Blood Oxygen Affinity Research Articles

Hemoglobin-oxygen affinity changes in neonatal blood transfusions: RBC selection insights.

Despite preterm newborns often requiring blood transfusions, we have an incomplete understanding of the impact of adult packed red blood cell (pRBC) transfusions on fetal red blood cell (RBC) oxygen affinity. We investigated the influence of adult pRBC on oxygen binding in fetal RBCs obtained from the umbilical cord of preterm newborns. This included exploring the influence of the biological age of adult pRBCs on the oxygen affinity of fetal blood. Cord blood samples from preterm infants were titrated with young (Y-RBC) and old (O-RBC) adult pRBCs using an in vitro transfusion model. Parameters, including oxygen affinity (p50), hemoglobin variants, and red blood cell indices, were measured. The titration of cord blood with adult pRBCs (n = 19) resulted in a concentration-dependent decrease in p50, indicating an increased oxygen affinity. Hemoglobin variant analysis revealed a shift in composition, with a decrease in fetal hemoglobin (HbF) and an increase in adult hemoglobin (HbA) following titration. The impact of biological age was evident, as O-RBCs had a more pronounced effect on p50 values compared to Y-RBCs. Understanding the physiologic implications of transfusing preterm infants with adult pRBCs is important for optimizing transfusion practices in newborns. In an in vitro model, titrating adult pRBC into fetal blood significantly affects oxygen binding Oxygen affinity of fetal blood is significantly increased after titration of adult pRBC. Compared to older RBC subpopulations of adult pRBC, oxygen-binding properties of younger RBC subpopulations of adult pRBC more closely approximate the oxygen affinity of fetal blood. This work highlights the importance of investigating the influence of adult pRBC transfusion on fetal RBC oxygen binding. This may have implications for morbidities in the newborn period related to oxygen physiology.

Pyruvate Kinase Thermostability Is Associated with Red Blood Cell Adhesion, Deformability and Oxygen Affinity in Patients with Sickle Cell Disease

Introduction: Sickle cell disease(SCD) is caused by a mutation in the beta globin gene that causes formation of abnormal hemoglobin S (HbS). Red blood cells (RBCs) from patients with SCD are less deformable and take on the shape of sickle upon deoxygenation. This results in multiple downstream effects including RBC adhesion, microvascular occlusion and chronic hemolysis. RBCs rely solely on anaerobic glycolysis to produce adenosine triphosphate (ATP), which is needed to maintain the cell's structure and function. Pyruvate kinase (PK) is a key enzyme involved in the last step of glycolysis. We have previously demonstrated a decrease in PK activity and stability as a novel pathophysiological feature of SCD. Currently there are several clinical trials ongoing investigating the efficacy of PK activation by small molecules such as mitapivat (NL8517, NCT04000165, NCT05031780), AG-946 (NCT04536792) and etavopivat (NCT04624659, NCT04987489). In this study we aim to investigate how PK properties correlate to sickle RBC properties in order to explore if impaired PK function is associated with specific features of the complex SCD pathophysiology. Methods: Homozygous HbS (HbSS) and HbS/ β0 thalassemia patients were eligible to participate. Patients who received blood transfusion (<3 months) were excluded. Dense RBCs (MCHC >41 g/dL, Advia 120/2120 Siemens) and HbF and HbS levels (Tosoh G8) were measured. PK and hexokinase (HK) activity were measured on purified RBCs. PK thermostability (reflected by % residual PK activity) was measured on purified RBCs lysates after one hour of incubation (53°C). RBC adhesion to laminin (Biolamina) was measured using a microfluidic device (IBIDI µ-Slide I 0.4). RBC hydration (Ohyper), RBC deformability (EImax) and point of sickling (PoS) were assessed by osmotic and oxygen gradient ektacytometry using the Laser-Optical Rotational Red Cell Analyzer (Lorrca, RR Mechatronics). Oxygen affinity (P50) was measured with the Hemox-Analyzer (TCS). Statistical analysis was performed in Graphpad Prism (Pearson's correlation). Results: Fifty-seven patients with SCD (53 HbSS, 4 HbS/β 0 thalassemia) were included of which 17 were children (median age 14 years [range 6-17]) and 40 were adults (median age 36 years [range 18-58]). Baseline characteristics are depicted in Table 1. Figure 1 shows a heatmap with correlation coefficients of all laboratory parameters from Table 1 as measured in this cohort. When focusing on PK properties we found that patients with SCD and lower PK thermostability had significantly higher absolute reticulocyte counts (ARC, p<0.0001), lower hemoglobin (p=0.012) and HbF (p=0.020) levels and increased RBC adhesion to laminin (p=0.007). Furthermore, PK thermostability was significantly correlated with EImax (p=0.017) and inversely correlated with PoS (p=0.011) and P50 (p=0.003), indicating that patients with less stable RBC PK had less deformable RBCs which sickled at a higher oxygen tension. RBC adhesion to laminin was significantly correlated with both ARC (p=0.0003) and dense RBCs (p=0.019). To explore whether our findings were a reflection of reticulocytosis we fractionated whole blood samples of HbSS patients according to cell density. We found that more RBCs from the most dense fraction adhered to laminin than from the less dense, reticulocyte enriched, fraction, even though RBCs from this less dense fraction had reduced PK-thermostability. Additionally, the most dense RBCs had the lowest PK activity, were less deformable and had a higher PoS (data not shown). Altogether these findings indicate that the reduced PK thermostability we found in our SCD cohort is associated with an overall functionally impaired RBC population with increased RBC adhesion and ARC. We found no correlation of markers of RBC dehydration (Ohyper and % dense RBCs) with PK thermostability which suggests that RBCs with reduced PK thermostability are not more dehydrated. Discussion/conclusion: This study shows for the first time a significant correlation between PK thermostability in sickle RBCs and RBC functions, such as adhesion, deformability and oxygen affinity. Our results suggest that enhancing the activity and stability of PK, with PK activators, is an attractive target in SCD that might improve other pathophysiological targets outside RBC metabolism. Future studies are needed to explore how activation of PK will affect these altered sickle RBC properties in vivo.

New insights into survival strategies to oxygen deprivation in anoxia-tolerant vertebrates.

Hypoxic environments pose a severe challenge to vertebrates and even short periods of oxygen deprivation are often lethal as they constrain aerobic ATP production. However, a few ectotherm vertebrates are capable of surviving long‐term hypoxia or even anoxia with little or no damage. Among these, freshwater turtles and crucian carp are the recognized champions of anoxia tolerance, capable of overwintering in complete oxygen deprivation for months at freezing temperatures by entering a stable hypometabolic state. While all steps of the oxygen cascade are adjusted in response to oxygen deprivation, this review draws from knowledge of freshwater turtles and crucian carp to highlight mechanisms regulating two of these steps, namely oxygen transport in the blood and oxygen utilization in mitochondria during three sequential phases: before anoxia, when hypoxia develops, during anoxia, and after anoxia at reoxygenation. In cold hypoxia, reduced red blood cell concentration of ATP plays a crucial role in increasing blood oxygen affinity and/or reducing oxygen unloading to tissues, to adjust aerobic metabolism to decrease ambient oxygen. In anoxia, metabolic rewiring of oxygen utilization keeps largely unaltered NADH/NAD+ ratios and limits ADP degradation and succinate buildup. These critical adjustments make it possible to restart mitochondrial respiration and energy production with little generation of reactive oxygen species at reoxygenation when oxygen is again available. Inhibition of key metabolic enzymes seems to play crucial roles in these responses, in particular mitochondrial complex V, although identifying the nature of such inhibition(s) in vivo remains a challenge for future studies.

Hemoglobin North Shore: A Variant Hemoglobin Associated With the Phenotype of β-Thalassemia

Hemoglobin (Hb) North Shore (beta 134 val leads to glu) is a mutant hemoglobin that is associated with the phenotype of mild heterozygous beta-thalassemia. Heterozygotes are characterized low normal hemoglobin levels or mild anemia, microcytosis, increased HbA2, and 34%-38% Hb North Shore. The mechanism of the anemia and microcytosis associated with Hb North Shore was explored by studies of hemolysate thermal instability, peripheral blood globin biosynthesis, and whole blood oxygen affinity. Hb North Shore was mildly heat unstable in comparison to normal adult hemolysate. Pulse labeling of reticulocytes with 3H-leucine showed an alpha/beta ratio of 1.35 (normal 1.0). The beta North Shore/alpha ratio was 0.22-0.27, which was less than expected on the basis of gene dosage and less than that seen for most beta-chain variants. The beta A/alpha ratio was 0.50, as would be expected. The beta North Shore/alpha ratio was 0.26 after a 15-min pulse and did not decrease during 120 min of chase. These findings suggest that suboptimal synthesis rather than posttranslational degradation is responsible for the thalassemic phenotype associated with this variant hemoglobin. These observations parallel the findings in heterozygous HbE. It is not presently known whether the thalassemia phenotype is conferred by the structural mutation itself or by another mutation cis to the beta North Shore gene.

Very high blood oxygen affinity and large Bohr shift differentiates the air-breathing siamese fighting fish (Betta splendens) from the closely related anabantoid the blue gourami (Trichopodus trichopterus)

The Siamese fighting fish, Betta splendens, and the blue gourami, Trichopodus trichopterus, are two closely related air-breathing anabantoid fishes. B. splendens is a sedentary facultative air breather frequenting often hypoxic waters, while T. trichopterus is a more active obligatory air-breather inhabiting better oxygenated waters. Despite their close taxonomic relationship, previous studies have shown inter-specific differences in both physiological and morphological plasticity. Consequently, we hypothesized that B. splendens would have the higher blood oxygen affinity characteristics typical of more hypoxia-tolerant fishes. Whole blood oxygen equilibrium curves were determined at 27 °C and pHs of 7.62, 7.44 and 7.25. At a pH of 7.62, the blood O2 affinity (P50) of B. splendens was just 2.9 mmHg, while that of T. trichopterus was ~5 times higher at 14.7 mmHg. There were no significant differences in P50 between males and females in either species. The Bohr coefficient in B. splendens and T. trichopterus was −1.79 and − 0.83, respectively. B. splendens, unlike T. trichopterus, showed a large Root effect. Hills cooperatively coefficient, n, was ~2 in both species, indicating a significant binding cooperative between oxygen and hemoglobin. Collectively, these differences in blood O2 transport characteristics in these two closely related species are likely correlated with the differing habitats in which they breed and inhabit as adults, as well as different activity levels. Finally, the very high blood O2 affinity of B. splendens is not extraordinary among air-breathing fish, as revealed by a review of the literature of blood oxygen affinity in air-breathing fishes.

Temperature effects on the blood oxygen affinity in sharks.

In fish, regional endothermy (i.e., the capacity to significantly elevate tissue temperatures above ambient via vascular heat exchangers) in the red swimming muscles (RM) has evolved only in a few marine groups (e.g., sharks: Lamnidae, Alopiidae, and teleosts Scombridae). Within these taxa, several species have also been shown to share similar physiological adaptations to enhance oxygen delivery to the working tissues. Although the hemoglobin (Hb) of most fish has a decreased affinity for oxygen with an increase in temperature, some regionally endothermic teleosts (e.g., tunas) have evolved Hbs that have a very low or even an increased affinity for oxygen with an increase in temperature. For sharks, however, blood oxygen affinities remain largely unknown. We examined the effects of temperature on the blood oxygen affinity in two pelagic species (the regionally endothermic shortfin mako shark and the ectothermic blue shark) at 15, 20, and 25°C, and two coastal ectothermic species (the leopard shark and brown smooth-hound shark) at 10, 15, and 20°C. Relative to the effects of temperature on the blood oxygen affinity of ectothermic sharks (e.g., blue shark), shortfin mako shark blood was less affected by an increase in temperature, a scenario similar to that documented in some of the tunas. In the shortfin mako shark, this may act to prevent premature oxygen dissociation from Hb as the blood is warmed during its passage through vascular heat exchangers. Even though the shortfin mako shark and blue shark occupy a similar niche, the effects of temperature on blood oxygen affinity in the latter more closely resembled that of the blood in the two coastal shark species examined in this study. The only exception was a small, reverse temperature effect (an increase in blood oxygen affinity with temperature) observed during the warming of the leopard shark blood under simulated arterial conditions, a finding that is likely related to the estuarine ecology of this species. Taken together, we found species-specific differences in how temperature affects blood oxygen affinity in sharks, with some similarities between the regionally endothermic sharks and several regionally endothermic teleost fishes.

Convective oxygen transport during development in embryos of the snapping turtle Chelydra serpentina.

This study investigated the maturation of convective oxygen transport in embryos of the snapping turtle (Chelydra serpentina). Measurements included: mass, oxygen consumption (V̇O2 ), heart rate, blood oxygen content and affinity and blood flow distribution at 50%, 70% and 90% of the incubation period. Body mass increased exponentially, paralleled by increased cardiac mass and metabolic rate. Heart rate was constant from 50% to 70% incubation but was significantly reduced at 90% incubation. Hematocrit and hemoglobin concentration were constant at the three points of development studied but arteriovenous difference doubled from 50% to 90% incubation. Oxygen affinity was lower for the early 50% incubation group (stage 19) compared with all other age groups. Blood flow was directed predominantly to the embryo but was highest to the chorioallantoic membrane (CAM) at 70% incubation and was directed away from the yolk as it was depleted at 90% incubation. The findings indicate that the plateau or reduction in egg V̇O2 characteristic of the late incubation period of turtle embryos may be related to an overall reduction in mass-specific V̇O2 that is correlated with decreasing relative heart mass and plateaued CAM blood flow. Importantly, if the blood properties remain unchanged prior to hatching, as they did during the incubation period studied in the current investigation, this could account for the pattern of V̇O2previously reported for embryonic snapping turtles prior to hatching.

Projections of climate-driven changes in tuna vertical habitat based on species-specific differences in blood oxygen affinity.

Oxygen concentrations are hypothesized to decrease in many areas of the ocean as a result of anthropogenically driven climate change, resulting in habitat compression for pelagic animals. The oxygen partial pressure, pO2 , at which blood is 50% saturated (P50 ) is a measure of blood oxygen affinity and a gauge of the tolerance of animals for low ambient oxygen. Tuna species display a wide range of blood oxygen affinities (i.e., P50 values) and therefore may be differentially impacted by habitat compression as they make extensive vertical movements to forage on subdaily time scales. To project the effects of end-of-the-century climate change on tuna habitat, we calculate tuna P50 depths (i.e., the vertical position in the water column at which ambient pO2 is equal to species-specific blood P50 values) from 21st century Earth System Model (ESM) projections included in the fifth phase of the Climate Model Intercomparison Project (CMIP5). Overall, we project P50 depths to shoal, indicating likely habitat compression for tuna species due to climate change. Tunas that will be most impacted by shoaling are Pacific and southern bluefin tunas-habitat compression is projected for the entire geographic range of Pacific bluefin tuna and for the spawning region of southern bluefin tuna. Vertical shifts in P50 depths will potentially influence resource partitioning among Pacific bluefin, bigeye, yellowfin, and skipjack tunas in the northern subtropical and eastern tropical Pacific Ocean, the Arabian Sea, and the Bay of Bengal. By establishing linkages between tuna physiology and environmental conditions, we provide a mechanistic basis to project the effects of anthropogenic climate change on tuna habitats.

A comparison of blood nitric oxide metabolites and hemoglobin functional properties among diving mammals

The ability of marine mammals to hunt prey at depth is known to rely on enhanced oxygen stores and on selective distribution of blood flow, but the molecular mechanisms regulating blood flow and oxygen transport remain unresolved. To investigate the molecular mechanisms that may be important in regulating blood flow, we measured concentration of nitrite and S-nitrosothiols (SNO), two metabolites of the vasodilator nitric oxide (NO), in the blood of 5 species of marine mammals differing in their dive duration: bottlenose dolphin, South American sea lion, harbor seal, walrus and beluga whale. We also examined oxygen affinity, sensitivity to 2,3-diphosphoglycerate (DPG) and nitrite reductase activity of the hemoglobin (Hb) to search for possible adaptive variations in these functional properties. We found levels of plasma and red blood cells nitrite similar to those reported for terrestrial mammals, but unusually high concentrations of red blood cell SNO in bottlenose dolphin, walrus and beluga whale, suggesting enhanced SNO-dependent signaling in these species. Purified Hbs showed similar functional properties in terms of oxygen affinity and sensitivity to DPG, indicating that reported large variations in blood oxygen affinity among diving mammals likely derive from phenotypic variations in red blood cell DPG levels. The nitrite reductase activities of the Hbs were overall slightly higher than that of human Hb, with the Hb of beluga whale, capable of longest dives, having the highest activity. Taken together, these results underscore adaptive variations in circulatory NO metabolism in diving mammals but not in the oxygenation properties of the Hb.

In vitro effects of increased temperature and decreased pH on blood oxygen affinity of 10 fish species of the Amazon.

Blood-O2 affinities (P50 ) were measured over a physiologically relevant pH range at 31 (highest temperature average of Rio Negro over the last 8 years), 33 and 35° C for 10 species of the Rio Negro, aiming to test the acute effects of temperature foreseen by the IPCC (Intergovernmental Panel on Climate Change) for coming years. The animals were collected during an expedition to the Anavilhanas Islands of the Rio Negro, 110 km upstream from Manaus (2° 23' 41″ S; 60° 55' 14″ W). Hoplias malabaricus showed higher blood-O2 sensitivity to pH changes (Bohr effect, Φ = Δlog10 P50 ΔpH(-1) ) at both 31° C (Φ = -0·44) and 35° C (Φ = -0·26) compared to Osteoglossum bicirrhosum (Φ = -0·54 at 31° C and Φ = -0·58 at 35° C), but lower P50 under most conditions, and a greater sensitivity of P50 to temperature. Two out of the 10 analysed species had significant increases of P50 (lower blood-O2 affinity) at the highest temperature throughout the pH range tested. For all other species, a minor increase of P50 over the assay-tested temperatures was observed, although all presented a normal Bohr effect. Overall, a diversity of intensities of pH and temperature effects on blood-O2 affinities was observed, which seems to be connected to the biological characteristics of the analysed species. Thermal disturbances in their habitats, likely to occur due to the global warming, would impair blood-O2 binding and unloading in some of the analysed fish species. Copyright © 2016 John Wiley & Sons, Ltd.

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.

Blood oxygen affinity increases during digestion in the South American rattlesnake, Crotalus durissus terrificus

Digesting snakes experience massive increases in metabolism that can last for many days and are accompanied by adjustments in the oxygen transport cascade. Accordingly, we examined the oxygen-binding properties of the blood in the South American rattlesnake (Crotalus durissus terrificus) during fasting and 24 and 48h after the snakes have ingested a rodent meal corresponding to 15% (±2%) of its own body mass. In general, oxygen–hemoglobin (Hb–O2) affinity was significantly increased 24h post-feeding, and then returned toward fasting values within 48h post-feeding. Content of organic phosphates ([NTP] and [NTP]/[Hb]), hemoglobin cooperativity (Hill's n), and Bohr Effect (ΔlogP50/ΔpH) were not affected by feeding. The postprandial increase in Hb–O2 affinity in the South American rattlesnake can be almost entirely ascribed by the moderate alkaline tide that follows meal ingestion. In general, digesting snakes were able to regulate blood metabolites at quite constant levels (e.g., plasma osmolality, lactate, glucose, and total protein levels). The level of circulating lipids, however, was considerably increased, which may be related to their mobilization, since lipids are known to be incorporated by the enterocytes after snakes have fed. In conclusion, our results indicate that the exceptional metabolic increment exhibited by C. d. terrificus during meal digestion is entirely supported by the aerobic pathways and that among the attending cardiorespiratory adjustments, pulmonary Hb–O2 loading is likely improved due to the increment in blood O2 affinity.

High blood oxygen affinity in the air-breathing swamp eel Monopterus albus

The Asian swamp eel (Monopterus albus, Zuiew 1793) is a facultative air-breathing fish with reduced gills. Previous studies have shown that gas exchange seems to occur across the epithelium of the buccopharyngeal cavity, the esophagus and the integument, resulting in substantial diffusion limitations that must be compensated by adaptations in others steps of the O2 transport system to secure adequate O2 delivery to the respiring tissues. We therefore investigated O2 binding properties of whole blood, stripped hemoglobin (Hb), two major isoHb components and the myoglobin (Mb) from M. albus. Whole blood was sampled using indwelling catheters for blood gas analysis and determination of O2 equilibrium curves. Hb was purified to assess the effects of endogenous allosteric effectors, and Mb was isolated from heart and skeletal muscle to determine its O2 binding properties. The blood of M. albus has a high O2 carrying capacity [hematocrit (Hct) of 42.4±4.5%] and binds O2 with an unusually high affinity (P50=2.8±0.4mmHg at 27°C and pH7.7), correlating with insensitivity of the Hb to the anionic allosteric effectors that normally decrease Hb-O2 affinity. In addition, Mb is present at high concentrations in both heart and muscle (5.16±0.99 and 1.08±0.19mg ∙ g wet tissue-1, respectively). We suggest that the high Hct and high blood O2 affinity serve to overcome the low diffusion capacity in the relatively inefficient respiratory surfaces, while high Hct and Mb concentration aid in increasing the O2 flux from the blood to the muscles.

Blood oxygen content and affinity in developing embryos of snapping turtle Chelydra serpentina (879.8)

In egg‐laying amniotic vertebrates gas exchange takes place over the chorioallantoic membrane (CAM). Oxygen diffuses from air in contact with the shell into blood perfusing the membrane where it combines reversibly with hemoglobin. The capacity of the blood to transport oxygen is determined by the functional characteristics of the hemoglobin. In this study we investigated changes in oxygen content and affinity during development in embryos of snapping turtle (Chelydra serpentina). Blood samples were taken from embryos at 50, 70 and 90% of incubation. Samples withdrawn from the CAM artery had statistically similar mean oxygen contents for all stages (combined mean ± SEM; 0.94 ± 0.13 mL O2/100 mL of blood). Mean CAM venous blood oxygen content was 2.74 ± 0.44 at 50%, 3.25 ± 0.26 at 70% and increased to 4.97 ± 0.47 mL O2/100 mL at 90% of development. Consequently, the arteriovenous difference increased by 72% late in incubation. Oxygen equilibrium curves were the same at 2% and 6% CO2 at 50% incubation but showed an increased affinity in 2% CO2 and a rightward shift in 6% CO2 at 70 and 90% of development. These results show that embryonic turtles increase oxygen content and affinity and show a Bohr shift late in development likely favoring hemoglobin loading at the CAM and unloading at the tissues, ensuring adequate O2 supply for the increased demand of later stages of development.Grant Funding Source: Supported by NSF CAREER IBN IOS‐0845741 (DAC) and FAPESP 2012/06938‐8 (EWT); 2013/05677‐9 (MRS)

Physiological insight into the high-altitude adaptations in domesticated yaks (Bos grunniens) along the Qinghai-Tibetan Plateau altitudinal gradient

The cold, hypoxic conditions of high-altitude habitats impose severe metabolic demands on domesticated yaks (Bos grunniens). Understanding how yaks cope with the combined effects of hypoxia and aerobic metabolism can provide important insights into the process of adaptive evolution. Here, hematological parameters, blood gases and blood volume, as well as enzyme activity, were determined in yaks from different altitudes. A total of 60 yaks, including 16 Tibet Alpine yaks (approximately 5100m a.s.l.), 23 Gannan yaks (3585m a.s.l.) and 21 Tianzhu white yaks (2960m a.s.l.) in the typically alpine meadows for the altitude of the Tibetan Plateau, were compared. The average red blood cell counts (RBC) value (9.34×1012/L) for the Tibet Alpine yaks was significantly higher than those of Gannan yaks (7.34×1012/L) and Tianzhu white yaks (6.77×1012/L) (P<0.05). Similar levels of significance were observed in the hematocrit (HCT), mean cell volume (MCV) and mean corpuscular hemoglobin concentration (MCHC) concentrations in Tibet Alpine yaks. Increases in hemoglobin (Hb) occurred in the Tibet Alpine yaks, with values of 13.21 and 10.67g/dl for the Gannan yaks and Tianzhu white yaks, respectively, which were lower than that of the Tibet Alpine yaks (P<0.05). Gas-tension and pH changes resulting from chronic hypoxia were evident in the reduced O2 and CO2 tensions in both venous and arterial blood and the slight but significant increase in the blood pH. The blood volume displayed a similar pattern of being greater in the Tibet Alpine yaks than in the Gannan yaks and Tianzhu white yaks. Lactate (LA) and lactate dehydrogenase (LDH) activity positively correlated with altitude (P<0.05). These results are interesting in respect to the low-oxygen environment in which yaks live and are consistent with the higher myoglobin (Mb) concentration in yak cardiac muscle, possibly permitting this type of yak muscle to be switched to aerobic metabolism when needed. These results suggest that Hb levels are not a totally conclusive explanation for the yak adaptation to high altitude, and the physiological adaptation was achieved in yaks perhaps by increases in RBC and blood oxygen affinity and decreases in MCV. In addition, enhanced enzymatic activity plays a key role in added metabolic cost and anaerobic metabolism.

PO2-DEPENDENT HORMONAL CONTROL OF AVIAN EMBRYONIC RED CELL CARBONIC ANHYDRASE II SYNTHESIS, ORGANIC PHOSPHATE PATTERN, AND OXYGEN AFFINITY

ABSTRACT Developmental changes of blood oxygen pressure are instrumental for the regulation of blood oxygen affinity (via ATP/2,3-diphosphoglycerate (2,3-DPG) concentration) and carbonic anhydrase (CA) synthesis in red cells of chick embryos. Hypoxia is the physiological stimulus for the coordinated onset of synthesis of CA and 2,3-DPG in late chick embryos, whereas the higher blood oxygen pressure prevailing in early—and midterm embryos suppresses CA and 2,3-DPG synthesis and increases red cell ATP. This mechanism guarantees flexible adaption of the oxygen affinity to the prevailing oxygen pressure. At least two hormonal effectors seem to be involved in the PO2-dependent regulation: (a) a heat-labile, low molecular weight compound of embryonic chick plasma that stimulates CA and 2,3-DPG synthesis and (b) an antagonistically-acting, short-lived metabolite present in the blood of normoxic and hyperoxic embryos, whose activity/concentration should be reduced with falling PO2. While the identity of the CAII ...

Effects of temperature on hardhead minnow (Mylopharodon conocephalus) blood-oxygen equilibria

Habitat perturbations, including dam construction with consequent temperature changes and the introduction of non-native species to California’s mid- to low-elevation streams, have negatively influenced some native fish populations’ historic distribution and abundance. Populations of hardhead, Mylopharodon conocephalus (Cyprinidae), have experienced such population declines, but environmental temperature effects on this large (to 60 cm SL), native species are poorly documented. We measured temperature effects on in vitro blood-oxygen affinity and equilibrium curve shape, key dynamics of the species’ oxygen-transport system, derived from blood collected from wild-caught hardhead. Over an 11–30 °C temperature range, the half-saturation value (P50, an inverse measure of affinity) increased with the temperature from 0.51 to 1.80 kPa for low-PCO2 (“arterial”) treatments and from 2.02 to 2.92 kPa for high-PCO2 (“venous”) treatments. The apparent heat of oxygenation (temperature effect) was higher at temperatures > (absolute value) 19 °C. Therefore, hardhead’s blood has a decreased ability to bind oxygen at its gills at temperatures ≥25 °C, compared to that at temperatures ≤19 °C. The hardhead’s Bohr factors (Ф), non-bicarbonate buffer values (β), nucleoside triphosphate (NTP) concentrations, blood oxygen capacities (CBO2), and mildly sigmoid-shaped oxygen equilibrium curves showed no relationship with temperature. Overall, their blood-oxygen equilibria suggest that hardhead can tolerate moderate hypoxia and temperature variations in its environment and that they have some capacity for sustained, high-aerobic activity.

Effect of Aes-103 Anti-Sickling Agent On Oxygen Affinity and Stability of Red Blood Cells From Patients with Sickle Cell Anemia

AbstractAbstract 85 Background:Aes-103 (5-hydroxymethylfurfural, 5-HMF) is a putative anti-sickling agent that has undergone pre-clinical testing for potential treatment for sickle cell anemia (SCA). It is an organic compound derived from dehydration of certain sugars, found commonly in small amounts in foods such as coffee and prunes. It binds to alpha subunits of hemoglobin and increases its oxygen affinity. At millimolar levels, it inhibits hypoxia-induced sickling in vitro and when dosed orally it protects sickle cell mice against hypoxia-induced death. We investigate the in vitro effects of a range of concentrations of Aes-103 on oxygen affinity and red cell stability in blood from healthy volunteers, and from patients with SCA with or without hydroxyurea treatment. Methods:Blood specimens from healthy control adults and adults with SCA were incubated in vitro with a range of concentrations of Aes-103 between 0.1 and 5 mM for one hour at 37 degrees C. Oxygen equilibrium curves were determined for each sample using the HemOx Analyzer. Samples were diluted in HemOx buffer and then loaded into the Analyzer, which exposed the samples to increasing partial pressure and then deoxygenated with nitrogen gas to produce the oxygen equilibrium curve. The P50 value for each curve was determined at the oxygen tension that produced 50% oxygen saturation. In additional experiments, samples of human control blood and SCA blood were treated with Aes-103 and incubated at 37°C for 1 hr, and then the samples in tubes were subjected to shear stress by rotation on a vertical rotator at 21 revolutions per minute for 3 hrs. The samples were centrifuged for 2 minutes and plasma was collected and free hemoglobin levels as an indicator of red cell membrane disruption were measured by ELISA. Results:Blood samples from SCA patients off hydroxyurea (n=6) without Aes-103 tended to have higher baseline p50 values than healthy controls (n=6)(30.3 ± 1.1 vs. 28.3 ± 0.8 torr, p=0.15), consistent with previous reports of high intracellular 2,3-DPG, known to increase P50. The P50 remained right shifted in SCA compared to controls at Aes-103 concentration below 1mM, converging with controls at higher concentrations (p=0.035). At baseline, P50 of SCA patients on chronic hydroxyurea (n=9) was significantly lower than SCA patients not on hydroxyurea (26.3 ± 0.8 vs. 30.3 ± 1.1 torr, p=0.008), compatible with the lower P50 contributed by fetal hemoglobin induced by hydroxyurea. At every concentration of Aes-103, P50 was lower for specimens SCA on hydroxyurea compared to those off hydroxyurea (p<0.001) (Figure 1A). Overall, the delta decrease in P50 from baseline in all subjects at all concentrations of Aes-103 was comparable, on regression analysis showing −2.16 torr for each mM increase in Aes-103 (r2=0.64, p<0.001).In vitro shear stress under normoxia promoted hemolysis in blood samples from patients with SCA compared to baseline (n=10, free hemoglobin 29.4 ± 3.4 vs. 8.4 ± 0.9 uM, p<0.001). Addition of Aes-103 at increasing concentrations reduced the extent of shear-stress induced hemolysis, by 15% at 1mM Aes-103; by 28% at 2mM Aes-103; and by 37% at 5mM Aes-103 (p<0.001, Figure 1B). Interestingly, although shear stress promoted less hemolysis in blood samples from healthy controls, Aes-103 at these concentrations also reduced this hemolysis to a comparable extent, suggesting a red cell stabilizing mechanism distinct from anti-sickling effect. Shear stress experiments under hypoxic conditions are underway. In pilot experiments using an imaging flow cytometry assay described in detail in a separate abstract, Aes-103 showed preliminary ability to repress sickling induced by hypoxia in vitro. Conclusions:Red cells from SCA adults treated with hydroxyurea have significantly higher affinity for oxygen than those from patients not treated with hydroxyurea, presumably related in part to the high affinity of fetal hemoglobin induced by the drug. Aes-103 increases oxygen affinity in sickle erythrocytes in a concentration-dependent fashion, and this effect is even more prominent when combined with that of hydroxyurea. Aes-103 at high concentrations stabilizes red cells against shear stress in vitro. With our collaborators at AesRx, LLC, a phase 1 safety and pharmacokinetics study of Aes-103 in healthy volunteers has been completed and we now are conducting a similar study at the NIH Clinical Center in adults with sickle cell anemia. [Display omitted] Disclosures:No relevant conflicts of interest to declare.

Interactions of acid–base balance and hematocrit regulation during environmental respiratory gas challenges in developing chicken embryos (Gallus gallus)

How the determinants of hematocrit (Hct) – alterations in mean corpuscular volume (MCV) and/or red blood cell concentration ([RBC]) – are influenced by acid–base balance adjustments across development in the chicken embryo is poorly understood. We hypothesized, based on oxygen transport needs of the embryos, that Hct will increase during 1 day of hypercapnic hypoxia (5%CO2, 15%O2) or hypoxia alone (0%CO2, 15%O2), but decrease in response to hyperoxia (0%CO2, 40%O2). Further, age-related differences in acid–base disturbances and Hct regulation may arise, because the O2 transport and hematological regulatory systems are still developing in embryonic chickens. Our studies showed that during 1 day of hypoxia (with or without hypercapnia) Hct increased through both increased MCV and [RBC] in day 15 (d15) embryo, but only through increased MCV in d17 embryo and therefore enhancement of O2 transport was age-dependent. Hypercapnia alone caused a ∼14% decrease in Hct through decreased [RBC] and therefore did not compensate for decreased blood oxygen affinity resulting from the Bohr shift. The 11% (d15) and 14% (d17) decrease in Hct during hyperoxia in advanced embryos was because of an 8% and 9% decrease, respectively, in [RBC], coupled with an associated 3% and 5% decrease in MCV. Younger, d13 embryos were able to metabolically compensate for respiratory acidosis induced by hypercapnic hypoxia, and so were more tolerant of disturbances in acid–base status induced via alterations in environmental respiratory gas composition than their more advanced counterparts. This counter-intuitive increased tolerance likely results from the relatively low M˙O2 and immature physiological functions of younger embryos.

Altitudinal Variation at Duplicated β-Globin Genes in Deer Mice: Effects of Selection, Recombination, and Gene Conversion

Spatially varying selection on a given polymorphism is expected to produce a localized peak in the between-population component of nucleotide diversity, and theory suggests that the chromosomal extent of elevated differentiation may be enhanced in cases where tandemly linked genes contribute to fitness variation. An intriguing example is provided by the tandemly duplicated β-globin genes of deer mice (Peromyscus maniculatus), which contribute to adaptive differentiation in blood-oxygen affinity between high- and low-altitude populations. Remarkably, the two β-globin genes segregate the same pair of functionally distinct alleles due to a history of interparalog gene conversion and alleles of the same functional type are in perfect coupling-phase linkage disequilibrium (LD). Here we report a multilocus analysis of nucleotide polymorphism and LD in highland and lowland mice with different genetic backgrounds at the β-globin genes. The analysis of haplotype structure revealed a paradoxical pattern whereby perfect LD between the two β-globin paralogs (which are separated by 16.2 kb) is maintained in spite of the fact that LD within both paralogs decays to background levels over physical distances of less than 1 kb. The survey of nucleotide polymorphism revealed that elevated levels of altitudinal differentiation at each of the β-globin genes drop away quite rapidly in the external flanking regions (upstream of the 5' paralog and downstream of the 3' paralog), but the level of differentiation remains unexpectedly high across the intergenic region. Observed patterns of diversity and haplotype structure are difficult to reconcile with expectations of a two-locus selection model with multiplicative fitness.