Lawrence T. McGrath

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

SummaryThe 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<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<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<0.05 for all). These data provide evidence for endothelial and smooth muscle dysfunction in diabetes which may have important therapeutic implications.

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

SummaryDecreased 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. NG monomethyl-l-arginine significantly reduced forearm blood flow from maximal stimulated values to acetylcholine when compared to the uninhibited decline in flow to acetylcholine infusions at comparable time points (p<0.01). Treatment with fish oils improved endothelium-dependent responses to acetylcholine without altering endothelium-independent responses to glyceryl trinitrate. By increasing stimulated nitric oxide release from the endothelium fish oils may afford protection against vasospasm and thrombosis in patients with diabetes mellitus.

Functional consequences of endothelial nitric oxide synthase uncoupling in congestive cardiac failure.

Background—Impaired endothelium-mediated vasodilatation (EMVD) in congestive cardiac failure (CCF) has been linked to decreased nitric oxide (NO) bioavailability because of its interaction with vascular superoxide (O2·−), derived predominantly from NAD(P)H-dependent oxidases. When uncoupled from essential cofactors, endothelial nitric oxide synthase (eNOS) produces O2·−. We studied the functional consequences of eNOS uncoupling in relation to EMVD in patients with CCF. Methods and Results—We employed the platelet as a compartmentalized ex-vivo model to examine O2·− and NO production. When eNOS is functioning normally, incorporation of N&ohgr;-Nitro-l-Arginine methyl ester (L-NAME, 1 mmol/L), results in increased O2·− detection, as inhibition of NO production prevents NO scavenging of O2·−. This was observed in controls and 9 of the CCF patients, in whom O2·− detection increased by 63% and 101%, respectively. In the remaining 9 CCF patients, incorporation of L-NAME reduced O2·− production by 39%, indicating O2·− production by eNOS uncoupling. Detection of platelet-derived NO was significantly greater in eNOS-coupled platelets compared with the uncoupled group (2.8±1.4 versus 0.9±0.4 pmol/108 platelets, P =0.04). Endothelium-dependent and -independent vasodilator responses to acetylcholine and sodium nitroprusside recorded using venous occlusion plethysmography were significantly impaired in patients exhibiting eNOS uncoupling. Conclusions—This study provides first evidence that platelet eNOS can become uncoupled in human CCF. Impaired endothelium-dependent and -independent vasodilator responses and diminished platelet-derived NO production occurred in association with enzyme uncoupling.

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.

Platelet nitric oxide and superoxide release during the development of nitrate tolerance: effect of supplemental ascorbate

Background—The therapeutic benefits that accompany the continuous administration of organic nitrates are attenuated by the development of tolerance to the compounds. Altered superoxide production and NO bioavailability have been implicated in contributing to the development of tolerance, an effect that may be ameliorated by the administration of antioxidants. Methods and Results—We studied the effect of 3 days of continuous transdermal administration of nitroglycerin (NTG) (10 mg/24 hours) on platelet free radical (NO and superoxide anion [O2·−] activity) with and without coadministration of supplemental ascorbate (2.4 g/24 hours). NAD(P)H oxidase activity, nitric oxide synthase (NOS) activity, and cyclic guanosine monophosphate (cGMP) content were also assessed. Radial artery pressure pulse waveforms were used to track the hemodynamic actions of NTG. Three days of NTG/placebo was associated with a significant increase in platelet NO and O2·− production from 1.0±1.17 to 2.52±0.88 pmol/108 platelets and 13.2±4.8 to 72.5±34.4 pmol/108 platelets, respectively (P <0.01 for both). These changes were accompanied by increased platelet NADH oxidase activity from 47.9±11.0 to 65.3±13.6 pmol O2·− min/mg protein and cGMP content from 0.60±0.10 to 0.89±0.16 pmol/109 platelets (P <0.05 for both). Administration of NTG/ascorbate attenuated both NO and O2·− release in platelets. Conclusions—Three days of continuous transdermal administration of NTG was accompanied by increased platelet NO and O2·− production and NADH oxidase activity that was suppressed by coadministration of oral ascorbate. Although a significant degree of tolerance would be expected during continuous nitrate administration, a residual hemodynamic action could be identified by arterial pulse contour analysis.

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.

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.

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.

Production of 8-epi prostaglandin F2α in human platelets during administration of organic nitrates

OBJECTIVES The objective of this study was, using isolated platelets as a surrogate for vascular cells, to examine the effect of nonintermittent organic nitrate administration on 8-epi prostaglandin F(2alpha) (8-epi PGF(2alpha)) content and the effect of concurrent oral ascorbate administration. BACKGROUND The long-term efficacy of organic nitrates is hampered by hemodynamic tolerance, which develops during continuous administration. This has been associated with altered production of superoxide and nitric oxide, as well as oxidative stress. This effect may be ameliorated by the co-administration of antioxidants. METHODS Ten healthy male subjects received nitroglycerin (NTG) transdermally at a dosage of 0.4 mg/h for 3 days with ascorbate or lactose (1.2 g/day). After two weeks washout, the treatment was repeated with reversed ascorbate/lactose. Platelets were prepared by centrifugation and esterified 8-epi PGF(2alpha) measured at the start and finish of each treatment by immunoassay. RESULTS Nitroglycerin, in the absence of supplemental ascorbate, was associated with a significant increase in platelet-esterified 8-epi PGF(2alpha), from 32.9 (95% confidence interval [CI] 11.8 to 54.0) to 51.0 (95% CI 16.3 to 85.7) pg/mg protein (p < 0.05). Co-administration of ascorbate with NTG resulted in a significant decrease in 8-epi PGF(2alpha) production, from 38.8 (95% CI 24.9 to 52.7) to 19.0 (95% CI 13.5 to 24.5) pg/mg protein (p < 0.05). CONCLUSIONS Continuous NTG administration results in an increase in platelet-esterified 8-epi PGF(2alpha), a free radical and cyclooxygenase-dependent compound. This is reversed by co-administration of the free radical scavenger ascorbate. Whether this increase is merely a marker for increased oxidative stress or a mediator of oxidative injury contributing to the hemodynamic changes observed in nonintermittent organic nitrate treatment has yet to be resolved.

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

BackgroundManipulation 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.MethodsTen 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.ResultsFollowing 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)ConclusionsThis 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.