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(Circulation. 1998;97:736-743.)
© 1998 American Heart Association, Inc.

Clinical Investigations and Reports

Coronary Artery Responses to Physiological Stimuli Are Improved by Deferoxamine but not by L-Arginine in Non–Insulin-Dependent Diabetic Patients With Angiographically Normal Coronary Arteries and No Other Risk Factors

Alain Nitenberg, MD; Frédéric Paycha, MD; Séverine Ledoux, MD; Régis Sachs, MD; Jean-Raymond Attali, MD; ; Paul Valensi, MD

From the Service de Physiologie et d'Explorations Fonctionnelles, INSERM, Hôpital Louis Mourier, CHU Xavier-Bichat, Colombes, France (A.N., F.P., S.L.), and Service d'Endocrinologie Diabetologue et Nutrition, Hôpital Jean Verdier, Bondy, France (R.S., J-R.A., P.V.).

Correspondence to Alain Nitenberg, MD, Service de Physiologie et d'Explorations Fonctionnelles, INSERM U.426, Hôpital Louis Mourier, CHU Xavier-Bichat, 178, rue des Renouillers, F-92700 Colombes, France.

Abstract

Background—Acetylcholine produces coronary artery (CA) constriction in diabetic patients, suggesting an impairment of endothelium-dependent dilation. In diabetes, multiple metabolic abnormalities may inactivate nitric oxide through oxygen free radical production.

Methods and Results—To examine the mechanism of this abnormal response, two physiological tests (ie, a cold pressor test [CPT] and coronary flow increase induced by an injection of 10 mg papaverine [PAP] in the distal left anterior descending CA) were performed before and after either intravenous L-arginine (625 mg/minx10 minutes) or intravenous deferoxamine (50 mg/minx10 minutes) in 22 normotensive nonsmoking diabetic patients with angiographically normal CAs and normal cholesterol. Coronary surface areas were measured with quantitative angiography. Before the administration of L-arginine or deferoxamine, CPT induced CA constriction in both groups (-14±10% and -15±11%, respectively; each P<.001), and PAP injection in distal LAD did not modify significantly proximal LAD dimensions. In the 10 diabetic patients receiving L-arginine, responses to CPT and PAP were not modified. Conversely, in the 12 patients receiving deferoxamine, CA dilated in response to the two tests (+10±9% after CPT and +22±7% after PAP, each P<.001). Intracoronary isosorbide dinitrate, an endothelium-independent dilator, produced similar dilation in the two groups (+47±19% and +41±15%, respectively; each P<.001).

Conclusions—This study shows that (1) responses of angiographically normal CAs to CPT and to flow increase are impaired in diabetic patients; (2) abnormal responses are not improved by L-arginine, suggesting that a deficit in substrate for nitric oxide synthesis is not involved; and (3) deferoxamine restores a vasodilator response to the two tests, suggesting that inactivation of NO by oxygen species might be partly responsible for the impairment of CA dilation in diabetic patients.

Key Words: antioxidants • coronary disease • diabetes mellitus • free radicals

Coronary atherosclerosis develops earlier in diabetic patients than in other subjects and accounts for excess morbidity and mortality in these patients.¹ It has been shown that diabetic patients without angiographically detectable coronary atherosclerosis² have abnormal coronary responses to acetylcholine, suggesting that the endothelium-derived nitric oxide system could be impaired before the development of overt atherosclerosis. Such an impairment of endothelium-dependent dilation also has been demonstrated in extracardiac arterial and arteriolar vessels in insulin- and non–insulin-dependent diabetes mellitus.^{3 4 5 6 7} However, the mechanisms by which diabetes mellitus might impair endothelium-dependent coronary vasodilation are unknown. The decreased availability of the substrate L-arginine⁸ or the inactivation of nitric oxide are two mechanisms by which endothelium-dependent coronary vasodilation might be impaired in diabetic patients. Thus, in hypercholesterolemic humans, it has been demonstrated that the administration of L-arginine could improve vasodilation of forearm resistance vessels.⁹ On the other hand, it has been proved that superoxide anions could inactivate nitric oxide,¹⁰ and metabolic abnormalities in diabetic patients could inhibit nitric oxide through free radical production by myocardium and endothelial cells.^{11 12}

The purpose of the present study was to determine whether impaired endothelium-dependent coronary vasorelaxation before the development of angiographically visible coronary atherosclerosis in response to two physiological stimuli (ie, cold pressor test [CPT] and increased blood flow) could be improved with L-arginine, a precursor of nitric oxide, or deferoxamine, an iron chelator that prevents iron-catalyzed generation of hydroxyl radicals.

Methods

Patient Selection
Twenty-two patients with type II diabetes mellitus who were undergoing diagnostic coronary angiography were included in this study. These patients were selected among 75 diabetic patients referred for coronary arteriography because of abnormal stress tests or SPECT stress thallium scintigraphy. Mean duration of diabetes was 12.5±8.1 years, and all patients manifested proper glucose homeostasis at the time of catheterization. Patients who had a history of arterial hypertension (blood pressure of >140/90 mm Hg), patients (untreated or with lipid-lowering therapy) with total cholesterol serum levels of >5.70 mmol/L (220 mg/dL) or LDL cholesterol of >3.70 mmol/L (143 mg/dL), smokers, patients older than 65 years, and postmenopausal women without substitutive hormonal therapy were excluded. None of the patients had a family history of premature coronary artery disease (defined as a first-degree relative at age <60 years with clinical evidence of coronary atherosclerosis). All patients had normal left ventricular systolic function and mass assessed by two-dimensional and M-mode echocardiography. Left ventricular dimensions and septal and posterior wall thicknesses were measured at end diastole according to American Society of Echocardiography guidelines.¹³ The left ventricular mass index was calculated according to the Penn convention.¹⁴

Patients with diabetes mellitus and no detectable coronary atherosclerosis were included in the present study by consensus of two experienced investigators on immediate review of the angiograms and only if coronary arteries were angiographically normal and completely smooth, without luminal irregularities. Patients were randomly allocated to one of two different groups before cardiac catheterization. The first group received L-arginine, and the second group received deferoxamine, with the investigators blinded as to the drug delivered to the patients. The study protocol was approved by the Institutional Review Committee of the University of Kremlin-Bicêtre. All patients gave written informed consent before cardiac catheterization.

Catheterization Protocol
Patients were studied in the fasting state. No premedication was administered, 1% lidocaine was used for local anesthesia, and 5000 U IV heparin was administered. After documentation of normal coronary arteries, an additional 5000 U IV heparin was given, and a 8F guiding catheter was positioned in the left coronary artery. Each patient then underwent the following study protocol. A 3F 20-MHz coronary Doppler catheter (Monorail Doppler 3; Schneider Europe AG) connected to a single-channel 20-MHz pulsed Doppler velocimeter (model MDV-20 Single Channel Velocimeter; Millar Instruments) was placed in the left anterior descending coronary artery (LAD). The proximal lumen of the Doppler catheter was placed in the midportion of the LAD through injection of contrast medium (Fig 1), and catheter position was adjusted to obtain an optimal audio signal and phasic tracing of coronary blood flow velocity. The use of this device to assess intracoronary blood flow velocity has been previously discussed in detail.¹⁵

View larger version (29K): [in this window] [in a new window]   Figure 1. Schematic of right anterior oblique projection of left coronary artery shows precise location of the three segments at which diameter measurements were made.

The protocol design is presented in Fig 2. Thirty minutes after the coronary arteriography, the first hemodynamic measurements and left coronary arteriography (Base 1) were carried out. Five minutes later, the cold pressor test (CPT 1) was performed. The patient's hands were immersed in ice water for 120 seconds. Then, after blood pressure and heart rate had returned to baseline values (Base 2), flow-dependent coronary dilation was assessed as previously described.¹⁵ Briefly, a bolus of 10 mg papaverine (PAP 1) was injected in the midportion of the LAD through the proximal lumen of the Doppler catheter, and the diameter of the proximal LAD (LAD 1) was measured. Papaverine reflux that might have caused direct dilation of the LAD 1 was excluded by verifying that injection of a bolus of 2 mL contrast medium through the Doppler catheter did not cause dye reflux to the LAD 1. The proximal circumflex artery (CX) segment served as control. Coronary angiograms were performed using an injection of 8 mL low osmolarity contrast medium (meglumine ioxaglate) in the left coronary artery, at Base 1, at the peak of the CPT 1 (immediately before removal of the hands from ice water), at Base 2, and 60 seconds after the peak blood flow velocity induced by PAP 1. Serial injections of the left coronary artery were performed at intervals of 5 minutes to exclude contrast-induced coronary dilation. Intracoronary blood flow velocity was measured in the distal LAD (LAD 2), near the tip of the Doppler catheter, just before each angiogram, to avoid the hyperemic effect of the contrast material. Heart rate, aortic pressure (through the guiding catheter), mean and phasic blood flow velocity (kilohertz shift), and ECG were continuously monitored throughout the protocol. Measurements of the diameters of the LAD 1, LAD 2, and CX arteries were made on each angiogram.

View larger version (9K): [in this window] [in a new window]   Figure 2. Protocol design. Hemodynamic measurements and quantitative coronary angiography were performed before L-arginine or deferoxamine administration, at Base 1, at the end of the CPT (CPT 1), at Base 2, and after intracoronary injection of papaverine (PAP 1). The same procedure was followed after intravenous infusion of L-arginine or deferoxamine (Base 3, CPT 2, Base 4, PAP 2) and finally after intracoronary injection of isosorbide dinitrate (ISDN).

After completion of the first series of measures, L-arginine or deferoxamine was administered intravenously. Ten patients were given L-arginine (Arginine-Glucose Veyron; Laboratoire Veyron et Froment) at a rate of 625 mg/min (10 mL/min) for a 10-minute period, and 12 patients received deferoxamine (Desferal; CIBA-GEIGY Laboratories) at a rate of 50 mg/min (10 mL/min) during 10 minutes (500 mg of deferoxamine was solved in 5 mL of distilled water, which was then diluted in 95 mL of 0.9% saline). The dose of deferoxamine was chosen according to experimental data in dogs that have shown that low doses of deferoxamine exhibited a cardioprotective action in the stunned myocardium.^{16 17 18} This procedure was then repeated (Fig 2). Left coronary angiograms, hemodynamic recordings, and measurements of blood flow velocity in LAD 2 were thus performed at Base 3, at the peak of the CPT 2, at Base 4, and 60 seconds after the peak blood flow velocity induced by papaverine (PAP 2). Last, hemodynamic measurements and coronary arteriography were repeated 4 minutes after the intracoronary infusion of a bolus of 2 mg of isosorbide dinitrate through the guiding catheter.

Quantitative Coronary Arteriography
Left coronary arteriograms were obtained by ECG-triggered digital subtraction at a rate of 6 frames/s on a 512-pixel matrix (CGR DG 300; General Electric). The angiographic system was set up in a right anterior oblique position with adequate cranial or caudal angulation allowing optimal view of the LAD 1, LAD 2, and CX segments on end-diastolic frames without overlap by side branches (Fig 1). Relations among focal spot, patient, and height of image tube were kept constant throughout the procedure. Analysis of coronary angiograms was performed by a previously validated technique.¹⁵

In this study, a segment of the guiding catheter filled with saline was placed close to the center of the image and used as a scaling device for calibration before the procedure was begun. A change in vessel diameter was defined as a minimum 6% variation, which corresponded to the highest error reported using the quantitative angiography validation technique (5.7%).²⁰ Cross-sectional area was calculated from diameters (d) assuming a circumferential model: Cross-sectional area=d²/4. Each angiogram was analyzed at random without knowledge of the sequence of the procedure (Base 1, CPT 1, Base 2, PAP 1, Base 3, CPT 2, Base 4, PAP 2, and ISDN) and the drug administered (L-arginine or deferoxamine).

Statistical Analysis
All data are expressed as mean±SD. Differences between the two groups of patients for clinical and biological characteristics and basal hemodynamic and echocardiographic parameters were compared with use of the nonparametric Mann-Whitney test. Statistical comparisons of hemodynamic parameters, coronary vessel dimensions under base, CPT, papaverine, before and after the administration of L-arginine or deferoxamine, and under post–isosorbide dinitrate conditions were made by two-way ANOVA with repeated measures for experimental condition factor, followed by the Fisher protected least-significant difference test. Statistical significance was assumed if the null hypothesis could be rejected at the .05 probability level.

Results

Clinical Data
Characteristics of the two groups of patients are summarized in Table 1. L-Arginine and deferoxamine groups were comparable for sex ratio, age, body mass index, and arterial pressure. Echocardiographic data did not show any differences in left ventricular end-diastolic diameter, fractional shortening, and left ventricular mass index, which were within the normal range in the two groups.¹⁴ Among the 22 patients, 6 of 10 in the L-arginine group and 8 of 12 in the deferoxamine group had lipid-lowering therapy. The lipid profile showed only a mild difference between the two groups for triglycerides plasma concentration. One woman in the L-arginine group and 3 women in the deferoxamine group had postmenopausal hormonal therapy.

View this table: [in this window] [in a new window]   Table 1. Characteristics of Study Population

Hemodynamic Parameters
The two groups of patients did not show any difference in heart rate and aortic pressures at each stage of the study (Table 2). CPT 1 and CPT 2 induced a significant increase in aortic pressures in the two groups. Heart rate was mildly increased only during the CPT after L-arginine administration. There was no difference between hemodynamic changes observed before and after drug administration. Intracoronary injection of papaverine produced a significant and comparable aortic pressure decrease and heart rate increase in the two groups of patients, without any difference between changes observed before and after drug administration. Intracoronary isosorbide dinitrate injection was followed by a comparable reduction in aortic pressures and heart rate increase in the two groups.

View this table: [in this window] [in a new window]   Table 2. Hemodynamic Changes Throughout the Study

Effects of L-Arginine on Coronary Artery Vasomotion
CPT induced a significant reduction in the surface area of coronary segments (n=30) either before (-14±10%, P<.0001) or after (-13±10%, P<.0001) L-arginine administration (Fig 3). Intracoronary papaverine was not followed by any change in cross-sectional area of proximal LAD (n=10) and CX (n=10) (Fig 3). A similar dilation of distal LAD, in which papaverine was injected, was observed either before or after L-arginine (Fig 3).

View larger version (28K): [in this window] [in a new window]   Figure 3. Changes in cross-sectional areas of the three segments measured throughout the protocol in patients receiving L-arginine. See legends to Figs 1 and 2 for abbreviations.

When diameter variations of >6% were considered, none of the segments dilated during CPT, and a majority constricted either before or after L-arginine (Fig 4). Similarly, intracoronary papaverine was not followed by any dilation in any proximal LAD segment, either before or after L-arginine administration (Fig 4).

View larger version (34K): [in this window] [in a new window]   Figure 4. Prevalence among the two groups of patients of dilation, no change, and constriction in coronary segment responses to CPT and to injection of papaverine in the distal LAD. Numbers at the bottom of each column indicate the number of segments for each type of response divided by the number of segments analyzed. All the segments were analyzed for the CPT; only dimensions of the proximal LAD were analyzed after papaverine injection.

Effects of Deferoxamine on Coronary Artery Vasomotion
CPT induced a significant reduction in cross-sectional area of coronary segments (n=36) before (-15±11%, P<.0001) deferoxamine administration (Fig 5). Conversely, after deferoxamine, segments dilated significantly (+10±9%, P<.0001). Flow-dependent dilation of proximal LAD (n=12) after intracoronary papaverine injection in the distal segment was absent before deferoxamine administration and present after it (+22±7%, P<.001) (Fig 5). A comparable dilation of distal LAD (n=12), in which papaverine was injected, was observed either before or after deferoxamine (Fig 5). There were no significant changes in CX dimensions after papaverine injection either before or after deferoxamine (Fig 5).

View larger version (30K): [in this window] [in a new window]   Figure 5. Changes in cross-sectional areas of the three segments measured throughout the protocol in patients receiving deferoxamine. See legends to Figs 1 and 2 for abbreviations.

The analysis of the prevalence of dilation, absence of change, or constriction showed that a majority of segments constricted and no segment dilated in response to CPT before deferoxamine (Fig 4). Conversely, after drug administration, a majority of segments dilated and none constricted (Fig 4). Likewise, no flow-dependent dilation was observed before deferoxamine, and almost all of the proximal LAD segments dilated after it (Fig 4).

Comparisons Between L-Arginine and Deferoxamine Responses
Comparisons between the coronary responses of the two groups showed a similar coronary constriction in response to CPT and an absence of flow-dependent dilation of the proximal LAD before drug administration (Fig 6). Conversely, dilation of coronary segments in response to CPT and to flow increase was observed after deferoxamine, but there was no modification of the abnormal response after L-arginine (Fig 6). Endothelium-independent coronary dilation evoked by intracoronary isosorbide dinitrate was comparable in the two groups of patients (L-arginine [n=30]: +47±19%, P<.0001; deferoxamine [n=36]: +41±15%, P<.0001) (Figs 3, 5, and 6).

View larger version (27K): [in this window] [in a new window]   Figure 6. Comparisons between the responses of the two groups of patients. Numbers in parentheses indicate the number of segments analyzed. All the segments were analyzed for the CPT; only dimensions of the proximal LAD were analyzed after papaverine injection. See legend to Fig 2 for abbreviations.

Discussion

Abnormal response to intracoronary acetylcholine in diabetic patients has suggested that nitric oxide–mediated endothelium-dependent dilation is impaired.² However, acetylcholine is not a physiological stimulus of endothelium-mediated coronary vasodilation, which raises the question of the physiological and clinical relevance of the abnormal response to acetylcholine. On the other hand, the mechanisms responsible for endothelial dysfunction in diabetes mellitus have not been elucidated. Indeed, nitric oxide dysfunction may lie at different levels: (1) decreased synthesis and release either because of insufficient substrate or abnormal nitric oxide synthase activity, (2) inactivation by oxygen-derived free radicals, or (3) decreased responsiveness of underlying smooth muscle cells.

The main results of the present study in type II diabetic patients with angiographically normal coronary arteries and no other coronary risk factor are that (1) coronary artery responses to physiological stimuli are impaired; (2) endothelium-dependent vasodilation is improved by administration of deferoxamine, an iron chelator, but not by administration of L-arginine, the substrate of nitric oxide synthesis; and (3) endothelium-independent dilation to nitrates is preserved.

Abnormal Endothelium-Dependent Dilation in Diabetic Patients
Impairment of coronary endothelium-dependent dilation assessed by the response to acetylcholine is a common finding in patients with coronary artery risk factors. Abnormalities have been documented in hypertensive patients^{15 19} or hypercholesterolemic patients²⁰ in either forearm or coronary circulations. In diabetes mellitus, abnormal responses have been demonstrated in extracardiac vessels.^{3 4} If Calver et al⁵ did not find any alteration in response to acetylcholine in type I diabetic patients, Williams et al²¹ showed an abnormal response to metacholine in type II diabetic patients. In a previous study, we did not find any difference in abnormal coronary response to acetylcholine between type I and type II patients.² However, intravascular acetylcholine is not a compound that plays a physiological role in vessel vasomotion. The present study shows that normal coronary dilation to two physiological stimuli, CPT and increased coronary flow, was impaired in diabetic patients with angiographically normal coronary arteries and no other risk factor.

CPT has been shown to increase coronary blood flow through the rise in myocardial oxygen demand²² resulting from sympathetic stimulation²³ and to dilate normal epicardial coronary arteries,²⁴ despite the -mediated constricting stimulus. Furthermore, blood flow velocity plays a major role in the dilator response of coronary arteries through the increased shear stress²⁵ that enhances the release of the endothelium-derived relaxing factor²⁶ and/or probably an endothelium-derived hyperpolarizing factor, which is different from nitric oxide.²⁷ In the present study, the absence of dilation in response to CPT cannot be explained by the absence of myocardial oxygen demand increase during the test. Indeed, the ratexpressure product, which is an estimate of myocardial oxygen demand, was significantly increased in the two groups (Table 2) either before (by +25±16% in the L-arginine group and by +21±10% in the deferoxamine group) or after drug administration (by +27±24% in the L-arginine group and by +21±10% in the deferoxamine group). In addition, flow velocity increase after papaverine injection in the distal LAD was comparable in the two groups either before (+375±98% in the L-arginine group and +421±128% in the deferoxamine group, all P<.0001) or after drug administration (+398±115% in the L-arginine group and +415±155% in the deferoxamine group, all P<.0001).

Because coronary artery dimension changes in response to isosorbide dinitrate, an exogenous donor of nitric oxide, were comparable in the two groups (Fig 6) and similar to results observed in normal patients,^{15 22} a decreased responsiveness of underlying smooth muscle cells can be ruled out. These results are at variance with those of Calver and Vallance,⁵ who found a reduced response of forearm vessels to sodium nitroprusside. In our study, we tested the hypothesis that the abnormalities in endothelium-dependent coronary dilation might be due to decreased availability of substrate L-arginine for endothelial synthesis of nitric oxide or inactivation of nitric oxide by superoxide anion generation.

Failure of L-Arginine to Improve Coronary Dilation in Diabetic Patients
Previous studies have shown that L-arginine administration augments the forearm endothelium-dependent dilation to acetylcholine in normal human beings²⁸ and that this compound may improve endothelium-dependent forearm dilation in hypercholesterolemic humans.⁹ Because endothelial nitric oxide synthesis and insulin sensitivity are positively related in healthy humans,²⁹ reduced nitric oxide production due to an insufficient amount of L-arginine linked to the decreased insulin sensitivity may be responsible for abnormal vasomotion in diabetes mellitus. These findings raised the possibility that supplying a supplement of substrate should improve endothelium-mediated coronary dilation in diabetic patients. Our results show that L-arginine did not improve coronary dilation in diabetic patients. Therefore, the abnormal vasomotion was not due to a decreased availability of the substrate for endothelial synthesis of nitric oxide. These results are comparable to those observed in the forearm of hypertensive patients.²⁸

Deferoxamine Improves Coronary Dilation in Diabetic Patients
There is growing evidence that the excess of vascular oxidative stress impairs nitric oxide function. In rat hypertension by angiotensin II, vascular relaxation is impaired because of smooth muscle O^-· production³⁰ ; hypercholesterolemia increases superoxide anion production.³¹ The release of superoxide anions can bind to and inactivate nitric oxide to NO^-₂ directly¹⁰ or stimulate oxidized LDL cholesterol formation,³² which may degrade nitric oxide.³³ Superoxide anions also could interfere with receptor-mediated stimulation of nitric oxide or signal transduction in the release of nitric oxide.^{34 35} Among reactive oxygen-derived free radicals, hydroxyl radical (OH·) is one of the more toxic generated through the Haber-Weiss and Fenton reactions (Fe³⁺+O₂^-·Fe²⁺+O₂, and Fe²⁺+H₂O₂Fe³⁺+OH^-+OH·), both of which require a reduced metal ion, the most abundant of which is iron.³⁶ Conversely, the antioxidant probucol preserves endothelium-dilating function in cholesterol-fed rabbits,³⁷ dietary lowering of cholesterol normalizes superoxide anion production and improves endothelium-dependent relaxation in rabbits,³⁸ and combination of cholesterol-lowering and antioxidant therapy improves coronary dilation in patients with atherosclerosis.³⁹ In diabetes, many metabolic abnormalities may inactivate nitric oxide through oxygen free radical production because superoxide generation is increased.⁴⁰ Indeed, induction of cellular oxidant stress by advanced glycation end products that are present in vessel wall has been shown in diabetes mellitus.⁴¹ In animal models of diabetes, antioxidant therapy can restore endothelium-dependent relaxation.⁴² Similar results have been demonstrated with the antioxidant vitamin C in diabetic patients.⁴³

In our study, because smokers,⁴⁴ hypertensive patients,¹⁵ postmenopausal women without substitutive hormonal therapy,⁴⁵ and patients with hypercholesterolemia²⁰ were excluded, abnormal responses of coronary arteries can be linked to diabetes. However, despite normal levels of cholesterol in our patients, increased permeability of endothelium⁴⁶ might have increased intracellular native LDL, which alters endothelial L-arginine metabolism and enhances superoxide anion generation by nitric oxide synthase.⁴⁷ In addition, oxidized LDL cholesterol could be increased in diabetic patients despite normal cholesterol levels because of increased production of hydroxyl radicals and accounted for by impairment of coronary vasomotion. Deferoxamine is an iron chelator that binds Fe³⁺ ions and prevents iron ions from catalyzing redox reactions leading to generation of OH·.^{16 17 18} This compound has been demonstrated to be protective for endothelial cells⁴⁸ and has been used to prevent myocardial stunning.^{17 18 19} Our results show that this antioxidant improves coronary artery dilation in response to CPT and to flow increase in diabetic patients without any other coronary risk factor. Thus, OH· generation may play an important role in the pathogenesis of vasomotion abnormalities in these patients. In our study, we used small doses of deferoxamine (500 mg infused for 10 minutes) because (1) it has been shown that free iron ions available to stimulate radical reactions are small (rarely >5 µmol/L⁴⁹ ) and that radical reactions promoted by such low levels of iron can be inhibited by deferoxamine at concentrations equimolar to that of iron⁵⁰ and (2) intravenous injection of 10 mg/kg of body weight of deferoxamine in humans gave plasma concentrations of 80 to 130 µmol/L, which fell rapidly (half-time, 5 to 10 minutes).⁵¹ Thus, opposite to vitamin C,⁵² the beneficial effect of deferoxamine cannot be accounted for by the scavenger effect of OH· and O₂^-· because its oxidation is a slow reaction that requires high plasma concentrations (millimolar) of deferoxamine.³⁶

Limitations and Clinical Implications
Although the diabetic patients we studied had no angiographic signs of atherosclerosis elsewhere in the coronary vasculature, we cannot absolutely exclude angiographically undetectable atherosclerosis. Intravascular ultrasound studies have shown that despite angiographically normal-appearing vessels, early coronary atherosclerosis can be present.⁵³ Nevertheless, it is unlikely that early atherosclerosis can completely account for the coronary vasomotor abnormalities observed.

This study provides arguments for an alteration in the endothelium-derived nitric oxide system due to hydroxyl radicals in diabetic patients. However, it does not determine whether hydroxyl radicals (1) directly inactivate nitric oxide, (2) alter the capacity of endothelial cells to uptake L-arginine, (3) impede nitric oxide synthase to synthesize nitric oxide, or (4) decrease the response of smooth muscle cells to nitric oxide at the receptor site or the intracellular levels as has been suggested in insulin-dependent diabetes.⁵ Moreover, the abnormality may involve other endothelium-vasodilating factors, such as hyperpolarizing factor.²⁷

Toxicity and short plasma half-life of deferoxamine do not make this compound useful in the prevention of deleterious effects of diabetes on endothelial cells. Nevertheless, this study provides new information about the consequences of diabetes mellitus on endothelial function and confirms that compounds preventing oxygen free radical generation might be potentially useful for the long-term management of coronary atherosclerosis in diabetic patients.

Endothelial cells play a key role in coronary vasomotion and vascular permeability, and vascular hyperpermeability is an important pathogenic process in the development of vascular disease in diabetic patients.⁴⁶ Nitric oxide modulates microvascular permeability,⁵⁴ and oxygen-derived free radicals might contribute to the initiation and development of coronary atherosclerosis in diabetic patients by increasing the endothelial cell permeability.⁵⁵

Conclusions
The present study suggests that the oxidative environment is a determinant of the coronary endothelial dysfunction in non–insulin-dependent diabetic patients with angiographically normal coronary arteries. Impairment of nitric oxide system by superoxide anions might be the cornerstone accounting for both abnormal vasomotion and increased atherosclerosis prevalence in diabetic patients. Therefore, further studies in diabetic patients are required to establish whether antioxidant therapy has long-term beneficial effects and whether it can be used as a possible prophylactic treatment against vascular dysfunction and the development of coronary artery disease.

Received July 30, 1997; revision received October 15, 1997; accepted October 30, 1997.

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