Changes In Hemoglobin-oxygen Affinity Research Articles

Dose- and Sex-Dependent Changes in Hemoglobin Oxygen Affinity by the Micronutrient 5-Hydroxymethylfurfural and α-Ketoglutaric Acid.

5-Hydroxymethylfurfural (5-HMF) is known to increase hemoglobin oxygen affinity (Hb–O2 affinity) and to induce a left shift of the oxygen dissociation curve (ODC). It is under investigation as a therapeutic agent in sickle cell anemia and in conditions where pulmonary oxygen uptake is deteriorated or limited (e.g., various clinical conditions or altitude exposure). The combination of 5-HMF and α-ketoglutaric acid (αKG) is commercially available as a nutritional supplement. To further elucidate dose effects, ODCs were measured in vitro in venous whole blood samples of 20 healthy volunteers (10 female and 10 male) after the addition of three different doses of 5-HMF, αKG and the combination of both. Linear regression analysis revealed a strong dose-dependent increase in Hb–O2 affinity for 5-HMF (R2 = 0.887; p < 0.001) and the commercially available combination with αKG (R2 = 0.882; p < 0.001). αKG alone increased Hb–O2 affinity as well but to a lower extent. Both the combination (5-HMF + αKG) and 5-HMF alone exerted different P50 and Hill coefficient responses overall and between sexes, with more pronounced effects in females. With increasing Hb–O2 affinity, the sigmoidal shape of the ODC was better preserved by the combination of 5-HMF and αKG than by 5-HMF alone. Concerning the therapeutic effects of 5-HMF, this study emphasizes the importance of adequate dosing in various physiological and clinical conditions, where a left-shifted ODC might be beneficial. By preserving the sigmoidal shape of the ODC, the combination of 5-HMF and αKG at low (both sexes) and medium (males only) doses might be able to better maintain efficient oxygen transport, particularly by mitigating potentially deteriorated oxygen unloading in the tissue. However, expanding knowledge on the interaction between 5-HMF and Hb–O2 affinity in vitro necessitates further investigations in vivo to additionally assess pharmacokinetic mechanisms.

High Altitude Genetic Adaptation In Tibetans Does Not Include Increased Hemoglobin-Oxygen Affinity

Increased hemoglobin-oxygen affinity has been shown to be an adaptive response to hypoxia in many high altitude animals such as the Andean goose, guinea pig and llamas (Reynafarje C. 1975; Hebbel R. 1978). It has been reported that people living in a high altitude, hypoxic environment have also developed a similar adaptation. Native Tibetans are known to have lived at an average of 3000-5000 meters on the Tibetan Plateau for more than 20,000 years, and have undergone genetic adaptations that have enabled them to thrive in this reduced oxygen environment. Most Tibetans are thus protected from polycythemia and other features of chronic mountain sickness. Several studies have reported higher arterial oxygen saturations among Tibetans as part of their genetic adaptation (Beall C. 1994; Moore L. 2001; Niermeyer S. 1995), thereby concluding that they have higher hemoglobin-oxygen affinity. Further, recent genomic studies have reported that beta-globin haplotypes (HBB and HBG2) have been selected in Tibetans, suggesting the presence of hemoglobin variants as a beneficial factor of Tibetan adaptation (Yi. 2010). However, some of the reports of increased hemoglobin-oxygen affinity are based on single readings of arterial oxygen measurements. Hemoglobin-oxygen affinity is more optimally measured by deriving the P50 value, which is the partial pressure of oxygen at which hemoglobin is 50% saturated with oxygen. A decreased P50 can be due to mutated globin genes resulting in high oxygen affinity hemoglobins, low 2,3 BPG, high pH or low temperature. The hemoglobin-oxygen dissociation is optimally derived by hemoximeter measurements of the percent saturation of hemoglobin at various partial pressures of oxygen. The resultant curve has a sigmoid shape due to the cooperative binding of oxygen to the four globins in the hemoglobin tetramer; this cooperative interaction can be enumerated as a Hill coefficient “n”. If a hemoximeter is not readily available, the P50 can be estimated from the venous blood gas using the measured pO2, hemoglobin oxygen percent saturation O2%, and pH (Lichtman M. 1976); however the Hill coefficient “n” cannot be derived by this method.To definitely establish whether the Tibetan adaptation to high altitude hypoxia involves increased hemoglobin-oxygen affinity, we conducted the following study of direct and indirect oxygen-hemoglobin affinity among Tibetans living at two different altitudes.We enrolled 14 healthy ethnic Tibetans and one closely related Nepalese Sherpa. There were 8 males and 7 females ages ranging 35-75 years. The first group consisted of 5 ethnic Tibetans living in Srinagar, India (1,600 meters), on whom venous blood gases were done and the P50 was derived using pH, PO2 and O2 saturation using the formula described by Lichtman and colleagues. Three were born in Tibet and two were offspring of Tibet-born parents. The second group consisted of 10 volunteers (9 Tibetans and one Nepalese Sherpa) residing in Salt Lake City, UT, (1,300 meters) whose peripheral blood was evaluated by Hemox Analyzer for obtaining P50 values and “n” Hill coefficients for hemoglobin oxygen binding. All the ethnic Tibetans in Salt Lake City were born in Tibet except for one, and the Nepalese Sherpa was born in Nepal.The results are depicted in Table. The P50 measured by venous blood gases on the Tibetan volunteers from Srinagar, India and those measured by Hemox Analyzer on the 10 volunteers from Salt Lake City, UT were normal, with values in the normal range (22-28 mmHg). No hemoglobin variants were detected by high pressure liquid chromatography in these 15 Tibetan volunteers.TableSubject IDP50 (mmHg)“n” Hill CoefficientS 0526.38n/aS 0825.95S 1326.55S 1523.68S 2722.72U 1926.962.97U 2025.162.83U 2124.202.89U 2225.462.84U 2322.502.89U 2424.062.87U 2524.282.83U 2622.352.82U 2723.292.79U 2825.992.75We report no evidence for the presence of high hemoglobin-oxygen affinity in Tibetans as a constituent of their genetic adaptation. Our data rule out the existence of hemoglobin variants and aberrant 2,3 BPG metabolism as possible features of Tibetan high-altitude adaptation; however acquired transient metabolic alterations at high altitudes, cannot be excluded to account for possible changes in hemoglobin-oxygen affinity but these are not evolved persistent features of Tibetan genetic adaptation. Studies of Tibetans living in these extreme hypoxic environment (>4,000m) are now planned. Disclosures:No relevant conflicts of interest to declare.

N-methyl-d-aspartate receptors in human erythroid precursor cells and in circulating red blood cells contribute to the intracellular calcium regulation

The presence of N-methyl-d-aspartate receptor (NMDAR) was previously shown in rat red blood cells (RBCs) and in a UT-7/Epo human myeloid cell line differentiating into erythroid lineage. Here we have characterized the subunit composition of the NMDAR and monitored its function during human erythropoiesis and in circulating RBCs. Expression of the NMDARs subunits was assessed in erythroid progenitors during ex vivo erythropoiesis and in circulating human RBCs using quantitative PCR and flow cytometry. Receptor activity was monitored using a radiolabeled antagonist binding assay, live imaging of Ca(2+) uptake, patch clamp, and monitoring of cell volume changes. The receptor tetramers in erythroid precursor cells are composed of the NR1, NR2A, 2C, 2D, NR3A, and 3B subunits of which the glycine-binding NR3A and 3B and glutamate-binding NR2C and 2D subunits prevailed. Functional receptor is required for survival of erythroid precursors. Circulating RBCs retain a low number of the receptor copies that is higher in young cells compared with mature and senescent RBC populations. In circulating RBCs the receptor activity is controlled by plasma glutamate and glycine. Modulation of the NMDAR activity in RBCs by agonists or antagonists is associated with the alterations in whole cell ion currents. Activation of the receptor results in the transient Ca(2+) accumulation, cell shrinkage, and alteration in the intracellular pH, which is associated with the change in hemoglobin oxygen affinity. Thus functional NMDARs are present in erythroid precursor cells and in circulating RBCs. These receptors contribute to intracellular Ca(2+) homeostasis and modulate oxygen delivery to peripheral tissues.

Effect of mild hypothermia during cardiopulmonary bypass on erythrocytic hemoglobin oxygen delivery

Objectives: To observe and define the degree of change in hemoglobin oxygen affinity induced by hypothermic extracorporeal circulation (ECC). Design: A prospective, nonrandomized, observational study. Setting: A single university medical center. Participants: Seventeen patients presenting for elective cardiac surgery. Interventions: Systemic hypothermia during ECC. Measurements and Main Results: During and after ECC, simultaneous arterial and mixed-venous whole-blood samples were obtained and immediately analyzed for gas tensions and hemoglobin saturation. Samples were obtained during the following times on ECC: initially after cardiopulmonary bypass onset during normothermia (37°C), after cooling to 32°C, and after rewarming to 37°C. A fourth sample was obtained 10 to 20 minutes after discontinuation of cardiopulmonary bypass. Extracorporeal pump flow and thermodilution-determined cardiac output were also recorded for calculation of oxygen delivery and consumption. Mixed-venous results were used to calculate in vivo the blood gas tension at which hemoglobin was 50% saturated with oxygen (P50). There were no differences in P50 for the 17 patients by analysis of variance (ANOVA) for repeated measures with paired t-test with Bonferroni correction. Furthermore, no change in P50 was observed during the course of cooling and rewarming in any individual patient's samples. Oxygen delivery decreased after hypothermia and rewarming from mild hypothermia; oxygen consumption was decreased after rewarming and markedly increased after discontinuation from ECC. Conclusion: Mild hypothermia to 32°C during ECC does not result in in vivo alterations in oxyhemoglobin dissociation and thus does not induce changes in oxygen delivery to peripheral tissues. However, oxygen usage appears to be markedly increased after cardiopulmonary bypass.

The ventilatory response of rodents to changes in arterial oxygen content

A strong correlation between the arterial oxygen partial pressure (Pa O 2 ) values for the threshold of the hypoxic ventilatory response (HVR) and the shoulder of the oxyhemoglobin equilibrium curve (OEC) is retained in heterothermic rodents as body temperature changes despite changes in hemoglobin-oxygen affinity. It has been suggested that this may reflect either temperature-induced changes in the response characteristics of arterial chemoreceptors or an ability to sense changes in arterial O 2 content (Ca O 2 ). This study examined the extent to which changing Ca O 2 independent of Pa O 2 with carbon monoxide could contribute to the HVR in heterothermic (golden-manteled groud squirrels) and non-heterothermic rodents (rats). The HVR of isocapnic, anaesthetized rodents was assessed during both hypoxic, which alters Pa O 2 and Ca O 2 simultaneously, and caarbon monoxide hypoxia, which alters Ca O 2 independent of Pa O 2 . While both species exhibited ventilatory responses to hypoxic hypoxia and carbon monoxide hypoxia, the HVR of the squirrel was consistently stronger than that of the rat. Reductions in Ca O 2 independetn of Pa O 2 could still produce 60% of the full HVR seen with hypoxic hypoxia in both species. Simultaneous changes in Pa O 2, however, were necessary to produce the full response. While it seems likely that the results can be explained by the changes in tissue P O 2 which would occur at receptor sites under the various conditions, such as an explanation is not totally supported by other studies.

Nitrogen binding to deoxyhemoglobin at high pressures and its relation to changes in hemoglobin-oxygen affinity.

The stoichiometric and thermodynamic properties of nitrogen (N2) binding to human deoxyhemoglobin (Hb) at N2 saturation pressures up to 400 atm were derived from measured N2 solubilities in protein-free buffers (pH 7.1) and in corresponding buffer + Hb (6.5% w/w) solutions at 20.0, 25.0, and 37.0 degrees C. At each temperature, approximately 3 N2 molecules bind per Hb tetramer at N2 pressures of 100 atm, while about 7 N2 molecules bind per tetramer at 400 atm N2 pressure, where available binding sites are still not fully saturated. Calculated N2-Hb binding isotherms are well described by a simple binding model with 12 independent and equivalent binding sites/Hb tetramer. N2 binding at each of the sites is hydrophobic, exhibiting the typical increase of the dissociation enthalpy with temperature. Enthalpies of dissociation are slightly more negative, while corresponding unitary entropies are somewhat less negative than those for the transfer of N2 from olive oil to water. Calculated partial molar volumes of N2 in Hb are positive but less than the corresponding partial molar volumes of N2 in buffer. Results indicate that N2 binding to Hb is accompanied by only small protein conformational changes which entail slight structural destabilization and loss of free volume in the protein that partially accommodates the volume of the N2 ligand. Results are related to previously reported effects of high pressure and high-pressure N2 on HbO2 affinity, illuminating essential features of the molecular mechanisms for these effects.

Effect of Inorganic Phosphate in Hypoxemic Chronic Obstructive Lung Disease Patients during Exercise

We have shown that normal subjects exercise more efficiently with increased plasma phosphate, presumably due to decreased hemoglobin-oxygen affinity and thus enhanced tissue O2 extraction. We subjected nine stable hypoxemic COLD patients to exercise at 0 (control), 24 (experimental) and 48 hrs (recovery) after phosphate infusion. Baseline variables were identical for each time period. Exercise responses at 0 and 48 hours were also indistinguishable. Exercise response at 24 hrs differed from those at 0 and 48 hours as follows: widening of A-V O2 content difference was more pronounced (28 +/- 6 vs 15 +/- 6 ml/L, p less than 0.03) and the increment in tissue O2 extraction was larger (14 +/- 3 vs 8 +/- 3 percent, p less than 0.03). P50 and related variables did not change during the course of the study. Thus, like normal subjects, hypoxemic patients stimulated with phosphate administration can exercise perhaps more efficiently; but, in contrast to normal subjects, this effect cannot be attributed to changes in hemoglobin-oxygen affinity. These data suggest that phosphate administration may be beneficial in hypoxemic states where adequate tissue oxygenation cannot be achieved by other more conventional methods. The mechanism of this effect remains to be elucidated.

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.

Changes in red blood cell oxygen transport in diabetic pregnancy

Parameters of red cell oxygen transport were studied in 23 nonsmoking, insulin-dependent diabetic women and 20 nonsmoking healthy women late during pregnancy. In the pregnant diabetic women, arterial oxygen saturation (SAT) and arterial oxygen tension were significantly decreased. SAT correlated inversely with hemoglobin AIc (Hb AIc) (diabetic women: r = −0.43, p < 0.05; total material: r = −0.66, p < 0.001). Red blood cell 2,3-diphosphoglycerate (2,3-DPG) was significantly increased in the pregnant diabetic women (p < 0.01), but the 2,3-DPG-induced change in hemoglobin-oxygen affinity was impaired. P50 (oxygen affinity, i.e., Po2 at 50% oxygen saturation) at actual pH correlated inversely with Hb AIc of the diabetic women (r = −0.45, p < 0.05). The suggestion is made that, in diabetic pregnancy particularly in poorly regulated cases, fetal hypoxia may be an important factor for the increased risk of intrauterine fetal death.

The Effect of Hemodialysis on Oxygen Transport in Chronic Uremics

Previous studies from our laboratory suggested a decrease in tissue oxygen delivered during hemodialysis of chronic uremic patients due to an increase in hemoglobin-oxygen affinity, i.e. decrease in P50. This current study was designed to determine whether the changes in cardiac index and/or tissue oxygen extraction could compensate for increases in hemoglobin-oxygen affinity previously observed, so that total tissue oxygen delivery was unchanged during hemodialysis. This study demonstrated in patients during a 6-hour hemodialysis that: 1) no change occurs in hemoglobin-oxygen affinity expressed as P50, with unchanging plasma inorganic phosphate and red blood cell pH; 2) increased extraction of oxygen from tissue, measured via (A-V) O2 difference compensates for decreased oxygen delivery due to decreased cardiac output; and although there was no demonstrable change in hemoglobin-oxygen affinity, it would appear that increased tissue extraction of oxygen via other mechanisms should be easily able to compensate for previously reported changes in P50.

Changes in blood glucose, glycosylated hemoglobin and hemoglobin-oxygen affinity following meals in diabetic children

As the concentration of total glycosylated hemoglobin (Hb AI) may be responsive to acute fluctuations in blood glucose, and as Hb AIc is known to have increased in vitro oxygen affinity, we evaluated whether acute variations in Hb AI were accompanied by changes in hemoglobin-oxygen affinity in insulin-dependent diabetic children.

Influence of temperature on hemoglobin-ligand interaction in whole blood.

Temperature-dependent change in hemoglobin-oxygen affinity was measured as a function of hemoglobin-oxygen saturation. In addition, the CO2 Bohr factor and fixed acid Bohr factor were measured as a function of saturation of temperatures of 23, 30, 37, and 44 degrees C. Measurements were made on normal blood and blood with reduced 2,3-diphosphoglycerate (DPG). The influence of temperature is greatest at low saturation and is enhanced slightly by DPG depletion. The CO2 Bohr factor is increased at high temperatures; this is primarily due to increased carbamino formation with rising temperature, especially at lower oxygen saturation. The effect of DPG on oxygen affinity is reduced at a high temperature and elevated at low temperature. These diverse effects of temperature on hemoglobin-ligand interaction require consideration in assessing oxygen delivery when temperature is increased or decreased.

Hemoglobin-Oxygen Equilibrium in Cystic Fibrosis

A study of 35 patients with cystic fibrosis demonstrated that increasing severity of pulmonary involvement was associated with a mild but definite increase in erythrocyte 2,3-diphosphoglycerate (2,3-DPG) and a decrease in hemoglobin affinity for oxygen. The predominant regulators of 2,3-DPG were blood pH, cardiac output, and systemic oxygen transport. No significant relationship was observed between erythrocyte 2,3-DPG content and arterial oxygen tension. Hypophosphatemia may have prevented a greater increase in erythrocyte 2,3-DPG content. The inadequate increase in 2,3-DPG and consequent insufficient change in hemoglobin-oxygen affinity, coupled with an insufficient compensatory erythrocytic response, may adversely affect tissue oxygenation in patients with severe cystic fibrosis.

The effect of cardiac disease on hemoglobin-oxygen binding.

The relation between degree of cardiac functional impairment and changes in hemoglobin-oxygen affinity and 2,3-diphosphoglycerate (2,3-DPG) has been studied in 39 patients with noncyanotic heart disease. A progressive decline in hemoglobin-oxygen affinity was found with worsening cardiac function as assessed by cardiac index, arteriovenous oxygen (A-V O(2)) difference, and cardiac symptoms; this alteration in hemoglobin-oxygen binding represents a significant mechanism for adaptation to the limited oxygen supply imposed by the cardiac lesion. The highly significant correlation of mixed venous blood oxygen saturation (S[unk]V(VO2)) with 2,3-DPG and the position of the oxygen dissociation curve suggests that the level of deoxygenated hemoglobin is an important in vivo regulator of hemoglobin-oxygen affinity.