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Am J Physiol Heart Circ Physiol 284: H2335-H2342, 2003; doi:10.1152/ajpheart.01030.2002
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Vol. 284, Issue 6, H2335-H2342, June 2003

Changes of RBC aggregation in oxygenation-deoxygenation: pH dependency and cell morphology

Iwona Cicha1, Yoji Suzuki1, Norihiko Tateishi2, and Nobuji Maeda1

1 Department of Physiology, School of Medicine, Ehime University, and 2 Department of Medical Informatics, Ehime University Hospital, Ehime 791-0295, Japan


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

The effects of the oxygenation-deoxygenation process on red blood cell (RBC) aggregation were examined in relation to morphological changes in RBCs and the contribution of CO2. A low-shear rheoscope was used to measure the rate of rouleaux (one-dimensional aggregate) formation in diluted autologous plasma exposed to gas mixtures with different PO2 and PCO2. RBC indexes and RBC suspension pH were measured for the oxygenated or the deoxygenated condition, and the cell shape was observed with a scanning electron microscope. In the oxygenation-deoxygenation process, the rate of rouleaux formation increased with rising pH of the RBC suspension, which was lowered in the presence of CO2. The rate increased with increasing mean corpuscular hemoglobin concentration (thus the cells shrank), which increased with rising pH and decreased in the presence of CO2. With rising pH, cell diameter increased and cell thickness decreased (thus the cell flattened). In addition, slight echinocytosis was induced in the presence of CO2, and the aggregation was reduced by the morphological change. In conclusion, RBC aggregation in the oxygenation-deoxygenation process is mainly influenced by the pH-dependent change in the surface area-to-volume ratio of the cells, and the aggregation is modified by CO2-induced acidification and the accompanying changes in mean corpuscular hemoglobin concentration and cell shape.

rouleaux formation; carbon dioxide; plasma; microcirculation; oxygen transfer


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

RED BLOOD CELLS (RBCs) spend much of their life span in a partially oxygenated-deoxygenated state, which may be exaggerated in certain pathophysiological conditions. The oxygenation-deoxygenation state greatly affects such factors as interactions between hemoglobin and cellular metabolites or membrane proteins (24), activation of some enzymes in the cells (8), and binding properties of membrane proteins (1). These changes may influence the rheological properties of RBCs as well as their morphological characteristics.

The capillaries represent the major site of O2 delivery from RBCs to tissues in the microcirculatory network, but tissue oxygenation occurs as a result of a complex exchange of O2 among microvessels (arterioles and venules) and capillaries by diffusion (6, 10). The flow behavior of RBCs in microvessels affects the diffusion processes of O2 from the cells to the tissues and even within the cells, with a close relation to microcirculation of the cells. This characteristic of the RBCs is modified by their rheological properties, such as hematocrit (Hct), deformability, and aggregability (30, 34). Our recent studies demonstrated that dextran-induced RBC aggregation (32) and acceleration-induced RBC accumulation (33) suppress O2 release from the cells, as evaluated using an O2-permeable narrow tube. However, little is known about the effect of the oxygenation-deoxygenation process on the rheological properties of RBCs.

The majority of the previous studies have demonstrated that RBC deformability is not altered by the oxygenation-deoxygenation process in the presence of CO2 (15, 27), despite slight cell swelling. It is well known that CO2 enters RBCs in peripheral tissues by diffusion. The excess bicarbonate ion formed in the cells enters plasma in exchange for the chloride ion, i.e., the chloride (or Hamberger's) shift. Each CO2 molecule added to the cell increases one osmotically active particle, either bicarbonate or chloride ion. Consequently, the cell takes up water and increases in size. On the other hand, deoxygenation of RBCs under nitrogen increases their filtration time; namely, it reduces RBC deformability (36). These controversial results may be due to different deoxygenation procedures, possibly in the presence or absence of CO2. There are no reports of the effect of the oxygenation-deoxygenation process on RBC aggregation. However, a close relation between rheological properties of RBCs and O2 transport has also been suggested in water-deprived rats: the dehydration causes significant elevation of RBC aggregation and reduces O2 supply to tissues (35).

The aim of the present study was to examine the effect of the oxygenation-deoxygenation process on RBC aggregation, to reveal the mechanism altering the RBC aggregation, and, finally, to understand the relation between flow behavior of the RBCs and O2 diffusion in the microcirculatory network. For these purposes, RBCs were exposed to extreme PO2 and PCO2 at extreme pH levels, because the experiments in physiological ranges of the gas tensions and pH levels may scarcely detect significant effects on RBC aggregation. The rate of rouleaux (i.e., 1-dimensional aggregate) formation of RBCs was kinetically and quantitatively measured in cells (Hct = 0.3%) from normal adult men suspended in 70% autologous plasma obtained.


    MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Preparation of RBC Suspension in Plasma

Blood samples from the cubital vein of healthy adult men were collected in test tubes treated with heparin [0.2 mg (equivalent to 20 IU)/ml blood] as an anticoagulant and immediately centrifuged at 1,200 g for 5 min at 4°C. After careful removal of plasma and the buffy coat, RBCs were washed three times with isotonic phosphate-buffered saline (PBS: 50 mM sodium phosphate, 90 mM NaCl, and 5 mM KCl, pH 7.4, 285 mosM) containing 0.1 g/dl glucose. Finally, a volume of isotonic PBS was added to the packed cells to yield an RBC suspension with an Hct of 30%. The plasma was centrifuged at 15,000 g for 15 min at 20°C. After removal of platelets, the plasma was diluted with isotonic PBS to provide 70% autologous plasma.

Measurement of Rouleaux Formation Rate of RBCs

RBC aggregation was evaluated by the rate of rouleaux (1-dimensionally extended aggregates of RBCs) formation. The kinetic and quantitative measurement was carried out using a modification of the apparatus of Shiga et al. (28). Briefly, a low-shear rheoscope consisting of a transparent cone-plate viscometer and an inverted microscope was combined with a videotape recorder (Hi-8 Video, model EVO-9650, Sony) through a charge-coupled device video camera (model DXC-101, Sony). Before the measurement, 70% autologous plasma was oxygenated or deoxygenated in an appropriate gas mixture (see below). The diluted plasma was most suitable for measurement of the process of rouleaux formation, and 30% PBS was added to allow stable measurement at constant pH. The washed RBC suspension (Hct = 30%) was added to the medium, and a pipette was used to apply 0.3 ml of the sample (Hct = 0.3%) to the gap between the cone and the glass plate of the viscometer. Very low Hct was required for the kinetic measurement to avoid overlapping of RBC images (28). Before application of the sample by the pipette, part of the cone plate was covered with a transparent box, the inside of which was flushed with the gas mixture. The flow of gas mixture was steadily directed in the vicinity of the cone plate during the measurement.

The measurements were carried out in triplicate at 25°C at a constant shear rate of 7.5 s-1. The count of particles (i.e., rouleaux and single cells) and the total area projected by the particles in a limited frame of the video image (corresponding to 200 × 267 µm in a microscopic field) were consecutively recorded on videotape, and the data were analyzed with a computer (Power Macintosh 8500/180, Apple Computer, Cupertino, CA) using an image analyzing program (NIH Image), as described elsewhere (31). The rate of rouleaux formation was expressed as the increment of area-to-count ratio per unit time (µm2/s) during the stage of long one-dimensional aggregates (i.e., rouleaux): in this stage, the count of particles decreased, but there was little change in the area projected by the particles (28). Therefore, the increase in the area-to-count ratio reflects a growing rate of rouleaux formation. However, in the stage of three-dimensional (spherical) aggregate formation, the measure underestimates the rate, because the cells overlap.

Oxygenation and Deoxygenation Procedures

The following gas mixtures were used for oxygenation and deoxygenation of the RBC suspension: 1) without CO2, i.e., air (Air, final PO2 = 155-160 mmHg under experimental conditions described above) for oxygenation with simultaneous CO2 elimination from plasma and N2 (final PO2 = 10-20 mmHg) for deoxygenation without CO2 uptake, and 2) with CO2, i.e., 95% O2-5% CO2 (O2/CO2, final PO2 = 450-500 mmHg) for oxygenation without CO2 elimination and 95% N2-5% CO2 (N2/CO2, final PO2 = 10-20 mmHg) for deoxygenation with simultaneous CO2 uptake. PO2 was measured using a PO2 monitor (model PO2-100, Inter Medical, Tokyo, Japan). The "final" PO2 values correspond to the PO2 of the RBC suspension after the suspension is flushed with the appropriate gas mixtures.

Before oxygenation and deoxygenation of the RBC suspension, the suspending medium, i.e., 70% autologous plasma, was oxygenated or deoxygenated by flushing prewetted gas mixtures for 15 min at 25°C. A small amount of the washed RBC suspension (Hct = 30%) was then added to the oxygenated or deoxygenated medium, and the diluted RBC suspension (final Hct = 0.3%) was further flushed for 2 min with gentle magnetic stirring before measurement of rouleaux formation rate. This procedure allowed fast oxygenation or deoxygenation of RBCs. The pH of oxygenated or deoxygenated RBC suspensions (Hct = 0.3%, as in experimental conditions for measurement of rouleaux formation) was measured at 25°C. If necessary, plasma pH was adjusted by addition of isotonic PBS or isotonic lactic acid solution.

Hematologic Examinations

Mean corpuscular hemoglobin concentration (MCHC) of RBCs was calculated from Hct (measured in duplicate with a microhematocrit centrifuge; model KH-120II, Kubota Manufacturing, Tokyo, Japan) and hemoglobin concentration (determined in triplicate by the cyanmethemoglobin method). Mean cell volume was calculated from Hct and RBC count (determined in triplicate with an automatic counter; model CC-110, Toa Medical Electronic, Tokyo, Japan). To adjust the MCHC (to 36 g/dl in this experiment and, thus, the mean cell volume), the osmolarity of plasma was changed by the addition of hypertonic or hypotonic PBS solution.

The shape of RBCs exposed to appropriate gas mixtures was observed using a scanning electron microscope (model S-800, Hitachi) after fixation with 1% glutaraldehyde in PBS (isotonicity of the fixative was adjusted with NaCl) and then with 1% OsO4. The morphological index (MI) was adopted to express the degree of echinocytic transformation of RBCs (14): MI = 0, 0.5, 1, and 2 correspond to diskocyte, echinocyte I, echinocyte II, and echinocyte III, respectively, according to the classification of Bessis (3). Furthermore, the diameter and maximum thickness of RBCs were determined for >20 cells on the photographs from the scanning microscope: diameter was measured in RBCs fixed in a horizontal position, and thickness was measured in RBCs fixed in a vertical position.

Statistical Analyses

Values are means ± SD; n is the number of measurements. According to the experimental setups, the differences between means in experiments without CO2 (Air vs. N2) and in experiments with CO2 (O2/CO2 vs. N2/CO2) were tested using the paired nonparametric Wilcoxon test. Differences between means in experiments obtained without and with CO2 (e.g., Air vs. N2/CO2) were tested using the nonparametric Mann-Whitney U-test. Analysis of covariance was used to compare the results obtained in the aggregation experiment with adjusted pH and MCHC. P < 0.05 was designated significant.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Effect of Oxygenation States on RBC Rouleaux Formation Rate

Effects of oxygenation and deoxygenation of RBCs on the rouleaux formation rate in 70% autologous plasma were examined by exposure of the RBCs to various gas mixtures (Fig. 1). Compared with Air, deoxygenation of RBCs in the absence of CO2 (N2) resulted in a significant increase in rouleaux formation rate (P < 0.05, Wilcoxon's test). However, RBCs deoxygenated in the presence of 5% CO2 (N2/CO2) showed a reduced rate of rouleaux formation compared with Air (P < 0.05, Mann-Whitney's U-test), and the rate was less in N2/CO2 than in RBCs oxygenated in O2/CO2 (P < 0.05, Wilcoxon's test).


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  		Fig. 1.   Rouleaux formation rate of red blood cells (RBCs) oxygenated or deoxygenated in the presence or absence of CO2 in 70% autologous plasma. Values are means ± SD; n, number of samples. *Significant differences (P < 0.05) between Air and N2 (Wilcoxon's test), Air and N2/CO2 (Mann-Whitney's U-test), and O2/CO2 and N2/CO2 (Wilcoxon's test). See Table 1 for pH of RBC suspensions.

Effect of Oxygenation State on RBC Suspension pH

The pH variation of RBC suspensions in autologous plasma as a result of exposure to various gas mixtures (Fig. 1) is shown in Table 1. There was a significant increase in pH of RBC suspensions deoxygenated with N2 compared with those oxygenated with Air (P < 0.05, Wilcoxon's test). On the contrary, the RBC suspensions deoxygenated in the presence of CO2 (N2/CO2) showed a marked decrease in pH compared with those oxygenated in Air (P < 0.01, Mann-Whitney's U-test). The pH of the RBC suspension deoxygenated with N2/CO2 was slightly less than that of the suspension oxygenated with O2/CO2, although it was not significant. The relation between rouleaux formation rate and pH in 70% autologous plasma is plotted in Fig. 2A. The pH of the RBC suspensions was well correlated to rouleaux formation rate: rouleaux formation rate was increased with increasing pH.

                              
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  		Table 1.   pH of RBC suspensions and MCHC of cells in 70% autologous plasma after exposure to various gas mixtures



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  		Fig. 2.   Relation between rouleaux formation rate and pH of RBC suspension (A) and mean corpuscular hemoglobin concentration (MCHC, B) after exposure of RBC suspension in 70% autologous plasma to various gas mixtures.

Effect of Oxygenation State on MCHC

The changes of MCHC in autologous plasma after exposure to various gas mixtures are shown in Table 1. With deoxygenation of RBCs in N2, MCHC was significantly increased compared with Air (P < 0.05, Wilcoxon's test). However, RBC suspensions deoxygenated in the presence of CO2 (N2/CO2) showed a significant decrease in MCHC compared with Air (P < 0.05, Mann-Whitney U-test). MCHC of cells deoxygenated with N2/CO2 was slightly less than that of cells oxygenated with O2/CO2, although it was not significant. The good correlation was observed between rouleaux formation rate and MCHC in 70% autologous plasma (Fig. 2B): the rouleaux formation rate was increased with increasing MCHC.

pH-Dependent Morphological Changes in RBCs

Morphological changes in RBCs at various pH levels were examined in isotonic PBS with a scanning electron microscope (Fig. 3). Changes in cell diameter and maximum thickness of the RBCs measured using the photographs are shown in Fig. 4, with MCHC change dependent on pH (determined in PBS). With increasing pH, MCHC increased, and the cell diameter of the biconcave diskocytes increased and the thickness decreased. These phenomena indicate that cell volume decreases and the cells flatten in alkaline pH; namely, the surface area-to-volume ratio increases.


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  		Fig. 3.   pH-dependent morphological change of RBCs as observed by scanning electron microscopy. A: pH 6.5; B: pH 7.1; C: pH 8.0.



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  		Fig. 4.   Morphological changes in RBCs at various pH levels in isotonic PBS. A: MCHC calculated from hemoglobin concentration and hematocrit. B and C: diameter and maximum thickness, respectively, determined from photographs obtained by scanning electron microscopy. Values are means ± SD, with 100% at pH 7.4.

RBC Rouleaux Formation Rate at Constant MCHC

To investigate the mechanisms for changes in rouleaux formation rate in the oxygenation-deoxygenation process in the presence or absence of CO2, MCHC of RBCs suspended in plasma was adjusted to 36 g/dl by the addition of an appropriate volume of hypertonic or hypotonic PBS. As shown in Fig. 5, the pH dependency of the rouleaux formation rate of RBC suspensions exposed to different gas mixtures was significantly reduced compared with the samples without MCHC adjustment (P < 0.05). However, the higher rate of rouleaux formation under N2 and the lower rate under N2/CO2 were preserved. Therefore, the MCHC changes in the oxygenation-deoxygenation process would partly account for the observed differences in rouleaux formation rate.


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  		Fig. 5.   pH-dependent changes in rouleaux formation rate of RBCs suspended in 70% autologous plasma before (open circle ) and after () adjustment to constant MCHC (36 g/dl). Measurements were carried out after oxygenation or deoxygenation with different gas mixtures. Significant difference between both relations was observed by simple regression analysis (P < 0.05).

The pH of plasma samples was varied with isotonic PBS or isotonic lactic acid solution, but MCHC of the RBCs was adjusted to a constant value (36 g/dl). Thus the rouleaux formation rate of the RBCs with constant MCHC was examined at different pH conditions in samples flushed with various gas mixtures. The results demonstrated that the rouleaux formation rate depended on the pH of the RBC suspension (Fig. 6). The significantly different pH dependency of the rate in the presence of CO2 at pH <7.5 (P < 0.05, analysis of covariance) suggested the contribution of other factors. The scanning electron microscopic analysis of RBCs demonstrated a slight echinocytic change in the cells in the presence of CO2, and the transformation was enhanced by deoxygenation and at more acidic pH: MI = 0.1 ± 0.06 in Air, 0.1 ± 0.07 in N2, 0.17 ± 0.08 in O2/CO2, and 0.24 ± 0.11 in N2/CO2 at pH 6.5. Therefore, the different pH dependency would be due to inhibition of rouleaux formation by the echinocytic transformation of RBCs.


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  		Fig. 6.   Rouleaux formation rate of RBCs suspended in 70% autologous plasma at various pH levels but at constant MCHC (36 g/dl). pH was varied with isotonic PBS or isotonic lactic acid solution. Measurements were carried out after oxygenation or deoxygenation with different gas mixtures. pH dependency of rouleaux formation rate in the presence of CO2 at pH <7.5 was significantly different from that in the absence of CO2 by analysis of covariance (* P < 0.05).


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

O2 transfer from RBCs to tissues in the microcirculation is essentially affected at a defined temperature by interactions between hemoglobin and various metabolites, such as hydrogen ion, CO2, and 2,3-diphosphoglycerate, in the cells. In addition to these factors affecting the O2 equilibrium curve, diffusional transfer of O2 from RBCs to tissues (6, 10) is greatly affected by flow behavior of the cells in microvessels and capillaries (22), which is modified by the morphological characteristics and the rheological properties of the cells (17, 23). Therefore, O2 transfer in the microcirculation changes dynamically in the oxygenation-deoxygenation process of RBCs, because changes in various metabolites in the process possibly modify the cell shape and the rheological characteristics as a result of the altered interactions with membrane proteins or other cell constituents (1). The aim of the present study was to examine the relation between the oxygenation-deoxygenation process of RBCs and the cell aggregation, which affects most strikingly the flow behavior of cells in the microcirculation.

The present study was carried out under extreme conditions of pH and PO2 to detect the changes in RBC aggregation in the process of oxygenation-deoxygenation of RBCs. The effects of pH, CO2, and MCHC on RBC aggregation were observed independently: 1) the rate of rouleaux formation increased with rising pH (Figs. 2, 5, and 6), 2) pH was lowered in the presence of CO2 (Table 1, Figs. 2 and 5), and 3) the rate of rouleaux formation increased with increasing MCHC (Fig. 2). In addition, slight echinocytosis was induced in the presence of CO2, and the rate of rouleaux formation was decreased by the shape change (Fig. 6). From these experimental results, it can be concluded that RBC aggregation is enhanced in alkaline pH, at low PCO2, and in cells with high MCHC (i.e., shrunken cells), whereas RBC aggregation is reduced in acidic pH, at high PCO2, and in cells with low MCHC (i.e., swollen cells).

Factors Affecting RBC Aggregation

RBC aggregation is induced in low shear regions, especially in venous regions, by interaction between RBCs and macromolecules in plasma, but the cells in aggregates are dispersed in high shear regions (5, 29). Therefore, RBC aggregation is greatly dependent on mechanical shearing under physiological conditions. RBC aggregation is necessarily affected by rheological and chemical characteristics of RBCs and macromolecules. However, the interaction is remarkably affected by the physical and chemical properties of the environment (i.e., suspension media of RBCs).

RBC factors. The biconcave disk shape of RBCs creates a large and stable contact area between adjacent cells in rouleaux formation (5, 29). As observed in the present experiment, the spherical shape change of RBCs in more acidic pH prevents stable rouleaux formation, whereas flattened RBCs promote stable rouleaux formation. The strong pH dependency of the rouleaux formation rate in the oxygenation-deoxygenation process is mainly due to the pH-dependent shape change of the RBCs (21). In this connection, the echinocytic transformation induced in the presence of CO2 prevents the stable cell-to-cell contact, leading to cell aggregation (28).

Membrane flexibility of RBCs is another important determinant of the cell aggregation (5, 29). Stiffening of the cell membrane results in decreased aggregation, because cell-to-cell contact is impeded (18). No alteration of cell deformability in the oxygenation-deoxygenation process in the presence of CO2 was reported (15, 27), despite slight changes in cell volume due to bicarbonate/chloride exchange (see above). Possibly, the countervailing effects of intracellular viscosity and RBC shape are working on cell deformability over a wide pH range (11). Certainly, the small surface area-to-cell volume ratio (i.e., swelling) of the cells in acidic pH suppresses cell-to-cell contact, leading to rouleaux formation (19, 21). However, the decrease in MCHC, which must be prominent in the presence of CO2 because of the entry of water into RBCs, may enhance cell-to-cell contact because of the decreased intracellular viscosity. The phenomenon will be reversed in alkaline pH. These opposing effects are working on RBC aggregation through simultaneous changes in the morphology and deformability of RBCs.

Recently, it has been demonstrated that deformability of RBCs with constant volume at various pH levels (6.2-8.0), which was achieved by varying osmolarity, significantly decreased at lower pH because of the change in membrane elastic properties (16). The pH-dependent change in RBC aggregation at constant MCHC in the present study (Figs. 5 and 6) may be due in part to the change in cellular deformability. However, it is unlikely that the pH change in the present experimental condition (pH 6.5-8.7) influences the surface charge of RBCs (9) or binding of fibrinogen to the cells (25), which are important factors affecting RBC aggregation.

Macromolecular factors. Measurements within the same experimental set were done using blood samples collected from the same donor (i.e., the same RBCs and plasma); thus rheological characteristics of the cells were constant, as was plasma viscosity, with constant concentrations of, e.g., albumin, fibrinogen, and immunoglobulins. It is well known that heparin used as an anticoagulant induces RBC aggregation. Heparin at 10 IU/ml enhances RBC aggregation slightly but significantly, as determined using an aggregometer (4). A statistically significant increase in RBC sedimentation rate was also induced by a low concentration of heparin (25 IU/ml), indicating a rise in RBC aggregation (12). Therefore, heparin used in the present study may contribute to the RBC aggregation, but the concentration was constant in all experiments (final concentration = 14 IU in 70% plasma).

Environmental factors. In the present study, all measurements were carried out under constant physical conditions (i.e., shear rate and temperature). For convenience in controlling the exposures of various gas mixtures, the present experiments were carried out at 25°C. RBC aggregation is enhanced by an increase in temperature (2, 20). Therefore, at physiological temperature (i.e., 37°C), RBC aggregation must be enhanced more as a result of the increased interaction between RBCs and macromolecules and/or flattening of the cells (20), possibly in all pH ranges. However, further studies of the effect of temperature on the pH dependency of RBC aggregation are required.

Briefly, the pH-dependent change in RBC aggregation in relation to the oxygenation-deoxygenation process of the cells is mainly due to morphological changes (i.e., surface area-to-volume ratio and echinocytosis in special conditions) in RBCs. The pH-dependent change in RBC deformability may contribute to RBC aggregation, but opposing effects of RBC shape (i.e., cell swelling and flattening) and internal viscosity (i.e., MCHC) are working on their deformability.

Physiological Relevance of RBC Aggregation

The normal pH of blood in the systemic circulation is between 7.35 and 7.45. However, the pH in acid-base disturbance compatible with life would probably be between 6.8 and 7.7. In a local microcirculatory region of peripheral tissues, further lowering of blood pH would be expected in the stasis of blood flow and/or in a metabolically active condition. Blood pH in a local microcirculatory region is influenced mainly through the Donnan membrane equilibrium effect by pH of interstitial (extracellular) fluid in the tissues. The reported values of extracellular pH measured with various techniques are quite variable (e.g., 7.2-7.8 in skeletal muscle and 8.0 in nervous tissue in extreme cases) (26). Intracellular pH values, which affect extracellular pH across the cell membrane, are also variable (e.g., 6.4 in brown adipose cells and 6.0 in skeletal muscle cells in extreme cases) (26), probably even under conditions of intracellular buffering and ion-exchange transports through the cell membrane. Therefore, the present experimental results at extreme pH levels may be relevant for understanding blood flow in some pathological conditions and in some peripheral tissues with high metabolic activities.

Effective O2 uptake and release in circulating RBCs depend on biconcave disk shape of the cells (ratio of surface area to cell volume for O2 diffusion) and internal viscosity (rate of O2 diffusion in the cell) (37). Aggregation of RBCs is one of several important factors affecting O2 release in microvessels. In enhanced cell aggregation, O2 diffusion from the inside to the outside of aggregates is reduced because of extension of the diffusion distance in the aggregates (32). A similar phenomenon was observed in accumulated cells induced by accelerational force (33). From the viewpoint of complex O2 transfer among microvessels and capillaries in the microvascular network (6, 10, 22), the study of RBC aggregation in the oxygenation-deoxygenation process and in various metabolic conditions may be physiologically relevant.

In metabolically active tissues, circulating RBCs are exposed to lower pH and higher PCO2. Therefore, the cells release more O2 to tissues by lowering the affinity of hemoglobin to O2 and take up more CO2. As a result, the cells swell more because of water entering the cell, accompanied by subsequent bicarbonate/chloride exchange. Therefore, deoxygenated cells have lower MCHC in more metabolically active tissues. On the other hand, in the hyperventilating lung, RBCs are exposed to higher pH and lower PCO2; thus oxygenated cells shrink and have higher MCHC. The present results suggest that changes in pH and CO2 in relation to these oxygenation and deoxygenation processes of RBCs affect RBC aggregation. Essentially, RBC aggregation greatly depends on mechanical shearing, and the degree is modified by several characteristics of interacting RBCs and macromolecules. Changes in pH and CO2 in blood may modify the interaction between RBCs and macromolecules to some extent, as discussed above. Therefore, in vivo effects of pH, CO2, and MCHC on RBC aggregation and thus O2 transfer to tissues, although the effects would be much smaller than in the present experiments, may be taken into consideration in the oxygenation-deoxygenation process of the cells. The present study suggests that any enhancement of RBC aggregation would be in highly oxygenated cells, as in lung circulation, whereas RBC aggregation is reduced in highly deoxygenated cells, as in metabolically active tissue circulation. In other words, blood flow in venules and/or veins of metabolically active tissues is enhanced by the reduction of RBC aggregation; thus the phenomenon may play an important role in removal of CO2 from tissues and supply of O2 to the tissues.

It is well known that the fluid shear stress stimulates endothelium of microvessels and blood flow changes by nitric oxide production (7). Arterioles and venules dilate in response to increases in wall shear stress by stimulating the release of endothelium-derived nitric oxide, and the responses are involved in regulation of microvascular resistance, especially in high flow conditions (13). Enhanced RBC aggregation in venules may increase wall shear stress and stimulate nitric oxide production to increase blood flow. However, the extent to which the mechanism works is not clear in the case of enhanced RBC aggregation because of low flow conditions.

In conclusion, changes in RBC aggregation in the oxygenation-deoxygenation process are mainly due to the pH-dependent change of surface area-to-volume ratio of the cells, and the aggregation is modified by further acidification and additional changes in MCHC and cell shape induced in the presence of CO2. The present study may suggest that the phenomenon facilitates blood flow in metabolically active tissues and thus enhancement of O2 supply and CO2 removal to some extent.


    ACKNOWLEDGEMENTS

This work was supported in part by grants from the Ministry of Education, Science, Sports, and Culture of Japan and the Ehime Health Foundation.


    FOOTNOTES

Address for reprint requests and other correspondence: N. Maeda, Dept. of Physiology, School of Medicine, Ehime University, Shigenobu, Onsen-gun, Ehime 791-0295, Japan (E-mail: nmaeda{at}m.ehime-u.ac.jp).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

10.1152/ajpheart.01030.2002

Received 26 November 2002; accepted in final form 17 February 2003.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

1.   Barbul, A, Zipser Y, Nachles A, and Korenstein R. Deoxygenation and elevation of intracellular magnesium induce tyrosine phosphorylation of band 3 in human erythrocytes. FEBS Lett 455: 87-91, 1999[Web of Science][Medline].

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Am J Physiol Heart Circ Physiol 284(6):H2335-H2342
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