Balz Frei

Ascorbic Acid Reverses Endothelial Vasomotor Dysfunction in Patients With Coronary Artery Disease

BACKGROUNDnIn the setting of atherosclerosis, endothelial vasomotor function is abnormal. Increased oxidative stress has been implicated as one potential mechanism for this observation. We therefore hypothesized that an antioxidant, ascorbic acid, would improve endothelium-dependent arterial dilation in patients with coronary artery disease.nnnMETHODS AND RESULTSnBrachial artery endothelium-dependent dilation in response to hyperemia was assessed by high-resolution vascular ultrasound before and 2 hours after oral administration of either 2 g ascorbic acid or placebo in a total of 46 patients with documented coronary artery disease. Plasma ascorbic acid concentration increased 2.5-fold 2 hours after treatment (46+/-8 to 114+/-11 micromol/L, P=.001). In the prospectively defined group of patients with an abnormal baseline response (<5% dilation), ascorbic acid produced marked improvement in dilation (2.0+/-0.6% to 9.7+/-2.0%), whereas placebo had no effect (1.1+/-1.5% to 1.7+/-1.5%, P=.003 for ascorbic acid versus placebo). Ascorbic acid had no effect on hyperemic flow or arterial dilation to sublingual nitroglycerin.nnnCONCLUSIONSnAscorbic acid reverses endothelial vasomotor dysfunction in the brachial circulation of patients with coronary artery disease. These findings suggest that increased oxidative stress contributes to endothelial dysfunction in patients with atherosclerosis and that endothelial dysfunction may respond to antioxidant therapy.

Reactive Oxygen Species and Antioxidant Vitamins: Mechanisms of Action

This article is a brief overview of the mechanisms of production of reactive oxygen species in biologic systems, and the various antioxidant defense systems that provide protection against oxidative damage to biologic macromolecules. The mechanisms of lipid peroxidation and antioxidant protection are explained using a specific example, viz., oxidative modification of human low density lipoprotein and its prevention by vitamin C, vitamin E, and beta-carotene.

Antioxidant Activity of Vitamin C in Iron-overloaded Human Plasma

Vitamin C (ascorbic acid, AA) can act as an antioxidant or a pro-oxidant in vitro, depending on the absence or the presence, respectively, of redox-active metal ions. Some adults with iron-overload and some premature infants have potentially redox-active, bleomycin-detectable iron (BDI) in their plasma. Thus, it has been hypothesized that the combination of AA and BDI causes oxidative damage in vivo. We found that plasma of preterm infants contains high levels of AA and F2-isoprostanes, stable lipid peroxidation end products. However, F2-isoprostane levels were not different between those infants with BDI (138 ± 51 pg/ml, n = 19) and those without (126 ± 41 pg/ml, n = 10), and the same was true for protein carbonyls, a marker of protein oxidation (0.77 ± 0.31 and 0.68 ± 0.13 nmol/mg protein, respectively). Incubation of BDI-containing plasma from preterm infants did not result in detectable lipid hydroperoxide formation (≤10 nm cholesteryl ester hydroperoxides) as long as AA concentrations remained high. Furthermore, when excess iron was added to adult plasma, BDI became detectable, and endogenous AA was rapidly oxidized. Despite this apparent interaction between excess iron and endogenous AA, there was no detectable lipid peroxidation as long as AA was present at >10% of its initial concentration. Finally, when iron was added to plasma devoid of AA, lipid hydroperoxides were formed immediately, whereas endogenous and exogenous AA delayed the onset of iron-induced lipid peroxidation in a dose-dependent manner. These findings demonstrate that in iron-overloaded plasma, AA acts an antioxidant toward lipids. Furthermore, our data do not support the hypothesis that the combination of high plasma concentrations of AA and BDI, or BDI alone, causes oxidative damage to lipids and proteinsin vivo.

Cardiovascular disease and nutrient antioxidants: Role of low‐density lipoprotein oxidation

Increasing evidence indicates that oxidative modification of low-density lipoprotein (LDL) is causally related to atherosclerosis. Oxidatively modified LDL (oxLDL), in contrast to native LDL, is taken up avidly by macrophages, leading to formation of lipid-laden foam cells. Foam cells are pathognomonic of the atherosclerotic fatty streak. Modified LDL may also promote atherosclerosis by many other mechanisms, such as recruitment and retention of monocyte-macrophages, T-lymphocytes, and smooth muscle cells in the arterial intima, and cytotoxicity toward endothelial cells and macrophage-derived foam cells. The oxidation hypothesis of atherosclerosis is supported by a number of in vivo findings, such as the presence of oxLDL in atherosclerotic lesions, and increased titers of autoantibodies against modified LDL in patients with atherosclerosis. As a corollary of the oxidation hypothesis of atherosclerosis, antioxidants that can inhibit LDL oxidation may act as antiatherogens. This conception is supported by animal studies showing that antioxidants such as probucol, butylated hydroxytoluene, and alpha-tocopherol can slow the progression of atherosclerosis. Epidemiological and clinical data indicate a protective role of dietary antioxidants against cardiovascular disease, including vitamin E, beta-carotene, and vitamin C. Likewise, basic research studies on LDL oxidation have demonstrated a protective role for antioxidants, present either in the aqueous environment of LDL or associated with the lipoprotein itself. More studies are needed to establish the effectiveness and determine the required doses of specific antioxidants to prevent and possibly treat cardiovascular disease.

Vascular Superoxide Dismutase Deficiency Impairs Endothelial Vasodilator Function Through Direct Inactivation of Nitric Oxide and Increased Lipid Peroxidation

Nitric oxide (NO) and superoxide are both constitutive products of the endothelium. Because NO is readily inactivated by superoxide, the bioactivity of endothelium-derived NO (EDNO) is dependent on local activity of superoxide dismutase (SOD). We examined the effects of chronic inhibition of copper-zinc SOD (CuZnSOD) using a rat model of dietary copper restriction. Male weanling Sprague-Dawley rats were fed a Cu-deficient diet and received either no Cu replacement (Cu-deficient) or Cu in the drinking water (Cu-sufficient). Compared with Cu-sufficient animals, Cu-deficiency was associated with a 68% reduction in CuZnSOD activity and a 58% increase in vascular superoxide as estimated by lucigenin chemiluminescence (both P < .05). Compared with Cu-sufficient animals, arterial relaxation in the thoracic aorta from Cu-deficient animals was 10-fold less sensitive to acetylcholine, a receptor-dependent EDNO agonist, but only 1.5-fold less sensitive to A23187, a receptor-independent EDNO agonist, and only 1.25-fold less sensitive to authentic NO (all P < .05). In contrast, acute inhibition of CuZnSOD with 10 mM diethyldithiocarbamate produced a more uniform reduction in sensitivity to acetylcholine (8-fold), A23187 (10-fold), and NO (4-fold; all P < .001). Cu-deficient animals demonstrated a 2.5-fold increase in plasma-esterified F2-isoprostanes, a stable marker of lipid peroxidation, that correlated inversely with arterial relaxation to acetylcholine (R = -.83; P < .0009) but not A23187 or authentic NO. From these findings, we conclude that chronic inhibition of CuZnSOD inhibits EDNO-mediated arterial relaxation through two mechanisms, one being direct inactivation of NO and the other being lipid peroxidation that preferentially interrupts receptor-mediated stimulation of EDNO.

Both Intracellular and Extracellular Vitamin C Inhibit Atherogenic Modification of LDL by Human Vascular Endothelial Cells

Oxidative modification of LDL by vascular cells has been proposed as a mechanism by which LDL becomes atherogenic. Antioxidants that can prevent LDL oxidation may therefore act as antiatherogens. We used endothelial cells (ECs) from human aortas (HAECs), human saphenous veins (HSECs), and bovine aortas (BAECs) to investigate the role of intracellular and extracellular vitamin C (ascorbate) in EC-mediated LDL modification. Incubation of LDL (0.1 mg protein per milliliter) with confluent HAECs in Hams F-10 medium led to time-dependent modification of the lipoprotein. In contrast, incubation of LDL with HAECs in medium 199, which does not contain redox-active transition metal ions, did not lead to LDL modification. Both HAEC-mediated and cell-free LDL modifications in Hams F-10 medium were strongly inhibited in a time- and dose-dependent manner by physiological concentrations of ascorbate. Confluent HAECs cultured under conventional conditions contained very little intracellular ascorbate (< 0.5 nmol/mg protein) but could be loaded with up to 20 nmol ascorbate per milligram protein in a time- and concentration-dependent manner. Ascorbate-loaded HAECs exhibited a lower capacity to modify LDL than did non-ascorbate-loaded control cells. When LDL was incubated with HSECs instead of HAECs, similar time- and concentration-dependent inhibitory effects on LDL modification of intracellular and extracellular ascorbate were observed. In contrast to human ECs, BAECs did not take up vitamin C and therefore only coincubation but not preincubation with ascorbate inhibited BAEC-mediated LDL modification. Our data show that enrichment of human vascular ECs with vitamin C lowers their capacity to modify LDL. In addition, extracellular vitamin C strongly inhibits EC-mediated, metal ion-dependent atherogenic modification of LDL.

Vitamin C prevents metal ion-dependent initiation and propagation of lipid peroxidation in human low-density lipoprotein

Lipid peroxidation and oxidative modification of low-density lipoprotein (LDL) have been implicated as causal factors in the pathogenesis of atherosclerosis, and prevention of LDL oxidation by antioxidants may be an effective strategy to inhibit the progression of the disease. We investigated the effects of the reduced form of vitamin C (L-ascorbic acid, AA) and its two-electron oxidation product (dehydro-L-ascorbic acid, DHA) upon metal ion-dependent oxidative modification of human LDL. We found that low micromolar concentrations of both AA and DHA protect LDL against oxidation induced by Cu2+ or by hemin and hydrogen peroxide. In a dose-dependent manner, AA and DHA prevented the initiation of lipid peroxidation in LDL, as determined by a sensitive and selective assay for lipid hydroperoxides utilizing HPLC with chemiluminescence detection. AA and DHA also preserved the LDL-associated antioxidants alpha-tocopherol, beta-carotene, and lycopene, but not ubiquinol-10. Furthermore, AA was able to stop propagation of lipid peroxidation in LDL, whereas DHA lacked this ability. The addition of 60 microM AA to LDL containing up to 38 nmol/mg protein of pre-formed lipid hydroperoxides led to their rapid disappearance; this activity of AA was dependent on the presence of redox-active copper, but did not lead to the formation of lipid hydroxides, the reduced form of lipid hydroperoxides. Our data show that in Cu(2+)-exposed LDL (i) vitamin C primarily spares, rather than regenerates, alpha-tocopherol and other endogenous antioxidants, except for AA and DHA prevent initiation of lipid peroxidation in LDL; and (iii) AA can terminate lipid peroxidation, thereby protecting partially oxidized LDL against further oxidative modification.

Increased plasma levels of lipid hydroperoxides in patients with ischemic stroke

A large body of experimental research indicates that the generation of free radicals leading to oxidative stress plays a role in the pathogenesis of ischemic brain injury, but evidence in humans is limited. We examined plasma levels of lipid hydroperoxides (measured as cholesteryl ester hydroperoxides, CEOOH) and ascorbic acid in 32 patients with cortical stroke, as compared with 13 patients with lacunar infarct. Patients with cortical stroke had significantly increased levels of CEOOH, which peaked on Day 5 after the ictus. Small decreases in ascorbic acid concentrations were not significant. There was a significant positive correlation of CEOOH with the NIH stroke scale, and a significant negative correlation with the Glasgow coma scale. Concentrations of CEOOH were significantly higher in patients with total anterior cerebral syndrome as compared with patients with partial anterior cerebral syndrome or posterior cerebral syndrome. Stroke volumes computed from CT or MRI scans were significantly correlated with plasma CEOOH levels. These findings implicate oxidative stress in ischemic brain injury in humans and suggest that measurements of CEOOH in plasma may be useful both prognostically as well as in monitoring therapeutic interventions.

Low Plasma Ascorbic Acid Independently Predicts the Presence of an Unstable Coronary Syndrome

OBJECTIVESnThis study sought to investigate the relations between plasma antioxidant status, extent of atherosclerosis and activity of coronary artery disease.nnnBACKGROUNDnPrevious studies indicate that increased antioxidant intake is associated with decreased coronary disease risk, but the underlying mechanisms remain controversial.nnnMETHODSnPlasma samples were obtained from 149 patients undergoing cardiac catheterization (65 with stable angina, 84 with unstable angina or a myocardial infarction within 2 weeks). Twelve plasma antioxidant/oxidant markers were measured and correlated with the extent of atherosclerosis and the presence of an unstable coronary syndrome.nnnRESULTSnBy multiple linear regression analysis, age (p < 0.001), diabetes mellitus (p < 0.001), male gender (p < 0.001) and hypercholesterolemia (p = 0.02) were independent predictors of the extent of atherosclerosis. No antioxidant/oxidant marker correlated with the extent of atherosclerosis. However, lower plasma ascorbic acid concentration predicted the presence of an unstable coronary syndrome by multiple logistic regression (odds ratio [OR] 0.59, 95% confidence interval [CI] 0.40 to 0.89, p = 0.01). The severity of atherosclerosis also predicted the presence of an unstable coronary syndrome (OR 1.7, 95% CI 1.14 to 2.47, p = 0.008) when all patients were considered. When only patients with significant coronary disease were considered (at least one stenosis >50%), ascorbic acid concentration (OR 0.56, 95% CI 0.37 to 0.85, p = 0.008) and total plasma thiols (OR 0.52, 95% CI 0.34 to 0.80, p = 0.004) predicted the presence of an unstable coronary syndrome, whereas the extent of atherosclerosis did not.nnnCONCLUSIONSnThese data are consistent with the hypothesis that the beneficial effects of antioxidants in coronary artery disease may result, in part, by an influence on lesion activity rather than a reduction in the overall extent of fixed disease.

Inhibition of copper-induced LDL oxidation by vitamin C is associated with decreased copper-binding to LDL and 2-oxo-histidine formation

Oxidatively modified low-density lipoprotein (LDL) has numerous atherogenic properties, and antioxidants that can prevent LDL oxidation may act as antiatherogens. We have previously shown that vitamin C (L-ascorbic acid, AA) and its two-electron oxidation product dehydro-L-ascorbic acid (DHA) strongly inhibit copper (Cu)-induced LDL oxidation. These findings are unusual, as AA is known to act not only as an antioxidant, but also a pro-oxidant in the presence of transition metal ions in vitro, and DHA has no known reducing capacity. Here we report that human LDL (0.4 mg protein/ml) incubated with 40 microM Cu2+ binds 28.0 +/- 3.3 Cu ions per LDL particle (mean +/- SD, n = 10). Co-incubation of LDL with AA or DHA led to the time- and concentration-dependent release of up to 70% of bound Cu, which was associated with the inhibition of LDL oxidation. Incubation of LDL with Cu and AA or DHA also led to the time-dependent formation of 2-oxo-histidine, an oxidized derivative of histidine with a low affinity for Cu. Addition of free histidine prevented the formation of the LDL-Cu complexes and inhibited LDL oxidation, despite the fact that Cu remained redox-active. Interestingly, histidine was more effective than AA or DHA at limiting Cu binding to LDL, but at low concentrations AA and DHA were more effective than histidine at inhibiting LDL oxidation. These data suggest that there are at least two types of Cu binding sites on LDL: those that bind Cu in a redox-active form critical for initiation of LDL oxidation, and those that bind Cu in a redox-inactive form not contributing to LDL oxidation. The former sites may be primarily histidine residues of apolipoprotein B-100 that are oxidized to 2-oxo-histidine in the presence of Cu and AA or DHA, thus explaining, at least in part, the unusual inhibitory effect of vitamin C on Cu-induced LDL oxidation.