LDL cholesterol–raising effect of low-dose docosahexaenoic acid in middle-aged men and women

¹ From the Nutrition Food and Health Research Centre (HET and TABS) and the Department of Clinical Pharmacology, St Thomas’ Hospital, Centre for Cardiovascular Biology and Medicine (PJC), King’s College London, and the Centre for the Genetics of Cardiovascular Disease, British Heart Foundation Laboratories, Royal Free and University College London Medical School (RW and SEH), London.

² Supported by Merck Darmstadt and the British Heart Foundation (RG 2000/015). HET was the recipient of a research studentship from King’s College London.

³ Reprints not available. Address correspondence to TAB Sanders, Nutrition Food and Health Research Centre, King’s College London, Franklin-Wilkins Building, 150 Stamford Street, London SE1 9NN, United Kingdom. E-mail: tom.sanders{at}kcl.ac.uk.

ABSTRACT  
Background: Long-chain n-3 polyunsaturated fatty acids have variable effects on LDL cholesterol, and the effects of docosahexaenoic acid (DHA) are uncertain.

Objective: The objective of the study was to determine the effect on blood lipids of a daily intake of 0.7 g DHA as triacylglycerol in middle-aged men and women.

Design: Men and women aged 40–65 y (n = 38) underwent a double-blind, randomized, placebo-controlled, crossover trial of treatment with 0.7 g DHA/d for 3 mo.

Results: DHA supplementation increased the DHA concentration in plasma by 76% (P < 0.0001) and the proportion in erythrocyte lipids by 58% (P < 0.0001). Values for serum total cholesterol, LDL cholesterol, and plasma apolipoprotein B concentrations were 4.2% (0.22 mmol/L; P = 0.04), 7.1% (0.23 mmol/L; P = 0.004), and 3.4% (P = 0.03) higher, respectively, with DHA treatment than with placebo. In addition, the LDL cholesterol:apolipoprotein B ratio was 3.1% higher with DHA treatment than with placebo (P = 0.04), which suggested an increase in LDL size. Plasma lathosterol and plant sterol concentrations were unaffected by treatment.

Conclusion: A daily intake of 0.7 g DHA increases LDL cholesterol by 7% in middle-aged men and women. It is suggested that DHA down-regulates the expression of the LDL receptor.

Key Words: Lipids • docosahexaenoic acid • LDL

INTRODUCTION  
The effect of mixtures of long-chain n-3 polyunsaturated fatty acids (n-3 LCPs) on plasma lipoproteins is well documented (1). Intakes > 1.5 g/d lower plasma triacylglycerol concentrations, but the effects on LDL cholesterol are variable. An increase in LDL cholesterol has repeatedly been observed in subjects with the Type IV and Type V Fredrickson lipoprotein phenotypes. However, it was argued that the LDL-elevating effects of n-3 LCPs were similar to those observed for other triacylglycerol-lowering drugs in subjects with these phenotypes (2). Minihane et al (3) in a post hoc analysis suggested that apolipoprotein E (Apo E) phenotype modulated the response to n-3 LCPs in subjects with the atherogenic lipoprotein phenotype. An increase in LDL cholesterol in subjects with the atherogenic lipoprotein phenotype of 15.9% in those carrying the 4 allele compared with 0.6% in those who were homozygous for the 3 allele was reported. It was suggested that subjects who were homozygous for the 3 allele did not show an increase in LDL after fish oil supplementation and that the increase in LDL cholesterol concentration was dependent on possession of the 4 allele.

Most studies of n-3 LCPs have generally used oils containing mixtures of eicosapentaenoic acid (20:5n-3; EPA) and docosahexaenoic acid (22:6n-3; DHA) in the range of 1–5 g/d. A few studies have examined the effects of DHA in hyperlipidemic subjects (4–7) in the dose range of 3–4 g/d, usually provided as ethyl esters. Single-cell oil sources of DHA, which contain only trace amounts of EPA and other n-3 fatty acids, have become available for food use. Previous reports of the effects of such oils on lipoprotein concentrations have studied intakes in the range of 1.25–6 g/d and are limited by the small sample used and the short duration of the interventions (8–10).

Prospective cohort studies (11, 12) and a trial (13) of secondary prevention of ischemic heart disease suggested that intakes of < 1 g n-3 LCPs/d offer protection from fatal ischemic heart disease. However, data from the study of the Gruppo Italiano per lo Studio della Sopravvivenza nell’Infarto miocardico (GISSI study; 13) suggested than an ethyl ester preparation providing 0.85 g of a mixture of EPA and DHA in a ratio of 1.2:1 raised the LDL cholesterol slightly (3–3.5%). The primary aim of the present study was to evaluate the effect of a low intake of a triacylglycerol providing long-chain n-3 fatty acids almost exclusively as DHA in middle-aged subjects. As a consequence of the observed LDL-raising effect of DHA, a post hoc evaluation of the response according to Apo E genotype was conducted.

SUBJECTS, MATERIALS, AND METHODS  
A randomized, double-blind, placebo-controlled, crossover design was chosen. Each treatment phase lasted 3 mo and was followed by a washout phase of =" BORDER="0"> 4 mo. Stratified randomization was used to allocate subjects to the 2 possible treatment sequences so that equal numbers of men and women were allocated to each sequence. The first phase was conducted between September 1999 and January 2000 and the second phase between April and August 2000. The study was conducted in a double-blind fashion, but the analysis of plasma apolipoprotein B (apo B), lathosterol, and plant sterol concentrations and Apo E genotype were conducted post hoc. The study subjects were recruited by E-mail from within the staff population of King’s College London, St Thomas’s, Guy’s, and King’s College hospitals. Exclusion criteria were history of myocardial infarction or diabetes mellitus; current pregnancy; current use of lipid- or blood pressure–lowering medication, immunosuppressive drugs, or hormone replacement therapy; body mass index (BMI; in kg/m²) > 35; serum cholesterol > 7.8 mmol/L; fasting serum triacylglycerol > 3.0 mmol/L; blood pressure > 160/105 mm Hg; abnormal results on hematologic or liver function test; and self-reported alcohol intake > 21 units/wk for women and > 28 units/wk for men (1 unit = 10 mL ethanol). Subjects received a small financial incentive for their participation in the study. A small fasting venous blood sample (17 mL) was obtained for measurement of plasma total cholesterol, serum HDL cholesterol, plasma triacylglycerol, and a complete blood count and for performance of liver function tests. The serum concentration of follicle-stimulating hormone was measured in postmenopausal women, and a urinary pregnancy test was performed in menstruating women to confirm menopausal and nonpregnant status, respectively. Habitual dietary intake was assessed by using a 3-d weighed dietary record, the data from which were converted by computer to nutrient intake on the basis of the UK Nutrient Databank (Norwich, United Kingdom: Her Majesty’s Stationery Office). Height without shoes and weight in minimum indoor clothing were measured with the use of a beam balance and stadiometer. The protocol was reviewed and approved by the Research Ethics Committee of King’s College London, and participants gave written informed consent before commencing the study. After analysis of the results of the study, informed written consent was obtained from the subjects to determine their Apo E genotype.

The DHA treatment was provided as 3 capsules/d; each capsule contained 500 mg of a refined triacylglycerol derived from Crypthecodinium cohnii (DHASCO; Martek Biosciences, Columbia, MD). Matching placebo capsules contained 500 mg refined olive oil (British Pharmacopoiea specification). To ensure that the treatments had the same antioxidant content, the concentrations in the oils were standardized before encapsulation so that each capsule contained 4 tocopherol equivalents, 0.073 mg ß-carotene, and 0.125 mg ascorbyl palmitate. Oils were flavored with peppermint to disguise the taste of the oil and were encapsulated in opaque gelatin capsules (RP Scherer, Swindon, United Kingdom). Capsules were stored at room temperature in a dry, dark place. Compliance with the treatment regimen was assessed by performing capsule counts and by measuring the incorporation of DHA into plasma and erythrocyte lipids.

At the start and at the end of each treatment period, subjects fasted overnight, and blood samples were obtained by venipuncture the following morning with the use of the evacuated-tube technique (Vacutainer; Vacutainer Systems, Becton Dickinson, Plymouth, United Kingdom). Blood for serum lipids and liver function tests was collected in an evacuated tube containing no anticoagulant (Vacutainer 17490; Becton Dickinson), serum was separated by centrifugation at 1500 x g for 15 min at 4 °C and kept at 4 °C until it was analyzed, within 3 d. For full blood counts, blood was collected into Vacutainer tubes containing 2 mL potassium EDTA (Vacutainer 368047; Becton Dickinson) and kept at room temperature until the count on the same day. Blood for fatty acid, sterol, apo B, and fat-soluble vitamin analyses was collected into an EDTA-coated tube (Vacutainer 17644; Becton Dickinson) chilled to 4 °C and centrifuged at 1500 x g for 15 min at 4 °C, and aliquots of plasma were frozen at -80 °C until they were analyzed. Packed erythrocytes were washed 3 times with 5 volumes of ice-cold saline (8.9 NaCl/L) containing 40 mg EDTA/L, and lipid extracts were prepared within 2 d of blood collection by using isopropanol:chloroform (11:7 by volume) containing 50 mg butylated hydroxytoluene/L. The lipid extracts were stored at -20 °C until they were analyzed. Blood (4.5 mL) for genetic analysis was collected into a Vacutainer tube containing 0.5 mL of 0.105 mol trisodium citrate/L (Vacutainer 367691; Becton Dickinson) and kept at room temperature until completion of centrifugation. Plasma was separated by centrifugation at 1500 x g for 15 min a 4 °C, and the remaining red and white blood cells were frozen at -40 °C until they were analyzed.

Total and HDL cholesterol and triacylglycerol concentrations were measured with the use of fully enzymatic procedures using reagents from Wako (Neuss, Germany), and assays were conducted on a Technicon DAX48 automated chemistry analyzer (Bayer Diagnostics, Newbury, United Kingdom). Frozen plasma samples were analyzed for apo B concentration with the use of a commercially available immunoturbidimetric assay (Immuno AG, Vienna) on a Cobas Mira analyzer (Roche Diagnostics, Lewes, United Kingdom) as described previously (14). Erythrocyte and plasma fatty acid concentrations were measured by using capillary gas chromatography (15). Plasma sterols were extracted, trimethylsilyl derivatives were prepared (16) and analyzed by using capillary gas chromatography–mass spectrometry, and plasma fat–soluble vitamins were measured by using HPLC (17). Genomic DNA was isolated from blood cells with the use of the "salting out" method, and the Apo E genotypes were determined (18).

Statistical power calculations were based on the completion by 32 subjects of both treatments. These power estimates were based on within-subject SDs of 0.11 mmol/L and 0.29 mmol/L for HDL and LDL cholesterol, respectively. The sample size gave 80% power for = 0.05 to detect a change of 0.08 mmol/L in HDL and a change of 0.2 mmol/L in LDL cholesterol. Forty subjects (20 men, 20 women) were recruited into each group to allow for dropouts. Data were analyzed by repeated-measures analysis of variance using SPSS/PC software (version 10; SPSS Inc, Chicago). Data for plasma triacylglycerol were log transformed before statistical analysis. Tests for treatment order effects were performed by using the technique described by Pocock (19). If the overall repeated-measures analysis of variance was statistically significant (P < 0.05), comparisons between active treatment and placebo were made by using a paired t test. Comparisons of the responses (active treatment - placebo) according to genotype were made by using analysis of covariance with adjustments for age, BMI, and sex.

RESULTS  
Of the 40 subjects recruited into the study, 39 completed the study; of this group, 20 were men and 19 were women. Fifteen of the women were premenopausal and 4 were postmenopausal. One subject (female) withdrew at the end of phase 1 for personal reasons. One subject (male) was excluded from the analysis because he was found to have a cardiac arrhythmia that was not treatment related and that thus violated the inclusion criteria for the study. Therefore, data on 38 subjects were available for analysis. The subjects’ characteristics and their habitual dietary intakes are shown in Tables 1 and 2, respectively.

View this table:
TABLE 1. Characteristics of the subjects of the study

 

View this table:
TABLE 2. Dietary intakes of the subjects before entry into the study¹

 
The DHA capsules were taken for a mean of 83.5 d (range: 76–99 d) and the placebo was taken for 84.9 d (78–104 d). Adherence to treatment as judged by capsule count was 93% for the DHA supplement and 95% for the placebo (NS). The estimated intakes provided by the supplement were 0.68 g DHA/d and < 0.005 g EPA/d; the remaining major fatty acid intakes provided by the supplement were 0.26 g 14:0/d, 0.23 g 16:0/d, and 0.20 g 18:1/d. The supplements were well tolerated, and blood counts and liver function tests remained within normal limits on both treatments (data not shown). Body weight was not affected by either treatment, and there were no time effects. However, over the yearlong study period, subjects showed a tendency to gain a small amount of weight ( Plasma concentrations of DHA increased by 76% during active treatment (from 59.9 ± 4.5 mg/L during placebo treatment to 105.3 ± 5.9 mg/L during DHA treatment; P < 0.001). The proportion of DHA in erythrocyte phosphoglycerides increased by 58% (from 5.2 ± 0.3% by wt during placebo treatment to 8.2 ± 0.2% by wt during DHA treatment; P < 0.001), and it is important that there was no evidence of any carryover effect between treatments (Figure 1). The plasma concentrations of EPA were unaffected by the DHA treatment (1.4 ± 0.1 mg/L during placebo treatment compared with 1.4 ± 0.1 mg/L during DHA treatment), as were the proportion in erythrocyte lipids (1.1 ± 0.1% by wt during placebo treatment compared with 1.2 ± 0.1% by wt during DHA treatment).

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FIGURE 1.. Mean changes in the proportions of docosahexaenoic acid (DHA) in erythrocyte lipids (n = 38). Error bars indicate 95% CIs. Statistically significant treatment effect, P < 0.0001 (paired t test).

 
The serum lipid and plasma apo B concentrations are shown in Table 3. Repeated-measures analysis of variance found significant treatment effects for total cholesterol (P = 0.001), LDL cholesterol (P = 0.0001), and plasma apo B (P = 0.002) concentrations. The increases during DHA treatment compared with those during placebo placebo were 0.22 mmol/L (95% CI: 0.01, 0.42 mmol/L; P = 0.04) for total cholesterol, 0.23 mmol/L (95% CI: 0.08, 0.38 mmol/L; P = 0.004) for LDL cholesterol, and 0.03 g/L (95% CI: 0.002, 0.055 g/L; P = 0.03) for apo B. The LDL:apo B ratio also showed a significant treatment effect (P = 0.004): the ratio was greater after DHA treatment than after placebo treatment (mean difference: 0.12 µmol/mg; 95% CI: 0.004, 0.24 µmol/mg; P = 0.04). HDL cholesterol concentrations showed a significant treatment x order effect (P = 0.005) and a significant treatment effect (P = 0.03). The 2 treatment sequences were thus analyzed separately. The values were significantly higher in the subjects who received DHA than in those who received placebo in the first treatment phase, which ran between fall (September and October) and midwinter (December and January; mean difference: 0.12 mmol/L; 95% CI: 0.02, 0.23 mmol/L; P = 0.021) but not in the subjects who received DHA for the second phase, which ran between spring (March and April) and summer (July and August; mean difference: 0.02 mmol/L; 95% CI: -0.08, 0.12 mmol/L). Posttreatment values for serum triacylglycerol (Table 3), plasma lathosterol, campesterol, and sitosterol concentrations did not differ significantly from pretreatment values (Table 4). The cholesterol-adjusted concentration of plasma -tocopherol increased slightly after supplementation with placebo treatment but remained unchanged after DHA treatment, but the posttreatment values with DHA treatment and with placebo treatment differed significantly. Plasma concentrations of retinol, ß-carotene, lycopene, and lutein were unchanged (data not shown).

View this table:
TABLE 3. Serum lipoproteins before treatment and after 3 mo of docosahexaenoic acid (DHA) and placebo treatment in all subjects

 

View this table:
TABLE 4. Serum lathosterol, ß-sitosterol, and campesterol concentrations and -tocopherol:cholesterol ratios adjusted for serum cholesterol concentrations before treatment and after 3 mo of docosahexaenoic acid (DHA) and placebo treatment in all subjects

 
The changes in LDL cholesterol in subjects according to Apo E genotype with adjustment for age, BMI, and sex are shown in Figure 2. The regression analysis did not show a significant genotype effect on the change in LDL cholesterol on treatment compared with placebo. The age-, BMI-, and sex-adjusted increase in LDL cholesterol for the subjects with the 3:3 genotype (n = 23) was 8.1% (95% CI: 1.4%, 14.8%), that for those with the 3:4 genotype (n = 10) was 6.7% (-4.2%, 17.6%), that for those with the 2:3 genotype (n = 4) was -1.6% (95% CI: -17.6%, 14.5%), and that for the single subject with the 2:4 genotype was 29.6%.

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FIGURE 2.. Mean percentage change in serum LDL cholesterol after docosahexaenoic acid (DHA) treatment compared with that after placebo treatment according to apolipoprotein E genotype (3:3 genotype, n = 23; 2:3 genotype, n = 4; 3:4 genotype, n = 10; 2:4 genotype, n = 1) after adjustment for age, sex, and BMI. Error bars indicate 95% CIs. Covariates in the model were a BMI of 24.0 and an age of 48.4 y. Analysis of covariance found no genotype effect.

 

DISCUSSION  
The primary aim of the study was to ascertain the effects of 0.7 g DHA/d on serum lipids in healthy middle-aged subjects. This relatively small amount of DHA had a remarkable effect on the plasma concentration of DHA and the proportion of DHA in erythrocytes. This finding is consistent with the recent study by Albert et al (20) that estimated both the intake of n-3 LCPs by using a food-frequency questionnaire and the proportions in total blood lipids in relation to the risk of sudden cardiac death.

Previous studies using DHA-rich oils have reported a significant reduction in triacylglycerol concentrations and an increase in HDL-cholesterol concentrations at DHA intakes of 1.62–6.0 g/d (4–7). In the present study, no such triacylglycerol-lowering effect was observed with an intake of 0.7 g/d, which is consistent with the view that the minimum dose of n-3 LCPs that effectively lowers triacylglycerol concentrations is 1.5 g/d (1). Furthermore, the subjects of this study did not have elevated serum triacylglycerol concentrations. The within-subject SD for serum triacylglycerol was 0.32 mmol/L, which gave power to detect a change of 0.21 mmol/L in serum triacylglycerol, which was greater than the 0.18 mmol/L difference observed. Mori et al (5) reported an 8% increase in LDL cholesterol with 4 g ethyl esters of DHA/d. However, Nestel et al (6) were unable to show a statistically significant increase in LDL cholesterol with 3 g DHA-enriched ethyl ester concentrate ken taken daily for 7 wk by 12 subjects. Grimsgaard et al (7) compared 3.8 g EPA/d, 3.6 g DHA as ethyl esters/d, and 4.0 g corn oil/d taken for 7 wk, but they did not find any effect of DHA on LDL cholesterol. Hamazaki et al (21) also found no difference between the effect of DHA-rich fish oil capsules containing 1.5–1.8 g DHA and that of a control oil capsule containing 97% soybean oil and 3% fish oil for 13 wk on LDL cholesterol in young men. However, Davidson et al (4) reported a statistically significant (13.6%) increase in LDL-cholesterol concentrations in male subjects with combined hyperlipoproteinemia with the provision of 2.5 g DHA/d as an algal triacylglycerol similar to that used in the present study and a nonsignificant increase of 9.3% with the provision of 1.25 g DHA/d. These workers also reported a reduction in serum triacylglycerol concentrations and an increase in LDL particle size.

Few controlled studies have examined the effects of n-3 LCP intakes < 1 g/d on plasma lipids. Although results from the GISSI study (13) suggest a 3–0.3.5% LDL-elevating effect with 0.85 g ethyl esters of DHA and EPA/d, these results are confounded by changes in lipid-lowering therapy during the study. Higgins et al (22) compared the effect of fish oil providing a daily intake of 0.3, 0.6, and 0.9 g n-3 LCPs for 16 wk with that of placebo, with the use of a parallel design, and they failed to note any significant changes in plasma LDL cholesterol in healthy men and women. The striking finding in the present study was the LDL cholesterol–raising effect of a relatively modest intake of DHA. The increase was 7% greater than that seen with placebo. This observation is not likely to be due to chance, because the study had sufficient power to detect the observed change. Furthermore, we have confirmed this observation in a subsequent study (TAB Sanders, GJ Miller, unpublished observations, 2002). Studies using purified EPA or EPA-rich oil have generally not reported increases in LDL cholesterol (1). This observation may suggest that, when DHA is fed alone as a triacylglycerol, it has effects that differ from those when it is fed in combination with EPA as in fish oil. Although most of the subjects of the present study were moderately hypercholesterolemic (serum cholesterol > 5 mmol/L), they were healthy men and women who were generally representative of this age group in the United Kingdom. Indeed, their BMI, blood pressure, and serum cholesterol concentrations were lower than the average (23). The results from this study, therefore, can be generalized to most of the British men and women in this age group. In support of this, the frequency of the 4 and 2 alleles did not differ significantly from that found by other UK studies (24). The increase in LDL was confirmed by the post hoc analysis of plasma apo B concentration. However, the LDL cholesterol:apo B ratio was greater after the DHA treatment, which may suggest that the LDL particle size was increased. This finding is in agreement with findings of a study using ethyl esters of DHA that made direct measurements of LDL size (5), a study using fish oil (3), and a study using an algal triacylglycerol preparation similar to that in the present study (6). Because small, dense LDL particles are associated with an increased risk of heart disease (25), it has been argued (3) that they are more susceptible to oxidation and more atherogenic than are those of a lower density (5). Consequently, the potentially greater risk associated with the increase in LDL-cholesterol concentration may be ameliorated by an increase in LDL particle size.

The reasons for variability in response to DHA are uncertain. Minihane et al (3) suggested that the increase in LDL cholesterol was greatest in men carrying the 4 allele but not significant in men who were homozygous for the 3 allele. However, their results did not differ significantly by genotype. In the present study, we were able to show that the LDL-cholesterol concentration increased with DHA supplementation in men and women who were homozygous for the 3 allele. However, the study was not powered to detect differences in response between genotypes, and further research using a much larger sample size is required to establish whether there is difference in response according to genotypes.

Plasma lathosterol and phytosterol concentrations were measured post hoc as indexes of cholesterol synthesis and absorption (26), but these concentrations were not found to be altered by DHA treatment. Consequently, it would appear that mechanisms other than cholesterol synthesis and absorption are likely to be responsible for the increase in LDL cholesterol. Feeding fish oil containing DHA to hamsters (27) and primates (28) results in decreased receptor-mediated clearance of LDL, which could be a result of either decreased binding of LDL to the LDL receptor or decreased expression of LDL receptor. The latter now seems a likely mechanism because, in vitro, DHA down-regulates LDL receptor expression in HepG2 cells (29). DHA is a physiologic ligand for several nuclear hormone receptors—in particular, the peroxysome proliferator–activated receptor and the liver X receptor—which play an important role as lipid sensors in regulating lipid metabolism (30–32). DHA may decrease the expression of LDL receptors by down-regulating the transcription or degradation of sterol regulatory element–binding protein. Alternatively, it may lead to the generation of oxysterols within the liver that stimulate the liver X receptor. Further research is needed to clarify the reasons for the increase in LDL cholesterol with low intakes of DHA.

ACKNOWLEDGMENTS  
We are grateful to Roy Sherwood for supervising the lipoprotein analyses and clinical chemistry.

TABS, HET, and PJC contributed to the design of the study. HET recruited the subjects for the study; analyzed the diet, vitamins, and fatty acids; and contributed to the analysis and interpretation of the study. RW and SEH performed the Apo E phenotyping and contributed to the discussion and interpretation. TABS acts a consultant to Seven Seas Ltd, a subsidiary of Merck Darmstadt. None of the other authors had any conflict of interest.

REFERENCES  

1. Harris WS. n-3 Fatty acids and serum lipoproteins: human studies. Am J Clin Nutr1997;65(suppl):1645S–54S.
2. Sanders TA. Polyunsaturated fatty acids and coronary heart disease. Baillieres Clin Endocrinol Metab1990;4:877–94.
3. Minihane AM, Khan S, Leigh-Firbank EC, et al. ApoE polymorphism and fish oil supplementation in subjects with an atherogenic lipoprotein phenotype. Arterioscler Thromb Vasc Biol2000;20:1990–7.
4. Davidson MH, Maki KC, Kalkowski J, Schaefer EJ, Torri SA, Drennan KB. Effects of docosahexaenoic acid on serum lipoproteins in patients with combined hyperlipidemia: a randomized, double-blind, placebo-controlled trial. J Am Coll Nutr1997;16:236–43.
5. Mori TA, BurkeV, Puddey IB, et al. Purified eicosapentaenoic and docosahexaenoic acids have differential effects on serum lipids and lipoproteins, LDL particle size, glucose, and insulin in mildly hyperlipidemic men. Am J Clin Nutr2000;71:1085–94.
6. Nestel P, Shige H, Pomeroy S, Cehun M, Abbey M, Raederstorff D. The n-3 fatty acids eicosapentaenoic acid and docosahexaenoic acid increase systemic arterial compliance in humans. Am J Clin Nutr2002;76:326–30.
7. Grimsgaard S, Bonaa KH, Hansen JB, Nordoy A. Highly purified eicosapentaenoic acid and docosahexaenoic acid in humans have similar triacylglycerol-lowering effects but divergent effects on serum fatty acids. Am J Clin Nutr1997;66:649–59.
8. Conquer JA, Holub BJ. Supplementation with an algae source of docosahexaenoic acid increases (n-3) fatty acid status and alters selected risk factors for heart disease in vegetarian subjects. J Nutr1996;126:3032–9.
9. Nelson GJ, Schmidt PC, Bartolini GL, Kelley DS, Kyle D. The effect of dietary docosahexaenoic acid on plasma lipoproteins and tissue fatty acid composition in humans. Lipids1997;32:1137–46.
10. Agren JJ, Hanninen O, Julkunen A, et al. Fish diet, fish oil and docosahexaenoic acid rich oil lower fasting and postprandial plasma lipid levels. Eur J Clin Nutr1996;50:765–71.
11. Hu FB, Bronner L, Willett WC, et al. Fish and omega-3 fatty acid intake and risk of coronary heart disease in women. JAMA2002;287:1815–21.
12. Albert CM, Hennekens CH, O’Donnell CJ, et al. Fish consumption and risk of sudden cardiac death. JAMA1998;279:23–8.
13. Anonymous. Dietary supplementation with n-3 polyunsaturated fatty acids and vitamin E after myocardial infarction: results of the GISSI-Prevenzione trial. Gruppo Italiano per lo Studio della Sopravvivenza nell’Infarto miocardico. Lancet1999;354:447–55.
14. Sanders TA, Dean TS, Grainger D, Miller GJ, Wiseman H. Moderate intakes of intact soy protein rich in isoflavones compared with ethanol-extracted soy protein increase HDL but do not influence transforming growth factor beta(1) concentrations and hemostatic risk factors for coronary heart disease in healthy subjects. Am J Clin Nutr2002;76:373–7.
15. Sanders TA, Oakley FR, Miller GJ, Mitropoulos KA, Crook D, Oliver MF. Influence of n-6 versus n-3 polyunsaturated fatty acids in diets low in saturated fatty acids on plasma lipoproteins and hemostatic factors. Arterioscler Thromb Vasc Biol1997;17:3449–60.
16. Salen G, Xu G, Tint GS, Batta AK, Shefer S. Hyperabsorption and retention of campestanol in a sitosterolemic homozygote: comparison with her mother and three control subjects. J Lipid Res2000;41:1883–9.
17. Thurnham DI, Smith E, Flora PS. Concurrent liquid-chromatographic assay of retinol, alpha-tocopherol, beta-carotene, alpha-carotene, lycopene, and beta-cryptoxanthin in plasma, with tocopherol acetate as internal standard. Clin Chem1988;34:377–81.
18. Bolla MK, Wood N, Humphries SE. Rapid determination of apolipoprotein E genotype using a heteroduplex generator. J Lipid Res1999;40:2340–5.
19. Pocock SJ. Clinical trials. A practical approach. Chichester, United Kingdom: Wiley & Sons, 1983.
20. Albert CM, Campos H, Stampfer MJ, et al. Blood levels of long-chain n-3 fatty acids and the risk of sudden death. N Engl J Med2002;346:1113–8.
21. Hamazaki T, Sawazaki S, Asaoka E, et al. Docosahexaenoic acid-rich fish oil does not affect serum lipid concentrations of normolipidemic young adults. J Nutr1996;126:2784–9.
22. Higgins S, Carroll YL, McCarthy SN, et al. Susceptibility of LDL to oxidative modification in healthy volunteers supplemented with low doses of n-3 polyunsaturated fatty acids. Br J Nutr2001;85:23–31.
23. Gregory J, Foster K, Tyler H, Wiseman M. The dietary and nutritional survey of British: adults a survey of the dietary behaviour, nutritional status and blood pressure of adults aged 16 to 64 living in Great Britain. London: Her Majesty’s Stationery Office, 1990.
24. Humphries SE, Talmud PJ, Hawe E, Bolla M, Day INM, Miller GJ. Apolipoprotein E4 and coronary heart disease in middle-aged men who smoke: a prospective study. Lancet2001;358:115–9.
25. Gardner CD, Fortmann SP, Krauss RM. Association of small low-density lipoprotein particles with the incidence of coronary artery disease in men and women. JAMA1996;276:875–81.
26. Miettinen TA, Tilvis RS, KesaniemiYA. Serum plant sterols and cholesterol precursors reflect cholesterol absorption and synthesis in volunteers of a randomly selected male population. Am J Epidemiol1990;131:20–31.
27. Lindsey S, Pronczuk A, Hayes KC. Low density lipoprotein from humans supplemented with n-3 fatty acids depresses both LDL receptor activity and LDLr mRNA abundance in HepG2 cells. J Lipid Res1992;33:647–58.
28. Surette ME, Whelan J, Lu GP, Broughton KS, Kinsella JE. Dependence on dietary cholesterol for n-3 polyunsaturated fatty acid-induced changes in plasma cholesterol in the Syrian hamster. J Lipid Res1992;33:263–71.
29. Schectman G, Boerboom LE, Hannah J, Howard BV, Mueller RA, Kissebah AH. Dietary fish oil decreases low-density-lipoprotein clearance in nonhuman primates. Am J Clin Nutr1996;64:215–21.
30. Ou J, Tu H, Shan B, et al. Unsaturated fatty acids inhibit transcription of the sterol regulatory element-binding protein-1c (SREBP-1c) gene by antagonizing ligand-dependent activation of the LXR. Proc Natl Acad Sci U S A2001;98:6027–32.
31. Xu J, Nakamura MT, Cho HP, Clarke SD. Sterol regulatory element binding protein-1 expression is suppressed by dietary polyunsaturated fatty acids. A mechanism for the coordinate suppression of lipogenic genes by polyunsaturated fats. J Biol Chem1999;274:23577–83.
32. Yoshikawa T, Shimano H, Yahagi N, et al. Polyunsaturated fatty acids suppress sterol regulatory element-binding protein 1c promoter activity by inhibition of liver X receptor (LXR) binding to LXR response elements. J Biol Chem2002;277:1705–11.