Arne Nordøy

Effects of n-3 polyunsaturated fatty acids on glucose homeostasis and blood pressure in essential hypertension : a randomized, controlled trial

Hypertension is a well-documented risk factor for coronary heart disease, but the widespread use of antihypertensive treatment has not resulted in the expected reduction in coronary heart disease mortality [1]. Persons with hypertension tend to have disturbances in glucose and lipid metabolism [2-4] that may contribute to their excess risk for coronary heart disease. Fish oils rich in polyunsaturated fatty acids of the n-3 family may protect against ischemic cardiovascular disease [5-7]. In hypertensive patients, a modest blood pressure-lowering effect has been shown after fish oil intake in some [8-12] but not all [13-15] studies. Fish oil may favorably affect platelet aggregation [16, 17], hepatic triglyceride and very-low-density lipoprotein (VLDL) cholesterol formation [18-21], and vascular prostaglandin production [9, 16, 22]. It has also been reported to suppress intimal smooth-muscle cell proliferation by inhibiting monocyte and neutrophil chemotaxis [23] and the vascular endothelial production of platelet-derived growth factor-like protein [24]. These antiatherosclerotic effects may be important in preventing the development of coronary heart disease in patients with hypertension [25]. Conflicting results have been published about the effects of fish oil on glucose homeostasis [26-41]. Some [26-31] but not all [32-41] studies have reported that fish oil has detrimental effects on glycemic control in glucose-intolerant persons and in persons with type 2 diabetes. The extent to which the findings from these studies can be generalized is constrained by limitations in study design. Only a few studies [26, 28, 30, 34, 35, 38] have used the classic glucose clamp technique to measure glucose and insulin dynamics, and no studies have examined the effects of fish oil on glucose homeostasis in nondiabetic persons with hypertension. Given the present gaps in the literature, the safety of fish oil supplementation for persons with hypertension has been disputed [42]. We therefore did a randomized, double-blind, placebo-controlled trial in 78 persons with untreated, stable hypertension to study the effects of n-3 polyunsaturated fatty acids on glucose and insulin kinetics, blood pressure, serum lipids, and the incorporation of fatty acid into plasma phospholipids. Methods Participants In 1986-1987, 21 826 persons (81.3% of the population [age range: men, 20 to 61 years; women, 20 to 56 years] living in the municipality of Tromso, Norway, participated in a health survey [43]. On the basis of that survey, 156 hypertensive persons were enrolled in a 10-week trial of dietary supplementation with n-3 polyunsaturated fatty acids [8]. The trial was completed in May 1988. In May 1991 and February 1992, the persons who had participated in the trial were invited to have physical examinations at the Clinical Research Unit of the University Hospital of Tromso as part of recruitment into our study. Of the persons invited, 103 volunteered. Each completed a questionnaire about previous and present illnesses, family history, medication, fish oil intake, physical activity, and smoking and alcohol habits, and each had a laboratory screening that included an oral glucose tolerance test and blood pressure measurements. Fifty-eight participants were receiving no medication and had systolic blood pressure measurements of less than 190 mm Hg and diastolic blood pressure measurements between 90 and 110 mm Hg on three separate occasions. Each had a body mass index of less than 32 kg/m2 body surface area and appeared otherwise healthy. They all participated in the study, as did 26 hypertensive persons recruited from the primary health care services using criteria identical to those described above. Four of these volunteers had been treated with antihypertensive drugs (atenolol, amlodipine, or felodipine); this therapy was discontinued at least 8 weeks before the trial. The 84 participants were encouraged not to change their diets or lifestyles during the study. Those who used cod liver oil supplements were instructed to discontinue this practice 12 months before the study started. The study was approved by the Regional Board of Research Ethics, and each participant gave written informed consent before participation. Study Design The participants were randomly assigned to receive either fish oil or corn oil. A person who was not involved in trial management did the randomization using a Statgraphic random number generator [44]. The list of randomization numbers and the codes were sent to the manufacturer of the fish oil and corn oil capsules (Pronova Biocare, Oslo, Norway). The boxes labeled with the randomization numbers were given to the participants in the sequence at which they met. The researchers doing the experiments were blinded to treatment assignments, and the randomization codes were not broken until all laboratory measurements had been done. The fish oil group received 85% eicosapentaenoic acid (C20:5n3) and docosahexaenoic acid (C22:6n3), 4 g/d, as ethyl esters (Omacor, Pronova Biocare, Oslo, Norway). To compensate for the extra daily energy intake received by those assigned to intervention with polyunsaturated fatty acids, the control group was given corn oil, 4 g/d, containing 56% linoleic acid (C18:2n6) and 26% oleic acid (C18:1n9). The fish and corn oils were given in indistinguishable soft gelatin capsules that each contained 1 g of oil. The intervention period lasted 16 weeks. Compliance was checked by counting leftover capsules and by measuring the concentrations of fatty acids in plasma phospholipids before and after intervention. Glucose tolerance studies were done during the last week before treatment and during the last week of intervention. A weight-maintenance diet was held 3 days before the experiments, and participants were asked to abstain from alcohol during this period. All studies were done at 0800 h after an overnight fast. Side effects, compliance, intercurrent diseases, and blood pressure were assessed by interview and physical examination every fourth week during treatment. Clinical and Laboratory Measurements Three blood pressure measurements were recorded on each of 2 separate days both before and after intervention by the same investigator using the same stethoscope and mercury sphygmomanometer. The mean of these measurements was used in the analysis. Measurements were done after each patient had rested, comfortably seated, for 10 minutes; Korotkoff test phases 1 and 5 were recorded as systolic and diastolic blood pressures, respectively. Mean arterial pressure was calculated as the diastolic pressure plus one third of the pulse pressure. Waist-to-hip ratio was calculated as the body circumference midway between the inferior border of the rib cage and the superior border of the iliac crest, divided by the maximal body circumference at the buttocks [45]. All participants had an oral glucose tolerance test with 1 g of dextrose per kg body weight or a maximum of 75 g of dextrose. The integrated increase of plasma glucose and insulin levels above baseline measurements after an oral glucose tolerance test was calculated as arbitrary incremental area units over the 2-hour sampling time. On a separate day, we used a standard hyperglycemic clamp technique to study both glucose-stimulated insulin secretion and insulin sensitivity [46, 47]. Dextrose was infused into an antecubital vein. We measured the blood sugar level every 5 to 10 minutes to keep it stable at 10 mmol/L for 3 hours by variable infusion rates. Blood was drawn from a cannulated dorsal hand vein without stasis; we arterialized the blood by keeping the hand in a heating device at 65 C [48]. Blood samples for insulin and C-peptide measurements were drawn at 30, 0, 2.5, 5, 7.5, 10, 15, 20, 40, 60, 80, 100, 120, 140, 160, and 180 minutes. First-phase insulin release, which reflects the early insulin peak secreted from the pancreatic -cells in response to glucose stimulation, was calculated as the area under the insulin curve over the initial 10-minute period of the hyperglycemic clamp technique. Second-phase insulin release, which is a measure of -cell function under sustained elevated glucose levels, was calculated as the area under the insulin curve from 120 to 180 minutes of the clamp period. On a third day, we used a euglycemic, hyperinsulinemic clamp technique [46, 47], which is the gold standard for measuring insulin sensitivity. However, this method does not give information about -cell function. In general, insulin is infused at a rate of 40 mU/m2 body surface area per minute for 180 minutes, inducing plasma insulin levels of about 400 pmol/L [46]. At this plasma insulin level, hepatic glucose production is zero. Plasma glucose level was maintained at 5 mmol/L by a variable glucose infusion. The glucose infusion rate therefore equals the uptake rate of glucose in the body. The insulin sensitivity index can be calculated by using both the hyperglycemic and the euglycemic clamp techniques by dividing the mean glucose infusion rate during the last hour of the clamp period (mol/kgmin) by the average plasma insulin level in the same period of time (pmol/L). The insulin sensitivity index measures how efficiently plasma insulin induces glucose uptake in insulin-sensitive tissues, such as fat and muscle. The insulin sensitivity index calculated by using the hyperglycemic clamp technique has been shown to be highly correlated with the insulin sensitivity index calculated by using the classic euglycemic, hyperinsulinemic clamp technique [46, 47]. To compare the insulin sensitivity indexes obtained with the two clamp techniques, we used the euglycemic, hyperinsulinemic clamp technique in 31 randomly selected participants on this third day. Plasma glucose levels were analyzed at the bedside with a Yellow Spring Instruments glucose analyzer (2300 STAT PLUS, Yellow Springs, Ohio). All other blood samples were stored at 70 C until study completion. Plasma in

Effects of highly purified eicosapentaenoic acid and docosahexaenoic acid on fatty acid absorption, incorporation into serum phospholipids and postprandial triglyceridemia

Fourteen healthy volunteers were randomly allocated to receive 4 g highly purified ethyl esters of eicosapentaenoic acid (EPA) (95% pure, n=7) or docosahexaenoic acid (DHA) (90% pure, n=7) daily for 5 wk in supplement to their ordinary diet. The n−3 fatty acids were given with a standard high-fat meal at the beginning and the end of the supplementation period. EPA and DHA induced a similar incorporation into chylomicrons which peaked 6 h after the meal. The relative uptake of EPA and DHA from the meal was >90% compared with the uptake of oleic acid. During absorption, there was no significant elongation or retroconversion of EPA or DHA in total chylomicron fatty acids. The concentration of EPA decreased by 13% and DHA by 62% (P<0.001) between 6 and 8 h after the meal. During the 5-wk supplementation period, EPA showed a more rapid and comprehensive increase in serum phospholipids than did DHA. DHA was retroconverted to EPA, whereas EPA was elongated to docosapentaenoic acid (DPA). The postprandial triglyceridemia was suppressed by 19 and 49% after prolonged intake of EPA and DHA, respectively, indicating that prolonged intake of DHA is equivalent to or even more efficient than that of EPA in lowering postprandial triglyceridemia. This study indicates that there are metabolic differences between EPA and DHA which may have implications for the use of n−3 fatty acids in preventive and clinical medicine.

n−3 Polyunsaturated fatty acids and cardiovascular diseases: To whom, how much, preparations

An expert round table discussion on the relationship between intake of n−3 polyunsaturated fatty acids (PUFA) mainly of marine sources and coronary heart disease at the 34th Annual Scientific Meeting of European Society for Clinical Investigation came to the following conclusions:1.Consumption of 1–2 fish meals/wk is associated with reduced coronary heart disease (CHD) mortality.2.Patients who have experienced myocardial infarction have decreased risk of total, cardiovascular, coronary, and sudden death by drug treatment with 1 g/d of ethylesters of n−3 PUFA, mainly as eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA). The effect is present irrespective of high or low traditional fish intake or simultaneous intake of other drugs for secondary CHD prevention, n−3 PUFA may also be given as fatty fish or triglyceride concentrates.3.Patients who have experienced coronary artery bypass surgery with venous grafts may reduce graft occlusion rates by administration of 4 g/d of n−3 PUFA.4.Patients with moderate hypertension may reduce blood pressure by administration of 4 g/d of n−3 PUFA.5.After heart transplantation, 4 g/d of n−3 PUFA may protect against development of hypertension.6.Patients with dyslipidemia and or postprandial hyperlipemia may reduce their coronary risk profile by administration of 1–4 g/d of marine n−3 PUFA. The combination with statins seems to be a potent alternative in these patients.7.There is growing evidence that daily intake of up to 1 energy % of nutrients from plant n−3 PUFA (α-linolenic acid) may decrease the risk for myocardial infarction and death in patients with CHD. This paper summarizes the conclusions of an expert panel on the relationship between n−3 PUFA and CHD. The objectives for the experts were to formulate scientifically sound conclusions on the effects of fish in the diet and the administration of marine n−3 PUFA, mainly eicosapentaenoic acid (EPA, 20∶5n−3) and docosahexaenoic acid (DHA, 22∶6n−3), and eventually of plant n−3 PUFA, α-linolenic acid (ALA, 18∶3n−3), on primary and secondary prevention of CHD. Fish in the diet should be considered as part of a healthy diet low in saturated fats for everybody, whereas additional administration of n−3 PUFA concentrates could be given to specific groups of patients. This workshop was organized on the basis of questions sent to the participants beforehand, on brief introductions by the participants, and finally on discussion and analysis by a group of ∼40 international scientists in the fields of nutrition, cardiology, epidemiology, lipidology, and thrombosis.

Sex hormones and high density lipoproteins in healthy males.

The serum concentrations of testosterone, androstenedione, oestradiol and total low polar oestrogens, mainly oestradiol and oestrone, were measured in 26 healthy male subjects. The subjects were divided into two groups each of them with low (less than 1.04 mmol/l) and high (greater than 1.56 mmol/l) levels of serum HDL cholesterol. The group with high HDL cholesterol had significantly higher testosterone than the other group. Positive correlations were established between HDL cholesterol and testosterone and total cholesterol and testosterone concentration in serum.

Tissue-factor pathway inhibitor and lipoproteins. Evidence for association with and regulation by LDL in human plasma.

Tissue-factor pathway inhibitor (TFPI) is a potent inhibitor of extrinsic coagulation, which is mainly associated with lipoproteins in circulating blood. Gel filtration of human plasma confirmed the presence of three peaks in which approximately 10%, 70%, and 20% of total TFPI activity was retained. Precipitation of very-low-density lipoproteins and low-density lipoproteins (LDLs) in plasma by polyethylene glycol almost completely abolished peaks and I and II. LDL isolated by ultracentrifugation revealed two peaks of TFPI after gel filtration that coeluted with peaks I and II, respectively, from gel filtration of total plasma. TFPI activity in peaks I and II was also precipitated by anti-apolipoprotein B antibodies. Fourteen patients with familial hypercholesterolemia had higher plasma TFPI activity than did age- and sex-matched normolipemic control subjects (1.45 +/- 0.27 U/mL versus 0.80 +/- 0.09 U/mL, P < .001). Plasma TFPI was correlated with LDL cholesterol (r = .73, P < .001) and apolipoprotein B (r = .69, P < .001). No association was found with high-density lipoprotein cholesterol or apolipoprotein A-I. In a double-blind, placebo-controlled trial among the familial hypercholesterolemia patients, lovastatin alone or in combination with fish oil concentrate lowered plasma TFPI in parallel with LDL cholesterol. Gel filtration of plasma from these patients demonstrated a specific drop in apolipoprotein B-TFPI complexes, whereas TFPI not associated with lipoproteins was unchanged. This study demonstrated that plasma TFPI was associated with and regulated by LDL in plasma from healthy subjects and patients with familial hypercholesterolemia.

Effect of ω-3 Fatty Acids and Simvastatin on Hemostatic Risk Factors and Postprandial Hyperlipemia in Patients With Combined Hyperlipemia

Patients with combined hyperlipemia have lipid abnormalities associated with an increased tendency to develop atherosclerosis and thrombosis. This tendency may be accelerated during postprandial hyperlipemia. In the present double-blind parallel study, 41 patients with combined hyperlipemia and serum triacylglycerols between 2.0 and 15.0 mmol/L and serum total cholesterol >5.3 mmol/L at the end of a 3-month dietary run-in period were treated with simvastatin at 20 mg/d for at least 10 weeks; patients were then randomized into 2 groups receiving simvastatin+omega-3 fatty acids at 3.36 g/d or placebo (corn oil) for an additional 5 weeks. Hemostatic variables that have been associated with increased thrombotic tendency were evaluated with subjects in the fasting state and during postprandial hyperlipemia before and after combined treatment. Supplementation of omega-3 fatty acid reduced tissue factor pathway inhibitor antigen (P<0.05) in the fasting state, reduced the degree of postprandial hyperlipemia (P<0.005), and reduced activated factor VII concentration appearing during postprandial hyperlipemia. In conclusion, omega-3 fatty acids given in addition to simvastatin to patients with combined hyperlipemia reduced the free tissue factor pathway inhibitor fraction in the fasting state and inhibited the activation of factor VII occurring during postprandial lipemia, thus representing a potential beneficial effect on the hemostatic risk profile in this patient group.

Serum ferritin, sex hormones, and cardiovascular risk factors in healthy women.

The protective effect of endogenous sex hormones is commonly believed to explain the gender gap in the risk of coronary heart disease and the diminished protection in women when menopause occurs. Recent reports indicate that iron overload, due to cessation of menstrual bleeding, may be an important factor. We therefore investigated iron stores by serum ferritin measurements in healthy premenopausal (n = 113) and postmenopausal (n = 46) women. Ferritin levels were higher in postmenopausal than in premenopausal women, both in blood donors (43.4 versus 23.1 micrograms/L, P < .001) and in nondonors (71.7 versus 32.8 micrograms/L, P < .001). Serum ferritin was positively correlated with age (r = .36, P < .001). After age adjustment, serum ferritin was positively correlated with hemoglobin, hematocrit, serum total cholesterol, and low-density lipoprotein (LDL) cholesterol. Total cholesterol was correlated with age (r = .66, P < .001), as were LDL cholesterol (r = .60, P < .01) and high-density lipoprotein cholesterol (r = .32, P < .01). Neither ferritin nor serum lipids were directly associated with female sex hormone levels. The mutual relation between ferritin, hemoglobin, and hematocrit probably only indicates their usefulness as measures of body iron. The parallel rise in serum ferritin, total cholesterol, and LDL cholesterol might contribute to the increased risk of coronary heart disease among postmenopausal women.

Reduction in Homocysteine by n-3 Polyunsaturated Fatty Acids after 1 Year in a Randomised Double-Blind Study following an Acute Myocardial Infarction: No Effect on Endothelial Adhesion Properties

We hypothesized that n–3 polyunsaturated fatty acids (n–3 PUFAs) as compared to corn oil administered for 1 year following an acute myocardial infarction (MI) may reduce plasma total homocysteine (p-tHcy), ultrasensitive C-reactive protein (µCRP), and the adhesive properties of the endothelium, expressed as soluble E-selectin (sE-selectin) and soluble intercellular adhesion molecule-1 (sICAM-1). In a prospective, randomised, double-blind study, 300 acute MI patients were allocated to highly concentrated n–3 PUFAs (n = 150) or corn oil (n = 150). After 1 year on treatment there was an intergroup difference in p-tHcy in favour of the n–3 group (n = 118), p = 0.022. However, sE-selectin, sICAM-1 and µCRP were unaffected by the treatment. In conclusion, reduction of p-tHcy by long-term n–3 PUFAs treatment was not associated with demonstrable effects on markers of endothelial adhesion properties.

Docosahexaenoic and eicosapentaenoic acids in plasma phospholipids are divergently associated with high density lipoprotein in humans.

The effect of fish oil rich in eicosapentaenoic (EPA) and docosahexaenoic (DHA) acids on serum lipoprotein concentrations is not clear, and it is not known whether EPA and DHA are similarly related to serum lipid or lipoprotein levels. We conducted a randomized, 10-week, dietary supplementation trial in which the effects of 6 g per day of 85% EPA and DHA were compared with 6 g per day of corn oil in 156 men and women. Multivariate analyses were used to assess independent relations between plasma phospholipid EPA and DHA and serum lipoprotein levels. In the fish oil group triglycerides fell 21% (p less than 0.001) and high density lipoprotein cholesterol (HDL-C) rose 3.8% (p less than 0.05). In the corn oil group triglycerides did not change, but HDL-C rose 6.1% (p less than 0.01). Compared with fish oil, apolipoprotein A-I (apo A-I) rose 5.1% after corn oil intake (p less than 0.05). Plasma EPA and DHA levels rose after fish oil intake and fell after corn oil intake (all p less than 0.001). The change (delta) in EPA was inversely correlated with delta triglycerides (p = 0.035) and positively correlated with delta HDL-C and delta apo A-I (both p less than 0.001) in the multivariate analyses. In contrast, delta DHA was not correlated with delta triglycerides but was inversely correlated with delta HDL-C and delta apo A-I (both p less than 0.001). Standardizing for DHA removed the difference in apo A-I levels between groups. This study suggests that EPA and DHA are divergently associated with HDL, possibly through different mechanisms.

Effect of Cholesterol Lowering on Intravascular Pools of TFPI and Its Anticoagulant Potential in Type II Hyperlipoproteinemia

Tissue factor pathway inhibitor (TFPI) inhibits the extrinsic coagulation system. A major pool of TFPI is associated with the vascular endothelium and can be mobilized into the circulation by heparin. In circulating blood, TFPI is mainly associated with LDL (80%), whereas 10% to 20% is carrier free. In this study, heparin administration caused a 2.2-fold and a 7.5-fold increase in TFPI activity and TFPI antigen, respectively, in 25 patients with phenotypes IIa and IIb hyperbetalipoproteinemia. Because the antigen determination of TFPI almost exclusively measures carrier-free TFPI, more than 90% of the heparin-induced increase in TFPI activity was caused by mobilization of carrier-free TFPI from the vascular endothelium. Therapeutic lowering of total cholesterol (a decrease of 31.1 +/- 11.6%, P < .001) by 40 mg/d lovastatin in 17 patients with hyperbetalipoproteinemia was accompanied by a parallel decrease in TFPI activity (of 27.7 +/- 24.2%, P < .001) because of a reduction in LDL-TFPI complexes. However, drug intervention did not affect carrier-free TFPI or the magnitude of the vascular pool of TFPI that could be mobilized into the circulation by heparin. Moreover, this reduction of LDL-TFPI complexes did not reduce the anticoagulant potency of TFPI in plasma or of the vascular endothelial pool. The results of this study may imply that the anticoagulant potency of TFPI is associated with its carrier-free form in plasma or on the endothelium and that downregulation of LDL affects neither the size nor the anticoagulant potency of the endothelial pool of TFPI.