Download Oxygen Transport Properties in Malaria-Infected

From www.bloodjournal.org by guest on June 18, 2017. For personal use only.
Oxygen Transport Properties in Malaria-Infected Rodents-A Comparison
Between Infected and Noninfected Erythrocytes
By W. Schmidt, R. Correa, D. Boning, J.H.H. Ehrich, and C. Kruger
This study was performed t o investigate oxygen transport
properties in whole blood (WB) of malaria-infected rats as
well as in infected erythrocytes(IE) and noninfected erythrocytes (NIE) separated by density centrifugation. One week
after inoculation with Plasmodiumberghei, mean parasitemia was 26.5% and high correlations were found between parasitemia and hemoglobinconcentration ([Hb]; r =
-.902), mean cellular Hb concentration (MCHC; r = -.712),
MetHb ( r = .923), and base excess ( r = -.922). Compared
with control animals (C), the oxygen affinity was lower in
WB under standard (pH 7.40) and simulated "in vivo" (pH
, 5.7 and 5.1 m m Hg, respec7.00) conditions (difference in,P
tively; 2P < .01,2P < .05). In IE Hb and2.3-biphosphoglycerate (2.3-BPG) concentrations were decreased (MCHC IE 14.6
? 1.0, NIE 33.1 f 1.7 g/lOO mL; [2,3-BPG]: IE 2.0 f 0.6, NIE 7.6
& 1.8 mmol/L), whereas [MetHb] and [ATP] were increased
([MetHbl: IE 19.0 f 3.7, NIE 0.7% & 0.8%;[ATP]: IE 33.5 f
2.4, NIE 6.2 f 1.0 p m o l l g Hb). At pH 7.40, half-saturation
oxygen tension P
(),
was reduced in IE (29.6 f 2.6, NIE 39.2
f 5.4 mm Hg, 2P < .001), which correlates with lower [2,3-
BPGI, increased MetHb content, and higher intrinsic Hb-0,
affinity. However, at pH7.00, the oxygen affinity was lower
in IE when compared with NIE, which was most likely due
t o high [ATPI in IE. The resulting Bohr coefficients (BC) calculated for COz and lactic acid were extremely high in IE and
low in NIE (at 50% 0,-saturation BCco,: IE -1.04 f 0.06,NIE
-0.26 f 0.10. 2P < .001; BCh,: IE -0.82 f 0.16, NIE -0.47 f
0.07, 2P < .001), which was caused by different [2,3-BPG]
and [ATPI as well as probably by structuralchanges of the
Hb molecule. The 0, capacity was 14.1 mL per 100 mL erythrocytes in IE compared with 44.4 mL/100 mL in NIE. On the
basis of the calculated arterio-venous O2 difference under
"in vivo" conditions, the infected red blood cell fraction
transports 30% of the Oz amount delivered to thetissues by
the noninfected cells (IE 8.0, NIE 26.9 mL/100 mL red blood
cells). We conclude that the0, transport in malaria infected
blood is notonly affected by thedegree of anemia but also
by thepercentage of infected erythrocytes.
0 1994 by The American Societyof Hematology.
0
blood, which wasreferred to decreased pH andlower cellular
concentration of the main allosteric factor 2,3-biphosphoglycerate (2,3-BPG).'",I2
Until now, the Hb-02 affinity was assumed to be similar
in all erythrocytes of the malaria-infected blood. However,
theoretical considerations suggest affinity differences between infected erythrocytes (IE) and noninfected erythrocytes (NIE). In the infected cells, the metabolic pathways
are disturbed, the Hb molecules are partly destroyed or digested, and the cellular milieu has been changed by accumulation of metabolic products of the parasite that decrease the
pH and the concentration of 2,3-BPG.'2,'3On the other hand,
the noninfected cells might be able to respondto the systemic
hypoxia with the "normal" anemic reaction, ie, a right shift
of the ODC by metabolic stimulation of the erythrocytic 2,3BPG produ~tion.'~
The ODC of the malaria-infected blood
therefore should be changed depending on the degree of the
parasitemia and anemia.
Furthermore, all ODCs determined until now from malaria-infected blood were recorded under prevailing conditions, ie, at low pH, and compared with the function of Hb
in noninfected blood at highpH. The aim of thepresent
study was therefore to investigate the ODC in the whole
blood (WB) of malaria-infected rats as well as in the separated IE and NIE under standardized conditions. Data obtained from the separated cell fractions would allow evaluation of the contribution of the infected erythrocytes to the
oxygen transport of the whole infected blood.
NE OF THE MOST obvious pathologic reactions to
the infection with malaria is the rapidly occurring
anemia and the related impairment of the oxygen transport
by the blood. The resulting tissue hypoxia is assumed to
be the main cause of death in rodents after infection with
Plasmodium berghei' or P yoelii2 and is one of the most
severe complications in humans infected with P falciparum,
contributing to the high mortality rate, especially in chilThe anemia is caused by immunopathologic mechanisms leading to cell elimination inthespleen
or in the
peripheral blood, by direct erythrocyte destruction bythe
parasite, and by a suppression of red bloodcell (RBC) formation despite higher erythropoietin concentration in the infected blood.'.'
The pathogenesis of the developing anemia and the obvious effects of lowered hemoglobin concentration [Hb] on
the oxygen transport capacity have been studied in various
experiments. However, there may also be implications for
Hb-oxygen binding properties and adaptive reactions to the
anemia, which have been only rarely investigated. Lowered
"in vivo" Hb-oxygen affinity in the blood of mice infected
with P berghei was first described in 1967.9 These results
could be confirmed later"."by
detection of right-shifted
oxygen dissociation curves (ODCs) in malaria-infected
From the Departments of Physiology and Pediatrics, Medical
School Hannover, Hannover, Germany.
Submitted February 12, 1993; accepted February 15, 1994.
Address reprint requests to Walter Schmidt, PhD, Institut fur
Sportmedizin (OHH),Freie Universitat Berlin, Clayallee 229, 14195
Berlin, Germany.
The publication costsof this article were defrayedin part by puge
charge payment. This article must therefore be hereby marked
"advertisement" in accordance with I 8 U.S.C.section 1734 solely to
indicate this fact.
0 I994 by The American Society of Hematology.
0006-4971/94/8312-0002$3.00/0
3746
MATERIALS AND METHODS
Rats
Female Lewis rats ( 5 to 7 weeks of age) bredin the Central
Animal Laboratory of the Medical School Hannover were used. The
rats were kept under standardized conditions at 22"C, 50% ? 5%
relative humidity, and artificial light under a 12 M12 h lightldarkness
rhythm with food and water ad libitum.
Blood, Vol 83, No 12 (June 15). 1994: pp 3746-3752
From www.bloodjournal.org by guest on June 18, 2017. For personal use only.
3747
02 TRANSPORT IN MALARIA-INFECTED RED CELLS
Parasites and Infection of Rats
Plasmodium berghei bergheiKSP II was originally obtained from
Prof Dr A. Haberkorn (Bayer A.G., Wuppertal, Germany) and was
maintained by regular passage to young female BXSBMPG mice
serving as donor for the investigated animals. The rats were inoculated intraperitoneally with about 3 X lo7 parasitized RBCs suspended in 2 mL saline solution. In both rats and mice, parasitemia
was controlled every second day in blood samples obtained from
blood vessels of the tail. The infected cells were counted using a
blood smear stained with Leishman solution (10 g Leishman dye,
Nr. 17125, 25 Q. George; T. Gun LTD, London, UK; in 1,500 mL
methanol).”
Blood Sampling and Sample Preparation
Between 7 and 10 days after inoculation, when parasitemia yielded
the highest ratio (26.5% 2 12.8%), blood samples were drawn from
a retrobulbar plexus after ether anesthesia and were heparinized
with 10 mUlmL sodium heparin (Liquemin N 25.000; HoffmannLa Roche, Basel, Switzerland). Immediately after the sampling procedure, lasting a total of 2.5 minutes, the animals were killed. One
milliliter of the sample was used for determinations in infected WB
and comparison with blood of noninoculated control animals (C).
The remaining blood (about 2 to 2.5 mL) was usedfor the separation
of IE and NIE.
Separation of the IE
The IE were separated by density centrifugation between discontinuous Percoll gradients (Pharmacia, Uppsala, Sweden) modified
according to the methods of Saul et all6 and Tosta et al.’’ A stock
solution was prepared from 9 v01 pure Percoll solution (22% wt/vol
silicondioxide, pH 8.9, density 1.23 g/mL, 25 mosmolkg H,O) and 1
v01 bicarbonate-free phosphate buffer (10-fold concentrated Hanks’
buffer solution with phenol red, Nr. 47251; Serva Feinbiochemica,
Heidelberg, Germany). The solution was adjusted to 310 mosmol/
kg and pH 7.4 by adding distilled water and HCl. The final gradients
were obtained by mixing this stock solution with bicarbonate containing Hanks’ buffer solution (Nr. 47250; Serva Feinchemica) in
the ratio 9:1, 8:2, 7:3, and 6:4, and adjusting the pH with HCl to
7.385 in each gradient.
Plasma from infected rats was separated by centrifugation and
stored at 4°C. The erythrocytes (400 pL suspended in 2 mL Hanks’
solution; Nr. 47250) were carefully layered on the top of the previously prepared percoll gradients (Perfusor V; B. Braun-Melsungen,
Melsungen, Germany) and centrifuged at25°C for 10 minutes at
2,000s after 2 minutes of slow acceleration. The NIE were collected
at the bottom between the 9: 1 and 8:2 gradients, whereas the infected
mostly mature trophocytes and schizontes) were found at
cells (E;
the top, between 7:3 and 6:4 gradients. The mean parasitemia in
these fractions was 2.2% % 1.9%in NE and 92.1% 2 3.4% in
E,respectively. Only few cells containing about 50% immature
trophocytes (ring forms) were separated between the mean bands
(8:2 and 7:3), but, because of low quantity, this fraction was not
used for further investigations. After washing twice in an equal
volume of Hanks’ solution, the infected and noninfected cells were
resuspended into their own plasma with a target hematocrit (Hct)
value of 30%.
Measurements
Hematologic quantities. In blood samples of all groups we determined the following quantities using the stipulated procedures: Hb
concentration (cyanhemiglobin method; test kit Nr. 3317, Merck,
Darmstadt, Germany); Hct value, microhematwrit centrifugation at
20,900s for 7 minutes; RBC count expressed as cells per microliter;
mean cellular Hb concentration (MCHC in grams per deciliter),
mean cellular Hb content (MCH; in picograms per cell), and mean
cellular volume (MCV; in cubic micrometers) were calculated using
the measured quantities mentioned above; 2,3-BPG concentration
(phospho-glycerate mutase reaction; test kit Nr. 148334; Boehringer
Mannheim, Mannheim, Germany; adenosin-triphosphate (ATP) concentration (phospho-glycerate kinase reaction: test kit Nr. 123897;
Boehringer Mannheim); and lactic acid (Lac) concentration (lactate
dehydrogenase reaction; test kit OSUA 50, 51; Behring, Marburg,
Germany).
Acid-base status was determined in whole blood from parasitefree and infected rats (C and WB) immediately after blood sampling.
Po>, Pco,, and Hb-0, saturation were measured by ABL 330 and
OSM 3 (Radiometer, Copenhagen, Denmark) and the base excess
(BE) was calculated for oxygenated blood. Concentrations of methemoglobin (MetHb), CO-hemoglobin (CO-Hb), and sulfhemoglobin
(Sulf-Hb) were measured in all blood samples using the OSM 3.
The principle of OSM 3 is spectrophotometric measurement at 6
wavelengths using a grating monochromator. To exclude any artefacts on MetHb measurement, [MetHb] was controlled spectrophotometrically at 630 nm according to Evelyn and Malloy,“ yielding
identical results (range between 0% and 97%, r = .97, P < .001, n
= 7).
ODC. The ODCs were obtained using a modified Hemoxanalyser system (TCS Medical Products CO, Huntingdon Valley, PA),
which allows simultaneous registration of Po, (Clark electrode) and
Hb-0, saturation (dual wavelength photometer) at constant pH and
Pco, in small blood ~amples.’~
To exclude possible influences of Hb
fragments on the registration of Hb-02saturation spectra of oxygenated and deoxygenated Hb from IE and NIE were recorded between
400 and 700 nm. But no signs of disturbing Hb fragments could be
detected.
Fifty microliters of blood were suspended in a plasma-like bicarbonate buffer solution (1 13.7 mmol/L NaCI, 26.6 mmol/L NaHC03,
4.4 mmom KH2FQ,, 1.2 mmol/L CaCI2, 1.1 mmol/L MgSO,, and
5.0 mmol/L glucose) and equilibrated with the following gas mixtures at 37°C. (1) To obtain the ODC under approximated standard
conditions, the equilibration gas consisted of 6% COz and 94% Oz
during oxygenation and 94% NZ during deoxygenation, yielding a
pHof about 7.40. (2) To estimate the effects of high Pco2 on the
ODC, the CO, content of the equilibration gas was increased to 10%
during oxygenation and deoxygenation corresponding to a pHof
about 7.23. (3) A curve under simulated “in vivo” conditions was
registered at a Pco2 value as during ( l ) and the pH was titrated to
about 7.00”’ using 1.0 N lactic acid (Nr. 0125440/035; Boehringer
Mannheim).
The Bohr coefficients (Alog Po,/ApH) were calculated at constant
saturation for acidification with COz (BCco,, curves 1 and 2 ) and
lactic acid (BCLac,curves 1 and 3). The individual BCLacvalues were
then used to calculate the exact ODCs under standard (pH 7.40, 6%
COz) and simulated “in vivo” conditions (pH 7.00, 6% COz).
Hill’s “n” as an indicator of the cooperativity of the Hb molecule
was calculated from the slope of the curves in the logarithmic diagram (log Po, v log [S0,/100 - SoJ) between 40% and 60% So,.
Statistics
All results are presented as means (X) and standard deviations
(kSD). Statistical comparisons were performed between C and W B
as well as between NIE and IE. A two-factorial analysis of variance
(ANOVA) was applied to compare the complete ODCs and saturation-dependent Bohr coefficients. Differences of single means between two groups were checked by Student’s r test (unpaired r-test
between C and WP, and paired t-test between IE and N E ) . In these
cases, the degree of significance was indicated by 2P (two-tailed).
From www.bloodjournal.org by guest on June 18, 2017. For personal use only.
3748
SCHMIDT ET AL
Linear regression analysis was used to investigate the correlation
between the degreeof parasitemia in the infected WB and the hematologic and acid-base properties.*’
Table 1. Hematologic Data of WB and Separated Cell Fractions
C
WB
X
0
SD
n
8
26.5
12.8
15
92.1
3.4
11
2.2
1.9
15
14.8
2.3
8
5.6’
1.1
15
4.5*
1.3
11
10.6
2.3
15
44.2
7.7
8
24.6*
2.9
15
29.5
8.1
10
32.0
5.9
15
8.2
0.7
6
4.2t
1.3
6
2.lt
0.4
7
4.9
1.6
7
33.2
1.4
8
22.7*
3.6
15
14.6*
1.o
10
33.1
1.7
15
13.9
0.4
6
13.6
2.4
6
11.5*
1.2
7
13.6
1.4
7
45.6
3.2
6
56.4
9.6
6
86.3’
8.0
7
40.3
4.6
7
0.3
0.2
8
4.8*
3.1
15
19.0’
3.7
11
0.7
0.8
9
7.0
1.5
8
3.9’
0.7
10
2.0*
0.6
9
7.6
1.8
12
20.9
4.4
8
17.6
4.1
11
13.2*
4.2
9
23.2
3.2
12
4.5
1.3
6
11.0t
4.5
6
33.5’
2.4
7
6.2
1.o
7
4.2
1.o
8
9.7‘
3.3
13
10.1*
2.3
11
6.9
2.2
13
IE
NIE
Parasitemia (%)
-
RESULTS
Hematologic Data
The infection of young female rats with P berghei b resulted in marked changes in hematologic parameters that
govern oxygen transport (Table 1). In WB, [Hb] was decreased by 62% and Hct by 44%, reflecting a decrease in
MCHC of 32%. The MCHC of NIE was in the normal range,
whereas it was dramatically reduced by 56% in IE. MCH
did not differ between the whole blood of infected and control animals and tended to decrease (15%) in IE. MCV was
slightly elevated in WB when compared with C andincreased more than twofold in IE.
MetHb concentration was clearly increased in WB, which
was caused by the oxidation of 19% of the Hb in the infected
fraction. No effects on CO-Hb or Sulf-Hb level could be
detected in WB or IE.
Despite lower erythrocytic 2,3-BPG concentration (mmoll
L) in WB than in C, the 2,3-BPG/Hb ratio (pmollg Hb)
was not significantly reduced. Comparing the separated cell
fractions, [2,3-BPG] was clearly decreased in IE bothin
regards to intraerythrocytic concentration and 2,3-BPG/Hb
ratio. In the NIE, there was no significant increase in [2,3BPG] as would be expected for anemic animals. [ATP] was
changed in opposite direction than [2,3 BPG], being normal
in NIE and increased fivefold in IE.
The acid base status observed in infected WB immediately
after blood sampling was characterized by a noncompensated
metabolic acidosis, which was indicated by the accumulation
of lactic acid (Table l), decreased pH (C: 7.34 +- 0.03, WB:
7.08 5 0.13, 2P < .Ol), and lowered base excess (C: -3.0
? 1.2, WB: -10.9 ? 5.7 mmol/L, 2P < .001).
[Hbl (g/dL)
-
X
SD
n
Hct (%)
X
SD
n
RBC (lo6cells/pL)
-
X
SD
n
MCHC (g/dL)
X
SD
n
MCH (pg/cell)
X
SD
n
MCV ( p m 3 )
X
SD
n
MetHb (%)
-
X
SD
n
12.3-BPGI (mmol/L En/)
X
SD
n
L2.3-BPGI (pmol/g Hb)
-
X
ODCs
The ODCs of the WB and of the separated fractions obtained under standard and under simulated ‘‘in vivo’ ’ conditions are depicted in Fig 1A and B. The standard curve of
WB was significantly shifted to right side (oxygen pressure
at 50% saturation of the Hb, P50, increased from 36.3 ? 2.6
mmHgin C to 42.0 +- 3.2 mm Hg in WB, 2P < .01).
Comparing the curves of the separated cell fractions, the
ODC of IE was markedly shifted to the left side (ANOVA
P < .001) and the P50 was correspondingly reduced (29.6 5
2.6 mm Hg in IE, 39.2 t 5.4 mm Hg in NIE, 2P < .001;
Fig 1B).
The slope “n” of the ODC at 50% saturation indicating
the cooperativity between the Hb subunits was similar in C
and WB (approximately 2.3), but was markedly reduced in
the infected cell fraction (IE 1.68 +- 0.13 U; NIE2.28 ?
0.22 U, 2P < .001).
When recording the ODCs under simulated “in vivo”
conditions (lactacidosis, pH 7.00), a similar difference in the
curves between WB and C could be detected as under standard conditions (ANOVA P < .05, Fig lA), ie, a higher P50
of the infected blood (62.6 ? 7.9 mm Hg in C, 67.7 +- 3.6
SD
n
[ATPI (pmol/g Hb)
X
SD
n
ILacl (mmol/L)
X
SD
n
Presented are means( 3 and standard deviations (SD) inC, WB, as
well as in the separated IE and NIE fractions resuspended in plasma.
Significance of differences (t-test) between C and WB as well as between IE and NIE are indicated by the symbols.
’2 P < ,001.
t 2 P < .01.
2P < .05.
*
mm Hg in WB, 2P < .05). However, the curve of IE was
more sensitive to acidosis than that of NIE, as evident from
the significantly right-shifted “in vivo” curve (ANOVA P
< .05, Fig 1B) and higher P50 in IE (68.1 ? 9.0 mmHg)
From www.bloodjournal.org by guest on June 18, 2017. For personal use only.
3749
O2 TRANSPORT IN MALARIA-INFECTED RED CELLS
A
B
100
pH 7.40
x
5
ii
601
m
2
O
0
!
~
20
"
I
40
~
I
60
.
1
"
80 120
100
I
~
'
02-pressure (rnrnHg)
02-pressure (rnrnHg)
Fig 1. ODCs for standard (pH 7.40) and simulated "in vivo" conditions (lactic acidosis resulting in pH 7.00) in (A) malaria-infected WB (n =
11) and C (n = 8 ) as well as in (B) separated IE (n = 6) and NIE In = 6). For statistical significance of differences, see text.
when compared with NIE at pH 7.00 (60.9 f 4.8 mm Hg,
2P < .OS).
This unexpected effect is expressed by the Bohr coefficients (BCs). The BCs determined when blood was acidified
with COz (BC,,,) and lactic acid (BC,) showed no significant differences between C and WB (Fig 2).However, a
strong increase of bothcoefficientsoccurredinIEand
a
significant decrease in NIE (Fig 2,ANOVA P < .001 in
both cases). This divergence was most pronounced at low
Hb-02saturation (So2 20%)when BC,,, andBCL,were
- 1.18 ? 0.15 and -0.76 2 0.26 in IE compared with -0.06
? 0.12 and -0.30 f 0.14 in NIE, respectively(in both cases,
2P < .001).
A
-1.2
-1.0
-0.8
0
-0.6
0
0
-0.4
(U
m
-0.2
0.0
DISCUSSION
I
0
Hematologic Data
All the hematologic quantities measured in WB samples
are influenced by the infection and are wellcorrelated to the
degree of parasitemia. Closest correlations were obtained
between parasitemia and BE ( r = -.922), [Hb] (I = -.902),
pH ( r = -.820, in all cases P < .001), [Lac] (I = +.758),
MCHC ( r = -.712), and Hct (-.643, in these cases P <
.Ol).
In IE, the decrease in [Hb] is more pronounced than that
in Hct. This is caused by lower erythrocytic [Hb] (MCHC)
and can be explained by the following two mechanisms.
(1) Hb is digested in the acidic food vacuoles expelled
by the parasite that contain acidic endopeptidase^.^"^^ By
this way the parasite destroys about 15% of the intracellular
Hb content, which is shown by the decrease in MCH (Table
1).
(2) The molecular changes of the erythrocyte membrane
as well as the decrease in intracellular pH" result in increased intracellular ion and water content reducing the intraerythrocytic [Hb].25 As is clearly shown by the increase
in MCV, this mechanism exerts more influence on MCHC
than thedirect destruction of the Hb molecule by the parasite.
Before the digestion by the parasite occurs, the Hb mole-
.
I
'
40
20
I
'
1
.
80
60
f
100
Hb-Op-saturation ( X )
B
-1.0
-0.8
l
-/
0.01
0
,o-o\o
-o-o-o"o
P
.
I
20
*
I
4080
.
,
60
.
,
.
,
100
Hb-02-saturation ( X )
Fig 2. BCs (Alog PoJApH. determined over the whole saturation
range) for CO, (A) and lactic acid (B) in the infected WB and in C as
well as in the separated IE and NIE. For the sake of clearness, the
standard deviation (STD) is not indicated in the figure. STD at 50%
saturation for BCco2:WB 0.04, C 0.22, IE 0.06, and NIE 0.10; for Bf&:
WB 0.06, C 0.10, IE 0.16, and NIE 0.07. (0)C; (A) WB; ( 4 ) NIE; 10)IE.
From www.bloodjournal.org by guest on June 18, 2017. For personal use only.
SCHMIDT ET AL
3750
cule is autoxidized to MetHb.26.27
This change is shown here
by the strong relationship between parasitemia and MetHb
concentration ( r = .932, P < .001) thatwasmeasuredby
two different methods and is also in accordance with previous data.” The transformation to MetHb only takes place in
the IE and blocks 19% of the Hb for the O2 transport.
Standard ODCs
The standard curve (pH 7.40, Pc,, 42 mm Hg) obtained
from the infected WB with a mean parasitemia of 26.5% is
shifted to the right side when compared with the standard
ODC of control animals (A5.7 mm Hg). In comparison to
these relatively small differences in Pso, there was a remarkable divergence in all Hb-0,-binding properties in IE and
NIE. Whereas the position of the ODC of NIE (Pso 39.2mm
Hg) did not differ from that of WB at pH 7.40,Hb-0, affinity
was markedly increased in the infected cells under standard
conditions (Pso 29.6 mm Hg). For the “mixed” ODC of
WB, one would expect the Pso to be between those of IE
and NIE, which cannot be demonstrated here. This is partly
caused by differences in sample size and could furthermore
be attributed to the separation procedure of IE and NIE that
may influence the O2affinity in both fractions. An additional
allosteric effector such as lactate might be washed out or the
Donnan ratio is changed by metabolic effects. The differences in Pso between IE and NIE may be explained by the
following mechanisms.
(1) The 2,3-BPG molecule is the main physiologic allosteric effector in mammals. Thus, a reduction of its concentration leads to higher Hb-0, affinity, resulting in a left shift
of the ODC and lower cooperativity of the Hb subunits. The
2,3-BPG/Hb (moVmol) ratio, being 1.36 in C, was clearly
reduced in IE (0.88) and slightly increased in NIE (1.48),
explaining a great part of the difference in the position of
the ODC in IE and NIE.
(2) According to the law of mass action, the interaction
between Hb and 2,3-BPG not only depends on their ratio
but also on their absolute concentration^.^^ In human blood,
a decrease in MCHC by 1 g/lOO mL erythrocytes reduces
Psoby 0.5 mmHg?’ This mechanism gains importance in
the IE, in which MCHC decreases by more than 50% to 14.6
gil00 mL (Table 1).
(3) In the IE, 19% of theHb is autoxidized to MetHb.
The oxidized subunits do not transport oxygen, and also
influence the position of the ODC by enhancing the oxygen
affinity of the remaining Hb subunits leading to a left-shifted
ODC.
(4) Part of the Hb in the infected cells is always being
digested by the parasite. It is not known whether the partly
destroyed molecule is able to transport oxygen and how it
influences the position and slope of the ODC. The results of
another repod’ indicate molecular changes in the Hb leading
to lower 2,3-BPG and temperature sensitivities and higher
intrinsic O2 affinity in Hb solutions isolated from infected
RBCs. Furthermore, dramatic changes in the composition of
the intraerythrocytic milieu (variation of the Donnan equilibrium, [lactic acid], [Cl-]) may exert various allosteric effects
on the Hb molecule that cannot be quantitatively estimated.
The reduced “n” value in IE can also be explained by
( l ) lower [2,3-BPG]19; (2) increased MetHb, because of the
interaction of only three or less subunits in a tetramer; and
(3) lower intrinsic cooperativity of Hb.”
BC
Because of common binding sites of COz and 2,3-BPG at
the deoxygenated @-chains, the specific COz (carbamatenegative) effect on Hb-Oz affinity is mostpronouncedat
low S,, in the absence of 2,3-BPG.32Therefore, particularly
the BCco,must be higher in IE than in NIE at low S,,,
which is clearly shown in this study. Whereas thelow
BCco, in NIE is caused by high 2,3-BPG, the extremely high
BC in IE cannot completely be explained by this factor. As
is shown in another report,” the BC for fixed acids is higher
in Hb solutions from IE than from NIE, indicating influences
of structural changes of the Hb molecule itself on this parameter. Furthermore, lower intracellular pH in IE than in
NIE24,33
and, therefore, different Donnan equilibrium hints
to different intracellular concentrations of small anions, eg,
Cl-, that are known to increase the BC by their pH-dependent
binding to specific amino acid residues in the a- and p-
chain^.'^^'^
In addition, in the infected cells, the concentration of the
allosteric factor ATP is controlled by the parasite.” ATP is
produced in the parasitic compartment and released into the
host cell by an adenylate translocator leading to similar concentrations in both compartments under “in vivo” condition~.’~
In our experiments, [ATP] is increased fivefold in
the parasite-cell complex (Table l), suggesting remarkable
ATP-influences on Hb-02 affinity in IE. This result of high
concentration of the allosteric factor ATP in the IE could
explain a number of puzzling features of the RBC oxygen
binding curves, including the surprisingly low oxygen affinity in RBCs under “in vivo” conditions despite containing
a large amount of MetHb and lowered [2,3-BPG]. Furthermore, the high ATP levels are likely to explain most of the
increase in the alkaline Bohr effect.
The BCs in WB represent the mean of the effects in IE
and NIE. Because of a higher count of NIE in the infected
WB, both BCs tend to lower values in WB than in the blood
of the control animals.
“In Vivo” ODC
The effects of the different BC are clearly shown by the
ODC measurements under “in vivo” conditions (Fig 1). For
pH values of 7.13 or lower, the ODC of IE is more right
shifted than that of N E . Thus, the right shift of the ODC of
the infected whole blood under “in vivo” conditions can be
referred to the infected and noninfected cells, despite the fact
that their oxygen-binding properties are absolutely different
under standard conditions.
Practical Importance
Tissue hypoxia of brain and kidneys are severe complications during malaria infections resulting from the anemia
and the impairment of the rheologic properties of the E.’
Using the present data, the Hb-02transport capacity is 19.8
mL 02/100 mL in the control animals and 7.1 mL Oz/lOO
From www.bloodjournal.org by guest on June 18, 2017. For personal use only.
3751
0,TRANSPORT IN MALARIA-INFECTED RED CELLS
v
0
od‘ 4 ° r20: -
l
40
’
I
60
-
I
80
I
100
m -
120
02-pressure (mrnHg)
Fig 3. Calculated arteriovenous O2 difference in 100 mLIE and
NIE. (Assumed arterial Poz:100 m m Hg, venous Po,: 20 m m Hg.) ( 01
NIE; (0)
IE.
mL in the infected WB. Under these circumstances, the O2
transport capacity and Hb-02binding properties of the single
erythrocyte gain importance. Because of lower intracellular
[Hb] and higher [MetHb], the maximal oxygen loading
amounts only to 14.l mL O2per 100 mL of the IE in comparison to 44.4 mL 02/100 mL in NIE. In addition, the right
shift of the ODC by the effects of low blood pH is disadvantageous for oxygen loading. At pH 7.00, the arterial saturation at Po, 100 mm Hg amounts to 70% in IE and 76% in
NIE and the theoretical arteriovenous O2difference (venous
Po, 20 mm Hg, equal pH) is only 8.0 and 26.9 mL per 100
mL RBCs in IE and NIE, respectively (Fig 3). It may furthermore be noted that O2 release from large erythrocytes as IE
is slower than that from smaller ones, suggesting slower
kinetics in the infected cells. Thus, wemay state that the
oxygen transport in malaria-infected blood is not only determined by the degree of anemia but also by the percentage
of IE that are characterized bylow
Hb content, high
[MetHb], and disadvantageous “in vivo” Hb-02 binding
properties.
The results of this animal model should be validfor human
malaria (Pfulcipurun),in which severe anemia ([Hb] lower
than 7 g/dL) is also widely distributed. Whereas parasitemia
is generally lower than 5% in repeatedly infected natives of
endemic malaria areas, high parasitemia up to 70% is observed in c a u c a s i a n ~because
~~
of late diagnosis. In combination with decreased renal and cerebral perfusion, deterioration in oxygen supply by disadvantageous O2 transport
properties in the infected cells may contribute to the bad
prognosis in these patients.39
ACKNOWLEDGMENT
We express our thanks to ProfHaberkom (Bayer A.G., Wuppertal,
Germany) for providing the parasite. We are also gratefully indebted
to Eva Schubert and P. Mertz for skillful technical assistance.
REFERENCES
1. Ehrich JHH, Beck EJ, Haberkom A, Meister G: Causes of
death in lethal rat malaria. Trop Med Parasitol 35:127, 1984
2. Yoeli M:Cerebral malaria-The quest for suitable experimental models in parasitic diseases of man. Trans R Soc Trop Med Hyg
70:24, 1976
3. Warrel DA, White NJ, Veal1 N,Looareesuwan S , ChanthavanichP, Phillips RE, Karbwang J, Pongpaew P: Cerebral anaerobic
glycolysis and reduced cerebral oxygen transport in human cerebral
malaria. Lancet 3534, 1988
4. Weatherall DJ, Abdalla S : The anemia of P. fakiparum. Br
Med Bull 38:147, 1982
S . Beaumelle BD, Vial HJ: Uninfected red cells from malariainfected blood: Alteration of fatty acid composition involving a serum protein: An in vivo and in vitro study. In Vitro Cell Dev Biol
24:711, 1988
6. Brinkmann V, Kaufmann HE, Simon MM, Fischer H: Role of
macrophages in malaria: 0,metabolite production and phagocytosis
by splenic macrophages during lethal Plasmodium berghei and selflimiting Plasmodium yoelii infection in mice. Infect Immun 44:743,
1984
7. Gupta CM: Red cell membrane alterations in malaria. Indian
J Biochem Biophys 25:20, 1988
8. Miller KL, Schooley JC, Smith U, Kullgren B: Inhibition of
erythropoiesis by a soluble factor in murine malaria. Exp Hematol
17:379, 1989
9. Palaceck F, Palacekova M, Aviado MD: Pathologic physiology
and chemotherapy of Plasmodium berghei 11. Oxyhemoglobin dissociation curve in mice infected with sensitive and resistent strains.
Exp Parasitol 21:16, 1967
10. Krishna S, Shoudbridge EA, White NJ, Weatherall DJ, Radda
GK: Plasmodium yoelii: Blood oxygen andbrain function in the
infected mouse. Exp Parasitol 56:391, 1983
11. Hioki A, Yoshino M, Kano S, Ohtomo H: Pathophysiology
of hypoxia in mice infected with Plasmodium berghei. Parasitol Res
73:298, 1987
12. Ali SN, Fletcher KA: The concentration of 2,3-diphosphoglycerate in malarial blood (P. berghei malaria). Int J Biochem 5:17,
1974
13. Sander BJ, Kruckeberg WC: Plasmodium berghei: Glycolytic
intermediate concentrations of the infected mouse erythrocyte. Exp
Parasitol 52:1, 1981
14. Boning D, Enciso G: Hemoglobin-oxygen affinity in anemia.
Blut S4:361, 1987
IS. Walker AJ: Manual of the Microscopic Diagnosis of Malaria.
Washington, DC, World Health Organization, 1960
16. Saul A, Myler P, Elliot T, Kidson C: Purification of mature
schizonts of Plasmodium fakiparum on colloidal silica gradients.
Bull WHO 60:755, 1982
17. Tosta CE, Sedegah M, Henderson DC, Wedderbum N: Plasmodium yoelii and Plasmodium berghei: Isolation of infected erythrocytes from blood by colloidal silica gradient centrifugation. Exp
Parasitol 50:7, 1980
18. Evelyn KA, Malloy HT: Microdetermination of oxyhemoglobin, methemoglobin, and sulfhemoglobin in a single sample of blood.
J Biol Chem 126:655, 1938
19. Schmidt W, Boning D, Braumann KM: Red cell age effects
on metabolism and 0,-affinity. Resp Physiol 68:215, 1987
20. Winer BJ: Statistical Principles in Experimental Design. New
York, NY, McGraw, 1971
21. Sherman IW: Metabolism and transport of parasitized erythrocytes in malaria, in Evered D, Whelan J (eds): Malaria and the
Red Cell. Ciba Foundation Symposium 94. London, UK, Pitman,
1983, p 206
22. Goldberg DE, Slater AFG, Beavis R, Chait B, Cerami A,
Henderson GB: Hemoglobin degradation in the human malaria
pathogen Plasmodium fakiparum:A catabolic pathway initiated by
a specific aspartic protease. J Exp Med 173:961, 1991
From www.bloodjournal.org by guest on June 18, 2017. For personal use only.
3752
23. Vander Jagt DL, Hunsaker LA, Campos NM, Scaletti JV:
Localisation and characterisation of hemoglobin-degrading aspartic
proteinases from the malarial parasite Plasmodium falciparum. Biochim Biophys Acta 1122:256, 1992
24. Mikkelsen RB, Tanabe K, Wallach D F Membrane potential
of plasmodium-infected erythrocytes. J Cell Biol 93:685, 1982
25. Tanabe K, Kageyama K, Takada S: An increase ofwater
content of erythrocytes infected with plasmodium yoelii. Trans R
Soc Trop Med Hyg 80:546, 1986
26. Sherman I W : Amino acid metabolism and protein synthesis
in malarial parasites. Bull WHO 55:265, 1977
27. Yamada K A , Sherman I W : Plasmodium lophurae: Composition and properties of hemozin, the malarial pigment. Exp Parasitol
48:61, 1979
28. Bhattacharya J, Swarup-Mitra S: Role of Plasmodium vivan
in oxidation of haemoglobin in red cell of the host. Indian J Med
Res 83:111, 1986
29. Gros G, Rollema HS, Jelkemann W, Gros H, Bauer C, Moll
W: Net charge and oxygen affinity of human hemoglobin are independent of hemoglobin concentration. J Gen Physiol 72:765, 1978
30. Bellingham AJ, Detter JC, Lenfant C: Regulatory mechanisms
of hemoglobin oxygen affinity in acidosis and alkalosis. J Clin Invest
50:700, 1971
SCHMIDT ET AL
31. Weber RE, Boning D, Schmidt W, Correa R, Fago A: Effects
of erythrocytic plasmodium infection on oxygenation characteristics
of rat hemoglobin. Blood (in press)
32. Duhm J: Dual effect of 2,3-diphosphoglycerate on the Bohr
effect of human blood. Pfluegers Arch 363:55, 1976
33. Sander BJ, Lowery MS, Kruckeberg W: Glycolytic metabolism in malaria infected red cells. The Red Cell: Fifth Ann Arbor
Conference. New York, NY, Liss, 1981, p 469
34. Imai K: Allosteric Effect in Haemoglobin. Cambridge, UK,
Cambridge, 1980
35. Weber RE: Use of ionic and zwitterionic (TrislSisTris and
HEPES) buffers in studies on hemoglobin function. J Appl Physiol
72:1611, 1992
36. Roth E: Plasmodium falciparum carbohydrate metabolism: A
connection between host cell and parasite. Blood Cells 16:453, 1990
37. Kanaani J, Ginsburg H: Metabolic interconnection between
thehumanmalarial parasite Plasmodium fakiparum and its host
erythrocyte. J Biol Chem 264:3194, 1989
38. Weber MW, Boker K, Horstmann RD, Ehrich JHH:Renal
failure is a common complication in non-immune Europeans with
Plasmodium falicparum malaria. Trop Med Parasitol 42: 1 15, 1991
39. Warell DA: Treatment of severe malaria. J R SOCMed 82:44,
1989
From www.bloodjournal.org by guest on June 18, 2017. For personal use only.
1994 83: 3746-3752
Oxygen transport properties in malaria-infected rodents--a
comparison between infected and noninfected erythrocytes
W Schmidt, R Correa, D Boning, JH Ehrich and C Kruger
Updated information and services can be found at:
http://www.bloodjournal.org/content/83/12/3746.full.html
Articles on similar topics can be found in the following Blood collections
Information about reproducing this article in parts or in its entirety may be found online at:
http://www.bloodjournal.org/site/misc/rights.xhtml#repub_requests
Information about ordering reprints may be found online at:
http://www.bloodjournal.org/site/misc/rights.xhtml#reprints
Information about subscriptions and ASH membership may be found online at:
http://www.bloodjournal.org/site/subscriptions/index.xhtml
Blood (print ISSN 0006-4971, online ISSN 1528-0020), is published weekly by the American
Society of Hematology, 2021 L St, NW, Suite 900, Washington DC 20036.
Copyright 2011 by The American Society of Hematology; all rights reserved.