MAJOR ARTICLE

Relationship of Cell-Free Hemoglobin to Impaired

Endothelial Nitric Oxide Bioavailability and Perfusion
in Severe Falciparum Malaria

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Tsin W. Yeo,1 Daniel A. Lampah,4 Emiliana Tjitra,5 Retno Gitawati,5 Enny Kenangalem,4 Kim Piera,1
Donald L. Granger,6 Bert K. Lopansri,6,a J. Brice Weinberg,7 Ric N. Price,1,2,8 Stephen B. Duffull,9
David S. Celermajer,3 and Nicholas M. Anstey1,2
1
International Health Division, Menzies School of Health Research and Charles Darwin University, and 2Division of Medicine, Royal Darwin
Hospital, Darwin, and 3Department of Medicine, University of Sydney and Department of Cardiology, Royal Prince Alfred Hospital, Sydney,
Australia; 4Menzies School of Health Research–National Institute of Health Research and Development Research Program, and District Ministry
of Health, Timika, Papua, and 5National Institute of Health Research and Development, Jakarta, Indonesia; 6Division of Infectious Diseases,
University of Utah and Veterans Affairs (VA) Medical Centers, Salt Lake City; 7Division of Hematology-Oncology, Duke and VA Medical Centers,
Durham, North Carolina; 8Centre for Vaccinology and Tropical Medicine, Nuffield Department of Clinical Medicine, John Radcliffe Hospital,
Oxford, United Kingdom; 9School of Pharmacy, University of Otago, Dunedin, New Zealand

Background. Hemolysis causes anemia in falciparum malaria, but its contribution to microvascular pathology
in severe malaria (SM) is not well characterized. In other hemolytic diseases, release of cell-free hemoglobin causes
nitric oxide (NO) quenching, endothelial activation, and vascular complications. We examined the relationship of
plasma hemoglobin and myoglobin to endothelial dysfunction and disease severity in malaria.
Methods. Cell-free hemoglobin (a potent NO quencher), reactive hyperemia peripheral arterial tonometry
(RH-PAT) (a measure of endothelial NO bioavailability), and measures of perfusion and endothelial activation
were quantified in adults with moderately severe (n p 78 ) or severe (n p 49 ) malaria and control subjects
(n p 16) from Papua, Indonesia.
Results. Cell-free hemoglobin concentrations in patients with SM (median, 5.4 mmol/L; interquartile range
[IQR], 3.2–7.4 mmol/L) were significantly higher than in those with moderately severe malaria (2.6 mmol/L; IQR,
1.3–4.5 mmol/L) or controls (1.2 mmol/L; IQR, 0.9–2.4 mmol/L; P ! .001 ). Multivariable regression analysis revealed
that cell-free hemoglobin remained inversely associated with RH-PAT, and in patients with SM, there was a signifi-
cant longitudinal association between improvement in RH-PAT index and decreasing levels of cell-free hemoglobin
(P p .047). Cell-free hemoglobin levels were also independently associated with lactate, endothelial activation, and
proinflammatory cytokinemia.
Conclusions. Hemolysis in falciparum malaria results in NO quenching by cell-free hemoglobin, and may
exacerbate endothelial dysfunction, adhesion receptor expression and impaired tissue perfusion. Treatments that
increase NO bioavailability may have potential as adjunctive therapies in SM.

Hemolysis of infected and uninfected red blood cells is
an important cause of anemia in falciparum malaria
[1], but its contribution to other pathophysiological
pathways in severe malaria (SM) is less well character-
ized. A central process in the pathogenesis of severe
Received 23 March 2009; accepted 12 June 2009; electronically published 2
October 2009.
Reprints or correspondence: Dr Anstey, International Health Division, Menzies
School of Health Research, PO Box 41096 Casuarina, Darwin, NT 0811, Australia
(anstey@menzies.edu.au).
The Journal of Infectious Diseases 2009; 200:1522–9
 2009 by the Infectious Diseases Society of America. All rights reserved.
0022-1899/2009/20010-0007$15.00
DOI: 10.1086/644641
falciparum malaria is microvascular obstruction re-
sulting from cytoadherence of parasitized erythrocytes
to activated endothelial cells, associated with impaired
Potential conflicts of interest: N.M.A., D.L.G., and J.B.W. are named as inventors
in a US patent for the use of L-arginine as treatment for severe malaria but have
transferred all their rights to their respective institutional malaria research
collaborations. This patent is issued for US rights only, and no rights are being
sought in other countries. All other authors report no other conflicting interests.
Presented in part: Annual Scientific Meeting of the American Society of Tropical
Medicine and Hygiene, New Orleans, December 2008 (abstract 1194).
Financial support: National Health and Medical Research Council (International
Collaborative Research Grant [ICRG] 283321 and practitioner fellowship to N.M.A.),
Wellcome Trust (ICRG GR071614MA and career development award 074637 to
R.N.P.), VA Research Service, National Institutes of Health (grants AI55982 and
AI041764), and the Tudor Foundation.
a
Present affiliation: Loyola University Medical Center, Maywood, Illinois.

1522 • JID 2009:200 (15 November) • Yeo et al

bioavailability of endothelial nitric oxide (NO) [2, 3]. Reduced
NO bioavailability in malaria contributes to increased endo-
thelial activation, increased cytoadherence of parasitized eryth-
rocytes, and impaired vasomotor regulation [3, 4], and it is
associated with increased mortality in murine malaria [5] and
disease severity in both adults and children [3, 6]. The etiology
of impaired NO bioavailability in malaria appears to be mul-
tifactorial [3, 6]. Decreased l-arginine (the substrate for NO
synthesis) is noted in SM. This hypoargininemia is considered
a major contributor to the low NO bioavailability in both adults
and children [3, 7, 8], but recent data suggest that hemolysis
may also be important.
Intravascular hemolysis plays a central role in the outcome
and pathogenesis of hemolytic diseases, such as sickle cell dis-
ease (SCD) and paroxysmal nocturnal hemoglobinuria [9, 10].
The proposed mechanism is a reduction in endothelial NO
bioavailability due to stoichiometric inactivation of NO by the
cell-free hemoglobin released with erythrocyte rupture [9, 11].
In SCD, cell-free hemoglobin is elevated and strongly correlated
with measurements of NO quenching, decreased NO-mediated
vascular flow, and increased endothelial activation [11]. He-
molysis also releases erythrocyte arginase, an enzyme that me-
tabolizes l-arginine. This reduces the amount of l-arginine
available for conversion to NO, further contributing to en-
dothelial dysfunction [7, 12]. In SCD, these mechanisms con-
tribute to complications, including pulmonary hypertension
and mortality [13].
Although most erythrocyte destruction in falciparum malar-
ia occurs extravascularly [14], a significant proportion of he-
molysis occurs in the intravascular compartment, with plasma
haptoglobin concentrations markedly decreased during active
disease [3, 14]. The clinical consequences of increased cell-free
hemoglobin levels in malaria and other infections causing in-
travascular hemolysis have not been well characterized. Muscle
breakdown during falciparum malaria increases plasma myo-
globin [15], which, like hemoglobin, can quench NO [16]. We
have described elsewhere an inverse association between en-
dothelial function and plasma lactate dehydrogenase (LDH)
levels in falciparum malaria [3]. However, LDH may not be a
specific measure of hemolysis in malaria, with extraerythrocytic
sources of LDH likely to be significant.
The association between cell-free hemoglobin, myoglobin,
and endothelial NO bioavailability is not known in malaria. In
a prospective observational study, we tested the following hy-
potheses in adult falciparum malaria: (1) levels of cell-free he-
moglobin and myoglobin (mediators of NO quenching) are
increased in proportion to disease severity; (2) these levels cor-
relate with impairment of both endothelial function and tissue
perfusion; and (3) these levels correlate with increases in en-
dothelial activation, proinflammatory cytokine levels, and par-
asite biomass.
Cell-Free Hemoglobin in Severe Malaria • JID 2009:200 (15 November) • 1523
PATIENTS, MATERIALS, AND METHODS
Study site. The study was performed at Mitra Masyarakat
Hospital in Timika, Papua, Indonesia, an area with unstable
transmission of multidrug-resistant malaria [17, 18]. Written,
informed consent was obtained from all patients or relatives;
ethical approval was obtained from the institutional review
boards of the National Institute of Health Research and De-
velopment and Menzies School of Health Research.
Patients. Patients were ⭓18 years old with moderately se-
vere or severe Plasmodium falciparum malaria without Plas-
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modium vivax infection and with a hemoglobin level of 160 g/
L; they had been prospectively enrolled in a study of endothelial
dysfunction, as reported elsewhere [3]. In brief, SM was defined
as P. falciparum parasitemia and ⭓1 modified World Health
Organization (WHO) criterion of severity (excluding severe
anemia) [3]; moderately severe malaria (MSM) was defined as
fever within the preceding 48 h, 11000 asexual P. falciparum
parasites/mL, no WHO warning signs or SM criteria, and a
requirement for inpatient parenteral therapy because of in-
ability to tolerate oral treatment. Healthy controls were unre-
lated hospital visitors with no history of fever in the last 48 h
and no parasites seen at microscopy, negative results of histi-
dine-rich protein 2 (HRP2) antigen testing, no evidence of
intercurrent illness, and no history of smoking in the preceding
12 h [3]. On recruitment, a standardized history was obtained,
physical examination was performed, and endothelial function
was measured. Heparinized blood was collected daily and cen-
trifuged within 30 min of collection, and plasma was stored at
⫺70C. Patients with MSM were treated with intravenous qui-
nine, and those with SM received either intravenous quinine
or artesunate, with both groups also receiving either doxycy-
cline or clindamycin. Parasite counts were determined from
microscopy of Giemsa-stained thick and thin films. Hemoglo-
bin, biochemistry, acid-base parameters, and lactate level (as a
measure of tissue perfusion) were measured with a bedside
analyzer (i-STAT), and LDH, creatine kinase, and creatinine
levels were measured with a COBAS analyzer (Roche).
Cell-free hemoglobin, cytokines, endothelial activation, ar-
ginase, and l-arginine. Plasma concentrations of cell-free he-
moglobin were measured by enzyme-linked immunosorbent
assay (ELISA) according to the manufacturer’s instructions
(Bethyl Laboratories). Plasma concentrations of the endotheli-
al activation markers, intercellular adhesion molecule 1 (ICAM-
1), E-selectin and angiopoietin 2 were measured by ELISA
(R&D), as described elsewhere [4]. Total parasite biomass was
quantified by measuring plasma HRP2 with ELISA, as described
elsewhere [3]. Tumor necrosis factor (TNF) and interleukin
(IL)–6 concentrations were measured by flow cytometry (BD
Cytometric Bead Array). Plasma arginase activity was measured
using a radiometric assay [3, 12], and plasma l-arginine was
 Table 1. Baseline Characteristics of Patients, According to Clinical Status

Patients with malaria

Healthy
control subjects Moderately severe Severe
Characteristic (n p 16) (n p 78) (n p 49)
Age, mean years (range) 25 (19–42) 28 (18–56) 29 (18–56)
No. (%) of male subjects 12 (75) 32 (41) 36 (74)
Weight, mean kg (range) 58 (45–85) 58 (43–77) 57 (45–70)
Ethnicity, no. (%) of Papuan highlandersa 15 (94) 59 (76) 27 (55)
No. (%) of current smokers 8 (50) 31 (40) 22 (45)
b
Duration fever before presentation, median days (IQR) … 2 (1–5) 4 (1–7)
b
Systolic blood pressure, mean mm Hg (range) 131 (116–145) 110 (80–134) 105 (60–154)

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Pulse rate, mean beats/min (range)b 65 (44–91) 81 (54–118) 98 (61–138)
b
Respiratory rate, mean breaths/min (range) 20 (18–24) 23 (16–32) 30 (16–60)
Temperature, mean C (range)b 35.6 (35–36.7) 36.5 (34.8–40.2) 37.2 (34.8–40.3)

NOTE. IQR, interquartile range.

a
P ! .01, by x2 test.
b
P ! .01, by analysis of variance or 2-sided t test.

measured by high-performance liquid chromatography (Shi-
madzu), using methods described elsewhere [3].
Endothelial function. Endothelial function was measured
noninvasively by using peripheral arterial tonometry (Endo-
PAT) to determine the change in digital pulse wave amplitude
in response to reactive hyperemia, giving a reactive hyperemia
peripheral arterial tonometry (RH-PAT) index. With this tech-
nique, the arterial pulsatile volume of the index finger at rest
is compared with that after an increase in shear stress induced
by 5 min of forearm ischemia (sphygmomanometer inflated to
200 mm Hg). Systemic influences were reduced by simulta-
neously performing peripheral arterial tonometry in the con-
tralateral index finger without reactive hyperemia. Patients were
assessed in a quiet, temperature-controlled environment, with
arms relaxed and supported by cushions, according to the man-
ufacturer’s instructions [3, 19]. The RH-PAT index is at least
50% dependent on endothelial NO production [20]. Endothe-
lial function was measured daily until death or discharge or
until the RH-PAT index was above an a priori cutoff value
(1.67) for 2 consecutive days [7].
Statistical methods. Statistical analysis was performed with
Stata software, version 9.2 (Stata). Intergroup differences were
compared by analysis of variance or Kruskal-Wallis test, where
appropriate. Pearson’s or Spearman’s correlation coefficients

Table 2. Baseline Laboratory and Physiological Measurements, According to Clinical Status

Patients with malaria

Healthy
control subjects Moderately severe Severe
Measurement (n p 16) (n p 78) (n p 49)
White blood cell count, ⫻ 103 cells/mL ND 5.9 (5.4–6.9) 9.5 (8.5–10.5)
Hemoglobin, mean g/L (range) ND 121 (70–170) 109 (60–163)
Cell-free hemoglobin, median mmol/L (IQR) 1.2 (0.9–2.4) 2.6 (1.3–4.5) 5.4 (3.2–7.4)
Plasma myoglobin, median mmol/L (IQR) 0.002 (0.001–0.0025) 0.002 (0.001–0.003) 0.015 (0.005–0.03)
RH-PAT index 1.77 (1.5–2.0) 1.82 (1.71–1.93) 1.37 (1.32–1.42)
Plasma creatinine, mmol/L ND 88 (82–94) 286 (207–365)
Lactate, mmol/L ND 1.4 (1.2–1.6) 2.93 (2.3–3.5)
Parasite density, geometric mean mL (range) ND 14,900 (850–127,000) 35,100 (125–725,000)
HRP2, mean loge ng/mL (range) ND 5.75 (1.34–8.79) 8.08 (1–10.98)
Soluble ICAM-1, pg/mL ND 569 (516–623) 938 (792–1084)
Soluble E-selectin, pg/mL ND 106 (95–118) 153 (113–193)
Plasma angiopoietin 2, pg/mL 2700 (2000–3300) 6500 (5000–8000) 17,000 (14,000–22,000)
Plasma arginase activity, mmol/mL/h 0.13 (0.07–0.16) 0.20 (0.14–0.24) 0.26 (0.22–0.31)
Plasma L-arginine, mmol/L 78 (67–89) 41 (37–44) 49 (43–56)

NOTE. Data are means (95% confidence intervals), unless otherwise indicated. P ! .01 for all variables, by analysis of variance (overall)
or 2-sided t test. HRP2, histidine-rich protein 2; ICAM-1, intercellular adhesion molecule 1; IQR, interquartile range; ND, not determined;
RH-PAT, reactive hyperemia peripheral arterial tonometry.

1524 • JID 2009:200 (15 November) • Yeo et al

 L (interquartile range [IQR], 1.9–8.4 mmol/L). These values
were not significantly different from those in patients with dys-
function of 2–4 organ systems. A patient with blackwater fever
and acute renal failure had a cell-free hemoglobin concentration
of 17 mmol/L. In patients with SM, there was no significant
difference in cell-free hemoglobin concentration between sur-
vivors and those who died. Plasma myoglobin was signifi-
cantly increased in patients with SM compared with those with
MSM and controls (P ! .001). In longitudinal analysis, there
was a significant decrease in plasma cell-free hemoglobin con-
centration with clinical recovery, with a median decrease

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of 1.50 mmol/L per day during the first 5 days of hospitali-
zation (P ! .001) (Figure 2).
Cell-free hemoglobin, myoglobin, and the RH-PAT index.
Patients with SM had significantly lower RH-PAT indexes at
enrollment than those with MSM and control subjects (P !
.001) (Table 2). Cell-free hemoglobin levels were inversely as-
Figure 1. Cell-free hemoglobin concentrations in patients with mod- sociated with RH-PAT indexes at univariate regression (Figure
erately severe and severe malaria and healthy controls (P ! .001, by 2 and Table 3) in all patients with malaria (r p ⫺0.36; P !
Kruskal-Wallis test). Horizontal lines indicate medians for each group.
.001) and in the subgroup with SM (r p ⫺0.29; P p .06). There
Differences among groups were compared using the Kruskal-Wallis
test, and the Mann-Whitney U test was used for post hoc pairwise was an inverse association between myoglobin and the RH-
comparisons. PAT index in all patients with malaria (rs p ⫺0.32 ; P ! .001)
but not in those with SM (Table 3). In the bivariate model that
were determined depending on normality of distributions. Mul- included plasma myoglobin and cell-free hemoglobin, myoglo-
tiple stepwise linear regression was used to adjust for confound- bin was no longer significantly associated with RH-PAT. For
ing variables. Mixed-effects modeling using generalized estimat- this reason and because concentrations of myoglobin were
ing equations was used to assess longitudinal associations. 1100-fold lower than those of cell-free hemoglobin, myoglobin
was excluded from subsequent regression analyses. After ad-
RESULTS justment for confounders (including plasma HRP2, angio-
poietin 2, plasma arginase, and disease severity), plasma argi-
Patients. Of the 177 subjects prospectively enrolled in the
original study, plasma was available for cell-free hemoglobin
assay in 49 (96%) of 51 patients with SM, all 78 with MSM
(100%), and 16 (33%) of 48 healthy control subjects. Baseline
characteristics of these 143 patients were not significantly dif-
ferent from those in the original group (Table 1). Of the patients
with SM, 26 (53%) had coma, 17 (35%) had acute renal failure,
23 (47%) had hyperbilirubinemia with either renal impairment
or high parasitemia (parasite load, 1100,000/mL), and 30 (61%)
had ⭓2 severity criteria. In total, 35 patients with SM (71%)
were treated with intravenous artesunate and 14 (29%) with
intravenous quinine, whereas 77 of the 78 patients with MSM
were treated with quinine (the other received artesunate). Eight
patients (16%) died in the SM group, and none in the MSM
group. Repeated measurements were possible in only 1 of the
8 fatal cases.
Cell-free hemoglobin, myoglobin, and clinical disease. Plas-
ma concentrations of cell-free hemoglobin were significantly Figure 2. Longitudinal course of cell-free hemoglobin concentrations
in patients with severe malaria. Median values (circles) and interquartile
elevated in patients with SM, compared with those with MSM
range (bars) are displayed for all time points. The x-axis values represent
and control subjects (overall, P ! .001 ) (Table 2 and Figure 1). time from the start of antimalarial therapy (day 0, 0–12 h; day 1, 13–
Among patients with SM, the median cell-free hemoglobin level 36 h; day 2, 37–60 h; day 3, 61–84 h; day 4, 85–109 h; and days 5–14,
in the 14 patients with isolated cerebral malaria was 5.4 mmol/ 1110 h).

Cell-Free Hemoglobin in Severe Malaria • JID 2009:200 (15 November) • 1525

 Table 3. Correlation between Cell-Free Hemoglobin Concentration and Physiological
Measures or Biomarkers of Severity

All patients with malaria Patients with severe malaria

a a
Measurement Correlation, rs P df Correlation, rs P df
RH-PAT index ⫺0.36 !.001 136 ⫺0.29 .06 48
Lactate 0.38 !.001 128 0.29 .04 47
HRP2 0.50 !.001 109 0.25 .10 45
ICAM-1 0.40 !.001 109 0.29 .04 47
E-selectin 0.34 !.001 117 0.19 .20 47
Angiopoietin 2 0.34 !.001 143 0.18 .20 48
TNFb

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… … … 0.33 .04 39
IL-6b … … … 0.41 .01 39

NOTE. df, degrees of freedom; HRP2, histidine-rich protein 2; ICAM-1, intercellular adhesion molecule
1; IL, interleukin; RH-PAT, reactive hyperemia peripheral arterial tonometry; TNF, tumor necrosis factor.
a
Correlation with cell-free hemoglobin level.
b
TNF and IL-6 levels were measured only in patients with severe malaria.

nase and cell-free hemoglobin remained significantly inverse-
ly associated with RH-PAT both in all patients with malar-
ia (P p .02 and P ! .001, respectively) and in those with SM
(P p .046 and P p .02, respectively). In a longitudinal mul-
tivariable, mixed-effects model in patients with SM who sur-
vived 124 h after enrollment, there were significant associa-
tions between improvement in RH-PAT index and both de-
creasing cell-free hemoglobin (P p .047) and increasing l-ar-
ginine (P p .001) concentrations.
Cell-free hemoglobin and endothelial activation. Relative
to patients with MSM, patients with SM had significantly higher
plasma concentrations of soluble ICAM-1, E-selectin, and an-
giopoietin 2 (Table 2). Cell-free hemoglobin was correlated with
all 3 parameters among all patients with malaria: ICAM-1
(r p 0.4; P ! .001), E-selectin (r p 0.33; P ! .001), and angio-
poietin 2 (r p 0.42; P ! .001) (Table 3). After adjustment for
plasma HRP2 and disease severity, cell-free hemoglobin re-
mained significantly associated with ICAM-1.
Cell-free hemoglobin and biomarkers of severity. Com-
pared with patients with MSM, those with SM had higher con-
centrations of lactate (P ! .001) and plasma HRP2 (P ! .001)
(Table 2). Blood lactate was correlated with cell-free hemoglo-
bin, both in all patients with malaria (rs p 0.38 ; P ! .001) and
those with SM (rs p 0.29; P p .04) (Table 3). Although plasma
HRP2 was correlated with cell-free hemoglobin (r p 0.5; P !
.001), this association was not significant in the subgroup with
SM. In multivariable analysis including all patients with ma-
laria and controlling for disease severity, lactate was associated
with plasma HRP2 (P ! .001) and cell-free hemoglobin (P p
.05). In a longitudinal mixed-effects model, the fall in lactate
during clinical recovery from SM was significantly associated
with the decrease in cell-free hemoglobin concentration (r p
⫺0.5; P ! .001).
TNF and IL-6 levels were measured only in patients with
SM, with median plasma concentrations of 2.4 pg/mL (IQR,
1.5–4.2 pg/mL) for TNF and 84 pg/mL (IQR, 15–501 pg/mL)
for IL-6. In patients with SM, cell-free hemoglobin level was
correlated significantly with both TNF level (r p 0.33; P p
.04) and IL-6 (r p 0.41; P p .01) (Table 3).
Cell-free hemoglobin, LDH, and markers of organ dys-
function. The LDH levels were correlated significantly with
levels of cell-free hemoglobin (rs p 0.65; P ! .001), creatinine
(rs p 0.68; P ! .001), and creatine kinase (rs p 0.48; P ! .001).
These associations remained significant after stratification by
disease severity.
Cell-free hemoglobin, plasma arginase activity, and markers
of hepatic function. Plasma arginase activity levels were in-
creased in patients with SM (P ! .001) (Table 3). Among all
patients with malaria, plasma arginase activity was correlated
with levels of cell-free hemoglobin (r p 0.29 ; P p .005) and
alanine transaminase (r p 0.29; P p .01), suggesting potential
contributions to circulating arginase from both erythrocytic
and hepatic sources.
DISCUSSION
The plasma cell-free hemoglobin concentration increases with
malaria disease severity, and levels are associated with impaired
endothelial NO bioavailability, endothelial activation, increased
parasite biomass, and impaired tissue perfusion, as measured
by blood lactate concentrations. Quenching of NO by cell-free
hemoglobin probably contributes to impaired endothelial ho-
meostasis with microvascular dysfunction, increased adhesion
receptor expression, increased microvascular sequestration of
parasitized erythrocytes, and tissue hypoxia. Endothelial dys-
function was independently associated with increases in plasma
cell-free hemoglobin and plasma arginase activity. This suggests
that, as in SCD, both of these consequences of hemolysis con-

1526 • JID 2009:200 (15 November) • Yeo et al

tribute to the impaired bioavailability of endothelial NO found
in malaria.
Similar to the findings in SCD, we found a significant as-
sociation between LDH and cell-free hemoglobin [21]. LDH
levels are much higher in malaria than in SCD, and they are
associated with creatinine and creatine kinase levels—measures
of renal and muscle damage [3]. Thus, unlike the mechanism
in SCD, nonerythrocytic sources probably contribute signif-
icantly to the elevated LDH concentrations. Cell-free hemo-
globin has been clearly shown to be a direct measure of NO
quenching in SCD [11] and therefore accurately reflects both
hemolysis and NO quenching capacity in malaria. The rate
constants for the NO dioxygenation reactions of myoglobin
and hemoglobin are similar. However, plasma myoglobin con-
centrations were ∼160-fold lower than cell-free hemoglobin
concentrations, indicating that the contribution of myoglobin
to NO scavenging in malaria is much less significant than that
of cell-free hemoglobin.
NO reacts with oxyhemoglobin, producing methemoglobin
and nitrate. The rapid rate of this reaction, together with the
large amount of erythrocytic hemoglobin, should theoretically
reduce NO to levels too low to function physiologically [22, 23].
In explaining this paradox, NO diffusion is reduced by up to
600-fold by an erythrocyte-free zone at the edge of the endo-
thelium and erythrocyte submembrane barriers [24, 25]. How-
ever, intravascular hemolysis with hemoglobin release into
plasma can reduce endothelial NO bioavailability dramatically
[9, 26]. Furthermore, cytoadherence of parasitized erythrocytes
to the endothelium with loss of the erythrocyte-free zone may
be an additional mechanism increasing NO quenching in malaria.
In vitro, NO decreases endothelial adhesion receptor ex-
pression [27] and cytoadherence of P. falciparum–parasitized
erythrocytes [28]. The association between plasma ICAM-1
concentrations and cell-free hemoglobin suggests that quench-
ing of endothelial NO in malaria contributes to increased ad-
hesion receptor expression in vivo. Endothelial activation in-
creases parasite sequestration, which is suggested by the in-
dependent associations observed between cell-free hemoglobin
and both ICAM-1 and HRP2. Increased blood lactate, a prog-
nostic marker in SM, is considered to be a result of reduced
tissue perfusion and oxygen delivery. In our study, both HRP2
and cell-free hemoglobin were independently associated with
increased lactate concentrations, suggesting that both parasite
sequestration and NO quenching contribute to impaired per-
fusion [29].
In both malaria and SCD, most erythrocyte destruction oc-
curs extravascularly through phagocytosis of erythrocytes by
macrophages, with a lesser contribution from intravascular he-
molysis [14, 30]. Nevertheless, hemolysis in the intravascular
compartment is sufficient to result in increased plasma he-
moglobin, with the concentrations we describe in malaria being
Cell-Free Hemoglobin in Severe Malaria • JID 2009:200 (15 November) • 1527
comparable to those reported from other studies of acute ma-
laria in both children and adults [14, 31, 32]. Although the
plasma hemoglobin concentrations are not markedly elevated,
the physiological effects on endothelial NO bioavailability, even
at these concentrations, have been shown to be highly signif-
icant in vitro [23].
The clinical consequences of hemolysis-related NO quench-
ing have been best studied in SCD, a disorder in which in-
creased plasma cell-free hemoglobin is associated with elevated
plasma NO-quenching capacity, decreased blood flow in re-
sponse to NO donors, and increased endothelial adhesion re-
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ceptor expression [10, 11]. NO quenching is also hypothesized
to explain the increases in blood pressure and mortality seen
with use of artificial hemoglobin solutions [33].
Although there are differences in the clinical features and
pathogenic mechanisms between SCD and severe falciparum
malaria, there may be similarities in the microvascular conse-
quences of increased plasma hemoglobin. Concentrations of
cell-free hemoglobin in SM were comparable to those found
in SCD [11], which are sufficient to result in increased ICAM-
1 and E-selectin levels and attenuate the blood flow response
to NO donors [11]. In vitro, 6 mmol/L hemoglobin, a level simi-
lar to the median values found in the patients with SM, inhibits
the vasodilator response to NO [23]. Furthermore, quantitative
models show that the micromolar amounts of cell-free hemo-
globin found in SCD (and now shown in SM) can reduce NO-
mediated vasodilation in arterioles [34]. Cell-free hemoglobin–
related NO quenching will therefore impair arteriolar vasodila-
tory regulation, the microcirculatory mechanism in which NO
is most effective in regulating organ perfusion [23]. In malaria,
this may cause decreased functional capillary density [35], adding
to the impaired microvascular flow and tissue hypoxia [36] al-
ready occurring from microvascular sequestration of parasitized
erythrocytes [29]. Decreased functional capillary density is as-
sociated with increased mortality in rodent malaria models [37]
and human sepsis [38]. Deleterious effects of decompartmen-
talized hemoglobin are minimized by binding to haptoglobin,
but this system is usually overwhelmed in malaria, with 190%
of adults with SM having undetectable plasma haptoglobin [3].
Increased levels of proinflammatory cytokines, including
TNF, are associated with a poor clinical outcome in African
children and Asian adults with falciparum malaria [39, 40]. In
vitro, hemoglobin impairs NO-mediated cytotoxic activity in
macrophages [41]. NO attenuates proinflammatory cytokine-
mia by inhibiting activation of nuclear transcription factor kB
[42]. The association between cell-free hemoglobin and TNF
and IL-6 in malaria suggests that NO quenching may increase
nuclear transcription factor kB–mediated production of pro-
inflammatory cytokines, endothelial activation, cytoadherence,
and consequent cellular damage.
Our study has several limitations. RH-PAT is at least 50%
NO dependent [20]. We cannot exclude a contribution to RH-
PAT in malaria from other vasodilators, such as prostacyclin
and endothelium-derived hyperpolarizing factor [20]. Assess-
ment of digital arteriolar function may not fully reflect micro-
circulatory NO bioavailability in malaria-affected organs. It is
possible that increased cell-free hemoglobin may be an epi-
phenomenon, with the pathological associations arising from
the effects of pathogenic parasite products released during
erythrocyte rupture. This is unlikely, however, because most
hemolysis in malaria arises from the destruction of nonpara-
sitized erythrocytes [1]. Furthermore, cell-free hemoglobin re-
mained inversely correlated with endothelial NO bioavailability
and perfusion even after adjustment for potential covariates,
including parasite biomass. Based on the strong relationship
between cell-free hemoglobin and NO quenching in SCD, we
postulate that this mechanism underlies the clear relationship
between cell-free hemoglobin and impaired endothelial NO
bioavailability and endothelial activation in malaria. Increased
cell-free hemoglobin also results in increased plasma heme,
which may also contribute to endothelial dysfunction and ac-
tivation. Heme has additional proinflammatory properties and
deleterious effects on endothelial cells, including breakdown of
endothelial integrity, with microvascular thrombus formation
and obstruction [43]. Because we excluded individuals with
hemoglobin concentrations of !60 g/L, we are unable to ex-
trapolate our results to severe malarial anemia. Although we
cannot exclude a degree of artifactual hemolysis during blood
sampling, contributing to the variability in cell-free hemoglobin
concentrations, the same method of collection was used in all
subjects and would not account for the increased levels in those
with severe disease nor for the associations with endothelial
NO. Furthermore, the plasma hemoglobin levels we observed
are comparable to findings of other studies [14, 31, 32]. Finally,
our study population was confined to adults, and additional
studies are required to determine whether similar pathogen-
ic processes occur in children with malaria. Because NO bio-
availability is also impaired in African children with cerebral
malaria [6] and cell-free hemoglobin levels are similarly in-
creased in children with malaria [14, 32], it is likely that NO
quenching also contributes to endothelial dysfunction in chil-
dren with SM.
In summary, hemolysis causes more than just anemia in
falciparum malaria. Cell-free hemoglobin increases in propor-
tion to disease severity in malaria and is associated with de-
creased endothelial NO bioavailability, increased endothelial
adhesion receptor expression, proinflammatory cytokinemia,
impaired tissue perfusion, and parasite sequestration. Therapies
considered for use in SCD to decrease the deleterious effects
of cell-free hemoglobin [44], such as haptoglobin infusion, may
have potential efficacy in SM. Treatments that increase NO
bioavailability (eg, l-arginine [3, 45] or nitrite infusion or in-
1528 • JID 2009:200 (15 November) • Yeo et al
haled NO) may also have potential as adjunctive therapies in
SM.
Acknowledgments
We thank Govert Waramori, Marlini Malisan, Margaretha Ferre, Fer-
ryanto Chalfein, Prayoga, Roesmini, and Yoshi Elvi for nursing, technical,
and logistical assistance; Mitra Masyarakat Hospital staff for clinical care;
and Jeanne Rini, Paulus Sugiarto, Mauritz Okeseray, and Lembaga Pen-
gembangan Masyarakat Amungme Kamoro for support. Purified P. falci-
parum HRP2 protein was kindly supplied by David Sullivan.
Downloaded from https://academic.oup.com/jid/article-abstract/200/10/1522/880289 by guest on 12 July 2019
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