Follow a blood cell from oxygenation through deoxygenation
In vertebrate,the blood system is a closed system, that is they are not open-ended. In the organs, the arteries divide to form arterioles, which is also divided to form numerous capillaries. The capillaries unite to form larger vessels, called the venules (i.e small veins). This venules join with other venules to form veins. Veins leave the organs and eventually join the venae cavae. It is thus clear that the arterial and venous bloods are transported by the means of capillaries in the tissue.
The pulmonary circulation, which is also known as lesser circulation, transport the deoxygenated blood through the pulmonary artery (which arises from the right ventricle) to the lungs. In the lungs the bloods are renewed and become oxygenated again. The four pulmonary veins return the oxygenated blood to the left atrium of the heart. This process is simply summarized as the movement of deoxygenated blood from the Right Ventricle – pulmonary artery – lungs – pulmonary veins – left atrium – left ventricle.
The branches of the aorta convey oxygenated blood from the left ventricle to all the tissues and organs of the body (except the lungs). The tissue cells are oxygenated and deoxygenated blood returned to the heart via the superior and inferior venae cavae. The blood then flows via the tricuspid valve into the right ventricle, from where it joins the pulmonary circulation. This is usually referred to as systemic circulation. The summary of the systemic circulation is thus: the movement of oxygenated blood from the Left ventricle – aorta – organs – venea cavea – right atrium – right ventricle.
The life span of Red Blood Cells (RBCs) is mostly spent in a partially oxygenated-deoxygenated state. The interactions between hemoglobin and cellular metabolites or membrane were greatly affected by oxygenation-deoxygenation state, it also affect the activation of some enzymes in the cells, and binding properties of membrane proteins. These changes may influence the rheological properties of RBCs as well as their morphological characteristics.
In the microcirculatory network, the capillaries represent the major site of O2 delivery from RBCs to tissues, but the complex exchange of O2 among micro vessels (arterioles and venules) and capillaries by diffusion result in tissue oxygenation. 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. The rheological properties modified the characteristics of the RBCs in the system, such as hematocrit (Hct), deformability, and aggregability. However, little is known about the effect of the oxygenation-deoxygenation process on the rheological properties of RBCs.
The measurement of oxygen uptake into intact and reconstituted human red blood cells is carried out using dual wavelength, stopped flow techniques. The rate of oxygen uptake by human erythrocytes is roughly 40 times slower (t 1/2 congruent to 80 ms at 0.125 mM O2, 25 degrees C) than the corresponding rate of oxygen combination with free hemoglobin. The difference is as a result of Oxygen transported through the red cell cytoplasm, which also predict the Half-time of uptake of about 15ms, which is 5 times smaller than that observed experimentally and the presence of unsaturated layer of solvent adjacent to the red cell surface. All these appear to be part of oxygen uptake limitation. These layers form immediately after mixing and become depleted of O2 due to uptake by the cells. This requires that the bulk of the oxygen molecules must diffuse over rather large distances, 1.0 to 5.0 micrometer, before they can penetrate the erythrocytes. A mathematical model was developed to take into account diffusion through an unstirred solvent layer, which increases in thickness with time.
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.
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: MI = 0, 0.5, 1, and 2 correspond to diskocyte, echinocyte I, echinocyte II, and echinocyte III, respectively, according to the classification of Bessis. 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.
The above statistical analysis 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.
The interaction between hemoglobin and various metabilites, such as hydrogen ion, CO2, and 2,3-diphosphoglycerate, in the cells at a defined temperature affect the O2 transfer from RBCs to tissues in the microcirculation. In addition to these factors affecting the O2 equilibrium curve, diffusional transfer of O2 from RBCs to tissues is greatly affected by flow behavior of the cells in microvessels and capillaries, which is modified by the morphological characteristics and the rheological properties of the cells. Therefore, O2 transfer in the system changes dynamically in the oxygenation-deoxygenation process of Red Blood Cell (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.
This process can account quantitatively both for the dependence of the apparent rate of uptake on O2 concentration and for the shape of the observed time courses. Thus, the developed diffuse parameters for the O2 reaction can also be used to describe quantitatively the rates and time courses of CO and ethyl isocyanide uptake and time courses of O2 release from cells in the presence of sodium dithionate.
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. And 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.
Barbul, A, Zipser Y, Nachles A, and Korenstein R.(1999) Deoxygenation and elevation of intracellular magnesium induce tyrosine phosphorylation of band 3 in human erythrocytes.
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