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(Circulation. 1997;95:2068-2074.)
© 1997 American Heart Association, Inc.

Articles

Biochemical Evidence for Impaired Nitric Oxide Synthesis in Patients With Peripheral Arterial Occlusive Disease

Rainer H. Böger, MD; Stefanie M. Bode-Böger, MD; Wolfgang Thiele; Wolfgang Junker; Klaus Alexander, MD; Jürgen C. Frölich, MD

From the Institute of Clinical Pharmacology and Department of Angiology (K.A.), Medical School, Hannover, Germany.

	Abstract

Top Abstract Introduction Methods Results Discussion References

 
Background We studied urinary nitrate and cGMP excretion rates, indices of systemic NO formation, and plasma concentrations of L-arginine and the endogenous NO synthase inhibitor asymmetrical dimethylarginine (ADMA) and its inactive stereoisomer, symmetrical dimethylarginine, in 77 patients with peripheral arterial occlusive disease (PAOD) in Fontaine stages IIb through IV and in 47 young and 37 elderly healthy control subjects.

Methods and Results Urinary nitrate excretion was 182.0±11.4 µmol/mmol creatinine and cGMP excretion was 186.2±13.0 nmol/mmol creatinine in young healthy control subjects. In elderly control subjects, both excretion rates were slightly lower (nitrate, 156.0±7.8 µmol/mmol creatinine; cGMP, 150.0±8.3 nmol/mmol creatinine; P=NS). In PAOD patients, there was a significant, progressive reduction of urinary nitrate (IIb, 138.4±11.9; III, 128.6±11.3; and IV, 91.9±8.0 µmol/mmol creatinine; P<.05) and cGMP (IIb, 139.9±25.2; III, 115.6±13.1; and IV, 76.9±7.9 nmol/mmol creatinine; P<.05) excretion rates related to the Fontaine stage of PAOD. These changes were independent of changes in renal excretory function. Plasma L-arginine concentrations were not significantly different between the groups, but ADMA concentrations were elevated in PAOD patients (young control subjects, 1.25±0.11; elderly control subjects, 1.01±0.05 µmol/L; IIb, 2.62±0.24; III, 3.06±0.48; and IV, 3.49±0.26 µmol/L; P<.05 for PAOD versus control subjects). There was a significant linear correlation between urinary nitrate and cGMP excretion rates and a significant negative linear correlation between plasma ADMA concentrations and urinary nitrate excretion.

Conclusions In PAOD patients, there is a progressive reduction in urinary nitrate and cGMP excretion rates, which may be caused in part by accumulation of ADMA, an endogenous inhibitor of NO synthase.

Key Words: hypercholesterolemia • atherosclerosis • endothelium-derived factors • risk factors

	Introduction

Top Abstract Introduction Methods Results Discussion References

 
Nitric oxide formed in the endothelium from the amino acid precursor L-arginine by the activity of the constitutive endothelial NOS isoenzyme^{1 2} has been shown to play an important role in the regulation of local vasomotor tone.¹ In hypercholesterolemia and atherosclerosis, the biological activity of NO is impaired.^{3 4} Decreased availability of biologically active NO in the vascular wall may be one of the earliest detectable findings in atherogenesis, leading to increased leukocyte adhesion^{5 6} and platelet aggregation.⁷ Exogenous administration of L-arginine has been shown to restore, at least in part, the physiological activity of NO in experimental hypercholesterolemia.^{4 8}

However, the mechanism leading to impaired NO activity in the atherosclerotic vascular wall has not yet been fully elucidated. One explanation for decreased NO formation, which can be restored by exogenous L-arginine, may be the accumulation of endogenous NOS inhibitors. ADMA has been characterized as an endogenously occurring NOS inhibitor.⁹ In hypercholesterolemic rabbits, plasma concentrations of endogenous DMAs have been reported to be elevated.¹⁰ Using a selective and specific HPLC method, we recently showed that ADMA plasma concentrations are elevated in hypercholesterolemic rabbits compared with healthy controls and that dietary supplementation with L-arginine restores NO formation by increasing the plasma L-arginine/ADMA ratio.¹¹

Nothing is known about DMA plasma concentrations in human hypercholesterolemia and atherosclerosis; however, if DMA concentrations are also elevated in this disease in humans, this might explain, at least in part, the decreased NO activity in the cardiovascular system in atherosclerotic patients. Systemic NO formation rates in vivo can be noninvasively assessed by use of the urinary excretion rates of NO₃^–_, the final metabolite of NO,¹² and of cGMP, its second messenger,¹ as index metabolites.^{13 14}

In the present study, we investigated whether urinary NO₃^– and cGMP excretion rates are altered in patients with PAOD of different severities compared with healthy humans and whether changed plasma concentrations of L-arginine and endogenous DMAs may contribute to these effects.

	Methods

Top Abstract Introduction Methods Results Discussion References

 
Subjects and Study Design
From January 1994 to November 1995, a total of 163 subjects were studied at the Departments of Clinical Pharmacology and Angiology of Hannover Medical School. Informed consent was obtained from all subjects in accordance with the Institutional Review Board. The study population consisted of three groups: Group 1 comprised 77 consecutively hospitalized patients with PAOD (Fontaine stages II through IV) of various causes (54 men, 23 women; mean age, 67.8±1.2 years; range, 49 to 88 years). In all patients, the presence of PAOD had been confirmed by angiography. The clinical characteristics and risk profiles of the patients are given in Table 1. Any vasoactive medication was stopped in this group at least 1 day before sample collection. Group 2 consisted of 37 consecutive nonhospitalized, elderly control subjects (24 men, 13 women; mean age, 68.0±1.2 years; range, 56 to 85 years) without any signs of clinically relevant atherosclerotic vascular disease or inflammatory diseases (exclusion criteria for this group were presence of peripheral vascular disease, carotid stenosis, or angina pectoris; history of smoking, hypercholesterolemia, hypertension, or previous stroke or transient ischemic attack; or any signs of inflammatory diseases as assessed by medical history, erythrocyte sedimentation rate, and C-reactive protein determinations). Group 3 was made up of 47 young healthy subjects (34 men, 13 women; mean age, 25.4±0.3 years; range, 22 to 31 years) who did not suffer from any concomitant disease and used no medication.

View this table: [in this window] [in a new window]   Table 1. Clinical Characteristics, Cardiovascular Risk Factors, and Parallel Diseases of PAOD Patients

In all subjects selected for the study, venous blood was collected in the morning after an overnight fast. The blood was separated within 1 hour after collection, and the plasma was kept frozen at –20°C until analysis. Twenty-four-hour urine samples were collected from all subjects at the same time. Isopropanol was added to the urine sampling containers in an amount to reach a final concentration of 10% to 15% (vol/vol) in urine to inhibit bacterial activity. We have previously demonstrated that this amount of isopropanol does not interfere with the NO₃^– and cGMP assays.^{13 14} In the patients, urine and plasma samples were obtained on day 2 after hospitalization, just before any active treatment was started, to exclude any influences from ambulatory medication or diet or from any interventional therapy.

Quantification of Urinary NO₃^– and cGMP
Urinary NO₂^–/NO₃^– was determined as its pentafluorobenzyl derivative by GC-MS as described previously.^{15 16} Briefly, aliquots of urine were spiked with [¹⁵N]NO₃^– (MSD Isotopes Merck Frosst) as internal standard, acidified, and treated with cadmium to reduce NO₃^– to NO₂^–. The suspension was then allowed to react with pentafluorobenzyl bromide, extracted with toluene, and dried over Na₂SO₄. Aliquots thereof (1 µL) were injected into the GC-MS. GC-MS was carried out on a triple-stage quadrupole mass spectrometer TSQ 45 interfaced with a gas chromatograph 9611 (Finnigan MAT). An OV-1 fused silica capillary column (25 mx0.25 mm ID, 0.25-µm film thickness) from Machery-Nagel was used with helium as the carrier gas (55 kPa). Negative ions were produced by chemical ionization with methane as the reactant gas (65 Pa) at an electron energy of 90 eV and an electron current of 0.2 mA. Quantification was performed by selected ion monitoring at m/z of 46 for endogenous NO₂^–/NO₃^– and m/z of 47 for the internal standard. The detection limit of the method was 20 fmol nitrite or nitrate. Intra-assay variability was below 3.8%.

For the determination of cGMP, urine samples were diluted 1:500 in PBS and acetylated with a mixture of acetic acid anhydride/triethylamine. cGMP content was measured by radioimmunoassay using ¹²⁵I-labeled cGMP as a tracer and globulin precipitation. The detection limit of the assay was 160 fmol/mL.

Urinary and plasma creatinine were determined spectrophotometrically with the alkaline picric acid method in an automatic analyzer (Beckman). The urinary excretion rates of NO₃^– and cGMP were corrected by urinary creatinine concentration to limit the variability due to differences in renal excretory function as described previously.^{14 17}

Determination of Plasma L-Arginine and DMA Concentrations
Plasma L-arginine and DMA concentrations were determined by HPLC using precolumn derivatization with OPA as described previously.¹¹ Plasma samples and standards were extracted on CBA solid-phase extraction cartridges (Varian). The eluents were dried over nitrogen and dissolved in bidistilled water for HPLC analysis. HPLC was carried out on a Gynkotek liquid chromatography system consisting of two HPLC pumps with a gradient controller (model M 480 HDG), a spectral fluorescence detector RF 1002, and an automatic injector (model GINA 160). Samples and standards were incubated for exactly 30 seconds with the OPA reagent (5.4 mg/mL OPA in borate buffer, pH 8.5, containing 0.4% 2-mercaptoethanol) before automatic injection into the HPLC. The OPA derivatives of L-arginine, ADMA and SDMA, were separated on a C₆H₅ column (Macherey and Nagel) with the fluorescence monitor set at ^ex=340 nm and ^em=455 nm. Samples were eluted from the column with 0.96% citric acid/methanol 2:1, pH 6.8, at a flow rate of 1 mL/min. The coefficients of variation of the method had previously been determined to be 5.2% within-assay and 5.5% between-assay; the detection limit of the assay was 0.1 µmol/L.

Calculations and Statistics
All values are given as mean±SEM. Statistical significance was tested with ANOVA followed by Fisher's protected least significant difference test for comparisons between treatment groups. Linear regression curves and correlation coefficients were obtained by the least-squares method. Statistical significance was accepted at the .05 level of probability.

	Results

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Urinary NO₃^– and cGMP Excretion Rates
Urinary NO₃^– excretion was 182.0±11.4 µmol/mmol creatinine (1861.2±113.1 µmol/24 h) in young healthy volunteers. In elderly control subjects, urinary NO₃^– excretion was 156.0±7.8 µmol/mmol creatinine (1717.2±46.0 µmol/24 h; P=NS versus young healthy subjects). In PAOD patients, urinary NO₃^– excretion was significantly lower (121.4±8.3 µmol/mmol creatinine, 1159.1±84.8 µmol/24 h; P<.05) (Fig 1A).

View larger version (21K): [in this window] [in a new window]   Figure 1. Urinary excretion rates of nitrate (A) and cGMP (B) in young healthy human volunteers, elderly control subjects, and patients with PAOD. Excretion rates of these index metabolites of endogenous NO formation were corrected for urinary creatinine concentration to exclude any variability due to differences in renal excretory function. Each point represents one subject; bars indicate mean±SEM. *P<.05.

Urinary cGMP excretion was 186.2±13.0 nmol/mmol creatinine in young healthy volunteers and 150.0±8.3 nmol/mmol creatinine in elderly control subjects (2399.1±155.9 and 2019.8±70.9 nmol/24 h, respectively; P=NS). In PAOD patients, urinary cGMP excretion was also significantly lower than in young or elderly control subjects (114.3±12.8 nmol/mmol creatinine, 1392.6±140.7 nmol/24 h; P<.05) (Fig 1B).

When PAOD patients were stratified according to the Fontaine classification of PAOD, a stage-dependent decrease in mean urinary NO₃^– and cGMP excretion rates was observed (Fig 2): Patients with intermittent claudication (Fontaine stage II) had only a slight reduction in both urinary NO₃^– and cGMP excretion rates, which was not significantly different from elderly control subjects, whereas in patients with resting pain (Fontaine stage III) or with peripheral necrosis (Fontaine stage IV), the excretion rates of these index metabolites decreased further and significantly. The mean percent decrease of cGMP excretion depending on the Fontaine stage of PAOD was larger than the relative decrease of NO₃^– excretion (Fontaine III: NO₃^–, –7.1%; cGMP, –17.4%; Fontaine IV: NO₃^–, –33.6%; cGMP, –45.1% versus Fontaine IIb patients). As a consequence, the mean individual nitrate-to-cGMP ratio was significantly elevated in PAOD patients compared with control subjects (young control subjects, 1.07±0.11; elderly control subjects, 1.09±0.05; Fontaine IIb, 1.71±0.33; III, 1.82±0.31; and IV, 1.88±0.56). There was a significant linear correlation between urinary nitrate and cGMP excretion rates (r=.356, P<.0001).

View larger version (21K): [in this window] [in a new window]   Figure 2. Urinary excretion rates of nitrate (A) and cGMP (B) in PAOD patients classified according to Fontaine grading of arterial disease compared with elderly control subjects. Horizontal line represents mean value in young healthy volunteers; dashed lines represent upper and lower limits of 95% CI in young healthy subjects. Each point represents one subject; bars indicate mean±SEM. *P<.05 vs young healthy control subjects. P<.05 vs elderly control subjects.

Interestingly, in all study groups, urinary NO₃^– and cGMP excretion rates were higher in female than in male subjects. Although the differences between the two sexes did not reach statistical significance in any single group of subjects, there was a significant overall trend toward higher values in women than in men (P<.05 in multiple regression analysis).

Creatinine clearance was 135.4±3.7 mL/min in young control subjects. It was significantly lower in elderly control subjects, but there was no significant difference between PAOD patients and elderly control subjects (Table 2). Plasma cholesterol levels are given in Table 2. None of the young or elderly control subjects had hypercholesterolemia, but hypercholesterolemia was a common risk factor in PAOD patients (see Table 1).

View this table: [in this window] [in a new window]   Table 2. Plasma Total Cholesterol Concentrations and Creatinine Clearances in Young Healthy Volunteers, Elderly Control Subjects, and PAOD Patients

Plasma L-Arginine and DMA Concentrations
Plasma L-arginine concentration was 83.2±4.9 µmol/L in young healthy subjects. It was not significantly different in elderly control subjects (75.5±3.9 µmol/L) or in PAOD patients (80.1±3.1 µmol/L; Table 3). Plasma ADMA and SDMA concentrations were 1.25±0.11 and 0.71±0.09 µmol/L in young healthy volunteers, respectively. DMA plasma levels were not significantly different in elderly control subjects (ADMA, 1.01±0.05 µmol/L; SDMA, 0.83±0.05 µmol/L) but were significantly elevated in PAOD patients (ADMA, 2.80±0.22 µmol/L; SDMA, 2.30±0.21 µmol/L; P<.05 versus control subjects). In PAOD patients, there was a significant, progressive increase in plasma ADMA concentrations related to the Fontaine stage of the disease (Table 3). Elevated ADMA plasma concentrations resulted in significantly decreased L-arginine/ADMA ratios in PAOD patients compared with young or elderly control subjects (Fig 3).

View this table: [in this window] [in a new window]   Table 3. Plasma L-Arginine, ADMA, and SDMA Concentrations in Young Healthy Volunteers, Elderly Control Subjects, and PAOD Patients Classified According to Fontaine Staging

View larger version (20K): [in this window] [in a new window]   Figure 3. Plasma L-arginine/ADMA ratios in young healthy volunteers, elderly control subjects, and PAOD patients according to Fontaine classification of disease. Data are given as mean±SEM. *P<.05.

In multiple regression analysis, ADMA plasma concentrations were dependent on plasma total cholesterol levels (r=.336; P<.001) and on creatinine clearance (r=.322; P<.01). However, when subgroups of elderly control subjects and PAOD patients with normal or impaired renal function were compared, there was only a slight, insignificant further increase in mean plasma ADMA that could be ascribed to impaired renal excretion (Fig 4). PAOD patients with normal renal function (creatinine clearance, 113.2±3.6 mL/min) had a mean ADMA plasma concentration of 2.63±0.28 µmol/L, whereas in PAOD patients with impaired renal function (creatinine clearance, 60.3±3.3 mL/min), mean plasma ADMA was 3.15±0.21 µmol/L (P=NS). Similarly, in elderly control subjects with normal renal function (creatinine clearance, 120.3±3.4 mL/min), ADMA concentration was 0.96±0.07 µmol/L, and in control subjects with impaired renal function (creatinine clearance, 77.6±2.1 mL/min), ADMA levels were 1.02±0.06 µmol/L (P=NS). There was a significant negative linear correlation of ADMA plasma concentrations with urinary nitrate excretion rate (r=.355; P<.0001) and a weak though significant negative linear correlation with urinary cGMP excretion rate (r=.200; P<.02).

View larger version (13K): [in this window] [in a new window]   Figure 4. Plasma ADMA concentrations in elderly control subjects (CS) and PAOD patients with normal (NRF) or moderately impaired (IRF) renal function. Data are mean±SEM. *P<.05 vs CS.

	Discussion

Top Abstract Introduction Methods Results Discussion References

 
Our present study suggests that systemic NO formation, as assessed by measurement of urinary NO₃^– excretion rates, is decreased in patients with PAOD. This decrease in urinary NO₃^– excretion is dependent on the severity of PAOD (as measured by the Fontaine classification); it is paralleled by decreased urinary cGMP excretion, an index of NO activity. Plasma concentrations of DMAs, endogenous NOS inhibitors, are concomitantly increased in PAOD.

It is well known that the biological activity of endothelium-derived NO is impaired in patients with atherosclerotic vascular diseases. This has usually been assessed by measurement of endothelium-dependent vascular dilatations in the coronary^{18 19} or forearm vascular beds.^{20 21} However, the cause leading to this defect has remained unclear (for review, see Reference 22²² ). Discussion has focused primarily on the question of whether there is a decreased NOS activity, which may be due to decreased endothelial NOS III gene expression or impaired NOS substrate availability^{10 23} or increased oxidative degradation of NO, eg, by superoxide anions^{24 25} in atherosclerotic blood vessels.²²

Our present data suggest that systemic NO production is decreased in patients with PAOD, because the urinary excretion rates of both NO₃^– and cGMP are significantly and stage-dependently decreased compared with healthy control subjects. These results are in line with our previous finding that urinary NO₃^– excretion progressively decreased during the induction of atherosclerosis in cholesterol-fed rabbits.⁴ However, some in vitro studies in isolated aortic rings from cholesterol-fed rabbits suggest that the production of nitrogen oxides is increased.²⁶

Our approach of noninvasively measuring NO₃^– and cGMP excretion rates allows us to differentiate between decreased NO production and enhanced NO inactivation, because urinary NO₃^– indicates the endogenous formation rate of NO irrespective of whether this NO was biologically active or whether it had been inactivated early (NO oxidatively inactivated by O₂^– is also converted into the final metabolite, NO₃^–27 ). In contrast, urinary cGMP excretion indicates the biological activity of NO, because only the portion of NO that exerts its biological effects on the soluble guanylyl cyclase will increase cGMP levels. Therefore, if oxidative degradation of NO were the main cause of decreased NO activity in PAOD patients, one should have expected a decreased urinary cGMP excretion in the presence of normal NO₃^– excretion.

In this respect, it is important to note that the relative decrease in urinary cGMP excretion with progression of PAOD was greater than for NO₃^–. Urinary NO₃^– excretion in PAOD Fontaine stage III was 92.9% of the value in Fontaine stage IIb and 66.4% in Fontaine stage IV. The respective data for urinary cGMP excretion were 82.6% in Fontaine stage III and 54.9% in Fontaine stage IV. This may be explained by an increasing contribution of oxidative inactivation of NO to its impaired activity in the more advanced clinical stages of PAOD. This is also suggested by the higher mean individual nitrate-to-cGMP ratio in PAOD patients compared with either of the control groups. The atherosclerotic vascular wall may release huge amounts of O₂^–, which have a strong impact on the accelerated inactivation of NO.²⁵ Moreover, leukocytes are capable of synthesizing NO by the iNOS isoenzyme.² There is evidence that iNOS is induced in atherosclerotic blood vessels.^{28 29} Enhanced iNOS-derived NO formation may thus be another explanation for relatively higher urinary nitrate excretion rates compared with cGMP excretion rates.

Decreased systemic NO formation in atherosclerosis may be due to decreased NOS gene expression or decreased NOS enzyme activity. Decreased enzyme activity may be related to decreased intracellular L-arginine availability in the vicinity of the NOS. We have recently shown that plasma concentrations of ADMA, an endogenous NOS inhibitor,⁹ are increased in hypercholesterolemic rabbits.¹¹ The ADMA plasma concentrations we found in our group of healthy young volunteers were very close to those previously reported by Vallance et al³⁰ for healthy humans (1.15±0.19 µmol/L). Our present study is the first to report elevated plasma concentrations of ADMA and SDMA in humans with atherosclerotic vascular disease. Elevated ADMA levels in atherosclerotic patients resulted in a disease stage–dependent deterioration of the plasma L-arginine/ADMA ratio. Vallance et al³⁰ reported elevated DMA plasma concentrations in patients with chronic renal failure and suggested that DMA levels increase in these patients because of diminished renal excretion. This finding was in line with biochemical evidence that DMAs are eliminated via the kidneys in healthy animals.³¹ However, ADMA plasma concentrations are elevated in hypercholesterolemic rabbits despite normal renal excretory function.^{10 11} In our present study, we found that the elevation of ADMA plasma concentrations in PAOD patients was also present in those patients with normal renal function, whereas elderly control subjects with moderately impaired renal function had no significant increase in ADMA plasma concentrations. The discrepancy with the findings of Vallance and coworkers³⁰ may relate to the fact that they studied patients with end-stage renal failure undergoing hemodialysis, in whom there may have been virtually no renal elimination of DMAs at all, whereas in our patients with moderately reduced creatinine clearances, residual DMA excretion may have been fairly high. Therefore, enhanced endogenous ADMA synthesis probably contributes at least in part to its elevated plasma concentrations in atherosclerosis. The ADMA plasma concentrations we found in PAOD patients were in the range of concentrations that have previously been shown to inhibit NO production in cultured macrophages,³¹ in rat mesentery tissue,³² and in rat brain.³³

The origin of the methyl groups of DMAs is currently unknown. Physiological DMA plasma concentrations in healthy animals stem from degradation of the corresponding methylated proteins.³⁴ It is not clear whether there is enhanced degradation of tissue proteins in PAOD, possibly caused by ischemia of peripheral skeletal muscles. This, however, would not explain why DMA concentrations are also elevated in hypercholesterolemic rabbits. DMA is metabolized by dimethylarginase to citrulline in cultured endothelial cells.³⁵ An impairment of this metabolizing pathway would also increase DMA concentrations. Another possible source of elevated DMA concentrations may be increased methylation of L-arginine, which has also been observed in cultured endothelial cells.³⁵ Utilization of L-arginine by a methylating metabolic pathway might also explain its decreased availability as a substrate for the NOS. Although the precise concentrations of DMAs within cells are not known, it has been reported that methylarginines are concentrated within cells.³⁵ Therefore, the relatively small increase in plasma ADMA concentration in patients with PAOD in our present study may mirror an even higher increase of this compound in the vicinity of the NOS.

Several studies have demonstrated in recent years that L-arginine induces NO-dependent vasodilatation in humans.^{17 36} However, because basal L-arginine plasma levels by far exceed the half-maximal substrate concentration for the endothelial NOS, whose K_m has been determined in vitro to be 2.9 µmol/L,³⁷ and the estimated intracellular concentration of L-arginine in early-passaged endothelial cells is likely to be in the low millimolar range,³⁸ the mechanism by which L-arginine might exert its hemodynamic effects in atherosclerotic patients has remained unclear. Impaired NOS activity due to accumulation of endogenous inhibitors like ADMA might explain in part the mechanism of action of L-arginine: It would act by competing with ADMA for the NOS and, by displacing it from the enzyme, restore NOS activity and normalize NO production.

In conclusion, our present study shows that systemic NO synthesis rates gradually decrease in patients with PAOD while the clinical symptoms increase. Decreased NO formation may be at least partly due to increased plasma concentrations of ADMA, an endogenous NOS inhibitor. These results may explain why L-arginine induces vasodilatation in atherosclerosis and thus solve the "L-arginine paradox" in patients with generalized atherosclerosis.

	Selected Abbreviations and Acronyms

 

ADMA = asymmetrical dimethylarginine DMA = dimethylarginine GC-MS = gas chromatography–mass spectrometry HPLC = high-performance liquid chromatography iNOS = inducible nitric oxide synthase NOS = nitric oxide synthase OPA = o-phthalaldehyde PAOD = peripheral arterial occlusive disease SDMA = symmetrical dimethylarginine

	Acknowledgments

 
This study was supported in part by grants from the Else-Kröner-Fresenius foundation and the Deutsche Forschungsgemeinschaft. The authors wish to thank A. Otten, F.-M. Gutzki, and K. Schnalle for their excellent technical assistance.

	Footnotes

 
Reprint requests to Rainer H. Böger, MD, Institute of Clinical Pharmacology, Hannover Medical School, 30623 Hannover, Germany.

Received September 30, 1996; revision received November 18, 1996; accepted November 25, 1996.

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