Functional aspects of hemoglobin evolution in the mammals

Summary Comparative studies of red cell 2,3 Diphosphoglycerate (DPG) and its effect on hemoglobin oxygen affinity from a taxonomically diverse set of mammals indicate two anomalous groups: members of the superfamilies Bovoidea (Actiodactyla) and Feloidea (Carnivora). In both taxa all of the individuals assayed had very low or unmeasurable quantities of DPG and red cell lysates with little, if any, DPG effect as measured by the change in oxygen affinity in the absence and presence of the phosphate. However, in both groups compensatory changes have occurred in hemoglobin structure and function so as to reduce the native oxygen affinity and thus cause them to resemble the hemoglobins of DPG-utilizing mammals as they occur in the setting of the red cell. We conclude that this parallelism of function is the result of convergent evolution.     

The red cell sodium, potassium, inorganic phosphate, ATP and 2,3DPG concentrations in chronic renal failure

In dialysis and non-dialysis patients with terminal renal failure the red cell ATP and 2,3 DPG concentrations are increased. Inorganic phosphate level in red cells is nearly normal in both groups of patients. The red cell potassium concentration is decreased in both groups. Sodium concentration in red cells is significantly lower in dialysis than in non-dialysis patients, the former being lower and the latter being higher, than the average normal value.     

Regulation of Hemoglobin Function in Mammals

This survey of hemoglobin function in mammals reveals a broadrange in oxygen affinity. The concentration of red cell 2,3-DPGvaries widely among groups of mammals. Those animals (feloidsand ruminants) that have very low levels of this intracellularmediator have hemoglobins of intrinsically low oxygen affinitywhich fail to respond to the addition of 2,3-DPG. Mammals whichhave adapted to various types of hypoxia tend to have increasedoxygen affinity, primarily mediated through reduced levels ofred cell 2,3 DPG. In contrast mammals who are experimentallysubjected to low oxygen tensions develop decreased oxygen affinityowing to increased red cell 2,3-DPG. Mammals employ one of threedifferent mechanisms for the maintenance of higher oxygen affinityof fetal red cells, compared to maternal red cells. Many of these phenomena can be satisfactorily explained at themolecular level but their adaptational significance is lessclear.     

Human red cell glycolysis in high altitude chronic hypoxia

We have found that glycolysis in human red blood cells under the hypoxic conditions found at high altitudes is connected with changes in enzyme activities and levels of various metabolic intermediates. The sensitivity of the four kinases to hypoxia results in 1) glycolytic hyperactivity leading to a higher intracellular energy state, and 2) accumulation of 2–3 DPG, whose role in the adaptation of red blood cell respiration to high altitude has been shown by previous research. PEP, 3PG, and G6P appear to be the main regulating intermediates in glycolysis in this system. The reason for the very large increase in G1-6DP is still not clear.     

Hematological changes and athletic performance in horses in response to high altitude (3,800 m)

This study had two goals: 1) measure hematologic changes with high-altitude acclimatization in horses; and 2) assess the effect of 9 days at high altitude on subsequent athletic performance at low altitude. Six horses performed standardized exercise tests on a dirt track (before and during time at altitude) and treadmill (pre- and postaltitude exposure). Resting and immediate postexercise blood samples were measured for blood volume, lactate, red cell number, packed cell volume, and 2,3-diphosphoglycerate (DPG) concentrations at 225 m, over a 9-day period at 3,800 m, and shortly after returning to 225 m. Acclimatization produced increases in total red cell volume (38.2 +/- 2.4 to 48.1 +/- 2.9 ml/kg, P = 0.004) and DPG/hemoglobin concentrations (19.4 +/- 1.7 increased to 29.4 +/- 0. 4 micromol/g, P = 0.004). Two performance variables, heart rate recovery postexercise and lactate recovery, were faster after acclimatization.     

Association between cholesterol and 2,3-diphosphoglycerate in genetically selected hooded rat lines

We have developed two strains of hooded rats with differing erythrocyte oxygen affinities by selection on red cell 2,3-diphosphoglycerate levels. Genetic studies have shown that these strains differ at one DPG-level-determining locus. This article reports the results of a study which involved measurement of plasma cholesterol levels in rats from the strains and the F₂ progeny of strain intercrosses. Low-DPG strain rats, with high oxygen affinity, had significantly higher mean cholesterol levels than High-DPG rats. Animals from the extremes of the F₂ distribution of DPG levels showed similar, significantly different mean cholesterol levels, indicating that the negative association between DPG and cholesterol levels in strain rats was not due to inadvertent fixation of unrelated genes during selection on DPG. The possibility is discussed that high oxygen affinity, brought about by low DPG levels, may be causative in increasing cholesterol levels.This work was supported by an NIH Training Grant (5-T01-GM-0071) and a Michigan Heart Association Grant.     

An expanded two-state allosteric model for interactions of human hemoglobin A with nonsaturating concentrations of 2,3-diphosphoglycerate

Oxygen binding curves (OEC) for red cell suspensions have a biphasic shape and reduced n50 values when the concentration of 2,3-diphosphoglycerate (DPG) is lowered by aging or experimental procedures. The mechanism for the abnormal shape of the OEC has been related to variations in the activity of free DPG. DPG binds to tetrameric Hb at a single site, and in red cells its normal concentration is equivalent to that of tetrameric Hb. This equivalence renders the oxygen affinity of Hb and the shape of the OEC very sensitive to small changes in the activity of DPG. The OEC for stripped Hb solutions in the presence of nonsaturating concentrations of DPG also exhibit a biphasic shape but with much larger changes in the n values than observed for red cells. Upon addition of chloride, a known competitor of DPG binding to Hb, the shape of the OEC becomes similar to that of red cell suspensions with the same DPG/Hb ratio. Studies on Hb solutions in the presence of varying concentrations of DPG, but without chloride, have revealed that the cofactor shifts the entire OEC to the right, including both its upper and lower asymptotes. This finding indicates that DPG lowers the intrinsic oxygen affinity for both the T and R states. Theoretical considerations leading to a successful modeling of OEC obtained under varying conditions of DPG and chloride require an expanded two-state allosteric model in which allowance is made for DPG-dependent variations in the dissociation constants of oxygen for both the T and R conformations.     

2,3-diphosphoglycerate and glycosylated hemoglobin in diabetes mellitus

Large amounts of intraerythrocyte 2-3 diphosphoglycerate (2-3 DPG) increase red cell oxygen-releasing capacity. Since glycosylated hemoglobins, found in higher percentages in diabetics, have an increased oxygen affinity, 2-3 DPG concentration was assayed in 12 diabetics (4 I.D.D., 8 N.I.D.D.) and 18 healthy volunteers. 2-3 DPG was related to glycemic fasting values and to glycosylated hemoglobins to evaluate if 2-3 DPG levels increase in diabetics as a compensatory mechanism to prevent peripheral hypoxia. 2-3 DPG values were significantly higher in diabetics than in normals: 11.4 mumol/gHb +/- 1.7 (= M +/- 1 SD) vs 9.8 mumol/gHb +/- 2.3 (p less than 0.05). 2-3 DPG did not correlate significantly to glycosylated hemoglobins or to glycemic values neither in diabetics nor in normals. These preliminary observations emphasize the usefullness of 2-3 DPG assay in evaluating peripheral oxygenation in diabetics: 2-3 DPG is higher in diabetics but does not correlate to glycemic equilibrium.     

Simultaneous determination of low free Mg2+ and pH in human sickle cells using 31P NMR spectroscopy

The concentrations of free magnesium, [Mg(2+)](free), [H(+)], and [ATP] are important in the dehydration of red blood cells from patients with sickle cell anemia, but they are not easily measured. Consequently, we have developed a rapid, noninvasive NMR spectroscopic method using the phosphorus chemical shifts of ATP and 2,3-diphosphoglycerate (DPG) to determine [Mg(2+)](free) and pH(i) simultaneously in fully oxygenated whole blood. The method employs theoretical equations expressing the observed chemical shift as a function of pH, K(+), and [Mg(2+)](free), over a pH range of 5.75-8.5 and [Mg(2+)](free) range 0-5 mm. The equations were adjusted to allow for the binding of hemoglobin to ATP and DPG, which required knowledge of the intracellular concentrations of ATP, DPG, K(+), and hemoglobin. Normal oxygenated whole blood (n = 33) had a pH(i) of 7.20 +/- 0.02, a [Mg(2+)](free) of 0.41 +/- 0.03 mm, and [DPG] of 7.69 +/- 0.47 mm. Under the same conditions, whole sickle blood (n = 9) had normal [ATP] but significantly lower pH(i) (7.10 +/- 0.03) and [Mg(2+)](free) (0.32 +/- 0.05 mm) than normal red cells, whereas [DPG] (10.8 +/- 1.2 mm) was significantly higher. Because total magnesium was normal in sickle cells, the lower [Mg(2+)](free) could be attributed to increased [DPG] and therefore greater magnesium binding capacity of sickle cells.     

The anemia of childhood revisited

The question of the mechanism of the physiological anemia of childhood was reexamined; we took into consideration the fact that microcytosis is a feature of this anemia as well as the evidence suggesting that the 2,3 DPG/hemoglobin ratio may depend in part on red cell size. In a group of normal, not-iron deficient children a high inverse correlation was obtained between the MCV and the 2,3 DPG/Hb ratio; this latter ratio did not correlate with serum phosphorus levels, previously incriminated in the indirect causation of the anemia of childhood.     

In vitro evaluation and study of the survival of erythrocytes preserved in a saline-adenine-glucose-mannitol medium for 35 days

The saline-adenine-glucose-mannitol (SAGM) solution for resuspension of red cells was evaluated on 30 blood units tested over 42 days and compared to 5 red cell concentrates collected on the conventional CPD medium. Total and extra-cellular hemoglobin, potassium, pH, ATP and DPG concentrations, osmotic fragility, schizocyte formation, and red cell antigenicity were studied through the storage period. Chromium survival studies of autologous donated red cells were performed in 10 donors. Red cell concentrates resuspended in SAGM solution showed at the 35th day of conservation at 4 degrees C, a mean storage hemolysis of only 0.66%, an ATP concentration of 67% of the initial value, a schizocyte proportion of less than 1.5%, a mean 24 hour posttransfusion viability of 88.33% and a mean red cell T 1/2 survival of 25 days 10 hours. No alteration of common blood group antigens could be found after storage of red cells for 42 days.     

Changes in mean erythrocyte volume and 2,3-diphosphoglycerate in two groups of diabetic subjects

Mean corpuscular volume and Red Blood Cell 2-3, diphosphoglycerate in insulin dependent diabetics and in non insulin dependent diabetics were evaluated. Only in insulin dependent diabetics an increased mean corpuscular volume was found while in non insulin dependent diabetics red blood cell 2-3, DPG level appears to be reduced. These findings were not correlated with the metabolic parameters neither they seemed to be dependent upon sex, age or vascular disease. The Authors suggest that the increase of mean corpuscular volume might be indicative of a tendency to macromegaloblastosis in insulin dependent diabetics; with regard to RBC 2-3, DPG level it seems to be extremely variable in dependence on oxygen request at the tissue level.     

Blood constituents during the estrous cycle and early pregnancy in dairy cows

The objective of this study was to determine if maternal platelet count, white blood cell count or other blood constituents undergo sustained alterations in concentration following fertilization. Blood samples from 17 Holstein females were collected over an 18-d period starting at estrus. Blood was analyzed for levels of platelets, white blood cells, red blood cells, hemoglobin and hematocrit. Results were analyzed for differences between nonpregnant and pregnant groups. Analysis of variance revealed a day-by-group interaction in the platelet count (P<0.01). White blood cell count showed both a day-by-group interaction and a difference between days (P<0.01). Red blood cell count, hematocrit and hemoglobin levels resulted in no significant difference between the two groups (P>0.05). While statistically significant differences were observed in platelet and white blood cell count, neither of these were sustained over a period longer than 2 d.     

Human red blood cells with normal or improved oxygen transport function prepared and frozen in the primary polyvinyl chloride plastic blood collection container.

This is a report a a new system for freezing human red blood cells in the same polyvinyl chloride plastic container in which the blood is collected and separated into components. This polyvinyl chloride plastic collection bag with integrally attached transfer packs for blood collection, component separation, red blood cell biochemical modification, freezing, storage, and post-thaw dilution before washing, represents a major advancement in the freeze-preservation process. The label with the donor's blood type and identification number affixed to the bag at the time of collection remains in place throughout the freezing and thawing process. The transfused red blood cells are of superior quality, and the processing cost is less than with other methods of freeze-preservation. There is a lower risk of contamination with these red blood cells because manipulation of the product is kept at a minimum. "Rejuvenation", a bioengineering process by which outdated red blood cells can be salvaged, can be incorporated into the preservation process using one of the attached transfer packs of the primary collection bag. This process has been introduced as a possible means of alleviating the dramatic blood shortages which occur periodically. Red blood cells may also be "rejuvenated" after storage in the liquid state to increase their 2,3 DPG and ATP levels to 150 to 200% of normal, and these red blood cells with improved oxygen transport function have been administered to anemic patients with and without cardiopulmonary insufficiency, patients undergoing cardiopulmonary bypass and treatment with hypothermia during cardiac surgery, and in instances where nonhemolytic transfusion reactions might be expected.     

Hemoglobin-oxygen affinity in anemia

In blood of 21 anemic patients and 8 normal subjects (N) three oxygen dissociation curves each were measured at different pH values to calculate Bohr coefficients after acidification with CO2 (BCCO2) or fixed acid (BCFA), and other important parameters of oxygen affinity. The patients had either low hemoglobin or red cell production (L: n = 11, 7.3 g/dl Hb) or high erythrocyte production combined with high loss (H: n = 10, 7.8 g/dl Hb). The standard half saturation pressure P50 (pH 7.4, 37 degrees C) was equally elevated in both anemic groups (L: 30.5, H: 30.8, N: 26.7 mmHg), as well as the diphosphoglycerate concentration (DPG) (L: 18.7, H: 18.6, N: 12.7 mumol/g Hb). The red cell pH of the anemics was lower than for the N (approximately 0.045 units) causing part of the difference in P50. Hill's "n" tended to high values in the anemics except at low O2-saturation in the H. For BCCO2 no significant difference among the groups was observed. BCFA, however, increased in the H at low SO2 compared to the N and L. The cause for most of the changes in hemoglobin oxygen affinity in anemics was the high [DPG]. The combination of high P50 and high "n" value as in the L seems to be most advantageous for tissue oxygenation.     

Reduced hemoglobin affinity for oxygen in venous blood following hemodilution, independent of changes in pH or PCO2.

A rapidly induced and readily reversible shift in the affinity of hemoglobin for oxygen has been demonstrated. The shift, similar to the Bohr effect, is independent of PCO2 or pH changes. It occurred within 30 min of hemodilution and was seen in portal venous blood but not arterial blood. A hypothesis is suggested involving a phasic alteration in levels of 2,3-diphosphoglycerate (DPG) or ATP binding to hemoglobin. It is proposed that, following hemodilution, the degree of these phosphates to hemoglobin increases on passage through the intestinal vascular bed. The increased DPG binding to hemoglobin results in displacement of additional oxygen. As the blood becomes reoxygenated, the levels of DPG-hemoglobin binding decline and DPG is displaced from the hemoglobin by oxygen.     

Structural features required for the reactivity and intracellular transport of bis(3,5-dibromosalicyl)fumarate and related anti-sickling compounds that modify hemoglobin S at the 2,3-diphosphoglycerate binding site

Bis(3,5-dibromosalicyl)fumarate (I) reacts preferentially with oxyhemoglobin to cross-link the two beta 82 lysine residues within the 2,3-diphosphoglycerate (DPG) binding site and as a result markedly increases the solubility of deoxyhemoglobin S. The cross-link acts by perturbing the acceptor site for Val 6 within the sickle cell fiber (Chatterjee, R., Walder, R. Y., Arnone, A., and Walder, J. A. (1982) Biochemistry 21, 5901-5909). In the present studies we have compared a large number of analogs of I to determine the structural features of the reagent required for specificity and for transport into the red cell. Both electrostatic and hydrophobic interactions contribute to the binding of these compounds at the DPG site. The optimal position for the negatively charged groups on the cross-linking agent for productive binding is adjacent to the ester as in the original salicylic acid derivatives. There is a direct correlation between the reactivity toward hemoglobin and the hydrophobicity of the substituent attached at the para position. Phenyl and substituted phenyl derivatives as in the analgesic, antiinflammatory drug diflunisal are particularly effective. These groups probably interact with hydrophobic residues of the amino-terminal tripeptide and the EF corner of the beta chains adjacent to the DPG binding site. Although bis(3,5-dibromosalicyl)fumarate is very reactive toward hemoglobin in solution, it is much less effective in modifying hemoglobin within the red cell. The reaction with intracellular hemoglobin was shown to be limited by competing hydrolysis of the reagent catalyzed at the outer surface of the erythrocyte membrane. Inactivation of the red cell membrane acetylcholinesterase with phenylmethylsulfonyl fluoride did not inhibit this reaction. Introduction of a single methyl group onto the carbon-carbon double bond of the fumaryl moiety decreases the lability of the ester 10-fold, due to steric effects, and allows the reagent to be taken up by the red cell and modify intracellular hemoglobin. The kinetics of transport of the methylfumarate derivative, bis(3,5-dibromosalicyl)mesaconate, are first-order, consistent with passive diffusion. The attachment of larger alkyl groups onto the cross-link bridge further enhances the transport of the reagent into the red cell. The solubility of deoxyhemoglobin S cross-linked with the butylfumarate derivative was found to be increased by almost 10% compared to the original fumarate diester.(ABSTRACT TRUNCATED AT 400 WORDS)     

Biochemical parameters to assess viability of blood stored for transfusional use

Blood collected in citrate-phosphate-dextrose-adenine (CPDA-1) containing transfusional bags, was weekly tested throughout a 35 day period. Biochemical assays included plasmatic glucose, electrolytes, free Hb, acid-base balance and hemogasanalysis. Intraerythrocytic ATP and 2,3 DPG were also determined. Results show that an almost total 2,3 DPG depletion occurs during the first three weeks, whereas intracellular ATP are about 50% of the initial values, at the same time. Lowering of pH is also maximal at the third week. pCO2 variation pattern is biphasic: an early increase due to HCO3- titration by lactic acid arising from red cells glycolysis, followed by a decrease probably due to plastic bag permeability to CO2 itself. The percentage of O2Hb also rises during blood storage: this might be the combined result of increase in pO2 and decrease in 2,3 DPG content. A rise of free Hb was obtained; extracellular K+ levels underwent a sixfold increase in 35 days. The mechanism of relative variations of these parameters and the gas transport are discussed. Some of these parameters might be used as routine controls to asses viability and functional status of stored red cells for transfusional use.     

Identification of a Major Locus Contributing to Erythrocyte 2,3-Diphosphoglycerate Variability in Hooded (Long-Evans) Rats

The erythrocyte glycolytic intermediate 2,3-diphosphoglycerate (DPG) and adenosine triphosphate (ATP) play an important role in oxygen transport and delivery by binding to hemoglobin (Hb) and reducing its affinity for oxygen. Considerable quantitative variability in the levels of DPG and ATP exists in human populations and in a population of hooded (Long-Evans) rats we have studied. This paper presents the results of studies on the genetic component of DPG-level variation in an outbred population of hooded rats. Beginning with about 100 rats, a two-way selection experiment was initiated. Pairs of rats with the highest DPG levels were mated to produce a High-DPG rat strain and animals with the lowest DPG levels were mated to produce a Low-DPG strain. Mean DPG levels responded rapidly to selection and, from generation 3 on, the differences between strain means were highly significant. Ten High-DPG strain rats were intercrossed with 10 Low-DPG strain rats of generation 10 to produce an F₁ generation in which the DPG levels were almost as high as those of High-DPG animals. This indicates partial dominance of High-DPG alleles. The F₂ DPG-level distribution showed two distinct subpopulations. The high DPG subpopulation contained three times as many animals as the low DPG subpopulation. From these results and the statistical analyses performed, it was concluded that the DPG differences between strains were due to an allelic difference at one major locus, the allele carried by the High-DPG strain showing partial dominance over the allele carried by the Low-DPG strain. It appears that this locus may also effect ATP levels to a large extent and is polymorphic in hooded rat populations. Identification of this locus gives us a useful tool for studies of the physiological effects of DPG variability, as well as providing an example of a major gene effect in a quantitatively varying trait.     

Is there a genetic control in man of red blood cell magnesium levels?

Three groups of human subjects underwent successive monthly examinations to determine red blood cell magnesium levels and plasma magnesium, potassium and sodium levels. From these data, a comparison between intra- and inter individual variances showed that red blood cell magnesium had a characteristically high intra individual stability and a wide inter individual variability. This characteristic, which was much more marked than for plasma cholesterol or serum gamma globulin levels, whose high coefficient of heritability has been demonstrated elsewhere, is in good keeping with the hypothesis of genetic control of red blood cell Mg content.