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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. 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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. 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