Control of oxygen affinity of hemoglobin in K562 cells induced by hemin

The oxygen affinity of hemoglobin in K562 cells induced by hemin and the relationship between levels of 2,3-diphosphoglycerate (2,3-DPG) and hemoglobin have been investigated. Absorption spectra of induced cells indicate that the hemoglobin is oxygenated; oxygen dissociation curves are symmetric, with a P50 of 20 +/- 0.9 mm Hg, Hill coefficient of 2.5, and a normal temperature dependence. The intracellular pH measured by phosphorus 31 nuclear magnetic resonance (NMR) was 7.3. The amount of 2,3-DPG was determined by an enzymatic method and by 31P NMR. The level of 2,3-DPG in uninduced K562 cells, containing 0.5 pg of hemoglobin per cell, was low (5 +/- 0.5 mumole/10(8) cells), but increased to 64 +/- 5 mumole/10(8) cells upon induction of hemoglobin accumulation (to a final level of 20 pg hemoglobin/cell). For several experiments, there was a closely coordinated relationship between 2,3-DPG and hemoglobin levels, at about 1:1 stoichiometry of the two molecules. The time course of induction of hemoglobin, and of 2,3-DPG levels, are very similar; both processes are reversible. These data suggest that induction of hemoglobin synthesis in K562 cells by hemin results in hemoglobin-containing cells with normal oxygenation properties and that 2,3-DPG and hemoglobin levels are coordinately controlled in these cells. Elucidation of the mechanism of this effect should be of importance in understanding the erythroid-like differentiation of these cells.     

Familial polycythemia caused by high oxygen affinity hemoglobin. 2nd world case of hemoglobin J Providence

The cas observed in Auvergne and reported here raises the aetiological problem of polycythaemias. Young subjects with polycythaemia should be investigated for congenital anomaly of oxygen transport by measuring P50 and 2,3-DPG, which provides information on the oxygen-carrying capacity of haemoglobin. When confronted with familial polycythaemia due to high oxygen affinity haemoglobin, clinicians must know that a cause-effect relationship is not always demonstrable since other factors, such as tobacco-smoking in this particular case, may intervene.     

The effect of short-term oxygen supplementation on oxygen hemoglobin affinity in patients with chronic obstructive pulmonary disease

In 12 hypoxemic patients with chronic obstructive pulmonary disease, the partial pressure of oxygen at which hemoglobin is 50% saturated (P50) and levels of 2,3-diphosphoglycerate (2,3-DPG) were determined under 3 study conditions: (1) while breathing room air, (2) during oxygen supplementation for 72 h sufficient to increase PaO2 above 70 mmHg, and (3) at 72 h after the period of oxygen supplementation. The data showed that in the control period in hypoxemic (PaO2, 52 +/- 6 mmHg), mildly hypercapnic (PaCO2, 47 +/- 6 mmHg) patients with a borderline elevation of pH (7.42 +/- 0.03), there was an increase in P50 (28.6 +/- 1.6 versus a normal value of 26.5 +/- 1; p less than 0.005), and a concomitant increase in 2,3-DPG (19.02 +/- 1.77 mg/g Hb versus a normal value of 13.52 +/- 1.27; p less than 0.005). Nine patients received oxygen for 24 h, and 5 received oxygen for 72 h. In these 5 patients, oxygen supplementation resulted in a shift in P50 to a normal value of 26.7 +/- 1.8 (this value was different from the patients' level while breathing room air and not different from that of the normoxemic control subjects) and a decrease in 2,3-DPG toward but not to a normal value (16.34 +/- 1.92; p less than 0.01). This shift in P50 to the left could be related to the decrease in 2,3-DPG. Accordingly, in patients with COPD who are treated with supplemental oxygen, the net effect on oxygen transport would be a function of the changes produced in PaO2 versus those in hemoglobin-oxygen affinity.(ABSTRACT TRUNCATED AT 250 WORDS)     

Increased thrombosis incidence in a family with an inherited protein S deficiency and a high oxygen affinity hemoglobin variant

Inherited protein S deficiency and the presence of a rare high oxygen affinity hemoglobin variant: Hb Rainier [β 145 (HC2) Tyr → Cys] were found in a family. Among 16 studied members, nine were found as carriers of protein S deficiency (type I with decrease of total, free, and activity levels). Six subjects carried the high-affinity hemoglobin variant, which displayed an increase of blood viscosity. Four members combined both abnormalities. Three had thrombotic accidents before the age of 30. We suggest the combination of protein S deficiency and the presence of this hemoglobin variant can lead to a severe primary hypercoagulable state with pathological consequences compared to each genetic defect alone. © 1994 Wiley-Liss, Inc.     

Hemoglobin Ohio (beta 142 Ala replaced by): a new abnormal hemoglobin with high oxygen affinity and erythrocytosis

Hemoglobin Ohio [beta 142 (H20) Ala replaced by Asp] was found in three members of a white family, all of whom showed erythrocytosis. The variant hemoglobin has a high oxygen affinity, a reduced Bohr effect, and diminished cooperativity. The functional abnormalities of Hb Ohio are explained by the proximity of the substituent beta 142 residue, both to beta 143 His, which is involved in the DPG binding site of hemoglobin, and to the critical C terminal region of the beta chain, which participates in the stabilization of the deoxy (T) conformation.     

Altered hemoglobin oxygen affinity in patients with acute myocardial infarction

Alterations in the oxyhemoglobin dissociation curve might affect calculations of arteriovenous oxygen differences and might contribute to maintenance of myocardial oxygénation in patients with acute myocardial infarction. Accordingly, we analyzed oxyhemoglobin dissociation curves by the dynamic scanning technique as well as red cell diphosphoglycerate and adenosine triphosphate (ATP) determinations in peripheral venous and arterial blood samples from 43 patients with acute myocardial infarction. The oxyhemoglobin dissociation curve in these patients was shifted to the right, as reflected by an increase in p₅₀ values beginning as early as 2 hours and as late as 10 days after the onset of infarction. The magnitude of the p₅₀ shift did not correlate with initial or prevailing levels of systemic or pulmonary arterial PO₂, red cell 2,3-diphosphoglycerate, ATP or cardiac index. The p₅₀ values in venous and simultaneously obtained arterial samples correlated closely. Peak P₅₀ was related to initial clinical class, size of infarct measured by serial serum creatine phosphokinase (CPK) analysis and acute mortality. Results obtained indicate that shifts in the oxyhemoglobin dissociation curve in. patients with acute infarction may facilitate oxygen availability to marginally perfused myocardium and must be taken into account when arteriovenous oxygen differences are calculated on the basis of PO₂ or hemoglobin oximeter determinations, or both.     

Human red cell age, oxygen affinity and oxygen transport

The [2,3-DPG]/[Hb] ratio and the P50 were found to be lower in the 10% denser (old) than in the 10% lighter (young) red blood cell (RBC) fractions (0.57 +/- 0.13 vs 0.96 +/- 0.13 and 23.02 +/- 0.85 vs 27.47 +/- 1.05 Torr, respectively, mean +/- SD, P less than 0.0005 for both, n = 6). The RBC aging processes appear thus to affect the RBC oxygen affinity. However, the [2,3-DPG] changes do not fully explain the drop of not fully explain the drop of P50 as measured at constant [H+], [CO2] and [HbCO]. It is therefore postulated that an additional factor is involved in the regulation of the oxygen affinity in the ageing RBC. The RBC density in 59 normal individuals matched for age (infants, adult, and aged) and for sex was found to be younger in adult females than in all other groups (P less than 0.0005), including an age-matched group of pregnant women. Correspondingly, the [2,3-DPG]/[Hb] ratio and the P50 are higher in adult females than in adult males (0.92 +/- 0.10 vs 0.82 +/- 0.09, P less than 0.009, and 29.03 +/- 1.07 vs 27.72 +/- 0.82 Torr, P less than 0.002, respectively). These data are evaluated in terms of the efficiency of the oxygen transport calculating the circulatory load required to transport a given amount of oxygen to the tissues. The results indicate that the lower oxygen affinity (due to the younger RBC population) in adult females partially compensates for their lower [Hb].     

Single-cell measurement of red blood cell oxygen affinity

Oxygen is transported throughout the body by hemoglobin (Hb) in red blood cells (RBCs). Although the oxygen affinity of blood is well-understood and routinely assessed in patients by pulse oximetry, variability at the single-cell level has not been previously measured. In contrast, single-cell measurements of RBC volume and Hb concentration are taken millions of times per day by clinical hematology analyzers, and they are important factors in determining the health of the hematologic system. To better understand the variability and determinants of oxygen affinity on a cellular level, we have developed a system that quantifies the oxygen saturation, cell volume, and Hb concentration for individual RBCs in high throughput. We find that the variability in single-cell saturation peaks at an oxygen partial pressure of 2.9%, which corresponds to the maximum slope of the oxygen–Hb dissociation curve. In addition, single-cell oxygen affinity is positively correlated with Hb concentration but independent of osmolarity, which suggests variation in the Hb to 2,3-diphosphoglycerate (2–3 DPG) ratio on a cellular level. By quantifying the functional behavior of a cellular population, our system adds a dimension to blood cell analysis and other measurements of single-cell variability.Red blood cells (RBCs) are the most common type of blood cell and constitute approximately one-half of the human body’s total cell count (1). They take up oxygen in the lungs and deliver it throughout the body, taking, on average, 20 s to complete one circuit through the circulation (2). Each cell is densely packaged with hemoglobin (Hb) that binds and releases oxygen based on the local oxygen partial pressure. The fraction of occupied binding sites relative to the total number of binding sites is called the oxygen saturation and can be described by the Hb–oxygen dissociation curve. Although it is known that several factors affect the oxygen affinity of Hb and consequently, shift the dissociation curve, such as pH, temperature, and 2,3-diphosphoglycerate (2–3 DPG) (3), it is not known how much variation these factors cause on a cellular level within individuals.Recently, there has been significant progress in developing biomimetic environments for studying physiological processes of cells in vitro. Analogs to the lung (4), heart (5), bone marrow (6), and gut (7) have all been developed in microfabricated chips that enable ex vivo studies to closely replicate an in vivo environment. In addition to adherent cell cultures, chips for studying flowing blood have been developed that enable control of oxygen partial pressure with high spatial resolution (8–12). We take advantage of this new technology and combine it with a recently developed microfluidic cytometry method that enables us to quantify cell volume and Hb mass for individual flowing cells in high throughput (13).Our system for measuring RBC mass is based on the optical absorption of Hb (14, 15). Unlike other recently developed single-cell mass measurements (16–20), however, it is straightforward to extend this method to resolving the mass of both oxygenated and deoxygenated species because of the well-known modification of the absorption spectra of Hb caused by oxygen binding (21). This method allows us to quantify RBC volume, Hb concentration (HbC), and oxygen affinity for cells while they are in a fluidic environment similar to the circulatory system. A few previous studies have explored the measurement of single-cell saturation (22–24), but to our knowledge, saturation measurements have never been performed on a large cell population or under accurate control of oxygen partial pressure.In this paper, we first describe the physical properties of the microfluidic chip followed by the optical measurement system for obtaining cell volume and Hb mass. We then discuss the spectroscopic measurement system and the quantification of single-cell saturation from the measured multispectral absorption. The temporal dynamics of the system are then characterized to better understand the time and length scales required for equilibrium deoxygenation. We use our system to capture an Hb–oxygen dissociation curve and an SD curve as a function of oxygen partial pressure. At each oxygen partial pressure, we can retrieve a full distribution of single-cell oxygen saturation and observe its correlation to total HbC. In addition, using the measured saturation values, we can retrieve the oxygen partial pressure where the saturation is 50% (P₅₀) value for each cell in the population using the Hill equation as a model.     

Hb south Milwaukee [beta 105 (G7) Leu----Phe]: a newly-identified hemoglobin variant with high oxygen affinity

Fifteen individuals among four generations of a family of English ancestry demonstrated elevated hemoglobin levels accompanied by leftward-shifted whole blood oxygen equilibrium curves. Five of the affected family members have required phlebotomies for relief of symptoms attributable to erythrocytosis. An abnormal hemoglobin or globin chain could not be isolated, but 43% of the beta chains of the affected individuals contained a Leu----Phe substitution at position 105 (G7). Oxygen equilibrium curves demonstrated a normal Bohr effect but decreased cooperativity.     

Venesection in Haemoglobin Yakima,a high oxygen affinity haemoglobin

High oxygen affinity haemoglobins result in polycythaemia and cardiovascular adaptation to maintain tissue oxygenation. The polycythaemia can cause symptoms of hyperviscosity and vaso-occlusive disease. We report a kindred with a high affinity haemoglobin (Haemoblobin Yakima) one of whose members gave birth to two infants with intra-uterine growth retardation and who suffered with symptoms of hyperviscosity which settled on reduction of the PCV by venesection.     

Hyperlipoproteinemia, diabetes, and oxygen affinity of hemoglobin.

Oxyhemoglobin dissociation curves (ODC) were performed on blood from diabetic and nondiabetic subjects with and without hypertriglyceridemia. P50 at in vivo pH was slightly lower than normal in normolipemic diabetics (25.7 versus 26.6 mmHg, p less than 0.05), in spite of increased red cell 2,3-diphosphoglycerate concentration (15.4 versus 14.4 mumole/g Hg, p less than 0.025). P50 at in vivo pH in diabetics with moderately elevated very low density lipoproteins (VLDL)--Type IV hyperlipoproteinemia (HLP)--was likewise found to be slightly lower than normal (25.5 versus 26.6 mmHg, p less than 0.05). In contrast, diabetics with pronounced hyperlipemia due to accumulation of chylomicrons (type I HLP) or due to accumulation of chylomicrons (type I HLP) or due to accumulation of chylomicrons as well as VLDL (type V HLP) showed markedly increased hemoglobin--oxygen affinity (P50:21.1 versus 26.6 mmHg, p less than 0.001). The change in the ODC of normolipemic diabetics is considered to be an expresssion of the presence of an increased proportion of a hemoglobin fraction (Hb Alc) with increased oxygen affinity. The additional change in the ODC of the hyperlipemic patients is thought to be secondary to accumulation of triglyceride-rich particles for the following reasons: (1) a similar increase in oxygen affinity of hemoglobin was demonstrated in familial type I HLP of nondiabetic subjects; (2) normal red cells increased their oxygen affinity when incubated in lactescent plasma; (3) in both acquired types I and V HLP the disappearance of HLP was followed by a normalization of the ODC.