Studies on Avian Erythrocyte Metabolism: VII. Effect of Inositol Pentapiiuspmte and Other Organic Phosphates on Oxygen Affinity of the Embryonic and Adult-Type Hemoglobins of the Turkey Embryo

The effects of 2.3-diphosphoglyceric acid (2,3-DPG), adenosine triphosphate (ATP), inositol tetraphosphate (ITP), inositol pentaphosphate (IPP), and inositol hexaphosphate (IHP) on oxygen affinity of whole “stripped” hemoglobin (WSH), hemcqlobin H (Hb-11; hatching hemoglobin), hemoglobin A (Hb-A), and hemoglobin D (Hb-D) isolated from erythrocytes (RBC) of the 25-day turkey embryo have been studied. The order of the decrease in oxygen affinity induced by these organic phosphates, at molar ratios of phosphate compound to hemoglobin (tetramer) between 2 and 4, is 2,3-DPG<ATP<TP<IPP<IHP. 2,3-DPG shows a slightly greater effect on reducing oxygen affinity of Hb-H than on either adult-type hemoglobin. The effect of IPP upon lowering the oxygen affinity of either WSII, Hb-H, Hb-A, or Hb-D is approximately 201 less than IHP. The effects of the various organic phosphates upon the Hill constant, n, of these purified hemoglobins is variable but appears to reach a maximum when the molar ratio of organic phosphate to hemoglobin (tetramer) is 2 or greater. None of the physiologically occurring organic prwsphates has a significant preferential interaction with any specific hemoglobin. These experiments strengthen and support our earlier conclusion, that the changes in whole blood oxygen affinity which occur during avian developtent result from the changes in composition of the intraerythrocytic organic ptwsphates.     

Oxygen binding of fetal and adult bovine hemoglobin in the presence of organic phosphates and uric acid riboside.

The P50 values of "stripped" fetal and adult bovine hemoglobin were 18.4 and 28.9 respectively. Neither the oxygen-hemoglobin dissociation curve nor the Hill coefficient, n, of fetal or adult bovine hemoglobin was affected by uric acid riboside (UAR), 2,3-diphosphoglyceric acid (2,3-DPG), adenosine triphosphate (ATP), or inositol pentaphosphate (IPP). Combinations of UAR and ATP with adult bovine hemoglobin or 2,3-DPG and ATP with fetal hemoglobin also had no effect. It was concluded that neither adult nor fetal bovine red cells contained an identifiable compound which affects the binding of oxygen to hemoglobin.     

The oxygen affinity of concentrated human hemoglobin solutions and human blood.

The log P50 of normal human blood at 37 degrees C, PCO2 = 0, 21, 42, AND 57 MM Hg in the absence and in the presence of 2,3-DPG and ATP, has been determined in the pH range 7.0 to 7.6. Similar data have been obtained for human hemoglobin isotonic solutions at different protein concentrations in the presence of various amounts of each of the cofactors which are known to affect hemoglobin oxygen affinity in blood. It has been found that the addition of KC1, organic phosphates, magnesium ions, and CO2 confers to a 32% human hemoglobin solution the same oxygen affinity (over the entire physiological pH range) of whole blood. Thus there is no room for significant effects caused by some other unidentified molecules or ions.     

Preparation of low-affinity red cells with dimethylsulfoxide-mediated inositol hexaphosphate incorporation: hemoglobin and ATP recovery using a continuous-flow method

Incorporation of IHP into red cells decreases oxygen affinity as a result of the binding of this compound to the 2,3-DPG site of hemoglobin. This investigation describes a continuous-flow method which utilizes the osmotic pulse technique to transport IHP into RBC. Using this procedure, it is possible to obtain a significant increase in P50 while maintaining in vitro cellular integrity. For example, IHP incorporation sufficient to cause an increase in the P50 of 20 mm Hg may be achieved with recovery of approximately 75% of the hemoglobin and with maintenance of ATP levels compatible with good viability. The continuous-flow method allows uniform treatment of large, unit-size volumes of red cells with a relatively small quantity of reagents. The final cell product is macrocytic/hypochromic with an increased number of stomatocytes.     

Effect of Pyridoxal 5''-Phosphate on the Oxygen Affinity of Human Erythrocytes

Pyridoxal 5'-phosphate (PLP), an allosteric effector for the oxygenation of haemoglobin, was incorporated readily into erythrocytes and disappeared from them by simple passive diffusion. The disappearance of PLP from the cells was accelerated by the generation of 2,3-DPG in a medium of inosine, pyruvate and phosphate. The oxygen dissociation curve measured at an extracellular pH of 7.4 demonstrated that PLP incorporated into the cells also lowered the oxygen affinity and that PLP functionally compensated for a metabolically reduced 2,3-DPG. However, the dependency of the oxygen affinity on the intracellular PLP concentration showed a different pattern from the observed for 2,3-DPG. On the other hand, the lowering of intracellular pH by organic phosphates accumulated in the cells was much larger with PLP than with 2,3-DPG. The peculiar relationship between the oxygen affinity of erythrocytes and the intracellular PLP concentration is discussed in detail. The present study may offer a new prospect for the preservation of blood with a normal function.     

Red cell organic phosphates and Bohr effects in house sparrow blood

CO2 and fixed acid Bohr effects (d log PO2/d pH) were determined for fresh whole blood of adult house sparrow (Passer domesticus) at 35, 41 and 45 degrees C. At each temperature, the effects of titrating blood with CO2 at constant base excess (CO2 Bohr effect) were similar at P50 (35 degrees, -0.48; 41 degrees, -0.49; 45 degrees, -0.49). The CO2 Bohr slopes were also reasonably saturation independent between 10 and 90% S. Fixed acid Bohr values, determined by titrating sparrow blood with HCl and NaHCO3 at 4% CO2, were significantly less than the corresponding CO2 coefficients at half saturation (35 degrees, -0.43; 41 degrees, -0.39; 45 degrees, -0.41). The difference between CO2 and H+ Bohr effects, assumed here to represent carbamino CO2 binding to hemoglobin, decreased in magnitude with increasing saturation at each temperature. Inositol pentaphosphate (IPP, 3.1 mumol/ml RBC) and ATP (7.7 mumol/ml RBC) were the major organic phosphates present in Passer erythrocytes. CO2 and organic phosphates are known to complete for common binding sites on the Hb molecule. Because of IPP's strong binding affinity and high concentration in most avian red cells, carbamate formation is generally suppressed in bird blood. The presence of a small but significant specific CO2 effect in Passer blood may indicate that one or both sparrow isohemoglobins has reduced affinity for IPP and/or ATP, permitting CO2 to compete more effectively in Hb-carbamate formation.     

The oxygen transport function and energy metabolism of erythrocytes in human aging

Age-related changes of hemoglobin affinity to oxygen and intraerythrocyte factors which determine it (pH, pCO2, rate of glycolysis, content of organic phosphates) were studied. The results obtained showed the decrease in aging of the affinity of hemoglobin to oxygen resultant from the decrease of pH of intraerythrocyte medium (Bohr effect), despite the inhibited energy metabolism and decreased content of organic phosphates in erythrocytes.     

Inhomogeneity of Hemoglobin. VI. The Minor Hemoglobin Components of Cord Blood

In a survey of over 300 cord bloods of Negro babies, some 54 cases wereselected at random to study the occurrence of the minor hemoglobin components Hb-Bart’s, Hb-H, and Hb-A₂. The relationship of these componentsto Hb-F is discussed. Seven cases with moderate amounts of Hb-Bart’s andHb-H were studied more intensively, along with the respective parents whenpossible. The findings suggest the absence of expression beyond infancy ofany abnormality responsible for chain-lacking hemoglobin types of cordblood.

The physiology of Hb-Bart’s reveals its high oxygen affinity to be approximately 8-10 times that of Hb-A, its lack of a Bohr effect and an oxygen dissociation curve showing no heme-heme interaction.

The discovery of a minute amount of a Hb-Gower II-like component in acord blood sample of a prematurely born white baby of 7.5 months is alsopresented.

Submitted on March 19, 1962 Accepted on May 12, 1962     

The Concentration Dependence of the Oxygen Affinity of Haemoglobin S

The effect of the concentration of haemoglobin S (Hb S) on its oxygen-dissociation properties was studied using either reconstituted Hb-S cells of different mean corpuscular haemoglobin concentrations (MCHCs) prepared by osmotic lysis, or cells in which Hb S is diluted by the presence of another haemoglobin. Only 4% (phosphate buffer) and 21%(bis Tris) of the low oxygen affinity of fresh Hb-S cells was found to be due to their slightly elevated intracellular 2,3-DPG concentrations since when the cells were depleted of 2,3-DPG most of the low affinity remained. The low affinity showed a marked dependence upon haemoglobin concentration which was absent for 2,3-DPG-depleted Hb-A cells and, by extrapolation, the MCHC at which the oxygen affinities of the Hb-S cells became identical to that of the Hb-A cells was 14.5 g/dl in phosphate buffer and 13.1 g/dl in bis Tris. Both fresh and 2,3-DPG-depleted cells containing another haemoglobin as well as Hb S (Hb-SA, Hb-SC and Hb-SF cells) were also found to have low oxygen affinities provided that the intracellular Hb-S concentration(MC(Hb-S)C) was above a certain level. These also showed a strong dependence upon the MC(Hb-S)C. The mean MC(Hb-S)C at which the low oxygen affinities of the DPG-depleted cells were abolished were 8.3 g/dl (phosphate) and 11.2 g/dl (bis Tris). Hb F in fresh Hb-SF cells brought about a much greater increase in oxygen affinity than the same amount of either Hb A or Hb C. In 2,3-DPG depleted cells Hb A showed a greater ability to 'dilute' the Hb S than did Hb C. The conditions for the low oxygen affinity of Hb S were therefore found to be very similar to those required for the gelling of both pure Hb S, and Hb S in haemoglobin mixtures. It was concluded therefore that the low oxygen affinity of the Hb S was caused by the polymerization and that the difference between the oxygen affinities of Hb-S and Hb-A cells may be used as a measure of the polymerization process.     

Hemoglobin Affinity for Oxygen in Chronic Renal Disease: The Effect of Hemodialysis

The affinity of hemoglobin for oxygen mayincrease significantly in subjects who arehypophosphatemic and alkalotic. Westudied the organic phosphate content andoxygen binding by hemoglobin of red cellsin subjects undergoing hemodialysis, during which time a decrease in plasma inorganic phosphate and an increase inblood pH may occur. Red cell 2,3-DPGwas not correlated with plasma inorganicphosphorus, whereas red cell ATP washighly correlated with plasma inorganicphosphorus when analyses were made onpredialysis samples. Predialysis red cellinorganic phosphorus was highly correlated with plasma inorganic phosphorus,supporting the concept that intraerythrocytic inorganic phosphorus is maintainedby a gradient from plasma to cell. Plasmainorganic phosphorus decreased by 45%during the period of hemodialysis, whereasred cell inorganic phosphorus did notchange. Red cell 2,3-DPG, ATP, and oxygen binding by hemoglobin at standardconditions of temperature, pH, and pCO₂were not altered after 6 hr of hemodialysis. Plasma pH and base excess increasedduring dialysis. The increase in base excess, an estimate of the non-pH-dependenteffect of CO₂ on oxygen binding by hemoglobin, counterbalanced a portion of theeffect of elevated pH on hemoglobin—oxygen affinity under in vivo conditions.Hence, only a slight increase in oxygenbinding by hemoglobin occurred. Moreover, late dialysis symptoms were notassociated with the degree of alkalosis orwith the extent of change in hemoglobin’saffinity for oxygen. Red cell 2,3-DPG content was lower and hemoglobin’s affinityfor oxygen was higher in subjects withchronic renal disease than in nonazotemicsubjects with similar hemoglobin deficits.Moreover, increased red cell ATP in chronicrenal disease patients did not influenceoxygen binding by hemoglobin.

Submitted on June 11, 1973 Revised on July 30, 1973 Accepted on September 13, 1973     

Hemoglobin Function in the Horse: The Role of 2,3-Diphosphoglycerate in Modifying the Oxygen Affinity of Maternal and Fetal Blood

The blood of the newborn horse was foundto have a higher affinity for oxygen thanthat of the mother. This difference was dueto the fact that the red cells of newbornfoals contained 36% lower 2,3-diphosphoglycerate (2,3-DPG) than red cells fromtheir respective mares. The ATP levels offoal and maternal red cells did not differsignificantly. Following birth a prompt risein the foal's red cell 2,3-DPG occurred,approaching normal (maternal) levelswithin 5 days. Unlike many other species,the hemoglobins of the newborn and adulthorse have been shown to be structurallyidentical. Furthermore, phosphate-freesolutions of newborn and maternal hemoglobins had identical oxygen saturationcurves in the absence and presence ofadded 2,3-DPG. This study demonstratesthat, in contrast to other species. the increased oxygen affinity of horse fetal redcells is due to a lower level of the cofactor2,3-DPG rather than to the presence offetal hemoglobin.

Submitted on November 28, 1972 Revised on February 17, 1973 Accepted on March 5, 1973     

The Control of Oxygen Affinity of Red Cells with Hb-Shepherds Bush

The red cells of the original patient with Hb-Shepherds Bush (Hb-ShB 25%, Hb-A 75%) have a physiologically significant increased oxygen affinity. This is one third due to the intrinsic raised oxygen affinity of Hb-ShB and two-thirds due to its diminished response to 2,3-DPG, there being no interaction between the two haemoglobins present in the cells.     

Functional Nonequivalence of α and β Hemes in Human Adult Hemoglobin

Nuclear magnetic resonance studies of the contact-shifted spectra of heme protons in deoxyhemoglobin A from human adults show conclusively that oxygen binds to the alpha hemes in preference to the beta hemes. The preferential binding is produced in 10% hemoglobin solution at neutral pH by either a 15-fold molar excess of 2,3-diphosphoglycerate or a 5-fold molar excess of inositol hexaphosphate. Preferential binding is not observable in the absence of the organic phosphates. The results indicate that the oxygenation of hemoglobin may be described by a sequential model, or by a concerted model that allows the alpha hemes to bind ligand first.     

Study of a kindred with partial deficiency of red cell 2,3-diphosphoglycerate mutase (2,3-DPGM) and compensated hemolysis.

A kindred with partial deficiency of red cell 2,3-diphosphoglycerate mutase (2,3-DPGM) was studied. The propositus presented with indirect hyperbilirubinemia, normal hemoglobin (15.8 g/dl), and elevated reticulocyte count (4.6%). The red cell 51Cr survival was decreased (tau1/2 16 days). Incubated osmotic fragility was normal; autohemolysis was increased and corrected with glucose and ATP. The P50 was 18.5 mm Hg (normal 25.5 +/- 3), but the stability, electrophoresis, and fingerprinting of hemoglobin were normal. The concentration of 2,3-diphosphoglycerate (2,3-DPG) was reduced to 43% of normal. Red cell 2,3-DPGM was decreased to 59% of normal; 2,3-DPG phosphatase was similarly decreased. All red cell glycolytic and hexose monophosphate shunt enzymes, glycolytic intermediates other than 2,3-DPG, and glucose consumption and lactate production were normal. Five family members showed similar hematologic findings. The deficiency appears to be secondary to decreased enzyme synthesis and to be inherited as an autosomal dominant trait in this family. Partial deficiency of 2,3-DPGM should now be considered in the differential diagnosis of compensated hemolysis associated with increased oxygen affinity.     

Evolution of mammalian hemoglobin function

Throughout their evolution, mammalian hemoglobins have acquired a broad repertoire of functional properties well suited to the internal milieu of the red cell. Mammals display a wide range in whole blood oxygen affinity dependent on three major factors: the intrinsic oxygen affinity of the hemoglobin, the level of red cell 2,3-DPG, and the response of the hemoglobin to 2,3-DPG. The concentration of 2,3-DPG varies among groups of mammals. Those animals (cats and ruminants) that have very low levels of this intracellular mediator have hemoglobins of intrinsically low oxygen affinity that fail to respond to the addition of 2,3-DPG. Mammals that have adapted to various types of hypoxia tend to have increased oxygen affinity, primarily mediated through reduced levels of red cell 2,3-DPG. In contrast, mammals who are experimentally subjected to low oxygen tensions develop decreased oxygen affinity owing to increased red cell 2,3-DPG. Mammals employ one of three different mechanisms for the maintenance of higher oxygen affinity of fetal red cells, compared to maternal red cells. Many of these phenomena can be satisfactorily explained at the molecular level but their adaptational significance is less clear.     

Red cell 2,3-diphosphoglycerate and oxygen affinity of hemoglobin in patients with thyroid disorders

We measured red blood cell 2,3-diphosphoglycerate (2,3-DPG), adenosine triphosphate (ATP), and the P50 value in vitro of the oxyhemoglobin dissociation curve, which is the oxygen tension at half saturation of hemoglobin, in order to quantitate red blood cell oxygen transport function in individuals who were diagnosed as hypothyroid, euthyroid, or hyperthyroid based on measurements of thyroxine (T4), triiodothyronine (T3), thyrotropin (TSH), and their clinical status. Hypothyroid (mean T4 2.8 microgram/dl, T3 49 ng/dl, TSH 37 microU/ml) and hyperthyroid (mean T4 14 microgram/dl, T3 271 ng/dl, TSH less than 0.7 microU/ml) patients had normal red cell 2,3-DPG and ATP levels and normal P50 values in vitro. The known changes in oxygen consumption produced by alterations in thyroid hormone levels in patients with hypothyroidism or hyperthyroidism did not affect red blood cell oxygen transport function.     

Oxygen and carbon dioxide transport in the blood of the muskrat (Ondatra zibethica)

We have investigated the oxygen and carbon dioxide transport properties of a small diving mammal, the muskrat (Ondatra zibethica), where the hemoglobin primary structure has been established by Duffy et al. (1978). While whole blood oxygen capacity, the Haldane effect and the buffer capacity are not different compared to non-diving mammals of similar size, the Bohr effect and the oxygen affinity are increased. The oxygen half saturation pressure (P50) was 26.1 mm Hg (3.5 kPa) at pH 7.4, and the Bohr effect -0.66 (related to plasma pH) and -1.07 related to cell pH. The high affinity of muskrat blood is caused by a comparatively small effect of 2,3 DPG and CO2 on muskrat hemoglobin, that is accentuated through a relatively low concentration of 2,3-DPG in the muskrat red cell. The increased Bohr effect is caused primarily through the pronounced pH dependence of oxygen-linked binding of 2,3-DPG. The weak interaction of muskrat hemoglobin with 2,3-DPG is not caused by substitutions at the binding site.     

Distribution of phosphorylated metabolites and magnesium in the red cells of a patient with hyperactive pyruvate kinase

The intracellular distribution of adenosine 5'-triphosphate (ATP) and 2,3-diphosphoglycerate (2,3-DPG) was studied in the red cells of a patient with a "high-ATP syndrome" by using 31P nuclear magnetic resonance. In this patient, red cell ATP was increased 2.5-fold, whereas 2,3-DPG was decreased fourfold due to the presence of a hyperactive pyruvate kinase. In oxygenated red cells, these abnormal concentrations were reflected to the same extent in all complexes in which ATP and 2,3-DPG take part. The diminished amount of 2,3-DPG bound to hemoglobin was almost completely replaced by ATP-hemoglobin complexes. Therefore, free hemoglobin was only slightly increased. In deoxygenated cells, the relative distribution of ATP and 2,3-DPG complexes was significantly disturbed. The main difference was a shift in the ratio of magnesium ATP (MgATP) over the ATP-hemoglobin complex; 74% of total ATP was complexed to hemoglobin (45% in normal cells), whereas the concentration of MgATP was only slightly increased with respect to normal. The shortage in 2,3-DPG bound to hemoglobin could partially be replenished by an increase in hemoglobin (Mg) ATP complexes. Therefore, the amount of uncomplexed hemoglobin raised from 15% in normal cells to 38% in the patient's cells. As a result, the oxygen-dissociation curve was only moderately shifted to the left. It is concluded that the regulatory role of 2,3-DPG in oxygen transport is taken over in part by (Mg) ATP in this patient. In both aerobic and anaerobic cells, the increase in magnesium bound to ATP, either free or bound to hemoglobin, exceeds the decrease in 2,3-DPG Mg complex. In spite of this, the amount of intracellular free Mg++ was normal or slightly lowered. This suggests the presence of a compensatory mechanism by which the amount of total cellular magnesium could be increased.     

Influence of the Cryoprotective Agents Glycerol and Hydroxyethyl Starch on Red Blood Cell ATP and 2,3-Diphosphoglyceric Acid Levels

Because hydroxyethyl starch (HAES) is used for volume replacement therapy and as a cryoprotectant for frozen red blood cells (RBCs), this compound, in contrast to glycerol, does not require labor-intensive removal from thawed cells prior to transfusion. We here report the effect of both glycerol and HAES on the RBC organic phosphates ATP and 2,3-diphosphoglyceric acid (2,3-DPG). The CPD-A1-stabilized RBCs of 20 healthy donors (3 females, 17 males) were separately frozen in either 40% glycerol or 6% HAES, of molecular weight 200,000. ATP and 2,3-DPG concentrations were determined in CPD-A1 RBCs before addition of cryoprotectant and in cryopreserved thawed RBCs after 24 h storage at -80 degrees C (glycerol) and -196 degrees C (HAES). It appears that HAES, but not glycerol, significantly reduces ATP concentrations whereas both lead to a reduction of 2,3-DPG concentrations; this reduction was more pronounced with glycerol than with HAES. Experiments with the blood of 6 donors demonstrated that HAES affects autohemolysis by 16%, in contrast to glycerol, after which cryoprotectant autohemolysis was affected by 3.1% only. RBC recoveries were comparable using glycerol or HAES as cryoprotectants. A distinct pattern of reduction of 2,3-DPG levels by glycerol and less by HAES, and of ATP levels by HAES but not by glycerol, emerges. Our findings may be of importance if HAES is to be introduced as a convenient cryoprotectant.     

The oxygen affinity of chicken haemoglobin in whole blood and erythrocyte suspensions.

The oxygen equilibrium of whole chicken blood has a P50 of 52.3 mm Hg and a Hill coefficient of 2.6 at pH 7.4 and 37 degrees C when determined with new microtechniques which are not vitiated by cellular respiration. The apparent failure of the haemoglobin to reach full saturation at arterial PO2 IS DISCUSSED IN RELATION TO THE HAEMoglobin-oxygen equilibrium concept. The low affinity observed is due to intraerythrocytic inositol pentaphosphate (IPP), and the affinity of a haemolysate "stripped" of IPP is greatly increased (deltalog P50 =1.25). Unlike the mamalian analogue 2, 3-diphosphoglycerate, IPP concentration of whole blood does not decrease after incubation for 10 hr at 37 degreesC.