Formation of a lipid gradient across the human aortic wall during ageing and the development of atherosclerosis.

The smooth muscle cell invasion and macrophage stimulation within the intima during prolonged exposure to high blood levels of cholesterol esters contribute to increased production of connective tissue matrix. The thickened intima in turn immobilising more LDL derived lipid from the plasma. With damage to the internal elastic lamellae, from essential hypertension, the absorbed lipid can move down a concentration gradient into the medial tissue. This model was supported by our laboratory finding of a lipid gradient across the aorta wall. The gradient commenced shortly after completion of body growth, when the transmedial gradient became detectable. The slope of the gradient progressively increased during ageing. Association of the lipid medial gradient with the degree of atherosclerotic involvement suggested that the gradient influenced the development of intimal lesions. Accumulation of lipid within the medial tissue may then reduce the inward lipid transfer rate from the intima, promoting increased intimal retention and cause the formation of atherosclerotic plaques from the fat saturated intima.

Effect of dietary fish oil supplementation on peroxidation of serum lipids in patients with non-insulin dependent diabetes mellitus.

Lipid peroxidation may be important in the development of cardiovascular disease, a common cause of mortality and morbidity in non-insulin dependent diabetes mellitus (NIDDM). We assessed the degree of lipid peroxidation by measuring plasma malondialdehyde, as thiobarbituric acid reacting substances (TBARS), in 23 non-insulin diabetic patients. Plasma levels of standardised alpha-tocopherol (vitamin E), lipid content of whole plasma and lipoprotein fractions, glycosylated haemoglobin, glycosylated low density lipoprotein (LDL) and fasting blood glucose were also measured. On completion of the baseline studies patients randomly received either fish oil or matching olive oil capsules in a double blind crossover fashion for 6 weeks followed by a 6 week washout period and a final 6 week treatment phase. Studies, identical to the initial baseline studies, were performed at the end of the of the active treatment periods at 6 and 18 weeks. Treatment with olive oil did not change levels of TBARS, vitamin E or indices of glycaemic control compared with baseline. Total cholesterol and triglyceride (TG) content of plasma and lipoprotein fractions were not significantly altered. Treatment with fish oil resulted in elevation of TBARS (P < 0.001) and reduction of vitamin E (P < 0.01) compared with baseline and olive oil treatment. Plasma cholesterol was unchanged. A reduction in plasma TG compared with baseline occurred but failed to reach significance (P =0.07). Changes in apo B containing lipoproteins induced by fish oil failed to reach significance. No significant changes were observed in concentration or composition of high density lipoprotein (HDL). Fish oil treatment showed no change in glycaemic control as assessed by glycosylated haemoglobin and LDL although a rise in fasting blood glucose just failed to reach significance (P = 0.06). Lipid peroxidation in NIDDM can be exacerbated by dietary fish oil. This potentially adverse reaction may limit the therapeutic use of fish oils in such patients.

Breath isoprene in patients with heart failure.

BACKGROUND: Chronic heart failure (CHF) is characterised by increased vascular resistance. This increased after load on the left ventricle contributes to the vicious cycle that leads to progression of myocardial failure, multiple organ failure and death. There is evidence for increased oxidative stress in heart failure, which will influence the myocardium but also peripheral vasculature endothelium. AIMS: The aim of the present study was to examine the production of isoprene, reputed to reflect oxidative stress, in patients with CHF compared to control subjects. METHODS: Twelve patients with CHF and thirty-one healthy control subjects free from heart disease were studied. Breath was collected via a two-way non-re-breathing valve into a 60-l gas collection bag. A sample of ambient air was collected at the same time. A measured aliquot of patient breath and ambient air (approx. 1.5 l) was adsorbed onto a gas adsorption tube packed with poropak-Q. Isoprene was measured using GC/MS and the production rate calculated. All samples of breath were collected at 10.00 h after subjects had been sitting at rest for 15 min. RESULTS: Breath isoprene production in subjects with CHF was significantly reduced compared to controls 83(23) vs. 168(20) pmol min(-1) kg(-1). CONCLUSION: Breath isoprene does not directly reflect oxidative stress in CHF.

Malondialdehyde and measures of antioxidant activity in subjects from the Belfast Elderly Longitudinal Free-living Aging Study.

Glutathione and glutathione peroxidase activity are important components in the complex body defense against oxidative damage. In this study, we have measured malondialdehyde (MDA) as a marker of oxidative stress, the antioxidant glutathione (GSH), and activity of the antioxidant enzyme (GSHPx), in a cohort of free-living elderly subjects from the Belfast Elderly Longitudinal Free-living Aging STudy (BELFAST), hypothesizing that free-living Senieur-approximated nonagenarians might demonstrate enhanced antioxidant defense mechanisms. The main finding in the BELFAST octo/nonagenarians was that plasma antioxidant glutathione increased in nonagenarian compared with septo/octogenarian subjects (P =.015), whereas conversely antioxidant glutathione peroxidase activity fell in the nonagenarian group (P <.0001). In the same subject group, malondialdehyde, a measure of lipid peroxidation, showed no change across the age groups (P =.73). These results might overall represent a situation in which elderly survivors in the BELFAST study have evolved a sort of free radical/antioxidant equilibrium as a mechanism of successful aging.

Effect of red wine consumption on lipoprotein (a) and other risk factors for atherosclerosis.

Epidemiological studies have pointed to the role of alcohol, and red wine in particular, in reducing the incidence of coronary heart disease. This study attempted to distinguish, in vivo, the effects of components specific to red wine and those of alcohol on lipoproteins, antioxidant status and membrane fluidity. Volunteers (n = 20) were given 200 ml of red wine per day for 10 days. Following a 6-week washout, this was repeated with white wine. Changes within treatment groups were analysed by paired t tests and repeated measures analysis of variance was used to distinguish effects of red wine components and alcohol. LDL was prepared by ultracentrifugation and all other assays were by conventional laboratory techniques. No effect with either treatment was detected on total cholesterol, triglycerides, HDL or measures of antioxidant status, including the susceptibility of LDL to oxidation. Red wine reduced LDL cholesterol (p < 0.01), and both treatments reduced LDL apo B (p < 0.01) and increased LDL chol:apo B ratio (p < 0.01), implying an increase in LDL size. Potential anti-atherogenic changes specific to red wine were reduction in lipoprotein (a) (p < 0.001) and increased membrane fluidity (p < 0.01). These results are not in keeping with the proposed role of red wine components in free-radical protection, but the reduction in lipoprotein (a) merits further investigation.

Increased oxidative stress in Alzheimer's disease as assessed with 4-hydroxynonenal but not malondialdehyde.

Oxidative stress is thought to play a major role in the pathogenesis of Alzheimer's disease (AD). Although there is strong post-mortem and experimental evidence of oxidative damage occurring in AD brains, the use of markers in the peripheral circulation to show oxidative stress is less convincing. We examined plasma from AD patients for markers of increased oxidative stress. We report elevated levels of 4-hydroxy-nonenal (4-HNE) in AD patients compared to controls (median 20.6, IQR 6.0-25.2 vs. 7.8, 3.3-14.5 micomol/l, respectively; p=0.001) but not malondialdehyde (MDA), and lower levels of ascorbate in AD plasma when compared to age-matched controls (9.9, 6.0-33.7 vs. 24.2, 13.9-48.6 micromol/l; p<0.05). Levels of 4-HNE in AD patients were inversely related to ascorbate (r=-0.337; p=0.07) and Folstein Mini-Mental State Examination (MMSE) (r=-0.474; p=0.015). The concentration of protein sulphydryls, free-radical scavengers, was directly related to the MMSE result (r=0.427; p=0.03). Increased production of 4-HNE indicates increased oxidative stress (lipid peroxidation), which is not evident using the more common marker MDA. This elevation of 4-HNE was related to the degree of cognitive impairment (MMSE).

Oxidative stress in cyclosporin and azathioprine treated renal transplant patients.

The major cause of death following transplantation is cardiovascular disease. Among the many processes involved in atherogenesis, oxidative stress and modification of low density lipoprotein has been assigned a major role. This in turn may be affected by the immunosuppressive regime used. We studied oxidative stress in 40 renal transplant patients receiving two different immunosuppressive regimens (20 on cyclosporin, 20 on azathioprine/prednisolone), and 19 normal controls. Changes in lipid peroxidation (assessed as thiobarbituric acid reacting substances, TBARS), antioxidant enzyme activities (glutathione reductase GSHPx, glutathione peroxidase GSHPx and superoxide dismutase SOD) vitamin E and antioxidant associated trace metals (selenium, copper, zinc) were studied. Alteration of erythrocyte membrane fluidity was examined using the fluorescent probe 1,6 diphenyl-1,3,5-hexatriene (DPH). Both transplant groups showed no difference in TBARS, lipid standardised vitamin E, copper or selenium compared to controls. Zinc was significantly increased in both the cyclosporin and azathioprine groups compared to controls (P < 0.05). SOD was reduced in both transplant groups compared to controls (P < 0.001). GSHPx was elevated in both groups compared to controls but only reached significance in the azathioprine treated group (P < 0.005). GSHRx was slightly elevated in both transplant groups but did not reach significance. Erythrocyte membrane anisotropy was decreased in the cyclosporin treated group (P < 0.05). There was no difference in the azathioprine group compared to controls. The present results suggest an adaptive response to increased oxidative stress in both transplant groups sufficient to minimise markers of oxidative stress (TBARS and anisotropy). The results also suggest no significant difference between the two immunosuppressive regimes with regard to oxidative stress.

Oxidative stress and erythrocyte membrane fluidity in patients undergoing regular dialysis.

Oxidative damage due to free radical production is increased in uraemic patients and has been suggested as a possible factor contributing to the anaemia of chronic renal failure (CRF) and the pathogenesis of atherosclerosis. Oxidative stress was assessed in 40 patients with CRF maintained by either haemodialysis (HD) or continuous ambulatory peritoneal dialysis (CAPD) and in 18 healthy controls. Lipid peroxidation (assessed as malondialdehyde, MDA), total glutathione (TG), antioxidant enzyme (glutathione reductase (GSHRx), glutathione peroxidase (GSHPx) and superoxide dismutase (SOD)) activity and antioxidant associated trace metal (selenium, copper, zinc) levels were studied. Erythrocyte membrane fluidity was examined using the fluorescent probe 1,6 diphenyl-1,3,5-hexatriene (DPH). The results indicate increased levels of oxidative stress and altered erythrocyte membrane fluidity in patients treated with CAPD compared with controls and patients treated with HD. Only minor changes were observed in patients treated with HD. Altered free radical activity, oxidative stress and altered erythrocyte membrane fluidity observed in patients with CRF may contribute to the increase in vascular disease in such patients and to the anaemia of CRF.

The effect on endothelial function of vitamin C during methionine induced hyperhomocysteinaemia.

BACKGROUND: Manipulation of total homocysteine concentration with oral methionine is associated with impairment of endothelial-dependent vasodilation. This may be caused by increased oxidative stress. Vitamin C is an aqueous phase antioxidant vitamin and free radical scavenger. We hypothesised that if the impairment of endothelial function related to experimental hyperhomocysteinaemia was free radically mediated then co-administration of vitamin C should prevent this. METHODS: Ten healthy adults took part in this crossover study. Endothelial function was determined by measuring forearm blood flow (FBF) in response to intra-arterial infusion of acetylcholine (endothelial-dependent) and sodium nitroprusside (endothelial-independent). Subjects received methionine (100 mg/Kg) plus placebo tablets, methionine plus vitamin C (2 g orally) or placebo drink plus placebo tablets. Study drugs were administered at 9 am on each study date, a minimum of two weeks passed between each study. Homocysteine (tHcy) concentration was determined at baseline and after 4 hours. Endothelial function was determined at 4 hours. Responses to the vasoactive substances are expressed as the area under the curve of change in FBF from baseline. Data are mean plus 95% Confidence Intervals. RESULTS: Following oral methionine tHcy concentration increased significantly versus placebo. At this time endothelial-dependent responses were significantly reduced compared to placebo (31.2 units [22.1-40.3] vs. 46.4 units [42.0-50.8], p < 0.05 vs. Placebo). Endothelial-independent responses were unchanged. Co-administration of vitamin C did not alter the increase in homocysteine or prevent the impairment of endothelial-dependent responses (31.4 [19.5-43.3] vs. 46.4 units [42.0-50.8], p < 0.05 vs. Placebo) CONCLUSIONS: This study demonstrates that methionine increased tHcy with impairment of the endothelial-dependent vasomotor responses. Administration of vitamin C did not prevent this impairment and our results do not support the hypothesis that the endothelial impairment is mediated by adverse oxidative stress.

Comparison of two fluorescent probes for the measurement of erythrocyte membrane fluidity in renal dialysis patients.

Two fluorescent probes were used for the measurement of membrane fluidity in patients on haemodialysis and continuous ambulatory peritoneal dialysis. 1,6-Diphenyl-1,3,5-hexatriene (DPH) anisotropy gives an indication of lipid order and pyrene measures lateral diffusion through the membrane. Pyrene dimer/monomer ratio was significantly lower than controls in both pre-dialysis and post-dialysis samples but DPH anisotropy was unchanged. Both methods showed an increase in membrane fluidity across a 4 hour haemodialysis session. There was an increase in membrane fluidity in CAPD patient samples which was more marked using DPH than pyrene. These results suggest that the two probes give different but complementary information about changes in membrane fluidity and may be more informative when used together rather than singly.

Calcification of the human thoracic aorta during aging.

The rate of calcification within the human thoracic aorta from completion of body growth to advanced old age was examined. Fifty-eight aortae, obtained at necropsy, were dissected into four layers: the complete intima and the separated media, which was subdivided into three tissue samples of equal thickness, defined as the media-inner, -middle, and -outer layers. The sampling sites selected for analysis were from regions of the aortic surface that were free of atherosclerotic plaques. The calcium content within each tissue layer of the aorta was determined. Arterial wall thickness and the cholesterol content of the four layers were also measured. Intimal calcification increased progressively during aging: from 1.6 micrograms Ca/mg tissue at 20 years of age to 5.2 micrograms Ca/mg tissue by 90 years of age. When intima calcium concentration was expressed by tissue volume (w/v), no significant change during aging was found. Medial calcification, as w/v and by w/w, increased throughout aging. Calcium accumulation was most marked in the middle, elastin-rich layer of the media, increasing from 1.4 micrograms Ca/mg tissue at 20 years of age to 49.50 micrograms Ca/mg tissue by 90 years of age. Calcium levels also increased in the other media layers, but at a slower rate than that found within the middle media.

Lipid analysis and fatty acid profiles of individual arterial atherosclerotic plaques.

A protocol for the analysis of the lipid profile of microsamples of aortic tissue was developed. Lipid extraction was from intact tissue using acetone and chloroform/methanol (2/1, v/v). The extract was analyzed for total lipid, esterified cholesterol, cholesterol, triacylglycerol, and phospholipid. The extract was then processed to separate cholesteryl esters, triacylglycerol, and phospholipid which were hydrolyzed and the fatty acid composition was determined by GLC of pentafluorobenzyl ester derivatives. A lipid profile could be obtained on samples with a wet weight of 5 mg.

Gas-liquid chromatographic analysis of volatile short chain fatty acids in fecal samples as pentafluorobenzyl esters.

A protocol was developed for the analysis of volatile short chain fatty acids in microsamples of feces, short chain fatty acid (SCFA) extraction was from fecal samples using ethanol incorporating n-hexanoic acid as an internal standard. The SCFAs were converted to pentafluorobenzyl esters with alpha-2,3,4,5,6-pentafluorotoluene and analyzed on a gas-liquid chromatograph equipped with an electron capture detector. One hundred milligrams of sample was routinely used but analysis could be carried out on 20 mg of sample.

Breath isoprene during acute respiratory exacerbation in cystic fibrosis.

Patients with cystic fibrosis (CF) experience a combination of chronic systemic oxidative stress, generation of free radicals in the lungs due to a hyperimmune response and a diminished ability to scavenge free radicals secondary to malabsorption and increased consumption. The authors asked the question, "Does breath isoprene content reflect systemic oxidative stress?" The study involved 12 CF patients and 12 matched healthy controls. The patients were sampled during acute respiratory exacerbation (increased respiratory symptoms, reduction in forced expiratory volume (FEV1) of >10%, and a decision to treat with intravenous antibiotics) and after two weeks of antibiotic treatment. Blood samples were examined for markers of oxidative stress. Breath samples were analysed for isoprene content. Malondialdehyde (MDA), erythrocyte membrane polyunsaturated fatty acids, protein sulphydryls and protein carbonyls all showed evidence of increased oxidative stress which was moderated by antibiotic treatment. Breath isoprene production rate was significantly lower in patients during exacerbation than in controls with a mean difference of-39 (95% confidence interval (CI) -11-57) pmol.min.kg(-1) and increased to normal values following treatment (mean change 63 (95% CI 42-84) pmol.min.kg(-1)). In conclusion, breath isoprene cannot be considered a reliable marker of oxidative stress.

The effects of oral methionine and homocysteine on endothelial function.

BACKGROUND: Raised homocysteine is a risk factor for vascular disease. Homocysteine is formed from methionine, and dietary manipulation of homocysteine in primates and humans with oral methionine is associated with endothelial dysfunction. A cause-effect relation has not been clearly established. AIM: To study the effect of oral methionine and then oral homocysteine on endothelial function. METHODS: 22 healthy adults were recruited for two randomised crossover studies, each containing 11 subjects. Endothelial function was determined by measuring forearm blood flow in response to intra-arterial infusion of acetylcholine (endothelium dependent) and sodium nitroprusside (endothelium independent). Subjects received methionine or placebo (study 1), or homocysteine or placebo (study 2). Methionine and homocysteine were determined at baseline and t = 4 hours. Endothelial function was determined at four hours. The responses to the vasoactive substances are expressed as the area under the curve of change in forearm blood flow from baseline. RESULTS: Study 1: plasma methionine and homocysteine concentrations increased significantly versus placebo. The increases were associated with a reduction of endothelium dependent responses (mean (95% confidence interval), arbitrary units), from 48.8 (95% CI 36.4 to 61.2) to 29.9 (95% CI 18.0 to 41.1), p < 0.04; endothelium independent responses were unchanged. Study 2: homocysteine concentration increased significantly while methionine remained unchanged. Endothelium dependent responses were reduced from 34.6 (95% CI 20.6 to 48.6) to 22.8 (95% CI 12.0 to 33.6), p < 0.03. CONCLUSIONS: Homocysteine and not methionine is responsible for the changes in endothelial function. This supports the hypothesis that homocysteine promotes atherosclerosis by inducing endothelial dysfunction.

Hyperhomocysteinaemia in young adults is not associated with impaired endothelial function.

A mild to moderate elevation of the total homocysteine concentration (tHcy) is now recognized as a risk factor for vascular disease. It is also associated with endothelial dysfunction in middle-aged and elderly individuals without overt atherosclerotic vascular disease. This is important, as endothelial dysfunction is a well recognized early and potentially reversible marker of the atherosclerotic process. We investigated whether mild hyperhomocysteinaemia was associated with endothelial dysfunction in otherwise healthy young males. We compared endothelial function, by measuring forearm blood flow, in 17 males with mild hyperhomocysteinaemia (defined as tHcy >10 micromol/l) and 14 controls with low tHcy (defined as <5 micromol/l). Forearm blood flow was measured in response to the intra-arterial infusion of acetylcholine (endothelial-dependent response) or sodium nitroprusside (endothelial-independent response). Responses to the vasoactive substances were expressed as the area under the curve of the change in forearm blood flow from baseline. Data are given as mean (95% confidence interval). The two groups were well matched for age, body mass index, pulse rate and blood pressure. tHcy was significantly different between the groups [12.3 (10.4-14.2) micromol/l compared with 4.9 (4.6-5.1) micromol/l; P<0.001]. Concentrations of vitamin B(12) and folate were significantly higher in the control group. There was no difference in basal forearm blood flow between the group with mild hyperhomocysteinaemia and the controls, and both the endothelial-dependent [37.5 (26.2-38.8) and 35.3 (26.1-44.4) arbitrary units respectively] and -independent [26.1 (22.2-29.9) and 25.9 (21.0-30.8) units respectively] responses were not significantly different between the groups. Thus the present study demonstrates that, in healthy adults, mild elevation of tHcy was not associated with impaired endothelial-dependent vasodilation. These data suggest an age effect with regard to homocysteine and endothelial dysfunction. The development of vascular disease in individuals with hyperhomocysteinaemia may only result with higher concentrations or after prolonged exposure.

Dietary fish oil augments nitric oxide production or release in patients with type 2 (non-insulin-dependent) diabetes mellitus.

Decreased release of nitric oxide from damaged endothelium is responsible for the impaired endothelium-dependent vasodilator responses found in animal models of vascular disease. Dietary supplementation with fish oils has been shown to augment endothelium-dependent relaxations, principally by improving the release of nitric oxide from injured endothelium. Using forearm venous occlusion plethysmography we studied vascular responses to 60, 120, 180 and 240 nmol/min of acetylcholine (an endothelium-dependent vasodilator) and 3, 6 and 9 nmol/min of glyceryl trinitrate (an endothelium-independent vasodilator) infused into the brachial artery in 23 patients with Type 2 (non-insulin-dependent) diabetes mellitus. NG monomethyl-L-arginine was employed to inhibit stimulated and basal release of nitric oxide from the endothelium. On completion of the baseline studies patients randomly received either fish oil or matching olive oil capsules in a double-blind crossover fashion for 6 weeks followed by a 6-week washout period and a final 6-week treatment phase. Studies, identical to the initial baseline studies, were performed at the end of the active treatment periods at 6 and 18 weeks. Fish oil supplementation significantly improved forearm blood flow responses to each dose of acetylcholine when compared to the vasodilator responses recorded at baseline and after olive oil administration (p < 0.01). Neither fish oil nor olive oil supplementation produced any significant changes in forearm blood flow to the incremental infusions of glyceryl trinitrate when compared with responses recorded during the baseline studies.(ABSTRACT TRUNCATED AT 250 WORDS)

Individuals with cystic fibrosis do not display impaired endothelial function or evidence of oxidative damage in endothelial cells exposed to serum.

Heightened systemic oxidative stress is increasingly recognized as a feature of cystic fibrosis (CF). The consequences of long-term exposure to free radical attack include a predisposition to diseases such as cancer and atherosclerosis. An increased incidence of malignancy among adult patients with CF has been reported, but the absence of atherosclerotic disease is well described. The aim of the present study was to assess endothelial function in vivo and relate this to the potential of serum from patients with CF to induce oxidative-mediated damage in cultured human endothelial cells. A group of 11 CF patients was matched with a group of healthy volunteers with regard to age and sex. Endothelial function was assessed as endothelium-dependent and -independent vasodilation by measuring forearm blood flow in response to infused acetylcholine and sodium nitroprusside respectively. Confluent monolayers of cultured human endothelial cells were exposed to serum from CF patients and control subjects. Following exposure, cell death was assessed by lactate dehydrogenase release, and the degree of lipid peroxidation in the membrane was assessed by measuring the content of lipid hydroperoxides, malondialdehyde and 4-hydroxynonenal. Endothelial monolayers exposed to serum from CF patients released significantly less lactate dehydrogenase following exposure than those exposed to serum from healthy controls (1.8% and 3.0% respectively; mean difference -1.2%; 95% confidence intervals -1.9% to -0.1%; P<0.05) and contained significantly less 4-hydroxynonenal (0.75 and 3.41 micromol/g of protein respectively; mean difference -2.66 micromol/g; 95% confidence intervals -5.10 to -0.22 micromol/g; P<0.05). There was no significant difference between patients and controls in the extent of serum-induced membrane peroxidation, as assessed by malondialdehyde or lipid hydroperoxides, or in endothelial function, as assessed by forearm blood flow. In conclusion, despite evidence for heightened systemic oxidative stress in CF, patients displayed no impairment of endothelial function, and their serum caused significantly less damage to human endothelial cells than that from matched controls.

Oxidative stress during acute respiratory exacerbations in cystic fibrosis.

BACKGROUND: Patients with cystic fibrosis experience chronic systemic oxidative stress. This is coupled with chronic inflammation of the lung involving bronchial polymorphonuclear neutrophil accumulation and activation. We hypothesised that, during periods of acute respiratory exacerbation, free radical activity and consequent damage would be most marked and that intensive treatment of the infection would result in improvement towards values found during stable periods. METHODS: Plasma and red blood cells were collected from 12 healthy normal volunteers and from 12 patients with cystic fibrosis with an acute respiratory exacerbation (increased respiratory symptoms, reduction in forced expiratory volume in one second (FEV1) of more than 10%, and a decision to treat with intravenous antibiotics). Further samples were collected from patients following two weeks of treatment. Samples were analysed for inflammatory markers, markers of free radical damage, and aqueous and lipid phase scavengers. RESULTS: During respiratory exacerbations FEV1 and forced vital capacity (FVC) were lower than in controls (mean differences -2.82 (95% CI -2.12 to -3.52) and -3. 79 (-3.03 to -4.55) l, respectively) but improved following treatment (mean change 0.29 (95% CI 0.18 to 0.40) and 0.33 (0.23 to 0.43) l, respectively). Inflammatory markers during exacerbations were significantly higher in patients than in controls with the following mean (95% CI) differences: C reactive protein (CRP), 46 (17 to 75) g/l; neutrophil elastase alpha1-antiprotease complexes (NEAPC), 4.4 (1.77 to 7.07) mg/l; white cell count (WCC), 5.3 (4.7 to 5.9) x 10(9)/l. These markers decreased significantly following treatment with the following mean (95% CI) changes: CRP -26 (-10 to -42) g/l; NEAPC -3.1 (-1.3 to -4.9) mg/l; WCC -1.5 (-1.3 to -1.7) x 10(9)/l. Malondialdehyde (MDA) as a marker of free radical activity was significantly higher in patients during exacerbations than in controls with a mean (95% CI) difference of 193 (107 to 279) which improved with treatment (mean change -56 (95% CI -28 to -84) nmol/mmol cholesterol). Red blood cell polyunsaturated fatty acids were significantly lower in patients than in controls with a mean difference of -4.4(95% CI -2.6 to -6.2) moles percent, but did not improve significantly after treatment. Protein carbonyls during exacerbations were not different from controls but did increase with treatment compared with levels during the exacerbation (mean change 0.39 (95% CI 0.11 to 0.67) micromol/g protein). Aqueous and lipid phase scavengers in patients during exacerbations were significantly lower than in controls with the following mean (95% CI) differences: ascorbate, -19.0 (-2.7 to -35.3) micromol/l; sulphydryls, -122 (-77 to -167) micromol/l; retinol, -237 (-47 to -427) nmol/mmol cholesterol; beta-carotene, -52.8 (-11.8 to -93.8) nmol/mmol cholesterol; luteine, -50.4 (-10.4 to -90.4) nmol/mmol cholesterol; lycopene, -90.1 (-30.1 to -150.1) nmol/mmol cholesterol. Treatment resulted in improvement with the following mean (95% CI) changes: sulphydryls, 50 (32 to 68) micromol/l; retinol, 152 (47 to 257) nmol/mmol cholesterol; alpha- and beta-carotene, 0.6 (0.0 to 1.2) and 7.6 (0.0 to 15.2) nmol/mmol cholesterol, respectively; alpha-tocopherol, 839 (283 to 1405) nmol/mmol cholesterol; and lycopene, 8.2 (0.0 to 16.2) nmol/mmol cholesterol. CONCLUSIONS: Abnormalities of markers of inflammation, free radical activity, and radical scavengers were significantly more extreme during acute respiratory exacerbations and showed improvement with treatment. The need to provide protection from inflammation and free radical damage should therefore be dynamic and related to the inflammatory and oxidative processes.

Impaired endothelium-dependent and independent vasodilation in patients with type 2 (non-insulin-dependent) diabetes mellitus.

The endothelium plays a pivotal role in modulating the reactivity of vascular smooth muscle through the formation of several vasoactive substances. We examined the effects of endothelium-dependent and independent vasodilators on forearm blood flow in 29 patients with Type 2 (non-insulin-dependent) diabetes mellitus and in 21 control subjects, using venous occlusion plethysmography. Via a brachial artery cannula, increasing amounts of acetylcholine and glyceryl trinitrate were infused in doses of 60, 120, 180 and 240 mmol per min and 3, 6 and 9 nmol per min respectively. NG monomethyl-L-arginine, a stereospecific inhibitor of endothelium derived relaxing factor, was infused to inhibit basal and stimulated release of this dilator substance. Reactive hyperaemic forearm blood flow did not differ between groups. Forearm blood flow responses to each dose of acetylcholine were significantly greater in control than diabetic subjects (p less than 0.01 for all doses). NG monomethyl-L-arginine attenuated forearm blood flow from maximal stimulated values when responses were compared with the natural decline to acetylcholine in forearm flow in both control and diabetic subjects (p less than 0.05 for both groups), but had no effect on basal blood flow responses. Forearm blood flow responses to each dose of glyceryl trinitrate were significantly greater in control than diabetic subjects (p less than 0.05 for all). These data provide evidence for endothelial and smooth muscle dysfunction in diabetes which may have important therapeutic implications.