Ole C. Ingebretsen

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

Determination of adenine nucleotides and inosine in human myocard by ion-pair reversed-phase high-performance liquid chromatography

An isocratic high-performance liquid chromatographic system for the quantitation of AMP, ADP and ATP is presented. The separations were achieved at room temperature by reversed-phase chromatography (Supelcosil LC-18). The standard solvent was 220 mM potassium phosphate, pH 6.9, 1% (v/v) methanol and 0.3 mM tetrabutylammonium hydrogen sulphate. A selective retention of the adenine nucleotides as a group relative to the mono-, di- and triphosphates of guanosine, uridine and cytidine was observed under these experimental conditions. The adopted procedure was applied to the separation of adenine nucleotides in biological extracts, i.e., human myocard. The adenine nucleotides in an extract of myocard were quantitated in less than 20 min. Only 5-10 mg (wet weight) of myocard were needed in order to determine the energy charge of a myocardial sample. Also inosine was easily quantitated in this liquid chromatographic system.

Cystatin C is not a better estimator of GFR than plasma creatinine in the general population.

Accurate measurement of glomerular filtration rate (GFR) is complicated and costly; therefore, GFR is commonly estimated by assessing creatinine or cystatin C concentrations. Because estimates based on cystatin C predict cardiovascular disease better than creatinine, these estimates have been hypothesized to be superior to those based on creatinine, when the GFR is near the normal range. To test this, we measured GFR by iohexol clearance in a representative sample of middle-aged (50-62 years) individuals in the general population, excluding those with coronary heart or kidney disease, stroke or diabetes mellitus. Bias, precision (median and interquartile range of estimated minus measured GFR (mGFR)), and accuracy (percentage of estimates within 30% of mGFR) of published cystatin C and creatinine-based GFR equations were compared in a total of 1621 patients. The cystatin C-based equation with the highest accuracy (94%) had a bias of 3.5 and precision of 18 ml/min per 1.73 m², whereas the most accurate (95%) creatinine-based equation had a bias of 2.9 and precision of 15 ml/min per 1.73 m² The best equation, based on both cystatin C and creatinine, had a bias of 7.6 ml/min per 1.73 m², precision of 15 ml/min per 1.73 m², and accuracy of 92%. Thus, estimates of GFR based on cystatin C were not superior to those based on creatinine in the general population. Hence, the better prediction of cardiovascular disease by cystatin C than creatinine measurements, found by others, may be due to factors other than GFR.

Estimated GFR Associates with Cardiovascular Risk Factors Independently of Measured GFR

Estimation of the GFR (eGFR) using creatinine- or cystatin C-based equations is imperfect, especially when the true GFR is normal or near-normal. Modest reductions in eGFR from the normal range variably predict cardiovascular morbidity. If eGFR associates not only with measured GFR (mGFR) but also with cardiovascular risk factors, the effects of these non-GFR-related factors might bias the association between eGFR and outcome. To investigate these potential non-GFR-related associations between eGFR and cardiovascular risk factors, we measured GFR by iohexol clearance in a sample from the general population (age 50 to 62 years) without known cardiovascular disease, diabetes, or kidney disease. Even after adjustment for mGFR, eGFR associated with traditional cardiovascular risk factors in multiple regression analyses. More risk factors influenced cystatin C-based eGFR than creatinine-based eGFR, adjusted for mGFR, and some of the risk factors exhibited nonlinear effects in generalized additive models (P<0.05). These results suggest that eGFR, calculated using standard creatinine- or cystatin C-based equations, partially depends on factors other than the true GFR. Thus, estimates of cardiovascular risk associated with small changes in eGFR must be interpreted with caution.

Impaired Fasting Glucose Is Associated With Renal Hyperfiltration in the General Population

OBJECTIVE Increased glomerular filtration rate (GFR), also called hyperfiltration, is a proposed mechanism for renal injury in diabetes. The causes of hyperfiltration in individuals without diabetes are largely unknown, including the possible role of borderline hyperglycemia. We assessed whether impaired fasting glucose (IFG; 5.6–6.9 mmol/L), elevated HbA1c, or hyperinsulinemia are associated with hyperfiltration in the general middle-aged population. RESEARCH DESIGN AND METHODS A total of 1,560 individuals, aged 50–62 years without diabetes, were included in the Renal Iohexol Clearance Survey in Tromsø 6 (RENIS-T6). GFR was measured as single-sample plasma iohexol clearance. Hyperfiltration was defined as GFR >90th percentile, adjusted for sex, age, weight, height, and use of renin-angiotensin system inhibitors. RESULTS Participants with IFG had a multivariable-adjusted odds ratio of 1.56 (95% CI 1.07–2.25) for hyperfiltration compared with individuals with normal fasting glucose. Odds ratios (95% CI) of hyperfiltration calculated for a 1-unit increase in fasting plasma glucose (FPG) and HbA1c, after multivariable-adjustment, were 1.97 (1.36–2.85) and 2.23 (1.30–3.86). There was no association between fasting insulin levels and hyperfiltration. A nonlinear association between FPG and GFR was observed (df = 3, P < 0.0001). GFR increased with higher glucose levels, with a steeper slope beginning at FPG ≥5.4 mmol/L. CONCLUSIONS Borderline hyperglycemia was associated with hyperfiltration, whereas hyperinsulinemia was not. Longitudinal studies are needed to investigate whether the hyperfiltration associated with IFG is a risk factor for renal injury in the general population.

Glycated hemoglobin level is strongly related to the prevalence of carotid artery plaques with high echogenicity in nondiabetic individuals: the Tromsø study.

Background—High levels of HbA1c have been associated with increased mortality and an increased risk of atherosclerosis assessed as carotid intima-media thickness or plaque prevalence. In the present population-based study, we examined the association between HbA1c and plaque prevalence with emphasis on plaque echogenicity in subjects not diagnosed with diabetes. Methods and Results—HbA1c measurements and ultrasonography of the carotid artery were performed in 5960 subjects (3026 women, 2934 men) 25 to 84 years of age. Plaque morphology was categorized into 4 groups from low echogenicity (soft plaque) to strong echogenicity (hard plaque). HbA1c was categorized into 5 groups: <5.0%, 5.0% to 5.4%, 5.5% to 5.9%, 6.0% to 6.4% and >6.4%. Carotid plaque prevalence increased with increasing HbA1c level (P for linear trend=0.002). The OR for hard plaques versus no plaques was 5.8 in the highest HbA1c group (>6.4%) compared with subjects in the lowest group (<5.0%) after adjustment for several possible confounders. The risk of predominantly hard plaques was also significantly associated with HbA1c levels, although the ORs at each level were somewhat lower than for hard plaques. With respect to the risk of soft plaques versus no plaques, no statistically significant relationship with HbA1c levels was found. Conclusions—Metabolic changes reflected by HbA1c levels may contribute to the development of hard carotid artery plaques, even at modestly elevated levels.

Subcellular distribution of ascorbate in bovine adrenal medulla: Evidence for accumulation in chromaffin granules against a concentration gradient

The subcellular distribution of ascorbate and catecholamines has been studied in homogenates of bovine adrenal medulla and cortex. 1. The recovery of the vitamin was found to be 4.10 +/- 0.22 and 9.57 +/- 1.37 mumol/g wet weight for the medulla and cortex, respectively. A major fraction (34.4%) of the vitamin was recovered in the particulate fraction of the medulla as compared to about 8% in the corresponding fraction of the cortex. In comparison, 78.9% of the catecholamines were found in the particulate fraction of the medulla. 2. Analytical differential centrifugation of medulla homogenates revealed a sedimentation profile of ascorbate which was identical to that obtained for noradrenalin and adrenalin. The co-sedimentation of these compounds indicates that ascorbate is an essential component of the heavy as well as the light population of chromaffin granules. The stoichiometry of catecholamines to ascorbate was approx. 25:1 in both subpopulations. 3. Based on an estimated volume fraction of approximately 13% for the chromaffin granules, as determined morphometrically (Kryvi, H., Flatmark, T. and Terland, O. (1979) Eur. J. Cell Biol. 20, 76-82), a concentration gradient (chromaffin granules:cytosol) of approx. 4 was estimated for ascorbate in the cells of adrenal medulla. 4. No ascorbate 2-sulfate was detected in any of the subcellular fractions isolated, and the content of dehydroascorbate in isolated chromaffin granules was less than 1% of the total ascorbate value.

Gender Differences in the Relationships Between Plasma Plasminogen Activator Inhibitor-1 Activity and Factors Linked to the Insulin Resistance Syndrome in Essential Hypertension

Impaired fibrinolysis due to elevated levels of plasma plasminogen activator inhibitor type 1 (PAI-1) is a risk factor for thromboembolic disease. Hypertension, obesity, derangements in lipid and glucose homeostasis, and elevated levels of PAI-1 are features of the insulin resistance syndrome. The interrelationships between PAI-1 and the metabolic disturbances seen in this condition are unsettled. We investigated the associations between PAI-1 activity and components of the insulin resistance syndrome in 53 men and 31 women with untreated hypertension. In men, PAI-1 activity correlated significantly with plasma glucose (r = .41, P = .002), insulin sensitivity (r = -.35, P = .01), and insulin-induced suppression of nonesterified fatty acid (NEFA) (r = -.43, P = .007). Plasma glucose and NEFA suppression were independently associated with PAI-1 activity in a multivariate analysis. In women, PAI-1 activity correlated with body mass index (r = .62, P = .0005), waist-to-hip ratio (r = .75, P = .0001), plasma glucose (r = .50, P = .007), insulin (r = .49, P = .009), proinsulin (r = .57, P = .002), C-peptide (r = .60, P = .0009), insulin sensitivity (r = -.74, P = .0001), NEFA suppression (r = -.64, P = .003), and triglycerides (r = .58, P = .001). In multivariate analyses, insulin sensitivity and NEFA suppression were independently associated with PAI-1 if waist-to-hip ratio was not included in the model. After introduction of waist-to-hip ratio into the model, waist-to-hip ratio was the only independent predictor of PAI-1 activity. We conclude that in women, waist-to-hip ratio, body mass index, and insulin-induced NEFA suppression are determinants for PAI-1 activity. In men, insulin-induced NEFA suppression and plasma glucose are independently associated with PAI-1 activity.

Direct measurement of free coenzyme A in biological extracts by reversed-phase high-performance liquid chromatography

A high-performance liquid chromatographic system was developed with baseline separation of coenzyme A (CoASH) from dephospho-coenzyme A and acetyl-coenzyme A using isocratic elution. The chromatographic separation was achieved in a reversed-phase system with a high concentration (220 mM) of potassium phosphate buffer at pH 4.0 and appropriate amounts of methanol (ca. 12%). The eluate was monitored with a UV detector at 254 nm with the limit of detection at ca. 5 pmoles. The system could be used without modification for the estimation of the content of CoASH in biological extracts, e.g. mitochondria.

Determination of CoASH by high-performance liquid chromatography and its application in the assay of long-chain acyl-CoA derivatives

Abstract A high-performance liquid chromatographic procedure is described for the determination of picomole amounts of CoASH, using a microparticulate, strong anion-exchange resin. The method was applied in a systematic study to optimize the conditions for alkaline hydrolysis of palmitoyl-CoA. The procedure, which ensures 100% recovery of CoASH by hydrolysis of paimitoyl-CoA, was found to be convenient also for the assay of the endogenous content of long-chain acyl-CoA derivatives in biological material.