Horizontal lines indicate mean values for each group. A, Arginine-ornithine
ratio in controls vs patients with sickle cell disease (SCD). B, Plasma arginase
activity in controls vs patients with SCD. C, Correlation of plasma arginase
activity to arginine-ornithine ratio.
Correlation of plasma arginase activity to cell free hemoglobin (n = 138)
in patients with sickle cell disease.
A, Red blood cell (RBC) lysate arginase activity in controls compared
with patients with sickle cell disease (SCD). Horizontal lines indicate mean
values for each group. B, Correlation of plasma arginase to RBC lysate arginase
activity in controls and patients with SCD (r = 0.43; P<.001). For purposes of comparison, horizontal and
vertical dotted lines are set at approximately the 80th percentile for arginase
activities of RBC lysates and plasma, respectively, for controls.
A, Mortality for 3 categories of arginine-ornithine ratio: highest quartile
(>0.8690), 25th to 75th percentiles (>0.4385 and ≤0.8690), and lowest quartile
(≤0.4385). B, Mortality for 3 categories of ratio of arginine to ornithine
plus citrulline: highest quartile (>0.6254), 25th to 75th percentiles (>0.3245
and ≤0.6254), and lowest quartile (≤0.3245).
Arginine is synthesized endogenously from citrulline, primarily in the
kidney via the intestinal-renal axis.46,47 Arginase
and nitric oxide synthase (NOS) compete for arginine, their common substrate.
In sickle cell disease (SCD), bioavailability of arginine and nitric oxide
(NO) are decreased by several mechanisms linked to hemolysis. The release
of erythrocyte arginase during hemolysis increases plasma arginase levels
and shifts arginine metabolism toward ornithine production, decreasing the
amount available for NO synthesis. The bioavailability of arginine is further
decreased by increased ornithine levels because ornithine and arginine compete
for the same transporter system for cellular uptake.26,27 Endogenous
synthesis of arginine from citrulline may be compromised by renal dysfunction,
commonly associated with SCD. Despite an increase in NOS,10- 12 NO
bioavailability in SCD is low due to low substrate availability,1- 3,16 NO
scavenging by cell free hemoglobin released during hemolysis,5 and
through reactions with free radicals such as superoxide.6,14,15 Superoxide
is elevated in SCD7 because of low superoxide
dismutase activity, high xanthine oxidase activity,6 and
potentially as a result of uncoupled NOS in an environment of low arginine
concentration.14 Endothelial dysfunction resulting
from NO depletion and increased levels of the downstream products of ornithine
metabolism (polyamines and proline) likely contribute to the pathogenesis
of lung injury and pulmonary hypertension.
Morris CR, Kato GJ, Poljakovic M, Wang X, Blackwelder WC, Sachdev V, Hazen SL, Vichinsky EP, Morris SM, Gladwin MT. Dysregulated Arginine Metabolism, Hemolysis-Associated Pulmonary Hypertension,
and Mortality in Sickle Cell Disease. JAMA. 2005;294(1):81–90. doi:10.1001/jama.294.1.81
Author Affiliations: Departments of Emergency
Medicine (Dr C. Morris) and Hematology-Oncology (Dr Vichinsky), Children’s
Hospital & Research Center at Oakland, Oakland, Calif; Vascular Therapeutics
Section, Cardiovascular Branch, National Heart, Lung, and Blood Institute
(Drs Kato, Wang, and Gladwin), Critical Care Medicine Department, Clinical
Center (Drs Kato, Wang, Blackwelder, and Gladwin), and Echocardiography Laboratory
(Dr Sachdev), National Institutes of Health, Bethesda, Md; Department of Molecular
Genetics and Biochemistry, University of Pittsburgh School of Medicine, Pittsburgh,
Pa (Drs Poljakovic and S. Morris); and Center for Cardiovascular Diagnostics
and Prevention, Departments of Cell Biology and Cardiovascular Medicine, Cleveland
Clinic Foundation, Cleveland, Ohio (Dr Hazen).
Context Sickle cell disease is characterized by a state of nitric oxide resistance
and limited bioavailability of L-arginine, the substrate for
nitric oxide synthesis. We hypothesized that increased arginase activity and
dysregulated arginine metabolism contribute to endothelial dysfunction, pulmonary
hypertension, and patient outcomes.
Objective To explore the role of arginase in sickle cell disease pathogenesis,
pulmonary hypertension, and mortality.
Design Plasma amino acid levels, plasma and erythrocyte arginase activities,
and pulmonary hypertension status as measured by Doppler echocardiogram were
prospectively obtained in outpatients with sickle cell disease. Patients were
followed up for survival up to 49 months.
Setting Urban tertiary care center and community clinics in the United States
between February 2001 and March 2005.
Participants Two hundred twenty-eight patients with sickle cell disease, aged 18
to 74 years, and 36 control participants.
Main Outcome Measures Plasma amino acid levels, plasma and erythrocyte arginase activities,
diagnosis of pulmonary hypertension, and mortality.
Results Plasma arginase activity was significantly elevated in patients with
sickle cell disease, with highest activity found in patients with secondary
pulmonary hypertension. Arginase activity correlated with the arginine-ornithine
ratio, and lower ratios were associated with greater severity of pulmonary
hypertension and with mortality in this population (risk ratio, 2.5; 95% confidence
interval [CI], 1.2-5.2; P = .006). Global
arginine bioavailability, characterized by the ratio of arginine to ornithine
plus citrulline, was also strongly associated with mortality (risk ratio,
3.6; 95% CI, 1.5-8.3; P<.001). Increased plasma
arginase activity was correlated with increased intravascular hemolytic rate
and, to a lesser extent, with markers of inflammation and soluble adhesion
Conclusions These data support a novel mechanism of disease in which hemolysis contributes
to reduced nitric oxide bioavailability and endothelial dysfunction via release
of erythrocyte arginase, which limits arginine bioavailability, and release
of erythrocyte hemoglobin, which scavenges nitric oxide. The ratios of arginine
to ornithine and arginine to ornithine plus citrulline are independently associated
with pulmonary hypertension and increased mortality in patients with sickle
L-Arginine, the substrate for nitric oxide (NO) synthesis, is deficient
in sickle cell disease (SCD).1- 4 Increased
NO consumption by cell free plasma hemoglobin5 and
reactive oxygen species6,7 leads
to decreased NO bioavailability8,9 that
is exacerbated by decreased availability of the NO synthase substrate L-arginine. This state of resistance to NO is accompanied by a compensatory
up-regulation of NO synthase and non–NO-dependent vasodilators.10- 13 Under
conditions of low arginine concentration, NO synthase is uncoupled, producing
reactive oxygen species in lieu of NO,14,15 potentially
further reducing NO bioavailability in SCD and enhancing oxidative stress.
Recent reports of elevated arginase activity in SCD16- 18 offer
another avenue for decreased arginine bioavailability. Arginase, an enzyme
that converts L-arginine to ornithine and urea, can limit NO
bioavailability through increased consumption of the substrate for NO synthase.19- 21 Arginase, which is
found predominantly in the liver and kidneys, is also present in human red
blood cells22,23 and can be induced
in many cell types by a variety of cytokines and inflammatory stimuli.20,24,25 Furthermore, since
arginine and ornithine compete for the same transport system for cellular
uptake,26,27 a decrease in the
ratio of arginine to ornithine resulting from increased arginase activity
could further limit arginine bioavailability for NO synthesis.
We have previously reported high plasma arginase activity in 10 SCD
patients with pulmonary hypertension.18 Death
within a year of enrollment in that study occurred in 2 patients with the
highest arginase activity. Pulmonary hypertension is common in both adults
and children with SCD28- 31 and
is an important predictor of early mortality.29 Pulmonary
hypertension also develops in most other hereditary and chronic hemolytic
anemias, including thalassemia,32 hereditary
spherocytosis,33 paroxysmal nocturnal hemoglobinuria,34 and other hemolytic disorders,35- 38 supporting
the existence of a clinical syndrome of hemolysis-associated pulmonary hypertension.29,39,40
Since endothelial dysfunction may contribute to the pathogenesis of
pulmonary hypertension through impaired production and bioavailability of
and responsiveness to NO,41- 44 we
hypothesized that elevated arginase activity and dysregulated arginine metabolism
may contribute to the endothelial dysfunction syndrome that occurs in SCD.
Consistent with this hypothesis, arginase activity and alterations in arginine
metabolic pathways have recently been implicated in the pathophysiology of
primary pulmonary hypertension.45 The goal
of this study was to identify the source of increased plasma arginase activity
in a large cohort of patients with SCD and to evaluate the contribution of
dysregulated arginine metabolism to morbidity and mortality.
The patient population was sequentially enrolled between February 2001
and March 2005 and comprised 228 patients with SCD hemoglobinopathies for
whom measurements of arginase (n = 140), arginine and ornithine
(n = 209), or both (n = 121) were available. This study
includes 188 of 195 patients who have been described in detail.29 Written
informed consent was obtained from each patient for an institutional review
board–approved protocol to obtain clinical information, echocardiography,
blood specimens, and prospective clinical follow-up data for research analysis.
All laboratory assays were performed using the blood specimens that were collected
prospectively at enrollment. In this population, right heart catheterization
studies have confirmed that a tricuspid regurgitant jet velocity less than
2.5 m/s corresponds to normal pulmonary artery pressures, tricuspid regurgitant
jet velocity of at least 2.5 m/s but less than 3.0 m/s corresponds to mild
pulmonary hypertension, and tricuspid regurgitant jet velocity of at least
3.0 m/s corresponds to moderate to severe pulmonary hypertension.29 Pulmonary hypertension was prospectively defined
as a tricuspid regurgitant jet velocity of at least 2.5 m/s on Doppler echocardiography.
Thirty-six African American individuals recruited from a list of volunteers
maintained at the National Institutes of Health were evaluated as controls
for comparisons of laboratory and echocardiographic data. Volunteers similar
in age and sex distribution to the sickle cell pulmonary hypertension screening
cohort were selected. An additional 9 African American controls were enrolled
specifically to obtain plasma and erythrocyte arginase measurements for comparison
of these 2 measures of arginase activity.
Plasma amino acids were quantified via ion exchange chromatography (Beckman
model 6300 amino acid analyzer, Fullerton, Calif) at the Mayo Clinic (Rochester,
Minn) by methods recommended by the manufacturer. In addition to arginine
and ornithine, citrulline, the endogenous precursor for de novo arginine synthesis,
which occurs primarily in the kidney,46,47 and
proline, which is synthesized from ornithine,46 were
After 153 patients had been enrolled, it was decided to obtain measurements
of plasma arginase activity in all patients for whom a sufficient quantity
of stored frozen plasma was available (n = 140). Arginase activity
was determined as the conversion of L-arginine that is carbon
14 (14C)–labeled on the guanidino carbon to 14C-labeled
urea, which was converted to 14C-labeled carbon dioxide by urease
and trapped as 14C-labeled sodium carbonate for scintillation counting,
as previously described.48 Briefly, aliquots
of plasma or red blood cell lysate were spun down on collection and frozen
at −80°C. Thawed samples were later incubated for 10 minutes at
55°C in complete assay mixture lacking arginine. The reaction was initiated
by addition of labeled arginine and incubation was continued at 37°C for
2 hours. The reaction was terminated by heating at 100°C for 3 minutes.
Samples were incubated with urease at 37°C for 45 minutes, and 14C-labeled sodium carbonate was trapped on sodium hydroxide–soaked
filters following acidification of the samples with hydrochloric acid to volatilize
the 14C-labeled carbon dioxide.
Plasma levels of endothelial and platelet-specific soluble (s) adhesion
molecules (sE-selectin, sP-selectin, vascular cell adhesion molecule 1 [sVCAM-1],
and intracellular adhesion molecule 1 [sICAM-1]) were measured using commercially
available enzyme-linked immunosorbent assay (ELISA) kits (R&D Systems,
Plasma levels of hemoglobin were measured by ELISA as previously described.49
Myeloperoxidase levels were measured by ELISA (PrognostiX, Cleveland,
Results are presented as mean and standard deviation, geometric mean
and 95% confidence interval (CI), or percentage of participants with characteristic,
as appropriate. Two-sided 2-sample t tests were used
to compare continuous variables in 2 groups (for example, amino acid values
in sickle cell patients and healthy controls). Linear regression on 3 categories
of tricuspid regurgitant jet velocity (<2.5, 2.5-2.99, and ≥3.0 m/s;
coded 0, 1, and 2, respectively) was used to evaluate relationships between
amino acid values or arginase and level of pulmonary hypertension in patients
with SCD. Since normal distributions provided poor approximations for many
of the variables of interest, bivariate correlations were assessed using the
Spearman rank correlation coefficient.
Multiple regression analysis of arginase activity used log10-transformed
values for arginase, as well as for laboratory correlates for which normal
distributions fit logarithms better than untransformed values; use of logarithms
also reduced the influence of extremely high values of independent variables.
This modeling used a stepwise procedure to add independent variables, beginning
with the variables most strongly associated with log10 arginase
and considering all potential covariates associated with log10 arginase
with P≤.15. Deletion of variables after initial
inclusion in the model was allowed. The procedure continued until all independent
variables in the final model had P≤.05, adjusted
for other independent variables, and no additional variable had P≤.05. The final model was confirmed by fitting a similar model
using ranks of all variables, including log10 arginase.
Proportional hazards (Cox) regression was used to study relationships
between mortality in patients with SCD and covariates of interest. The proportional
hazards assumption was evaluated by assessing whether scaled Shoenfeld residuals50 showed a trend with time.
In all regression modeling, observations with missing values for any
variable included in a particular model were deleted; no imputation of missing
values was performed.
P<.05 was considered statistically significant;
no adjustment for multiple comparisons was made. Analysis was performed using
NCSS 2004 software (Number Cruncher Statistical Systems, Kaysville, Utah).
The analyses described above were preplanned. Additional analyses evaluated
arginase activity within erythrocytes and the association between arginase
activity in plasma and red blood cell lysate. These analyses involved an unselected
subgroup of 16 patients, as well as 45 controls (the original 36 and the additional
9 for whom plasma and erythrocyte arginase measurements were made).
General characteristics of the study population of patients with SCD
and controls are shown in Table 1.
Plasma amino acid levels in SCD patients were compared with those in
African American control participants without SCD (Table 2). An abnormal amino acid profile was observed in patients
with SCD that is consistent with altered arginine metabolism. The observed
dysregulation of the arginine-to-NO metabolism was greatest in SCD patients
with pulmonary hypertension. Although mean (SD) plasma arginine concentrations
were low in patients with SCD compared with healthy controls (41  vs 67
 μmol/L; P<.001), these levels were similar
in patients with and without pulmonary hypertension. However, plasma ornithine
levels were elevated in patients with SCD and severe pulmonary hypertension
(tricuspid regurgitant jet velocity ≥3.0 m/s), and the elevation was significant
compared with patients with SCD without pulmonary hypertension (mean [SD],
81  vs 63  μmol/L; P<.001 by 2-sided t test) and is likely the result of elevated arginase activity.
The ratio of arginine to ornithine, an indirect measure of arginase activity
and relative arginine bioavailability, was low in patients with SCD compared
with controls (mean [SD], 0.71 [0.39] vs 1.20 [0.49]; P<.001) (Table 2 and Figure 1A) and was particularly low in the group
with severe pulmonary hypertension (mean, 0.49 [SD, 0.18]). Ratios were clearly
lower even in patients with SCD without evidence of pulmonary hypertension
than in controls (mean [SD], 0.74 [0.41] vs 1.20 [0.49]; P<.001 by 2-sided t test). The association
between arginine-ornithine ratio and level of pulmonary hypertension was significant
by linear regression of the ratio on category of tricuspid regurgitant jet
velocity (P = .03) (Table 2), but ratios were low only in patients with severe pulmonary
hypertension compared with patients with no evidence of pulmonary hypertension
by 2-sided t test adjusted for unequal variances
(mean [SD], 0.49 [0.18] vs 0.74 [0.41]; P<.001).
Plasma proline concentrations were also significantly increased in patients
with SCD compared with controls (mean [SD], 210  vs 154  μmol/L; P<.001 (Table 2).
Patients without pulmonary hypertension had higher proline levels than controls
(mean [SD], 202  vs 154 ; P<.001 by 2-sided t test), and even higher levels occurred in patients with
severe and moderate pulmonary hypertension (mean [SD], 245  vs 219 
vs 202  μmol/L for tricuspid regurgitant jet velocity ≥3.0, 2.5-2.99,
and <2.5 m/s, respectively; P = .01
by linear regression analysis; Table 2).
This possibly indicates increased conversion of ornithine to proline in SCD
that is amplified in patients with pulmonary hypertension (Table 2) and may well account for the fact that plasma ornithine
levels did not increase in most patients with SCD, 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 methods.
Although citrulline levels tended to increase slightly with level of
pulmonary hypertension in patients, mean levels were almost identical in patients
and controls (Table 2). Citrulline was
significantly correlated with creatinine level (ρ = 0.51; P<.001), which is consistent with impaired renal function.
The ratio of arginine to ornithine plus citrulline, 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-ornithine
ratio (Table 2). In aggregate, these
data indicate significant modulation of L-arginine metabolism
in SCD that is associated with the development of pulmonary and renal vasculopathy.
To understand the mechanism responsible for dysregulation of L-arginine metabolism, plasma arginase activity was measured in patients
with SCD and controls. Plasma arginase activity was significantly elevated
in patients with SCD (n = 140) (mean [SD], 2.1 [2.1] μmol/mL
per hour) compared with controls (n = 36) (mean [SD], 0.4 [0.2] μmol/mL
per hour; P<.001 by t test
on log10 arginase) (Figure 1B).
In patients with SCD, arginase activity tended to increase with level of pulmonary
hypertension (mean [SD], 1.9 [1.8], 2.6 [2.8], and 2.8 [2.0] μmol/mL per
hour for tricuspid regurgitant jet velocity <2.5, 2.5-2.99, and ≥3.0,
respectively), although the association was not significant in linear regression
of log10 arginase on the 3 categories of tricuspid regurgitant
jet velocity, coded 0, 1, or 2 (R2 = 0.017; P = .13). However, even in patients without pulmonary
hypertension, patients with SCD had significantly higher arginase activity
compared with controls (mean [SD], 0.4 [0.2] vs 1.9 [1.8]; P<.001). Arginase activity was significantly correlated with arginine-ornithine
ratio (ρ = −0.34; P<.001)
(Figure 1C); however, it likely is only
one of several factors affecting this ratio and arginine bioavailability in
patients with SCD.
The relationship between arginase activity and clinical laboratory markers
of disease severity was evaluated to identify mechanisms for increased enzymatic
activity and associated effects on organ function (Table 3). Plasma arginase activity was significantly associated
with several markers of increased hemolytic rate, including cell free plasma
hemoglobin (ρ = 0.56; P<.001) (Figure 2), lactate dehydrogenase (ρ = 0.35; P<.001), aspartate aminotransferase (ρ = 0.34; P<.001), and hematocrit (ρ = −0.20; P = .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,
endothelial and platelet-specific soluble adhesion molecules (sE-selectin,
sP-selectin, sVCAM-1, and sICAM-1), triglycerides, and cholesterol (Table 3). No association of arginase activity
with age (ρ = −0.09; P = .31) or sex (P = .63 by t test on log10 arginase) was identified. There
was no evidence of association between elevated arginase activity and markers
of renal function (Table 3).
In multiple regression analysis of log10 arginase activity,
all variables associated with log10 arginase with P≤.15 in Table 3 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<.001 for all independent variables). Although the strongest association
was with hemolysis, these data also demonstrate a link between arginase activity
and abnormal lipid metabolism as well as adhesion. The low-variance inflation
factors (≤1.11 for all 3 covariates in the model) suggest that there was
no multicollinearity problem in this model and that each of the 3 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<.001 for ranks of cell free hemoglobin and sP-selectin
and P = .001 for rank of triglycerides).
No adjustment was made for multiple comparisons in these analyses. This
seems appropriate, since the objective of this study was 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 Table 3 by the number of correlations shown) the major associations
would still have P<.05. Furthermore, regression
modeling enabled 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 patients
with SCD is associated with intravascular hemolysis, endothelial activation,
To further identify the source of increased plasma arginase activity,
arginase activity was also determined for red blood cell lysates of 45 controls
and a subset of 16 patients with SCD in whom both frozen plasma and red blood
cell lysates were available for comparison (Figure
3A). Specific activities of arginase in red blood cell lysate of
patients with SCD were significantly higher than those of controls (mean [SD],
37.7 [2.9] vs 23.5 [1.7] nmol/mg per min; P<.001
by t test on log10 arginase). For purposes
of comparison, “normal-range” boundaries for controls were set
arbitrarily at approximately the 80th percentile for arginase activities of
both red blood cell lysates and plasma. Two thirds of all control values fell
within these boundaries, while, in striking contrast, 94% of all values for
plasma and erythrocyte arginase activities of patients with SCD fell outside
of these boundaries (Figure 3B).
Information on deaths was collected during a follow-up period of up
to 49 months. Between enrollment in the study and March 2005, 18 patients
with SCD had died, with a 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 a tricuspid regurgitant jet
velocity of at least 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; 95% CI, 2.4-22.4; P<.001) (Table 4). 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 plus citrulline were associated with mortality in proportional hazards
regression (Table 4 and Figure 4). After adjustment for high tricuspid regurgitant jet velocity
and log10 creatinine level, the ratios of arginine to ornithine
and arginine to ornithine plus citrulline remained significantly related to
mortality; thus, either of these ratios may be an independent risk factor
for death in patients with SCD. 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 = .02) for ratio of arginine to ornithine and 2.9 (P = .007) for ratio of arginine to ornithine
plus citrulline (Table 4). 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.
The assumption of proportional hazards (ie, constant risk ratio) for
ratios of arginine to ornithine and arginine to ornithine plus citrulline
seemed reasonable, since scaled Shoenfeld residuals50 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
toward ornithine-dependent pathways, increased arginase activity may contribute
to events that put patients at risk of early death.
This large cohort study of patients with SCD not only confirms previous
reports of elevated arginase activity in SCD16- 18,23 but,
more importantly, also demonstrates important associations among dysregulated L-arginine metabolism, pulmonary hypertension, and prospective mortality.
Additionally, these data suggest that elevated plasma arginase activity in
SCD is primarily the consequence of erythrocyte arginase release during intravascular
hemolysis, with some possible contribution from endothelial cell activation
associated with inflammation, and liver injury in some patients. Global arginine
bioavailability is diminished further through impairment of de novo arginine
synthesis in patients with renal dysfunction. These observations support a
novel mechanism of disease that links oxidative stress,6,7 chronic
organ damage,13,51 and hemolytic
endothelial dysfunction and pulmonary hypertension.
Arginase is an intracellular enzyme that appears in plasma only after
cell damage or death. Thus, inflammation, chronic end-organ damage and hemolysis
are all potential sources of elevated arginase activity in SCD. Arginase activity
is higher in immature red blood cells and reticulocytes,23 both
of which are plentiful in SCD. Nearly 94% of the patients with SCD had arginase
values outside the normal-range boundaries, with two thirds of the SCD population
exhibiting elevated arginase activity in both plasma and red blood cell lysate
(Figure 3). Elevated erythrocyte arginase
activity has also been reported in patients with megaloblastic anemia23 and thalassemia patients.53,54 In
addition to NO scavenging by cell free plasma hemoglobin, erythrocyte arginase
release during hemolysis may represent a mechanistic link between the pulmonary
hypertension syndrome that develops in SCD and other hemolytic disorders,
such as thalassemia, hereditary spherocytosis, and paroxysmal nocturnal hemoglobinuria,29,39 and warrants further investigation.
These combined mechanisms support a model whereby hemolysis produces a state
of reduced endothelial NO bioavailability that might contribute to endothelial
dysfunction, intimal and smooth muscle proliferation, oxidant stress, and
Association of arginase activity with common markers of inflammation
such as white blood cell count and myeloperoxidase is not unexpected because
arginase is induced in monocytes in response to helper T-cell type 2 cytokines,24 inflammatory mediators that are elevated in SCD.55 Elevated arginase activity has also recently been
discovered in asthma,56- 59 another
T-cell type 2 cytokine–related disorder60,61 that
may be relevant to the pathophysiology of SCD since 30% to 70% of pediatric
patients with SCD have reactive airways.62,63
An association of arginase activity with plasma soluble adhesion molecules
is a novel observation, although also not unexpected since endothelial cells
assume an inflammatory phenotype in SCD.64,65 In
particular, endothelial sP-selectin is thought to contribute to the microcirculatory
abnormalities in SCD.66- 68 Significant
correlations with sE-selectin, sVCAM-1, and sICAM-1 were also identified;
however, only an independent relationship of arginase activity to sP-selectin
was maintained in multiple regression analysis. This may also represent a
link between arginase activity and platelet activation. It remains to be determined
whether these findings represent a causal relationship between arginase and
adhesion molecules or reflect a common response to endothelial injury or proinflammatory
Equally intriguing is the association of arginase activity with triglycerides
and cholesterol, given growing support for triglyceride involvement in endothelial
dysfunction.69- 71 Although
triglyceride levels are generally lower in SCD,72 they
are increased by tumor necrosis factor α, interleukin 1, and other cytokines,73 which are elevated in SCD.74 Although
the true significance of this finding remains to be determined, this relationship
may be a reflection of a broader common pathway between arginase activity
and abnormal lipid metabolism in endothelial dysfunction and warrants further
Plasma arginine concentration in SCD is approximately 40 to 50 μmol/L
at baseline, well below the arginine concentration at which the rate of cellular
uptake via the arginine transport system is half-maximal (Km, approximately
100 μmol/L26,27). Thus, even modest reductions in plasma arginine
concentration can significantly affect cellular arginine uptake and bioavailability.
In the current study, we find that the ratio of arginine to ornithine is associated
with arginase activity. Because arginine and ornithine compete for uptake
via the same transport system,26,27 decreases
in the arginine-ornithine ratio in patients with SCD also represent decreases
in arginine bioavailability. Consistent with a metabolic marker of arginine
bioavailability, a low arginine-ornithine ratio was associated with worsening
severity of pulmonary hypertension and independently associated with mortality.
Because conversion of citrulline to arginine occurs primarily within the kidney,46,47 the increased mortality risk ratio
observed after citrulline was included in the Cox regression analysis probably
reflects effects of renal dysfunction on arginine bioavailability. Indeed,
citrulline levels trended higher in SCD patients with pulmonary hypertension
and correlated with rising creatinine levels (Spearman ρ = 0.51; P<.001).
The alterations in arginine metabolism in SCD and their implications
for clinical complications are summarized in Figure 5. As arginase and cell free hemoglobin correlate strongly
with one another and are both released from erythrocytes as they undergo hemolysis,
the independent contribution of each toward decreasing bioavailability of
NO cannot be determined in this clinical study, and causality cannot be assumed.
However, increased catabolism of arginine via arginase may not only compromise
the ability to synthesize NO but also may contribute to the pulmonary vascular
remodeling that occurs in pulmonary hypertension through increased production
of ornithine, a precursor for synthesis of proline and polyamines46 (Figure 5),
which are required for the collagen synthesis and cell proliferation, respectively,
that occur in vascular remodeling.57,59 Analogous
to its proposed roles in asthma,57,59 elevated
proline levels in SCD demonstrated in this and other studies3,75 may
contribute to pulmonary fibrosis and lung pathogenesis by promoting collagen
These findings suggest that dysregulated arginine metabolism is associated
with intravascular hemolysis, inflammation, and endothelial cell activation.
Alterations in the normal balance of arginine and its catabolic byproducts,
ornithine, citrulline, and proline, are associated with pulmonary hypertension
and prospective risk of death. These easily measured ratios may provide clinicians
with an objective index of disease severity that could identify patients at
risk and allow for earlier and more aggressive therapeutic intervention.
Corresponding Author: Claudia R. Morris,
MD, Department of Emergency Medicine, Children’s Hospital & Research
Center at Oakland, 747 52nd St, Oakland, CA 94609 (firstname.lastname@example.org).
Author Contributions: Dr C. Morris had full
access to all of the data in the study and takes responsibility for the integrity
of the data and the accuracy of the data analysis.
Study concept and design: C. Morris, Kato,
Vichinsky, S. Morris, Gladwin.
Acquisition of data: C. Morris, Kato, Poljakovic,
Wang, Sachdev, Hazen, Vichinsky, S. Morris, Gladwin.
Analysis and interpretation of data: C. Morris,
Kato, Poljakovic, Blackwelder, Hazen, S. Morris, Gladwin.
Drafting of the manuscript: C. Morris, Kato,
Critical revision of the manuscript for important
intellectual content: C. Morris, Kato, Poljakovic, Wang, Sachdev, Hazen,
Vichinsky, S. Morris, Gladwin.
Statistical analysis: C. Morris, Kato, Poljakovic,
Obtained funding: Hazen, Vichinsky, S. Morris,
Administrative, technical, or material support:
Poljakovic, Wang, Sachdev, Vichinsky, S. Morris, Gladwin.
Study supervision: C. Morris, Kato, Vichinsky,
Financial Disclosures: None reported.
Funding/Support: This study was supported in
part by National Institutes of Health (NIH) grants M01-RR01271 (Pediatric
Clinical Research Center) and HL-04386-05 (to Dr C. Morris); Dr S. Morris
was supported by NIH grant R01 GM57384, Dr Hazen was supported by NIH grant
P01 HL076491, and Drs Kato and Gladwin were supported by NIH Intramural Research
Role of the Sponsor: The funding agency had
no role in the design and conduct of the study, the collection, analysis,
and interpretation of the data, or the preparation, review, or approval of
Acknowledgment: We acknowledge the clinical
contributions of Oswaldo Castro, MD, and Wynona Coles, RT, and the patients
with SCD who participated in these studies. Mary Hall provided invaluable
protocol support and Inez Ernst, RN, provided echocardiography technical support.