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FJ
EXPRESS SUMMARY ARTICLE The Full-length version of this article is also available, published online February 6, 2006 as doi:10.1096/fj.05-5122fje. |
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* Department of Biomedical Engineering, Lerner Research Institute, The Cleveland Clinic Foundation, Cleveland, Ohio, USA;
Institute of Pathology, Case Western Reserve University School of Medicine, Cleveland, Ohio, USA;
Department of Chemical and Biomolecular Engineering, The Ohio State University, Columbus, Ohio, USA; and
The Center for Global Health and Diseases, Case Western Reserve University, Cleveland, Ohio, USA
1Correspondence: Department of Biomedical Engineering/ND20, The Cleveland Clinic Foundation, 9500 Euclid Ave., Cleveland, OH 44195, USA. E-mail: zborowm{at}ccf.org
SPECIFIC AIMS
The aim of the study was to measure of the malaria parasite-infected erythrocytes induced by the applied magnetic field and test it for correlation with the parasite development stage on synchronized Plasmodium falciparum cultures. We postulated that as the proportion of high-spin ferriheme sequestered in the crystalline malarial pigment (hemozoin) increases as a result of the parasite metabolism, it could serve as an intrinsic magnetic label of the intra-erythrocytic parasite development, with potential applications to investigations of the parasite toxic heme management and anti-malarial drug development.
PRINICIPAL FINDINGS
1. The magnetophoretic mobility of P. falciparum infected erythrocytes increases with development from ring to schizont form of the parasite but does not exceed mobility of the deoxygenated erythrocyte
P. falciparum HB3 clone in human erythrocytes were cultivated at 5% hematocrit in albumax II complete medium (RPMI-1640 supplemented with 25 mg/mL HEPES, 2 mg/mL sodium bicarbonate, 5% albumax II) at 37°C in an atmosphere of 5% CO2, 5% O2, and 90% N2, with once or twice daily media changes. Parasitized erythrocytes with ring forms were treated with 5% D-sorbitol. This procedure was repeated 4 h after the first treatment to eliminate other forms and synchronize the culture. After treatment, the cells were washed twice and resuspended in the complete media and cultured for 24, 36, 38, and 45 h to allow continued development to early trophozoite, late trophozoite, early schizont, and late schizont stages, respectively (Fig. 1
). A maximum expected level of parasitemia for these conditions is 
10%. Parasitized erythrocytes were counted per 104 erythrocytes. Five hundred parasitized erythrocytes were observed and evaluated percentage of the following 5 forms: rings, early trophozoites, late trophozoites, early schizonts, and late schizonts. Listed by the experiment number, the corresponding percentages and parasitemias (in parentheses) were: 1) 51.8, 48.2, 0, 0, 0 (12.0%); 2) 0, 7.2, 73.6, 12.4, 6.8 (7.5%); 3) 0, 0, 70.0, 25.0, 5.0 (4.0%); 4) 0, 0, 19.2, 55.6, 25.2 (6.0%); and 5) 6.5, 0, 10.0, 0, 83.5 (6.6%), for a total of 5 experiments. Experiments were performed using a microscope observation area of 1.72 mm x 1.27 mm in an isodynamic magnetic field of 1.26 T, and mean field gradient of 0.140 T/mm. The cell motion was observed with a x5 microscope objective and x2.5 photo eyepiece coupled to and recorded by a Cohu CCD 4915 camera with a µ-Tech Vision 1000 PCI Bus Frame Grabber run from a personal computer. We first measured the mobility distribution of a control, uninfected erythrocyte sample and used it to define a cut-off mobility, m0, to discriminate for the magnetic effect in the infected erythrocytes. The cut-off mobility was defined as the 95th percentile mobility of the cumulative mobility frequency distribution. The magnetic erythrocyte population with mobilities higher than m0 was further divided into quartiles and characterized by the minimum (the lowest mobility that is higher than m0), 25th percentile, median, 75th percentile, and the maximum mobility of the magnetic erythrocyte fraction.
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The uninfected erythrocytes magnetophoretic mobilities and the corresponding volume magnetic susceptibilities were distributed normally (Fig. 2
A). A small, negative value of the mean peak net susceptibility indicated that the erythrocytes were oxygenated and in equilibrium with the ambient air. A highly symmetrical distribution around the mean indicated contribution of random errors of measurement not related to the magnetic field, such as the effects of gravitational sedimentation. The cut-off mobility was m0 = 0.75 x 10–6 mm3 s/kg. Predominantly ring and predominantly schizont cultures differed markedly in their magnetophoretic mobility and the net magnetic susceptibility distributions, Fig. 2B, C
. In the predominantly rings and early trophozoites sample (Expt. 1), 89% of the cells had mobility below the cut-off mobility, m0. The "magnetic" cell fraction, 11% in Fig. 2B
, was comparable to the infected cell fraction in the sample, 12% as determined by differential cell counting. The culture with predominantly late schizont forms (Expt. 5) contained 62% of cells that showed increased magnetophoretic mobility as compared with control, Fig. 2C
. That percentage was higher than expected from the sample parasitemia, 6.6% (Table 1). This was attributed to the known difference in gravitational sedimentation rate between uninfected and schizont-laden erythrocytes causing disappearance from the CTV field of view of the low magnetic mobility but fast sedimenting, uninfected erythrocytes and the apparent enrichment in the high magnetic mobility but slowly sedimenting infected erythrocytes. The 25–75 percentile mobility range was from 1.35 x 10–6 to 2.95 x 10–6 mm3 s/kg (Fig. 2C
) and was significantly higher than that in the sample devoid of schizonts, 0.85 x 10–6 to 1.25 x 10–6 mm3 s/kg (Fig. 2B
). Cultures from experiments 2–4 populated by increasing percentages of mature trophozoites and schizonts were also analyzed. The results were consistent with the results of experiments 1 and 5. The magnetophoretic mobility of the schizont form did not exceed that of the normal, deoxygenated erythrocytes and that of the erythrocytes with the hemoglobin converted to high-spin methemoglobin, as indicated in Fig. 2
.
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2. The fraction of hemoglobin converted to hemozoin, calculated from magnetophoretic mobility of live erythrocytes and the known magnetic susceptibilities of their constituents, agrees with the fractions measured by biochemical and crystallographic methods on fixed specimens
The calculations of erythrocyte magnetic susceptibility were based on the published data on hemoglobin susceptibilities by Pauling et al. in the 1930s using Guoy balance and, more recently, from nuclear magnetic resonance spectroscopy, superconducting quantum interference device, Mössbauer spectroscopy, and recently available information on hemozoin crystal structure and susceptibility. Here we assume, after Pauling, that there are no unpaired electrons in the oxyhemoglobin heme group, and that the hemozoin heme electron configuration corresponds to that of a ferriheme with 5 unpaired electrons, S = 5/2. The various contributions to the erythrocyte volume magnetic susceptibility are:
 | (1) |
H2O and Hb are the volume fractions of water and hemoglobin in a normal (uninfected) erythrocyte, and 
H2O and globin are the volumetric magnetic susceptibilities of water and the protein (globin) part of hemoglobin, respectively. Also, z is the fraction of hemoglobin heme converted to hemozoin heme in the infected erythrocyte and ferri is the magnetic susceptibility of ferriheme. For an erythrocyte suspension in equilibrium with ambient air, the intact hemoglobin is fully saturated with oxygen and its 4 heme groups do not contribute to the erythrocyte magnetic susceptibility; therefore, the expected changes in the erythrocyte magnetic susceptibility with the parasite development stage depend on the hemozoin fraction alone, z. Also, Hb ferri = nHb 'ferri, where 'ferri = 57,428 x 10–6 cm3/mol, and nHb = 5.5 mM is the intracellular molar concentration of hemoglobin. H2O = 1 – nHb 
Hb, where Hb = 48,277 cm3/mol is the molar volume of hemoglobin, and Hbglobin = nHbMHb'globin, where MHb = 64,450 is the molecular weight of hemoglobin, and 'globin = –0.580 x 10–6 cm3/g is the specific susceptibility of globin in the hemoglobin molecule. The experimentally established relationship between the net magnetic susceptibility and the erythrocyte mobility, m, is
 | (2) |
= RBC – H20 and H2O = –0.719x10–6 is the magnetic susceptibility of water. By comparing the measured and calculated net magnetic susceptibilities, one finds the fraction of hemoglobin converted to hemozoin in the infected erythrocyte, z, from Eq. 1
. Thus, at the 75th percentile mobility of the schizont-rich magnetic fraction, equal to m = 2.94 x 10–6 mm3 s/kg, Fig. 2C
, one obtains = 1.80 x 10–6, corresponding to the converted hemoglobin fraction of z = 0.50. In other words, the results show that 75% of schizont-rich erythrocytes that move in the magnetic field faster than the uninfected control contain at least 50% hemoglobin heme groups converted to a high-spin form characteristic of hemozoin. The respective values calculated for the ring/early trophozoite culture, 15% (Expt. 1) and cultures containing maturing trophozoites/schizonts (19–21% for experiments 2–4) indicate increase of the high-spin heme fraction associated with hemozoin, consistent with the schematics shown in Fig. 1
. CONCLUSIONS AND SIGNIFICANCE
Published data on blood stage parasite physiology show that within infected erythrocytes, P. falciparum consumes 50–80% of the cytosolic hemoglobin (molar concentration of 5 mM in the erythrocyte’s volume of 88.4 µm3) during a 48 h period releasing an equivalent of 10–16 mM free heme, a potent biological toxin. For survival, the parasite compartmentalizes free heme in the digestive vacuole (volume of 4 µm3, resulting in an estimated 22-fold increase in concentration reaching 350–400 mM), and polymerizes this toxin into insoluble hemozoin. The magnetic properties of hemozoin have been determined using electron paramagnetic resonance and Mössbauer spectroscopy showing that the Fe atom exists in a high-spin Fe(III) state, S = 5/2. The hemozoin heme electron configuration with 5 unpaired electrons corresponds to a ferriheme that is known to be a part of another high-spin hemoglobin species, methemoglobin. Methemoglobin-rich erythrocytes have been shown by us to possess higher magnetophoretic mobility than low-spin, oxygenated hemoglobin erythrocytes (S=0). Here we demonstrate that changes of the magnetic properties of live, infected erythrocytes are consistent with the conversion of low-spin heme associated with oxyhemoglobin to high-spin heme associated with hemozoin in the course of parasite development from ring to schizont forms. Our results suggest that upon further refinement the method could be used to study the molar ratio of free heme to hemozoin-sequestered heme in live erythrocytes, a parameter of vital importance to parasite survival inside the erythrocyte. Indeed, without conversion to hemozoin, free heme would be increasing to an intra-erythrocytic concentration of at least 3 mM in culture analyzed in Expt. 1, and 10 mM in culture analyzed in Expt. 5, which would greatly exceed the 100 µM toxic level in other studies. The anticipated refinement to the strategies presented here may lead to magnetic fractionation tools of live erythrocytes that are sensitive to hemoglobin conversion to hemozoin during P. falciparum ring and early trophozoite stages and improve our understanding of parasite heme management in relation to antimalarial drug susceptibility and resistance.
FOOTNOTES
To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.05-5122fje;
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