Pilot study of combined therapy with ω-3 fatty acids and niacin in atherogenic dyslipidemia

Journal of Clinical Lipidology (2007) 1, 211–217

Pilot study of combined therapy with ␻-3 fatty acids and niacin in atherogenic dyslipidemia William L. Isley, MD*, John M. Miles, MD, William S. Harris, PhD† Endocrine Research Unit, Division of Endocrinology, Nutrition and Metabolism, Mayo Clinic, 200 First Street SW, Rochester, MN 55905, USA (Drs. Isley and Miles) and Department of Medicine, University of Missouri-Kansas City, Lipid and Diabetes Research Center, Saint Luke’s Hospital, Kansas City, MO 64111, USA (Dr. Harris) KEYWORDS: Fish oils; Flushing reaction; HDL-cholesterol; LDL subfractions; LDL-cholesterol; Lipoproteins; Nicotinic acid; Triglyceride

Abstract. BACKGROUND: Niacin and ␻-3 fatty acids (␻-3 FA) are both nutrients that reduce serum triglyceride and raise high-density lipoprotein cholesterol (HDL-C) levels when used at pharmacological doses. The possibility that these two agents, given in combination, might have additive effects on these lipid parameters has not been examined previously. In addition, the combination might prevent the rise in low-density lipoprotein (LDL)-C levels frequently seen with ␻-3 FA treatment. OBJECTIVE: To determine the effect of therapy of niacin and ␻-3 FA alone and in combination versus placebo on lipid parameters in subjects with atherogenic dyslipidemia. METHODS: In a pilot parallel group study, we studied 29 patients with atherogenic dyslipidemia who were assigned to either dual placebo (n ⫽ 7), ␻-3 FA (3.4 g/d; n ⫽ 8), crystalline niacin (3 g/d; n ⫽ 7), or the combination (n ⫽ 7) for 12 weeks. Fasting lipid profiles were assessed before and after treatment. RESULTS: Changes in serum triglyceride levels from baseline were 10%, ⫺2%, ⫺17%, and ⫺52%, respectively, while HDL-C concentrations rose by 4%, 10%, 18%, and 33%, respectively. Both of these results were statistically significantly different for combination therapy compared to changes with placebo and ␻-3 FA monotherapy (analysis of variance with Tukey’s post-hoc test). No statistically significant changes were seen for LDL-C. Addition of ␻-FA to niacin had no effect on niacin-induced flushing. CONCLUSION: From this small pilot study, we conclude that combined therapy with niacin and ␻-3 FA has beneficial effects on triglyceride and HDL-C levels, and use of these two agents in combination prevents the ␻-3 FA-induced rise in LDL-C. Larger studies of this combination therapy are clearly warranted in patient populations with atherogenic dyslipidemia to assess not only lipid effects, but also potential coronary heart disease benefits. © 2007 National Lipid Association. All rights reserved.

Atherogenic dyslipidemia is characterized by elevated triglyceride and reduced high-density lipoprotein cholesterol (HDL-C) levels and is a common feature of the insulin-resistance syndrome. Insulin resistance, which is also

* Corresponding author. E-mail address: [email protected] Submitted February 2, 2007; Revised May 1, 2007; Accepted for publication May 13, 2007.

often accompanied by essential hypertension and hyperglycemia,1 is recognized as a risk factor for coronary heart disease.2 Both niacin and the ␻-3 fatty acids (FA) from fish oils have been reported to lower triglycerides and raise HDL-C,3-5 and in addition ␻-3 FA may reduce blood pressure.6,7 However, niacin lowers low-density lipoprotein cholesterol (LDL-C) concentrations8,9 while ␻-3 FA can actually raise LDL-C.10,11 Moreover, patient acceptance of niacin is limited by a characteristic flushing reaction.9,12 In

1933-2874/$ -see front matter © 2007 National Lipid Association. All rights reserved. doi:10.1016/j.jacl.2007.05.002

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contrast, ␻-3 FA have aspirin-like, antiprostaglandin effects13,14 that could theoretically diminish the niacin “flush.” The present studies were undertaken to determine whether combined niacin and ␻-3 FA therapy in patients with atherogenic dyslipidemia results in favorable changes in triglyceride and HDL-C without an increase in LDL-C, but with decreases in niacin-associated flushing.

Methods Subjects Men and postmenopausal or surgically sterile women ages 21 to 70 years were studied according to a protocol approved by the Institutional Review Boards of Saint Luke’s Hospital and the University of Missouri-Kansas City. Stable doses of transdermal estrogen were permitted. To be eligible for the study, it was necessary for fasting serum triglyceride levels to be between 150 and 500 mg/dL. A normal chemistry screen was required to exclude secondary hypertriglyceridemia. HDL-C had to be ⬍40 mg/dL for men and ⬍50 mg/dL for women. LDL-C inclusion criteria were based on 1993 National Cholesterol Education Program guidelines,15 which take into account the presence of coronary heart disease (CHD) and CHD risk factors: ⬍130 mg/dL with CHD; ⬍160 mg/dL with two or more risk factors; and ⬍190 mg/dL with one risk factor. Subjects with diabetes mellitus, peptic ulcer disease, gouty arthritis, or hyperuricemia; or known hepatic, renal, autoimmune, or gastrointestinal diseases were excluded. Subjects were also excluded if they were taking warfarin, chronic nonsteroidal anti-inflammatory agents, or any medication known to affect lipid metabolism. Written informed consent was obtained from all participants prior to study initiation.

Protocol The protocol had a randomized, placebo-controlled, prospective, parallel-group design, beginning with a dual placebo run-in period of 4 weeks duration followed by a 2-week ␻-3 FA/placebo run-in period (Fig. 1). Subjects were instructed to follow a low-fat, low-cholesterol diet per the National Cholesterol Education Progam, abstain from alcohol excess, and maintain their usual exercise habits throughout the study. The ␻-3 FA/placebo run-in period

was included prior to randomization to niacin or placebo to ensure that the ␻-3 FA were well-incorporated into cell membranes before the first niacin dose was administered. This was done to permit an assessment of the effects of ␻-3 FA on the niacin flush. The second randomization thus produced four study groups: a dual placebo group, an ␻-3 FA/placebo group, a niacin/placebo group, and an ␻-3 FA/ niacin group (see Fig. 1). The ␻-3 FA were provided as Omacor (Pronova Biocare, Oslo, Norway). This product contains 85% eicosapentaenoic (EPA) plus docosahexaenoic acids (DHA), respectively, as FA ethyl esters. Four 1-g capsules (two capsules twice a day with meals for a total of 3.4 g of ␻-3 FA) per day were given. The Omacor placebo was corn oil ethyl esters, which at the doses used in this study have no effect on serum lipid levels.3 Full doses (4 g/d) of the oils were used throughout the entire 4.5-month study. Niacin was given as 500 mg, immediate-release niacin tablets (Rugby Laboratories, West Hempstead, NY). Patients took niacin (or placebo) in escalating doses to reach, within 6 weeks, a final dose of 3 g/d (1000 mg three times a day with meals). The placebo for the niacin tablets were tablets of calcium gluconate USP (Roxane Laboratories, Columbus, OH). This was chosen for the placebo because at the highest dose (six tablets per day) it provided only 279 mg elemental calcium, a dose far below that which would be expected to influence lipid metabolism.16,17 Niacin (and its placebo) were dosed in a stepwise manner: 500 mg/d for the first 2 weeks; 1000 mg/d for the third week; 2000 mg/d for the fourth week; and 3000 mg/d from the fifth week onward. Liver enzymes were determined at monthly intervals, and any patient whose liver enzymes exceeded twice the upper limit of normal was retested. If the levels remained elevated above twice the upper limit of normal, the niacin dose was decreased to the next lower level, but not ⬍2 g/d. If enzymes were still elevated at this dose, the patient was dropped from the study. When niacin is used clinically, patients are often advised to take aspirin 30 minutes before the niacin dose to diminish the flush.12 To avoid the confounding effect of uncontrolled aspirin use on the assessment of flush severity, and because some patients were taking aspirin for CHD prevention, all patients were instructed to take one 325-mg aspirin tablet after breakfast each day, but no other aspirin during the study. For pain or fever relief, ibuprofen or acetaminophen were allowed as needed.

Laboratory methods Lipids and lipoproteins

Figure 1

Study schema.

Serum total cholesterol, triglyceride, and HDL-C were measured enzymatically on a Cobas Fara II (Roche) using enzymatic reagents and procedures standardized by the Lipid Standardization Program of the Centers for Disease Control and Prevention/National Institutes of Health. HDL-C was determined in the serum supernate after pre-

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Combination of ␻-3 fatty acids and niacin

213 by the Tukey’s post-hoc test. A P value ⬍0.05 was required for statistical significance. Demographic data between men and women were compared by Student’s t-test.

cipitation of very-low– density lipoprotein (VLDL) and LDL with dextran sulfate-magnesium chloride as described by Warnick and Albers.18 VLDL-C and LDL-C concentrations were determined directly by ultracentrifugation as described previously.19 LDL from fasting plasma was separated into three subfractions by density gradient ultracentrifugation.20 Intermediate density lipoprotein cholesterol (IDL-C) was determined by ultracentrifugation at serum density 1.019 g/mL. Lp(a) cholesterol was assayed using lectin capture with a kit from Genzyme (Cambridge, MA).21 A 12-analyte chemistry profile, including tests of liver function, was performed by the hospital’s clinical laboratory. A questionnaire to assess the extent of the niacin-induced flush was administered during 1 week during the dual placebo run-in period, the first week of niacin/placebo treatment, the first week of full dose, and the last week of the study. Patients were asked to rank their symptoms after each dose (ie, three times a day) from 0 (no noticeable flush) to 3 (warm and red or itchy for about 15 minutes) to 6 (red, hot, swollen or intense itch for 1 or more hours).

Results Subjects Of approximately 100 potential subjects screened, 36 subjects enrolled, with 29 completing the protocol. Three withdrew because they could not tolerate the flushing associated with niacin, while four withdrew for other reasons. No subjects were withdrawn because of liver test abnormalities. Baseline characteristics of those completing the study are given in Table 1. There were no significant differences in age, gender, body mass index, or lipid values among the groups.

Lipids and lipoproteins

Plasma phospholipid FA composition

Effects of each agent alone and in combination are presented in Table 2. Total cholesterol increased significantly (by 13%) in the ␻-3 FA group compared to the niacin and combination therapy groups, whereas a 52% decrease in serum triglycerides was observed in the combination niacin plus ␻-3 FA group (P ⬍ 0.05 compared to ␻-3 FA and P ⬍ 0.01 compared to placebo). No significant effect in serum triglycerides was observed with either agent alone. Consistent with the decrease in triglycerides was a 60% decrease in VLDL-C seen with the combination (P ⬍ 0.05 vs the placebo group). There were no significant increases in LDL-C with any therapy. Combination niacin and ␻3 therapy raised HDL-C by 33% (P ⬍ 0.05 vs ␻-3 FA and P ⬍ 0.01 versus placebo). No statistically significant effects were noted on IDL-C levels among the four groups. The only LDL subfraction to change significantly was LDL2-C, which increased from baseline by 68% in the group treated with combination niacin and ␻-3 therapy (P ⬍ 0.05 compared to all other

Compliance with ␻-3 FA supplementation was monitored by measuring the increase in phospholipid FA EPA and DHA levels.22 The plasma lipid was extracted with methylene chloride:methanol and separated by thin layer chromatography on silica gel G in hexane:ethyl ether: formic acid (70:30:1). The phospholipid fraction was isolated, methylated, and analyzed by gas chromatography (Shimadzu GC-14A, Columbia, MD) using a 30 m. SP2330 capillary column. Major FA were identified by comparison with known standards and reported as percent of total FA.

Statistical analysis Data were analyzed by determining the change from baseline to end of treatment for each parameter of interest and then asking whether the changes were different across groups using a single factor analysis of variance followed

Table 1

Baseline characteristics of subjects

Placebo (n ⫽ 7) Niacin (n ⫽ 7) ␻-3 FA (n ⫽ 8)# Niacin ⫹ ␻-3 FA (n ⫽ 7)

Age (y)

BMI*

⫾ ⫾ ⫾ ⫾

30.3 31.7 32.2 29.9

58 48 48 46

10 14 17 7

⫾ ⫾ ⫾ ⫾

2.1 1.5 2.8 3.4

M/F†

TG‡ (mg/dL)

3/4 5/2 7/1 5/2

281 268 287 265

⫾ ⫾ ⫾ ⫾

76 111 58 133

LDL-C§ (mg/dL) 137 108 106 110

⫾ ⫾ ⫾ ⫾

59 41 28 25

HDL-C¶ (mg/dL) 38 41 36 37

⫾ ⫾ ⫾ ⫾

4 7 5 8

*BMI, body mass index; calculated as kg/m2. Male subjects weighed more female subjects (98.8 ⫾ 11.2 vs 86.6 ⫾ 13.6 kg, P ⬍ 0.02), but BMI was similar for male and female subjects (30.7 ⫾ 2.6 vs 31.8 ⫾ 2.7, respectively). †M/F, male/female. ‡TG, triglycerides. §LDL-C, low-density lipoprotein cholesterol. ¶HDL-C, high-density lipoprotein cholesterol. #␻-3 FA, ␻-3 fatty acid.

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Table 2

Effects of niacin, ␻-3 fatty acid, and the combination on serum lipids and lipoproteins*

Total cholesterol Pre Post % change Triglycerides Pre Post % change VLDL-C¶ Pre Post % change LDL-C** Pre Post % change HDL-C†† Pre Post % change

Placebo (mg/dL) (n ⫽ 7)

Niacin (mg/dL) (n ⫽ 7)

␻-3 FA† (mg/dL) (n ⫽ 8)

␻-3 FA ⫹ niacin (mg/dL) (n ⫽ 7)

229 ⫾ 61 224 ⫾ 57 ⫺2

197 ⫾ 35 185 ⫾ 18 ⫺6

194 ⫾ 37 220 ⫾ 48‡ 13

205 ⫾ 37 194 ⫾ 29 ⫺5

281 ⫾ 76 309 ⫾ 97 10

268 ⫾ 111 222 ⫾ 102 ⫺17

271 ⫾ 51 265 ⫾ 105 ⫺2

265 ⫾ 133 128 ⫾ 72§ ⫺52

55 ⫾ 22 56 ⫾ 26 2

48 ⫾ 17 33 ⫾ 16 ⫺32

53 ⫾ 20 48 ⫾ 29 ⫺8

58 ⫾ 33 23 ⫾ 17# ⫺60

137 ⫾ 38 129 ⫾ 47 ⫺6

108 ⫾ 41 104 ⫾ 17 ⫺4

106 ⫾ 28 133 ⫾ 39 25

110 ⫾ 25 121 ⫾ 23 11

38 ⫾ 4 39 ⫾ 4 4

41 ⫾ 17 48 ⫾ 14 18

35 ⫾ 5 39 ⫾ 5 10

37 ⫾ 8 49 ⫾ 10§ 33

*Values are mean ⫾ SD unless otherwise indicated. †FA, fatty acid ‡P ⬍ 0.05 ␻-3 FA vs niacin and combination therapy. §P ⬍ 0.05 combination therapy vs ␻-3 FA and P⬍.01 combination therapy vs placebo. ¶VLDL-C, very-low– density lipoprotein cholesterol. #P ⬍ 0.05 combination therapy vs placebo. **LDL-C, low-density lipoprotein cholesterol. ††HLD-C, high-density lipoprotein cholesterol.

groups). The concentrations of HDL2-C and HDL3-C increased approximately equally in the combination therapy group. Lp(a)-C concentrations were unaffected by any treatment regimen.

There was no evidence for a reduction in flushing in the group taking ␻-3 FA plus niacin compared to those taking niacin alone.

Serum phospholipid fatty acid composition

Discussion There was significant incorporation of all three longchain ␻-3 FA into serum phospholipids in both of the ␻-3 FA supplementation groups (data not shown). EPA rose approximately fivefold, DHA by twofold, and docosapentaenoic acid by about 30%. The content of arachidonic acid (20:4␻6) decreased by about 15%, and its metabolic precursors di-homo-␥-linolenic acid (20:3␻6) and linoleic acid (18:2␻6) by 20% to 40% and 15%, respectively. There was no effect of placebo or niacin alone on the FA distribution.

Flushing reaction Patients completed 1-week “flushing” diaries four times throughout the study. Flushing score was 0 at baseline, approximately three during the first week of 1 g/d, and decreased to less than two after 6 to 12 weeks of 3 g/d.

The principal finding of this study was that combined treatment with 3.4 g ␻-3 FA plus 3 g crystalline niacin lowered serum triglyceride levels by ⬎50% and raised HDL-C by ⬎30%. LDL-C levels did not change significantly with any therapy. Addition of ␻-3 FA to niacin treatment had no effect on the niacin flush. It was expected that serum triglycerides would decrease in the mono- and dual-therapy groups because both niacin and fish oil are effective in this regard. However, there was virtually no effect of ␻-3 FA alone on serum triglycerides (⫺2%), a finding that is at variance with other studies showing reductions in serum triglycerides of ⬃25% in similar patient populations.3 Of the eight patients in that group, triglyceride levels decreased in five, did not change in one, and increased (by an average of 130 mg/dL) in two (data not shown). The three subjects in this group without a decrease in triglycerides gained a mean of 3.5 kg during the study,

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whereas the other subjects had weight stability. This gain in weight likely explained to some degree this anomalous finding. However, the extreme variability of triglycerides, the exclusion of patients with extreme hypertriglyceridemia, and the small sample size likely played a role as well. Furthermore, there might be heterogeneity in the response of individuals to ␻-3 therapy.23 Triglyceride-lowering effects of ␻-3 FA have been explored in cell culture, perfused livers, animal and human kinetic trials. These studies have generally shown that ␻-3 FA slow the rate of secretion of VLDL triglyceride into the blood from the liver. In addition, recent studies from our laboratory have demonstrated an increase in circulating lipoprotein lipase activity in patients fed ␻-3 FA.24 The 17% decrease in triglycerides observed in the niacin group was also somewhat smaller than the 20% to 25% expected.25 Other than small sample size, we have no ready explanation for this finding. Niacin acutely reduces the release of nonesterified fatty acids from adipose tissue,26 the primary substrate for hepatic triglyceride synthesis.27 Niacin may cause hepatocytes to increase apolipoprotein (Apo) B degradation and thereby reduce VLDL secretion.8 The combination of ␻-3 FA and niacin produced a marked decrease in serum triglycerides (⫺52%), more than twice the additive effect of the two monotherapies, and also proved to be effective in raising HDL-C levels (32%). Such an increase (from 37 to 49 mg/dL) may possibly reduce risk for CHD.28 The mechanism behind the ␻-3 FA effect on HDL-C has not been studied. However, the increase in HDL-C with niacin therapy appears to be due to a decrease in the fractional clearance rate of HDL-C.29 A significant decrease in VLDL triglycerides may have contributed to the observed effect on HDL-C. Reduced levels of triglyceride-rich lipoproteins diminish the substrate pool for cholesteryl ester transfer protein action. This enzyme exchanges triglyceride in the former particles with cholesterol in HDL, producing a cholesterol-depleted, triglyceride-enriched HDL.30,31 Lower serum triglyceride levels would allow for less of this exchange, raising HDL-C concentrations. The reason for the reduction in LDL-C with niacin has not been explored in depth. It is likely, however, to be a consequence of reduced VLDL output because VLDL is the precursor of LDL, and kinetic studies have suggested that niacin affects LDL production, not clearance, rates.32 The increase in LDL-C levels with ␻-3 FA is also poorly understood. Data from pig studies33 and trials in diabetic patients given high doses of ␻-3 FA34 suggest that these FA increase conversion of VLDL to IDL and LDL; they not do not appear to affect LDL catabolism. On the other hand, other data have shown that LDL-receptor affinity for its ligand may be diminished by ␻-3 FA feeding.35–37 Thus, ␻-3 FA could affect both production and clearance of LDL. The fourth issue of interest in this study was to determine whether the niacin flush could be diminished by increased

215 tissue ␻-3 FA levels. It is well-known that prior administration of cyclooxygenase inhibitors (aspirin, indomethacin) will blunt the reaction,38,39 an observation that led to the discovery that prostaglandins (primarily PGD2) play a central, causal role in the flush.40 Because EPA often gives rise to eicosanoids of lesser biological activity than those derived from arachidonic acid,41,42 it seemed reasonable that ␻-3 FA supplementation might partially eliminate this bothersome side effect of niacin. However, we found no reduction in subjective perception of flushing in the patients taking ␻-3 FA plus niacin compared to those taking the latter alone. The dose of ␻-3 FA used in this study (3.4 g) may have been too little to shift membrane arachidonate (PGD2 precursor) to EPA (PGD3 precursor) ratios sufficiently. Indeed, plasma phospholipid levels of the former fatty acid only decreased from 13% to 11%, and final levels of EPA only reached 4%. In addition, PGD2 and PGD3 have equipotent vasoactive effects in some animal models,43,44 although in the rabbit uvea, the 2-series PG induced hyperemia while the 3-series PG did not.45 Niacin is consistently effective at raising HDL-C and lowering triglycerides when used in combination with other lipid-lowering agents, including bile acid binding resins,46 statins,47 and even oat bran.48 ␻-3 FA have been used successfully in combination with statins49,50 and oat bran.51 The clinical utility of the combination of these two agents in patients with atherogenic dyslipidemia seems promising, as both have shown efficacy not only in improving dyslipidemias, but more importantly, in reducing risk for death from cardiovascular disease.52,53 In addition, larger population studies providing balance in subjects’ age, gender, cardiovascular risk factors, glucose tolerance, previous lipid-lowering drug use, dietary composition, and individual variability in nutraceutical response will be needed to define the full spectrum of effect.

Acknowledgments This work was supported by grants from the American Heart Association (Heartland Affiliate), the University of Missouri Research Board, and the Saint Luke’s Hospital Foundation for Research and Education. The authors wish to thank the following colleagues for their excellent contributions to this project: Sheryl Windsor, Jeff Lickteig, Bart Damron, Rebecca Acuff, Tania Mahinda, and Qing Lan Zhang.

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