Randall K. Harada

l-Arginine Supplementation in Peripheral Arterial Disease No Benefit and Possible Harm

Background— l-Arginine is the precursor of endothelium-derived nitric oxide, an endogenous vasodilator. l-Arginine supplementation improves vascular reactivity and functional capacity in peripheral arterial disease (PAD) in small, short-term studies. We aimed to determine the effects of long-term administration of l-arginine on vascular reactivity and functional capacity in patients with PAD. Methods and Results— The Nitric Oxide in Peripheral Arterial Insufficiency (NO-PAIN) study was a randomized clinical trial of oral l-arginine (3 g/d) versus placebo for 6 months in 133 subjects with intermittent claudication due to PAD in a single-center setting. The primary end point was the change at 6 months in the absolute claudication distance as assessed by the Skinner-Gardner treadmill protocol. l-Arginine supplementation significantly increased plasma l-arginine levels. However, measures of nitric oxide availability (including flow-mediated vasodilation, vascular compliance, plasma and urinary nitrogen oxides, and plasma citrulline formation) were reduced or not improved compared with placebo. Although absolute claudication distance improved in both l-arginine– and placebo-treated patients, the improvement in the l-arginine–treated group was significantly less than that in the placebo group (28.3% versus 11.5%; P=0.024). Conclusions— In patients with PAD, long-term administration of l-arginine does not increase nitric oxide synthesis or improve vascular reactivity. Furthermore, the expected placebo effect observed in studies of functional capacity was attenuated in the l-arginine–treated group. As opposed to its short-term administration, long-term administration of l-arginine is not useful in patients with intermittent claudication and PAD.

β2-Microglobulin as a Biomarker in Peripheral Arterial Disease Proteomic Profiling and Clinical Studies

Background— Peripheral arterial disease (PAD) is common but commonly unrecognized. Improved recognition of PAD is needed. We used high-throughput proteomic profiling to find PAD-associated biomarkers. Methods and Results— Plasma was collected from PAD patients (ankle brachial index of <0.90; n=45) and subjects with risk factors but without PAD (n=43). Plasma was analyzed with surface-enhanced laser desorption/ionization time-of-flight mass spectrometry to quantify 1619 protein peaks. The peak intensity of a 12-kDa protein was higher in PAD patients. Western blot analyses and immunoaffinity studies confirmed that this protein was &bgr;2-microglobulin (B2M). In a validation study, B2M was measured by ELISA in plasma in age- and gender-matched PAD (n=20) and non-PAD (n=20) subjects. Finally, we studied a larger cohort of subjects (n=237) referred for coronary angiography but without known PAD. Plasma B2M levels were higher in PAD patients than in non-PAD patients with coronary artery disease. Plasma B2M correlated with ankle brachial index and functional capacity. Independent predictors of PAD were diabetes mellitus, age, and the combination of B2M and C-reactive protein level. Conclusions— In PAD patients, circulating B2M is elevated and correlates with the severity of disease independent of other risk factors. These findings might provide a needed biomarker for PAD and new insight into its pathophysiology. Further studies in other populations are needed to confirm the utility of measuring B2M in cardiovascular disease risk assessment.

Limb hemodynamics are not predictive of functional capacity in patients with PAD

To the practicing clinician, it seems obvious that limb hemodynamics would be the primary determinant of walking distance. However, other determinants, such as skeletal muscle metabolism, may play a role. Accordingly, in the current study, we examined the relationship between measures of limb hemodynamics and walking capacity in patients with peripheral arterial disease (PAD). We measured toe and ankle pressures for calculation of toe-(TBI) and ankle (ABI)-brachial indices; basal and hyperemic calf blood flow (CBF; by plethysmography); and initial (ICT) and absolute (ACT) claudication time using the Skinner-Gardner protocol. As expected, PAD patients had impaired limb hemodynamics with reduced TBI, ABI and a reduction in ABI post-exercise. However, there was no relationship between any of the hemodynamic variables (including ABI, ABI reduction post-exercise, TBI, baseline or maximal CBF) and walking distance as assessed by ICT or ACT. A subset of PAD patients with an ACT >750 s (n =16; ‘long claudicators’) were compared with a subset of PAD patients with an ACT <260 s (n = 16; ‘short claudicators’). The average ACT in the long claudicants was over fivefold greater than the short claudicators. Surprisingly, there were no differences between the two groups in any of the hemo-dynamic variables. There was also no relationship between the initial ABI, TBI, toe pressure, baseline or hyperemic CBF, and the improvement in ACT over the 3-month course of the study. This study found little relationship between hemodynamic variables and functional capacity in PAD. Accordingly, to assess the response to therapeutic interventions, exercise performance and functional status need to be directly measured, and cannot be predicted from hemodynamic measurements.

Asymmetric dimethylarginine Correlates with Measures of Disease Severity, Major Adverse Cardiovascular Events and All-Cause Mortality in Patients with Peripheral Arterial Disease

Peripheral arterial disease (PAD) is associated with major cardiovascular morbidity and mortality. Abnormalities in nitric oxide metabolism due to excess of the NO synthase inhibitor asymmetric dimethylarginine (ADMA) may be pathogenic in PAD. We explored the association between ADMA levels and markers of atherosclerosis, function, and prognosis. A total of 133 patients with symptomatic PAD were enrolled. Ankle—brachial index (ABI), walking time, vascular function measures (arterial compliance and flow-mediated vasodilatation) and plasma ADMA level were assessed for each patient at baseline. ADMA correlated inversely with ABI (r = —0.238, p = 0.003) and walking time (r = —0.255, p = 0.001), independent of other vascular risk factors. We followed up 125 (94%) of our 133 initial subjects with baseline measurements (mean 35 months). Subjects with ADMA levels in the highest quartile (> 0.84 μmol/l) showed a significantly greater occurrence of a major adverse cardiovascular event (MACE) compared to those with ADMA levels in the lower three quartiles (p = 0.001). Cox proportional-hazards regression analysis revealed that ADMA was a significant predictor of MACE, independent of other risk factors including age, sex, blood pressure, smoking history, diabetes and ABI (hazard ratio = 5.1, p < 0.001). Measures of vascular function, such as compliance, flow-mediated vasodilatation (FMVD) and blood pressure, as well as markers of PAD severity, including ABI and walking time, were not predictive. In conclusion, circulating levels of ADMA correlate independently with measures of disease severity and major adverse cardiovascular events. Agents that target this pathway may be useful for this patient population. Clinical Trial Registration — URL: http:// www.clinicaltrials.gov. Unique identifier: NCT00284076

Response to Letter Regarding Article, “L-Arginine Supplementation in Peripheral Arterial Disease: No Benefit and Possible Harm”

We thank Dr Teerlink for his comments regarding the unexpected results of our study.1 Pre-clinical studies and short-term trials in patients with coronary or peripheral arterial disease showed that supplemental L-arginine improved endothelium-dependent vasodilation and increased nitric oxide (NO) production. Accordingly, we hypothesized that long-term administration of L-arginine would improve vascular function and enhance collateral blood flow, thereby increasing walking distance in patients with peripheral arterial disease. To our surprise, long-term L-arginine supplementation tended to impair vascular function and to limit the …

Response to Letter Regarding Article, “β2-Microglobulin as a Biomarker in Peripheral Arterial Disease: Proteomic Profiling and Clinical Studies”

Schroecksnadel and collaborators seem a bit skeptical about our recent publication on β2 microglobulin (β2M)1 and about the laudatory editorial2 that accompanied it. Our article described the discovery that blood levels of β2M correlate with the severity of peripheral arterial disease (PAD) as assessed by the ankle-brachial index or treadmill testing. This novel finding arose from an agnostic high-throughput proteomic profiling effort using surface-enhanced laser desorption and ionization time-of-flight mass spectroscopy. This is a candidate-generating approach, in contrast to the more common candidate-based approach to proteomic profiling. The major advantage of the candidate-generating approach is that it provides for the discovery of new biomarkers for disease and potentially new insights into pathobiology. We hypothesized that repeated bouts of ischemia–reperfusion in the lower …