The evaluation of arginase activity during acute vasoocclusive sickle cell crisis

Abstract

Study objectives: Nitric oxide is an endothelium-derived vasodilator that plays a significant role in the pathophysiology of sickle cell acute vaso-occlusive crisis (VOC) and may play an important role in new therapeutic strategies. Nitric oxide is formed from the conversion of the substrate l-arginine to citrulline by the enzyme endothelial nitric oxide synthase (eNOS). Our previous studies have demonstrated low nitric oxide and, more recently, low l-arginine levels during adult VOC. Although it seems intuitive that low substrate levels would explain low product, the low l-arginine levels in our study were still 10-fold higher than the k_m (3 μM) of eNOS and were more than sufficient to saturate eNOS, yet nitric oxide production was diminished, thereby suggesting a derangement in the normal l-arg-nitric oxide pathway. L-arginine supplementation, in other conditions, even in the face of this low but enzyme-saturating concentration, has been shown to enhance nitric oxide production. This phenomenon has been called the "arginine paradox." One explanation for this paradox involves the enzyme arginase. Arginase, a ubiquitous intra- and extracellular enzyme, catalyzes l-arginine to ornithine. Arginase activity has been found to be enhanced in certain pathologic states and may thus compete for available l-arginine, lessening l-arginine available to NOS. A pediatric study previously demonstrated enhanced arginase activity, as demonstrated by increased ornithine levels during VOC, which has not been examined in adult VOC. We hypothesize that arginase activity is enhanced during adult VOC and may thus play an important role in the l-arginine-nitric oxide pathway during adult VOC. The specific aim of this study is to measure arginase activity by l-arginine and ornithine measurement in VOC. Methods: Included were patients older than 18 years and with a chief complaint of typical VOC pain (defined as patient-described pain consistent in duration, severity, quality, and distribution with previous episodes of VOC and no evidence of coexistent illness), sickle cell patients not in VOC, and healthy controls. Subjects with sickle cell SS, SC, and β-thalassemia disease as documented by previous hemoglobin electrophoresis were eligible. Five milliliters of blood was obtained by standard venipuncture for l-arginine and ornithine analysis before standard analgesic therapy. Blood was immediately centrifuged at 10,000 RPM at 4°C for 10 minutes. The serum was separated and stored at –20°C. L-arginine and ornithine levels were measured by Molecular Structure Facility (University of California, Davis, CA) for an α equal to .05 and power of 0.9, and based on an l-arginine level expected SD of 3.0 μM/L. Results: The study included 42 sickle cell patients in typical VOC, 14 sickle cell controls not in crisis, and 8 healthy controls. As in our previous study, l-arginine was significantly low in VOC (median 23.160 μM/L, <i>P</i><.05) versus healthy sickle cell controls (median 38.560 μM/L) and healthy controls (median 73.010 μM/L). There was no significant difference between citrulline measurements in any of the groups. When ornithine was measured, a statistically significant difference (<i>P</i><.5) was found between sickle cell patients in VOC (median 22.610) and sickle cell healthy controls (36.780). No statistical difference was found between either sickle cell group and healthy controls (median 26.910). Conclusion: We found low ornithine levels in healthy sickle cell controls and VOC subjects. This finding does not support increased arginase activity in sickle cell disease. Interestingly, ornithine levels are significantly lower in VOC than in the healthy sickle cell controls, which may simply be explained by the low l-arginine levels already found in VOC. The significance of this, however, is unknown.