Markku Ahotupa

Weight Reduction With Very-Low-Caloric Diet and Endothelial Function in Overweight Adults: Role of Plasma Glucose

Objective—Obesity is associated with endothelial dysfunction that may contribute to the development of atherosclerosis. We studied whether weight reduction improves endothelial function in overweight individuals. Methods and Results—Flow-mediated endothelium-dependent vasodilation of the brachial artery was measured in 67 adults (age: 46±7 years, body mass index: 35.2±5.4 kg/m2) before and after a 6-week weight reduction program induced by very-low-calorie diet (daily energy: 580 kcal/2.3 MJ). Caloric restriction reduced body weight from 101±18 to 90±17 kg. Flow-mediated vasodilation increased from 5.5%±3.7 to 8.8%±3.7% (P <0.0001). Nitrate-mediated vasodilation was not significantly affected. The improvement in flow-mediated dilation was associated with the reduction in plasma glucose concentration (P =0.0003). This relationship was independent of changes in weight, serum lipids, oxidized LDL, C-reactive protein, adiponectin, blood pressure, and insulin. Conclusions—Weight reduction with very-low-calorie diet improves flow-mediated vasodilation in obese individuals. This improvement is related to the reduction in plasma glucose concentration. These observations suggest that changes in glucose metabolism may determine endothelial vasodilatory function in obesity.

Lipid Peroxidation and Antioxidants in Normal and Pre-eclamptic Pregnancies

Lipid peroxidation has been suggested as a pathogenetic factor of pre-eclampsia. In this study we measured lipid peroxidation products and the counteracting antioxidant functions in maternal serum and placental tissue in normal pregnancy and pre-eclampsia. Placentae and maternal serum from 15 normal and 15 pre-eclamptic pregnancies were collected. Lipid peroxidation was measured as peroxidation potential, thiobarbituric acid reacting substances (TBARS) and conjugated diene onuble bonds. The antioxidative capacity was measured as the activity of superoxide dismutase, glucose 6-phosphate-dehydrogenase, glutathione peroxidase and glutathione transferase and the concentration of placental vitamin E. Placental lipid peroxidation was higher in pre-eclampsia than in normal pregnancy, when measured by peroxidation potential and TBARS (P = 0.002 and P = 0.027, respectively). The activity of placental superoxide dismutase (P = 0.003) and glucose 6-phosphate-dehydrogenase (P = 0.019) was significantly lower in pre-eclampsia than in normal pregnancy. There were no significant differences in the activity of glutathione peroxidase, glutathione-S-transferase or vitamin E level between the study groups. The peroxyl radical trapping capacity (TRAP) was higher (P = 0.013) in the serum of pre-eclamptic than control patients. Lipid peroxidation is increased and the activity of antioxidant enzymes superoxide dismutase and glucose 6-phosphate-dehydrogenase are decreased in pre-eclamptic placenta. The TRAP is high in the serum of pre-eclamptic patients.

Constantly low HDL-cholesterol concentration relates to endothelial dysfunction and increased in vivo LDL-oxidation in healthy young men

To test the hypothesis that low HDL-C concentration interferes with vascular endothelial function and lipoprotein oxidation, we measured endothelium-dependent flow mediated dilatation (FMD, %) of the brachial artery in young men (n=20) classified prospectively into two groups on basis of having either low or high HDL-C concentration over the past 2 years. As an estimate of in vivo low-density lipoprotein oxidation (ox-LDL), we measured LDL diene conjugation. FMD was present in the group with high HDL-C concentration, but impaired in the group with low HDL-C (5.5+/-3.2 vs 0.2+/-1.2%, P<0. 001). The group with high HDL-C level had significantly lower levels of ox-LDL compared to low HDL-C group (18.0+/-1.8 vs 22.9+/-4.4, P</=0.01). In all subjects, FMD correlated with HDL-C (r=0.59, P=0. 006), HDL(2)-C (r=0.62, P=0.004) and ox-LDL (r=-0.56, P=0.013) but not with HDL(3)-C (r=0.16, P=0.52). We conclude that constantly low HDL-C concentration is related with endothelial dysfunction and increased oxidative stress in healthy young men, consistent with the idea that HDL particles may protect endothelium and inhibit the oxidation of LDL. These findings may offer insight into increased atherosclerosis associated with low HDL-C levels.

Simple methods of quantifying oxidation products and antioxidant potential of low density lipoproteins

OBJECTIVES The present study describes new methods for the measurement of oxidation products and antioxidant potential of low density lipoproteins (LDL). DESIGN AND METHODS LDL is isolated by precipitation with buffered heparin. The assay for LDL oxidation products (LDL-BDC) is based on determination of baseline levels of conjugated dienes (BDC) in lipids extracted from LDL. The assay for antioxidant potential of LDL (LDL-TRAP) is based on the ability of LDL to trap peroxyl radicals. RESULTS LDL-BDC was found to increase linearly over a range from 100 to 1750 microL, LDL-TRAP from 250 to 1750 microL of serum taken for precipitation. For LDL-BDC, the CV was 4.4% and 4.5% for within- and between-assay precision, respectively. For the LDL-TRAP, the CV was 8.1% and 8.7% for within- and between-assay precisions, respectively. Freezing of the serum (2 weeks at -70 degrees C) did not affect LDL-BDC or LDL-TRAP levels. A negative correlation was found to exist between the LDL-BDC and LDL-TRAP values. LDL-BDC and LDL-TRAP values were at the same level in both sexes. The LDL-BDC was found to increase with age. Short-term intervention with antioxidants increased LDL-TRAP substantially, but did not affect the LDL-BDC level. CONCLUSIONS These methods are fast and simple to perform, and can, therefore, be applied to clinical purposes.

Increased Arterial Intima-Media Thickness and In Vivo LDL Oxidation in Young Men With Borderline Hypertension

We used borderline hypertension as a model for prehypertension to examine the early influences of elevated blood pressure on subclinical atherosclerosis, lipoprotein oxidation, and cardiac adaptation. Healthy men (age 37±4 years) were classified prospectively into 2 groups on the basis of having either borderline hypertension (systolic 130 to 140 mm Hg or diastolic 85 to 89 mm Hg, n=16) or normal (<130/85 mm Hg, n=22) blood pressure values during the previous 2 years. The groups were matched for age, body size, and serum cholesterol levels. High-resolution ultrasound was used to measure intima-media thickness (IMT) of the carotid and brachial arteries, cardiac dimensions, and brachial artery endothelial function. Baseline low-density lipoprotein (LDL)-diene conjugation was measured as an estimate of in vivo LDL oxidation (ox-LDL). Compared with normotensive controls, men with borderline hypertension had higher IMT of the carotid artery (0.58±0.06 versus 0.75±0.07 mm, P <0.001) and IMT of the brachial artery (0.45±0.05 versus 0.57±0.07 mm, P <0.001), and increased levels of ox-LDL (29±9 versus 47±17 mol/L, P <0.001), but similar endothelial function. Left ventricular mass was similar in both groups, but there were significant differences in left ventricular geometry. In multivariate analyses, the predictors of carotid IMT were 24-hour systolic blood pressure (P <0.001) and ox-LDL (P =0.10). The current study demonstrates evidence of increased subclinical atherosclerosis and ox-LDL in borderline hypertension. These results are consistent with the idea that enhanced ox-LDL may be one of the pathophysiological events related to development of atherosclerosis in men with borderline elevated blood pressure.

Baseline diene conjugation in LDL lipids as a direct measure of in vivo LDL oxidation.

OBJECTIVES To additionally test validity of the recently developed method (LDL baseline diene conjugation, LDL-BDC) for determination of circulating oxidized LDL. DESIGN AND METHODS A detailed comparison between the ultracentrifugation and heparin precipitation methods for LDL isolation was performed to test suitability of the fast precipitation method. Validity of LDL-BDC as an indicator of circulating oxidized LDL was tested by comparing LDL-BDC to results obtained by the immunological autoantibody method. RESULTS BDC values in LDL isolated by heparin precipitation did not differ from those isolated by sequential ultracentrifugation. While highest amount of diene conjugation was found in LDL (40% of that in serum), substantial amounts were also found in VLDL (31%) and HDL (25%). When analyzed in the same samples, assays for the titer of autoantibodies against oxidized LDL and LDL-BDC were found to show good correlation (r = 0.57, p = 0.001, n = 29). CONCLUSIONS These results, together with thus far conducted studies on clinical applicability of the method, indicate that LDL-BDC is a promising candidate in search for a method for estimation of LDL oxidation in vivo.

Large-Artery Elastic Properties in Young Men Relationships to Serum Lipoproteins and Oxidized Low-Density Lipoproteins

Measures of arterial elasticity have been proposed as surrogate markers for asymptomatic atherosclerosis. We investigated the relations of serum lipoproteins, oxidized low-density lipoprotein (ox-LDL), and familial hypercholesterolemia (FH) to arterial elasticity among young men. As a marker of arterial elasticity we measured compliance in the thoracic aorta by using magnetic resonance imaging and in the common carotid artery by using ultrasound. LDL diene conjugation was used as a marker of ox-LDL. In study I, 25 healthy men (aged 29 to 39) were classified into 2 extreme groups according to previously measured high-density lipoprotein cholesterol to total cholesterol ratio (HDL-C/TC ratio). In study II, the healthy men were used as controls for 10 age matched asymptomatic patients with FH. In healthy men, the group with low HDL-C/TC ratio had decreased carotid artery compliance (2. 3+/-0.4% versus 1.9+/-0.5%/10 mm Hg, P=0.034). In univariate analysis, the compliance of the carotid artery associated with ox-LDL (r =-0.49, P=0.016) and HDL-C/TC ratio (r=0.41, P=0.040). In multivariate regression analyses, ox-LDL was the only independent determinant for compliance of the carotid artery (P=0.016). Aortic elasticity was not related to standard lipid variables, but the compliance of the ascending aorta associated with ox-LDL (r=-0.44, P=0.030). In FH patients, arterial elasticity was similar to that in controls. We conclude that elasticity of the common carotid artery is affected by serum lipid profile in young men. The current study demonstrates for the first time an in vivo association between ox-LDL and arterial elasticity suggesting that oxidative modification of LDL may play a role in the alteration of arterial wall elastic properties.

Antioxidant enzyme activities and oxidative stress in human breast cancer

We have analysed products of lipid peroxidation reactions and activities of antioxidant enzymes in cancerous breast tissue and in corresponding reference tissue. In addition, the serum lipid peroxidation and peroxyl-radical-trapping capacity of breast cancer patients were compared to those of healthy subjects. A total of 23 patients with breast cancer participated in this study. In the cancerous tissue, catalase activity was lower than in the reference tissue, while the activities of superoxide dismutase, glutathione peroxidase and the hexose monophosphate shunt were elevated. The content of thiobarbituric-acid-reactive material was slightly lower in the cancerous tissues, but the levels in serum were found to be elevated in patients with breast cancer. The amounts of conjugated diene double bonds were essentially equal both in the cancerous and in the reference tissue. Moreover, in breast cancer patients the serum levels of diene conjugation and the peroxyl-radical-scavenging capacity did not differ from those measured in healthy subjects. This study indicates that the antioxidant defence system is altered in cancerous breast tissues, but does not support the hypothesis suggesting that formation of lipid peroxides in the tumour tissue itself is of primary importance in the carcinogenesis.

Oxidized LDL and thickness of carotid intima-media are associated with coronary atherosclerosis in middle-aged men: lower levels of oxidized LDL with statin therapy

We investigated the relation between serum lipids including oxidized LDL and the severity of coronary atherosclerosis. Serum lipids and oxidized LDL was measured in 62 men (33-66 years), who underwent diagnostic coronary angiography and sonography to measure the carotid intima-media thickness. LDL oxidation was found in chemical analyses to be due to conjugated fatty acids in cholesteryl esters and triglycerides. Regression analysis indicated that the carotid intima-media thickness and the ratio of LDL diene conjugation to LDL cholesterol (the ox-LDL:LDL ratio) were the only factors associated independently with the severity of coronary atherosclerosis. The patients with multi-vessel disease who did not use lipid lowering therapy had a 50% thicker carotid intima media (P = 0.030) and a 41% higher ox-LDL:LDL ratio (P = 0.020) than patients with normal vessels. Further, patients with multi-vessel disease on statin therapy had a 24% lower ox-LDL:LDL ratio than the subjects with multi-vessel disease who did not use lipid lowering drugs (P = 0.027), although the concentration of LDL cholesterol did not differ between the groups. This study supports the hypothesis that lipid oxidation plays a role in the development of atherosclerosis.

Oxidative stress has a role in malignant transformation in Barrett's oesophagus

Mechanisms underlying the development of oesophageal adenocarcinoma are poorly understood. To discover the role of oxidative stress and radical scavenger capacity in the malignant transformation of Barretts oesophagus, we measured myeloperoxidase activity, superoxide dismutase activity, glutathione content and total aromatic DNA adducts. Mucosal specimens came from 52 patients in 6 groups: symptomatic gastro‐oesophageal reflux disease (GORD) without and with endoscopic oesophagitis, Barretts epithelium without and with dysplasia, adenocarcinoma in the oesophagus and controls. In the GORD‐oesophagitis‐metaplasia‐dysplasia‐adenocarcinoma sequence, glutathione content was progressively lower and myeloperoxidase activity higher than in controls, plateauing at Barretts epithelium without dysplasia. Only in Barretts epithelium with dysplasia was SOD activity significantly increased. In all patient groups, DNA adduct levels were significantly higher than the control level. Though these levels between patient groups did not differ significantly, the level was highest in Barretts epithelium without dysplasia and progressively lower in Barretts with dysplasia and adenocarcinoma. Pooled data showed a negative correlation between glutathione content and DNA adducts (−0.28, p = 0.05). Simultaneous formation of DNA adducts, increased myeloperoxidase‐related oxidative stress, decreased antioxidant capacity (glutathione content) and the negative correlation between glutathione content and DNA adducts in the GORD–oesophagitis–metaplasia–dysplasia–adenocarcinoma sequence of Barretts oesophagus indicate a role in the pathogenesis and malignant transformation related to oxidative stress.