Patients with sickle cell disease
General characteristics of the study population of patients with sickle cell disease and controls are shown in .
Plasma Amino Acid Levels
Plasma amino acid levels in sickle cell disease patients were compared to those in African-American control subjects without sickle cell disease (). An abnormal amino acid profile was observed in patients with sickle cell disease that is consistent with altered arginine metabolism. The observed dysregulation of the arginine-to-nitric oxide metabolism was greatest in sickle cell disease patients with pulmonary hypertension. Although plasma arginine concentrations were low in sickle cell disease compared to normal controls [41 (16) vs. 67 (18) μM, p<0.001], these levels were similar in patients with and without pulmonary hypertension. However, plasma ornithine levels were elevated in sickle cell patients with severe pulmonary hypertension (tricuspid regurgitant jet velocity ≥ 3.0), and the elevation was significant compared to sickle cell disease patients without pulmonary hypertension [81 (24) vs. 63 (21) μM, p < 0.001 by two-sided t-test], and is likely the result of elevated arginase activity. The arginine-to-ornithine ratio, an indirect measure of arginase activity and relative arginine bioavailability, was low in patients with sickle cell disease compared to controls [0.71 (0.39) vs. 1.20 (0.49), p<0.001; , ] and was particularly low in the group with severe pulmonary hypertension [0.49 (0.18)]. Ratios were clearly lower even in sickle cell patients without evidence of pulmonary hypertension than in controls [0.74 (0.41) vs. 1.20 (0.49), p <0.001 by two-sided t-test.). The association between arginine-to-ornithine ratio and level of pulmonary hypertension was significant by linear regression of the ratio on category of tricuspid regurgitant jet velocity (, ), but ratios were low only in patients with severe pulmonary hypertension when compared to patients with no evidence of pulmonary hypertension by two-sided t-test adjusted for unequal variances [0.49 (0.18) vs. 0.74 (0.41), p < 0.001].
Table 2 Distribution (mean ± SD) of amino acids linked to the L-arginine-nitric oxide pathway in sickle cell disease patients with tricuspid regurgitant jet velocity (TRV) < 2.5 m/s, 2.5 – 2.99 m/s, and ≥ 3.0 m/s, and in normal (more ...)
The arginine-to-ornithine ratio as a surrogate marker for arginase activity, its association with pulmonary hypertension (PHT) and correlation with arginase activity
Plasma proline concentrations were also significantly increased in patients compared to controls [210 (75) vs. 154 (59) μM, p<0.001; ]. Patients without pulmonary hypertension had higher proline levels than controls [202 (70) vs. 154 (59), p<0.001 by two-sided t-test], and even higher levels occurred in patients with severe and moderate pulmonary hypertension [245 (88) vs. 219 (80) vs. 202 (70) μM, tricuspid regurgitant jet velocity ≥ 3.0, 2.5-2.99, and < 2.5 m/s, respectively, p=0.01 by linear regression analysis; ]. This possibly indicates increased conversion of ornithine to proline in sickle cell disease that is amplified in patients with pulmonary hypertension () and may well account for the fact that plasma ornithine levels did not increase in most sickle cell patients, even as plasma arginase activity increased and plasma arginine levels declined. However, the possibility that reductions in proline catabolism also may contribute to the elevated proline concentrations cannot be ruled out. More precise explanations of the changes in amino acid levels in this patient population will require further studies using isotopic tracer methodology.
Although citrulline levels tended to increase slightly with level of pulmonary hypertension in patients, mean levels were almost identical in patients and controls (). Citrulline was significantly correlated with creatinine level (ρ=0.51, p<0.001), which is consistent with impaired renal function. Arginine-to-(ornithine plus citrulline) ratio, which takes into account the impact of renal dysfunction on global arginine bioavailability, showed very similar relationships among controls and patients to those for arginine-to-ornithine ratio (). In aggregate, these data indicate significant modulation of L-arginine metabolism in sickle cell disease that is associated with the development of pulmonary and renal vasculopathy.
Arginase Activity in Plasma
In order to understand the mechanism responsible for dysregulation of L-arginine metabolism, plasma arginase activity was measured in patients and controls. Plasma arginase activity was significantly elevated in patients with sickle cell disease [2.1 (2.1), n=140] compared to controls [0.4 (0.2), n=36, p<0.001 by t-test on log10 arginase]; (). In patients, arginase activity tended to increase with level of pulmonary hypertension [1.9 (1.8), 2.6 (2.8), and 2.8 (2.0) for tricuspid regurgitant jet velocity < 2.5 m/s, 2.5-2.99 m/s, and ≥ 3.0 m/s, respectively], although the association was not significant in linear regression of log10 arginase on the three categories of tricuspid regurgitant jet velocity, coded 0, 1, 2 (R2 = 0.017, p=0.13). However even in patients without pulmonary hypertension, sickle cell disease patients had significantly higher arginase activity compared to control subjects [0.4 (0.2) vs. 1.9 (1.8), p<0.001). Arginase activity was significantly correlated with arginine-to-ornithine ratio (ρ=−0.34, p<0.001; ); however, it is likely only one of several factors affecting this ratio and arginine bioavailability in patients with sickle cell disease.
Arginase Activity and Associations with Biochemical and Clinical Markers
The relationship between arginase activity and clinical laboratory markers of disease severity was evaluated in order to identify mechanisms for increased enzymatic activity and associated effects on organ function (). Plasma arginase activity was significantly associated with several markers of increased hemolytic rate, including cell-free plasma hemoglobin (ρ=0.56, p<0.001; ), lactate dehydrogenase (LDH, ρ=0.35, p<0.001), aspartate aminotransferase (AST, ρ=0.34, p<0.001), and hematocrit (ρ=−0.20 p=0.02). The lack of correlation between arginase and reticulocyte count in this cohort likely reflects the suppressive effects of transfusions, renal impairment and hydroxyurea therapy on reticulocytosis in the most severely affected patients. Other significant associations included oxygen saturation, white blood cell count, myeloperoxidase, alanine aminotransferase (ALT), endothelial and platelet specific soluble adhesion molecules (sE-selectin, sP-selectin, sVCAM-1 and sICAM-1), triglycerides and cholesterol (). No association of arginase activity with age (ρ=−0.09, p=0.31) or gender (p=0.63 by t-test on log10 arginase) was identified. There was no evidence of association between elevated arginase activity and markers of renal function ().
Association of arginase activity with hemolytic rate
In multiple regression analysis of log10 arginase activity, all variables associated with log10 arginase with p ≤ 0.15 in were considered in a stepwise model fitting process. In the final model, log10 arginase activity was related to log10 cell-free hemoglobin, log10 sP-selectin and log10 triglycerides (n = 110, R2=0.40, adjusted p<0.001 for all independent variables; ). The low variance inflation factors (≤ 1.11 for all three covariates in the model) suggest that there was no multicollinearity problem in this model and that each of the three covariates was independently related to log10 arginase. However, since the residuals were not well fit by a normal distribution, the analysis for the final model was repeated using ranks for all the variables. The results were similar; R2=0.43, adjusted p<0.001 for ranks of cell-free hemoglobin and sP-selectin and 0.001 for rank of triglycerides.
Table 3 B
Associations with Log10 Arginase Activity in Multiple Regression Analysis.
No adjustment has been made for multiple comparisons in these analyses. This seems appropriate, since the objective of this study is to detect potentially important associations involving arginase, not to control the overall type I error rate. Even if we made a conservative adjustment, however – for example, multiplying p-values in by the number of correlations shown – the major associations would still have p < 0.05. (Note that p-values reported here as < 0.001 are often actually < 0.0001.) Further, regression modeling enables us to estimate the number of variables, among those considered, that are independently associated with arginase activity and with mortality.
These data indicate that increased plasma arginase activity in sickle cell disease patients is associated with intravascular hemolysis, endothelial activation and inflammation.
Arginase Activity in Red Blood Cells
In order to further identify the source of increased plasma arginase activity, arginase activities were determined also for red blood cell lysates of normal controls and a subset of 16 patients with sickle cell disease in whom both frozen plasma and red blood cell lysates were available for comparison (). Specific activities of arginase in red blood cell-lysate of patients with sickle cell disease were significantly higher than those of controls [37.7 (2.9), n=16 vs. 23.5 (1.7) nmol/mg/min, n=45, p<0.0001 by t-test on log10 arginase]. For purposes of comparison, “normal range” boundaries were set arbitrarily at approximately the 80th percentile for arginase activities of both red blood cell-lysates and plasma of control patients. Two-thirds of all control values fall within these boundaries, while in striking contrast, 94% of all values for plasma and erythrocyte arginase activities of sickle patients fall outside these boundaries ().
Arginase activity in red blood cell lysate vs. plasma
Relationship of Dysregulated Arginine Metabolism to Mortality Rate
Information on deaths was collected during a follow-up period of up to 49 months. Since enrollment in the study, eighteen sickle cell patients had died as of March 2005, with median survival time of 14 months (range 2-41 months). Median follow-up was 33 months for the 210 patients who survived (range 7-49 months). Nine patients had not responded to attempts to contact them and were considered lost to follow-up. Confirmation of all deaths with death certificates and the absence of available death certificates in the United States for patients lost to follow-up suggest that we have not missed any deaths and all patients lost to follow-up were alive at the time of data analysis.
Fourteen of the 18 patients who died had tricuspid regurgitant jet velocity ≥ 2.5 m/s, and by proportional hazards regression analysis the presence of pulmonary hypertension by this definition was the most significant risk factor for death (risk ratio 7.4, [2.4,22.4], p<0.001, ). Plasma amino acid concentrations and plasma arginase activities were available for all 18 who died. Low ratios of plasma arginine-to-ornithine and arginine-to-(ornithine+citrulline) were associated with mortality in proportional hazards regression (, ). After adjustment for high tricuspid regurgitant jet velocity and log10 creatinine, the arginine-to-ornithine and arginine-to(ornithine+citrulline) ratios remained significantly related to mortality ; thus, either of these ratios may be an independent risk factor for death in sickle cell patients. Age was not significantly related to mortality after adjustment for the above variables. Estimated adjusted risk ratios (for 25th percentile relative to 75th percentile) were 2.2 (p=0.023) for arginine-to-ornithine and 2.9 (p=0.007) for arginine-to-(ornithine+citrulline) (). There was no evidence that risk ratios were different for patients with low (< 2.5 m/s) and high (≥ 2.5 m/s) tricuspid regurgitant jet velocity.
Proportional Hazards (Cox) Regression Analysis of Mortality
Association of arginine bioavailability ratios with mortality in sickle cell disease: Kaplan-Meier survival plots
The assumption of proportional hazards (i.e., constant risk ratio) for arginine-to-ornithine and arginine-to-(ornithine+citrulline) seemed reasonable, since scaled Shoenfeld residuals 50
for these variables did not show a strong trend with follow-up time.
Arginase activity was not directly associated with mortality, however in shifting L-arginine metabolism away from NO production and towards ornithine-dependent pathways, increased arginase activity may contribute to events that put patients at risk for early death.