions supported by results with biases and limitations taken into consideration? Yes
  9.1. Is there a discussion of findings? Yes
  9.2. Are biases and study limitations identified and discussed? Yes
10. Is bias due to study's funding or sponsorship unlikely? Yes
  10.1. Were sources of funding and investigators' affiliations described? Yes
  10.2. Was the study free from apparent conflict of interest? Yes