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(Circulation. 1997;96:2802-2807.)
© 1997 American Heart Association, Inc.

Articles

Dietary Supplementation With Marine Fish Oil Improves In Vitro Small Artery Endothelial Function in Hypercholesterolemic Patients

A Double-Blind Placebo-Controlled Study

Grahame K. Goode, MB, MRCP; Susana Garcia, BSc; ; Anthony M. Heagerty, MD, FRCP

From the Department of Medicine, Manchester (UK) Royal Infirmary.

Correspondence to Dr Grahame K. Goode, Department of Medicine, Manchester Royal Infirmary, Oxford Rd, Manchester, M13 9WL UK. E-mail Tony.Heagerty{at}man.ac.uk

	Abstract

Top Abstract Introduction Methods Results Discussion References

 
Background Marine fish oils improve vascular function, but the mechanism of benefit is unclear. We conducted a study to examine the effects of fish oils given to hypercholesterolemic patients on small artery function in vitro.

Methods and Results In a randomized, double-blind, placebo-controlled trial, subcutaneous gluteal fat biopsies were taken from 16 hypercholesterolemic patients (serum total cholesterol, 7.97±0.16 mmol/L [mean±SEM]) and 12 age- and sex-matched control subjects (mean cholesterol, 5.11±0.34 mmol/L). Small arteries were mounted on a wire myograph for isometric tension experiments. Patients and control subjects were randomized to receive fish oil (Maxepa 5 capsules BID) or placebo for 3 months. A second biopsy was taken and the studies were repeated. Relaxation to acetylcholine was significantly improved in the hypercholesterolemic group given Maxepa but not in the placebo group (mean maximum relaxation before and after, 48±6.2% and 68.83±2.19%, P=.0054). The dysfunction was not restored to control values (84.3±5.2%, P=.0002). There was also a smaller but significant impairment in endothelium-independent relaxation provoked by sodium nitroprusside (P<.01). A good correlation between the increase in eicosapentanoic acid (n=3) in red cell membrane and improvement in relaxation in the hypercholesterolemic group given fish oils was seen (r=.781, P<.02).

Conclusions Marine fish oil significantly improved endothelial function in peripheral small arteries in hypercholesterolemia patients. This may provide a mechanism for the beneficial effects of these fatty acids in coronary heart disease.

Key Words: hypercholesterolemia • endothelium • arteries • marine fish oil

	Introduction

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There has been much interest in the possible early processes that lead to the development of atherogenesis and in this context how serum cholesterol interacts with the vascular wall. In the human heart, recent evidence has been reported that hypercholesterolemia causes impairment of endothelium-dependent dilatation in intramyocardial small blood vessels.¹ In addition, a study of forearm blood flow has demonstrated blunted responses to both methacholine and nitroprusside in hyperlipidemia.² Both investigations would indicate that hyperlipidemia can compromise endothelium-dependent dilatation and have a direct action on smooth muscle function. It has been suggested that these phenomena precede atheroma formation in medium and large arteries while representing a widespread abnormality in the peripheral vasculature that persists and interferes with vascular tone and pressure homeostasis.³ There is evidence for changes in aortic compliance⁴ and endothelial function in young adults and children with hypercholesterolemia⁵ and some evidence that blood pressure is higher in patients with hyperlipidemia, which may represent the influence of this process on small artery tone in the resistance vasculature.³ Recent studies of in vitro responses of human peripheral small arteries in hypercholesterolemia have confirmed that there is a widespread abnormality of endothelium-dependent dilator function in the human circulation and that the degree of impairment is proportional to the level of hypercholesterolemia.⁶ Furthermore, correction of the abnormal lipid profile results in the restoration of endothelial integrity.⁶

There has been much interest in the role of marine fish oil supplementation for the prevention and treatment of coronary heart disease.^{7 8 9 10 11} Several mechanisms have been proposed for the beneficial results that have been obtained. These include improving the serum lipid profile,¹² lowering blood pressure,¹³ inhibiting platelet aggregation, and prolonging bleeding time,¹⁴ as well as producing vasodilator prostaglandins, which of course may improve vascular function.¹⁵ Because of the recognized impairment of vascular relaxation in patients with hypercholesterolemia, it was decided to examine the effects of marine fish oil supplements on small artery endothelial function in vitro to try to establish whether this dietary manipulation would influence vascular tone.

	Methods

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Sixteen patients with a serum total cholesterol >7.5 mmol/L were recruited from the Lipid Clinic of the hospital. They had all had 3 months of an American Heart Association step 1 diet. Care was taken to exclude secondary hyperlipidemia and patients receiving lipid-lowering drugs, hormone supplements, or vasoactive medication. No subject taking proprietary medications such as vitamins, antioxidants, or fish oils was included. No subject had familial hypercholesterolemia.

The study population was compared with 12 healthy age- and sex-matched control subjects selected on the basis of having a total serum cholesterol of 5.2 mmol/L. These individuals responded to an advertisement placed in a local newspaper.

Patients and control subjects underwent full clinical examination and had blood drawn for a fasting lipid profile and red blood cell membrane fatty acid analysis. Blood pressure was recorded from the right arm of patients in the seated position following 15 minutes of rest and using a standard mercury sphygmomanometer. All patients were seen between 8:30 and 11:00 am, and the mean of three successive readings was noted.

Small Artery Study
A sample of skin and underlying gluteal fat was taken under local anesthetic as previously described.¹⁶ The specimen was immediately placed into a cold physiological salt solution of the following composition (in mmol/L): NaCl 119, NaH₂CO₃ 25, KCl 4.7, KH₂PO₄ 1.18, MgSO₄ 1.17, CaCl₂ 2.5, glucose 5.5, and EDTA 0.026.

Intact small arterial segments 2 mm long were then dissected while under a light microscope and mounted as ring preparations in a wire myograph.¹⁶ Vessels were kept at 37°C in physiological salt solution gassed with 95% 0₂/5% CO₂ to maintain pH constant at7.45.

The vessel was set to an internal circumference (L_o) as determined previously to allow isometric tension experiments.¹⁷ The circumference that the vessels would have had in vivo when relaxed and under a transmural pressure of 100 mm Hg (L₁₀₀) was found with the use of the law of Laplace (P=T/r, where P is transmural pressure, T is tension, and r is radius). L_o was then taken as 0.9L₁₀₀, and the normalized internal diameter was taken as L_o/ (Fig 1).

View larger version (24K): [in this window] [in a new window]   Figure 1. Wire myograph setup and dimensions.

Fish Oil Supplementation
Patients and control subjects were randomized to receive either marine fish oil or placebo, five capsules twice daily for 3 months (Seven Seas). The fish oil capsules (Maxepa) contained the n-3 fatty acids eicasapentanoic acid (EPA) 18% and docosahexanoic acid (DHA) 12%. The placebo capsules contained olive oil. Both contained dodecylgallate BP to prevent in vitro lipid peroxidation and vitamin A <100 IU/g and vitamin D <10 IU/g. The small quantity of dodecylgallate does not have an effect in vivo. Subjects were asked to start a new container of capsules at the beginning of each week and to return used containers for a count to check compliance.

At the end of the study period, all subjects underwent repeated blood testing examination, a second skin biopsy was taken, and functional studies were repeated.

Fatty Acid Analysis
Separation of red blood cells from plasma was carried out within 3 hours of the samples being taken. Extraction and esterification of fatty acids from erythocyte membrane phospholipids were then carried out before separation and measurement by gas chromatography as described elsewhere.¹⁸ Quantification was carried out by flame ionization detection.

Pharmacological Protocol
Vasodilation was assessed by constructing dose-response curves to acetylcholine (endothelium dependent) and sodium nitroprusside (endothelium independent) in increasing concentrations (10^-9 to 10^-5 mol/L) after stable preconstriction with the thromboxane analogue U46619 10^-6 mol/L. Dose-response curves to acetylcholine were then repeated in the presence of indomethacin 0.1 mol/L to block prostaglandin synthesis.

All patients and control subjects were fully informed of the nature of the study and gave written consent. The study was approved by the local Ethics Committee.

Statistical Analysis
Data were expressed as mean±SEM. ANOVA for repeated measures was used to compare dose-response curves. Student's t tests were used to compare paired or unpaired data. Linear regression analysis was performed for selected variables. A value of P<.05 was considered significant.

	Results

Top Abstract Introduction Methods Results Discussion References

 
Demographic Data
The groups of patients and control subjects were well matched for age and sex, but the patients had significantly higher levels of total and LDL cholesterol (Table 1). There was no significant difference in HDL cholesterol or triglyceride levels between the two groups. There were no differences between systolic or diastolic blood pressures or weight (Table 1).

View this table: [in this window] [in a new window]   Table 1. Demographic Data at Baseline

Pretreatment Small Artery Study
The small artery internal diameter was similar in both groups of individual (309±8 and 310±13 µm for hypercholesterolemic subjects and control subjects).

The arteries from hypercholesterolemic patients had markedly impaired endothelium-dependent relaxation compared with responses for vessels from control subjects (Fig 2). The mean maximum relaxation to acetylcholine was 48.0±6.2% in the hypercholesterolemic group and 84.3±5.2% in the control population (P<.0001). There was also a smaller but significant impairment in endothelium-independent relaxation provoked by sodium nitroprusside (Fig 3) (P<.01) as previously reported.⁷

View larger version (12K): [in this window] [in a new window]   Figure 2. Dose-response curve demonstrating relaxation to acetylcholine after preconstriction with U46619 (10-6 mol/L). Data (expressed as mean±SEM) are for 16 hypercholesterolemic (HC) and 12 control subjects. *P<.0001 by ANOVA. Max indicates maximum.

View larger version (14K): [in this window] [in a new window]   Figure 3. Dose-response curve demonstrating relaxation to sodium nitroprusside after preconstriction with U46619 (10-6 mol/L). Data are for 16 hypercholesterolemic (HC) patients and 12 control subjects. *P<.01 by ANOVA. Max indicates maximum.

Posttreatment Small Artery Study
All hypercholesterolemic patients and control subjects were randomized to receive Maxepa or placebo. They all underwent a second biopsy to permit further in vitro examination of peripheral small arteries after 3 months. The compliance was considered satisfactory after a tablet count on the returned bottles (mean, 94.5±4.3% for control subjects and 93.4%±6.8 for hypercholesterolemic patients).

Examination of the responses of preconstricted small arteries to incremental concentrations of acetylcholine demonstrated improvement in relaxation (mean maximum relaxation, 68.83±2.19%; P=.0054) in patients given fish oil supplements but did not restore the dysfunction to control values (P=.0002) (Fig 4). There was no significant difference in relaxation after the addition of indomethacin (Fig 5).

View larger version (21K): [in this window] [in a new window]   Figure 4. Dose-response curve demonstrating relaxation to acetylcholine for hypercholesterolemic (HC) patients before and after fish oil supplementation (n=8). Plot shows significant improvement in relaxation (P=.0054) with treatment. Dysfunction is not restored to control values, however (P=.0002). Max indicates maximum.

View larger version (16K): [in this window] [in a new window]   Figure 5. Dose-response curve demonstrating relaxation to acetylcholine in hypercholesterolemic (HC) group before and after fish oil supplementation. Plot also shows no impairment in relaxation after the addition of indomethacin 0.1 mol/L. Max indicates maximum.

There was no significant difference in the responses of preconstricted arteries to sodium nitroprusside in the hypercholesterolemic group before and after fish oil supplementation (Fig 6). Responses in vessels from control subjects were not influenced by Maxepa (Fig 7).

View larger version (21K): [in this window] [in a new window]   Figure 6. Dose-response curve demonstrating relaxation to sodium nitroprusside in the hypercholesterolemic (HC) group before and after fish oil supplementation (n=8). Plot demonstrates no significant difference in relaxation with treatment. Max indicates maximum.

View larger version (16K): [in this window] [in a new window]   Figure 7. Dose-response curve demonstrating relaxation to acetylcholine in the hypercholesterolemic (HC) group vs control subjects. There are no differences in relaxation in the control group before and after maxepa. Max indicates maximum.

Lipid Values
There was no significant changes in lipid values in either the hypercholesterolemic group or the control group given fish oil (Table 2). There was a small but nonsignificant fall in serum triglycerides in the hypercholesterolemic group given fish oil. There also were no differences in the groups before and after placebo.

View this table: [in this window] [in a new window]   Table 2. Lipid and Blood Pressure Values Before and After Treatment With Maxepa or Placebo

Blood Pressure
There was no significant change in blood pressure in either the hypercholesterolemic group or normocholesterolemic group given fish oil (Table 2).

Erythrocyte Membrane Fatty Acid Analysis
The absolute values of EPA and DHA are given in Table 3. There was a significant increase in both EPA and DHA in the group given fish oil supplementation with a concomitant decrease in arachidonic acid, confirming compliance. There appeared to be good correlation between the magnitude of increase in the red cell membrane EPA and improvement in relaxation to acetylcholine in the hypercholesterolemic group given fish oils (r=.781, P<.02). There was also a weaker but nonsignificant correlation between the increase in red cell DHA and improvement in relaxation (r=.536, P=.09).

View this table: [in this window] [in a new window]   Table 3. Absolute Values for Red Blood Cell Membrane Fatty Acids in All Groups Before and After Maxepa or Placebo

	Discussion

Top Abstract Introduction Methods Results Discussion References

 
In this double-blind, placebo-controlled study, we have found an improvement in endothelial function in middle-aged patients with high serum cholesterol given marine fish oil supplementation for 3 months, despite no significant changes in their lipids. This is the first in vitro examination of this phenomenon in humans, but it has been reported in porcine coronary arteries¹⁹ and in human forearm resistance arteries in vivo.²⁰ Chin et al²¹ previously demonstrated that marine fish oils attenuate vasoconstrictor responses to norepinephrine and angiotensin II in forearm resistance vessels without affecting vasodilator responses to acetylcholine or sodium nitroprusside. The same group also examined responses in subcutaneous small arteries in vitro,²² finding that Maxepa supplementation failed to attenuate vasoconstrictor or vasodilator responses. These studies were performed in healthy volunteers, dosed with 10 capsules for just 28 days. They postulated that the fish oils produced the effects in vivo alone because of modification of circulating factors, possibly prostaglandin products of platelet origin. This was supported by the fact that the in vivo effects were abolished by indomethacin. More recently, Chin and Dart²⁰ studied a small group of hypercholesterolaemic patients in vivo using forearm venous occlusion plethysmography given dietary supplementations of fish oil in a nonrandomized fashion. They demonstrated an augmentation of acetylcholine-induced relaxation in these subjects, who had endothelial dysfunction.

The mechanism of improvement in our patients is unclear but would appear not to involve vasodilator prostaglandins because the improvement in vascular reactivity was not altered by indomethacin. This finding is in keeping with results from animal studies. Shimokawa et al¹⁹ found that supplementing the diet of Yorkshire pigs with fish oil facilitated the endothelium-dependent relaxations to bradykinin, serotonin, ADP, and thrombin. The facilitation was not altered by indomethacin but was significantly inhibited by the guanylate cyclase inhibitor methylene blue. These results are consistent with the possibility that the improvement in relaxation is caused by endothelium-derived nitric oxide. Interestingly, there was no improvement in vasorelaxation to the endothelium-independent agent sodium nitroprusside. This would further support a beneficial effect of fish oils on the endothelium, although we have provided no direct evidence. The mechanism of impairment in relaxation to sodium nitroprusside is not certain. Ultimately, the vasodilation brought about by endothelial factors is through the same mechanism activated by the endothelium-independent against sodium nitroprusside. Guanylate cyclase is stimulated within vascular myocytes by formation of a nitrosylporphyrin complex.²³ It is known that guanylate cyclase activity can be directly affected by hypercholesterolemia by a change in vascular smooth muscle redox state.²⁴ Other studies of peripheral vasculature have also shown impairment in relaxation to muscarinic agonists,^{2 25 26} and one has shown that the forearm vascular response to sodium nitroprusside was blunted response to acetylcholine, which is related to a reduced release of nitric oxide by forearm studies using N^G-monomethyl-L-arginine to inhibit nitric oxide synthesis.²⁶

We have shown also that the greatest improvement in endothelial function occurred in those patients who had the greatest increase in membrane EPA and DHA as reflected by increases in these fatty acids in red cell membrane. Therefore, it is a possibility that an increase in dietary fish oils may change the membrane fluidity of endothelial cells, promoting either increased synthesis and/or release of endothelium-derived nitric oxide in response to acetylcholine. However the direct effects of raised lipids on guanylate cyclase would be unaffected.

There is evidence that the endothelial dysfunction associated with hypercholesterolemia may be produced by increased nitric oxide degradation by free radical species such as superoxide anions generated from endothelial cells.²⁷ This degradation may be reduced by the powerful antioxidant vitamin E, which is present in most fish oil preparations but not in our Maxepa preparations, which had no systemic antioxidant. Certainly there is evidence that vitamin E supplementation prevents oxidized LDL-mediated vascular injury in vitro.²⁸ The capsules contained an in vitro antioxidant that is unlikely to have a systemic effect. However, we have no direct evidence for this.

The beneficial effects also do not appear to be secondary to any change in lipid profile. Marine fish oils are well known to reduce raised serum triglycerides, but our population had relatively normal triglyceride levels because hypertriglyceridemia was excluded. There was a small fall in triglyceride levels in the hypercholesterolemia group given fish oils, but this was not a statistically significant change.

Clinical Importance
The endothelial dysfunction associated with hypercholesterolemia has important clinical implications. There is loss of flow-dependent dilation in coronary vessels,¹ and this coronary vasomotor dysfunction has been implicated in the pathogenesis of unstable coronary syndromes.²⁹ The endothelium also has an important role in combating thrombosis and promoting fibrinolysis. Patients with hypercholesterolemia have reduced fibrinolytic activity,³⁰ and endothelial cells secrete more plasminogen-activator inhibitor.³¹ Marine fish oils appear to augment the function of the endothelium as we have shown, and this may provide a mechanism by which cardiac events are reduced. There is certainly evidence that a close relationship exists between endothelial function in human coronary arteries and the peripheral circulation.³²

There was no significant effect of fish oil supplements on blood pressure, but the study did not have the power to detect small changes in blood pressure in this normotensive population. A meta-analysis of 31 placebo-controlled trials showed a dose-dependent reduction in both systolic and diastolic pressures, but this reduction was small.¹³ The greatest reduction was in those subjects with hypertension, especially with clinical atherosclerosis or hypercholesterolemia. Our patients had normal blood pressures because hypertension was carefully excluded. However, the improvement in endothelial function in our patients may provide a mechanism to explain the blood pressure–lowering effect of marine fish oils in hypertensive patients.

In summary, we have demonstrated an improvement in endothelial function in hypercholesterolemic patients given high-dose fish oil supplements over 3 months. This provides further evidence of a beneficial effect of fish oils and may in part provide a mechanism by which these fatty acids reduce blood pressure and help to prevent acute coronary events.

	Acknowledgments

 
This work was funded by the British Heart Foundation (FS/93044). We are indebted to Seven Seas (UK) for providing the Maxepa and placebo capsules and to Sue Scobie and Dr Brian Faragher for randomization. Special thanks go to Tracy Bent for preparation of the manuscript.

Received March 10, 1997; revision received June 11, 1997; accepted June 14, 1997.

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