Bohr Effect Research Articles

Nitric Oxide Ameliorates the Effects of Hypoxia in Mice by Regulating Oxygen Transport by Hemoglobin.

Xiaoying Zhou, Wenting Su, Quanwei Bao, Yu Cui, Xiaoxu Li, Yidong Yang, Chengzhong Yang, Chengyuan Wang, Li Jiao, Dewei Chen, and Jian Huang. Nitric oxide ameliorates the effects of hypoxia in mice by regulating oxygen transport by hemoglobin. High Alt Med Biol. 25:174-185, 2024.-Hypoxia is a common pathological and physiological phenomenon in ischemia, cancer, and strenuous exercise. Nitric oxide (NO) acts as an endothelium-derived relaxing factor in hypoxic vasodilation and serves as an allosteric regulator of hemoglobin (Hb). However, the ultimate effects of NO on the hematological system invivo remain unknown, especially in extreme environmental hypoxia. Whether NO regulation of the structure of Hb improves oxygen transport remains unclear. Hence, we examined whether NO altered the oxygen affinity of Hb (Hb-O2 affinity) to protect extremely hypoxic mice. Mice were exposed to severe hypoxia with various concentrations of NO, and the survival time, exercise capacity, and other physical indexes were recorded. The survival time was prolonged in the 5 ppm NO (6.09 ± 1.29 minutes) and 10 ppm NO (6.39 ± 1.58 minutes) groups compared with the 0 ppm group (4.98 ± 1.23 minutes). Hypoxia of the brain was relieved, and the exercise exhaustion time was prolonged when mice inhaled 20 ppm NO (24.70 ± 6.87 minutes vs. 20.23 ± 6.51 minutes). In addition, the differences in arterial oxygen saturation (SO2%) (49.64 ± 7.29% vs. 42.90 ± 4.30%) and arteriovenous SO2% difference (25.14 ± 8.95% vs. 18.10 ± 6.90%) obviously increased. In ex vivo experiments, the oxygen equilibrium curve (OEC) left shifted as P50 decreased from 43.77 ± 2.49 mmHg (0 ppm NO) to 40.97 ± 1.40 mmHg (100 ppm NO) and 38.36 ± 2.78 mmHg (200 ppm NO). Furthermore, the Bohr effect of Hb was enhanced by the introduction of 200 ppm NO (-0.72 ± 0.062 vs.-0.65 ± 0.051), possibly allowing Hb to more easily offload oxygen in tissue at lower pH. The crystal structure reveals a greater distance between Asp94β-His146β in nitrosyl -Hb(NO-Hb), NO-HbβCSO93, and S-NitrosoHb(SNO-Hb) compared to tense Hb(T-Hb, 3.7 Å, 4.3 Å, and 5.8 Å respectively, versus 3.5 Å for T-Hb). Moreover, hydrogen bonds were less likely to form, representing a key limitation of relaxed Hb (R-Hb). Upon NO interaction with Hb, hydrogen bonds and salt bridges were less favored, facilitating relaxation. We speculated that NO ameliorated the effects of hypoxia in mice by promoting erythrocyte oxygen loading in the lung and offloading in tissues.

Translational insights into the hormetic potential of carbon dioxide: from physiological mechanisms to innovative adjunct therapeutic potential for cancer.

Carbon dioxide (CO2), traditionally viewed as a mere byproduct of cellular respiration, plays a multifaceted role in human physiology beyond simple elimination through respiration. CO2 may regulate the tumor microenvironment by significantly affecting the release of oxygen (O2) to tissues through the Bohr effect and by modulating blood pH and vasodilation. Previous studies suggest hypercapnia (elevated CO2 levels) might trigger optimized cellular mechanisms with potential therapeutic benefits. The role of CO2 in cellular stress conditions within tumor environments and its impact on O2 utilization offers a new investigative area in oncology. This study aims to explore CO2's role in the tumor environment, particularly how its physiological properties and adaptive responses can influence therapeutic strategies. By applying a structured translational approach using the Work Breakdown Structure method, the study divided the analysis into six interconnected work packages to comprehensively analyze the interactions between carbon dioxide and the tumor microenvironment. Methods included systematic literature reviews, data analyses, data integration for identifying critical success factors and exploring extracellular environment modulation. The research used SMART criteria for assessing innovation and the applicability of results. The research revealed that the human body's adaptability to hypercapnic conditions could potentially inform innovative strategies for manipulating the tumor microenvironment. This could enhance O2 utilization efficiency and manage adaptive responses to cellular stress. The study proposed that carbon dioxide's hormetic potential could induce beneficial responses in the tumor microenvironment, prompting clinical protocols for experimental validation. The research underscored the importance of pH regulation, emphasizing CO2 and carbonic acid's role in modulating metabolic and signaling pathways related to cancer. The study underscores CO2 as vital to our physiology and suggests potential therapeutic uses within the tumor microenvironment. pH modulation and cellular oxygenation optimization via CO2 manipulation could offer innovative strategies to enhance existing cancer therapies. These findings encourage further exploration of CO2's therapeutic potential. Future research should focus on experimental validation and exploration of clinical applications, emphasizing the need for interdisciplinary and collaborative approaches to tackle current challenges in cancer treatment.

Convergent High O2 Affinity but Distinct ATP-Mediated Allosteric Regulation of Hemoglobins in Oviparous and Viviparous Eremias Lizards from the Qinghai-Tibet Plateau.

The functional adaptation and underlying molecular mechanisms of hemoglobins (Hbs) have primarily concentrated on mammals and birds, with few reports on reptiles. This study aimed to investigate the convergent and species-specific high-altitude adaptation mechanisms of Hbs in two Eremias lizards from the Qinghai-Tibet Plateau. The Hbs of high-altitude E. argus and E. multiocellata were characterized by significantly high overall and intrinsic Hb-O2 affinity compared to their low-altitude populations. Despite the similarly low Cl- sensitivities, the Hbs of high-altitude E. argus exhibited higher ATP sensitivity and ATP-dependent Bohr effects than that of E. multiocellata, which could facilitate O2 unloading in respiring tissues. Eremias lizards Hbs exhibited similarly low temperature sensitivities and relatively high Bohr effects at lower temperatures, which could help to stably deliver and release O2 to cold extremities at low temperatures. The oxygenation properties of Hbs in high-altitude populations might be attributed to varying ratios of β2/β1 globin and substitutions on the β2-type globin. Notably, the Asn12Ala in lowland E. argus could cause localized destabilization of the E-helix in the tetrameric Hb by elimination of hydrogen bonds, thereby resulting in its lowest O2 affinity. This study provides a valuable reference for the high-altitude adaptation mechanisms of hemoglobins in reptiles.

Hemoglobin concentration and blood shift during dry static apnea in elite breath hold divers.

Elite breath-hold divers (BHD) enduring apneas of more than 5min are characterized by tolerance to arterial blood oxygen levels of 4.3kPa and low oxygen-consumption in their hearts and skeletal muscles, similar to adult seals. Adult seals possess an adaptive higher hemoglobin-concentration and Bohr effect than pups, and when sedated, adult seals demonstrate a blood shift from the spleen towards the brain, lungs, and heart during apnea. We hypothesized these observations to be similar in human BHD. Therefore, we measured hemoglobin- and 2,3-biphosphoglycerate-concentrations in BHD (n = 11) and matched controls (n = 11) at rest, while myocardial mass, spleen and lower extremity volumes were assessed at rest and during apnea in BHD. After 4min of apnea, left ventricular myocardial mass (LVMM) determined by 15O-H2O-PET/CT (n = 6) and cardiac MRI (n = 6), was unaltered compared to rest. During maximum apnea (∼6min), lower extremity volume assessed by DXA-scan revealed a ∼268mL decrease, and spleen volume, assessed by ultrasonography, decreased ∼102mL. Compared to age, BMI and VO2max matched controls (n = 11), BHD had similar spleen sizes and 2,3- biphosphoglycerate-concentrations, but higher total hemoglobin-concentrations. Our results indicate: 1) Apnea training in BHD may increase hemoglobin concentration as an oxygen conserving adaptation similar to adult diving mammals. 2) The blood shift during dry apnea in BHD is 162% more from the lower extremities than from the spleen. 3) In contrast to the previous theory of the blood shift demonstrated in sedated adult seals, blood shift is not towards the heart during dry apnea in humans.

Histidine Protonation States Regulate the State Transition from R State Hemoglobin.

The objective of our work is to investigate the impact of pH on the structural changes of hemoglobin that affect its O2 affinity, known as the Bohr effect. We conducted molecular dynamics (MD) simulations to explore the transition between various hemoglobin states based on the protonation states (PSs) of two histidine residues (βHis143 and βHis146). We conducted the MD simulations from the R and R2 states with three sets of PSs assuming pH values of 7.0, 6.5, and 5.5, aiming to investigate the influence of pH on hemoglobin behavior. Our results demonstrated that the protonated His residues promote the state transition from the R state to the R2 state and encourage elongation of the distance between the β1-β2 subunits by weakening the inter-subunit interactions in the R state. These observations, aligning with the experimental evidence, indicate that the R2 state typically crystallizes under low pH conditions. Our findings suggest that the relationship between the PSs and the structural stability of the R state plays a role in the acid and alkaline Bohr effect.

An investigation of gill and blood carbonic anhydrase characteristics in three basal actinopterygian species: alligator gar (Atractosteus spatula), white sturgeon (Acipenser transmontanus) and Senegal bichir (Polypterus senegalus).

Many teleosts possess a unique set of respiratory characteristics allowing enhanced oxygen unloading to the tissues during stress. This system comprises three major components: highly pH sensitive haemoglobins (large Bohr and Root effects), rapid red blood cell (RBC) intracellular pH (pHi) protection, and a heterogeneous distribution of membrane-bound plasma-accessible carbonic anhydrase (paCA; absence in the gills). The first two components have received considerable research effort; however, the evolutionary loss of branchial paCA has received little attention. In the current study, we investigated the availability of branchial membrane-bound CA, along with several other CA-related characteristics in species belonging to three basal actinopterygian groups: the Lepisosteiformes, Acipenseriformes and Polypteriformes to assess the earlier hypothesis that Root effect haemoglobins constrain branchial paCA availability. We present the first evidence suggesting branchial membrane-bound CA presence in a basal actinopterygian species: the Senegal bichir (Polypterus senegalus) and show that like the teleosts, white sturgeon (Acipenser transmontanus) and alligator gar (Atractosteus spatula) do not possess branchial membrane-bound CA. We discuss the varying respiratory strategies for these species and propose that branchial paCA may have been lost much earlier than previously thought, likely in relation to the changes in haemoglobin buffer capacity associated with the increasing magnitude of the Bohr effect. The findings described here represent an important advancement in our understanding of the evolution of the unique system of enhanced oxygen unloading thought to be present in most teleosts, a group that encompasses half of all vertebrates.

Photo-Responsive Ascorbic Acid-Modified Ag2S-ZnS Heteronanostructure Dropping pH to Trigger Synergistic Antibacterial and Bohr Effects for Accelerating Infected Wound Healing.

Nonantibiotic approaches must be developed to kill pathogenic bacteria and ensure that clinicians have a means to treat wounds that are infected by multidrug-resistant bacteria. This study prepared matchstick-like Ag2S-ZnS heteronanostructures (HNSs). Their hydrophobic surfactants were then replaced with hydrophilic poly(ethylene glycol) (PEG) and thioglycolic acid (TGA) through the ligand exchange method, and this was followed by ascorbic acid (AA) conjugation with TGA through esterification, yielding well-dispersed PEGylated Ag2S-ZnS@TGA-AA HNSs. The ZnS component of the HNSs has innate semiconductivity, enabling the generation of electron-hole pairs upon irradiation with a light of wavelength 320 nm. These separate charges can react with oxygen and water around the HNSs to produce reactive oxygen species. Moreover, some holes can oxidize the surface-grafted AA to produce protons, decreasing the local pH and resulting in the corrosion of Ag2S, which releases silver ions. In evaluation tests, the PEGylated Ag2S-ZnS@TGA-AA had synergistic antibacterial ability and inhibited Gram-negative Escherichia coli and Gram-positive methicillin-resistant Staphylococcus aureus (MRSA). Additionally, MRSA-infected wounds treated with a single dose of PEGylated Ag2S-ZnS@TGA-AA HNSs under light exposure healed significantly more quickly than those not treated, a result attributable to the HNSs' excellent antibacterial and Bohr effects.

Rapid measurement of hemoglobin-oxygen dissociation by leveraging Bohr effect and Soret band bathochromic shift.

Oxygen (O2) binds to hemoglobin (Hb) in the lungs and is then released (dissociated) in the tissues. The Bohr effect is a physiological mechanism that governs the affinity of Hb for O2 based on pH, where a lower pH results in a lower Hb-O2 affinity and higher Hb-O2 dissociation. Hb-O2 affinity and dissociation are crucial for maintaining aerobic metabolism in cells and tissues. Despite its vital role in human physiology, Hb-O2 dissociation measurement is underutilized in basic research and in clinical laboratories, primarily due to the technical complexity and limited throughput of existing methods. We present a rapid Hb-O2 dissociation measurement approach by leveraging the Bohr effect and detecting the optical shift in the Soret band that corresponds to the light absorption by the heme group in Hb. This new method reduces Hb-O2 dissociation measurement time from hours to minutes. We show that Hb deoxygenation can be accelerated chemically at the optimal pH of 6.9. We show that time and pH-controlled deoxygenation of Hb results in rapid and distinct conformational changes in its tertiary structure. These molecular conformational changes are manifested as significant, detectable shifts in Hb's optical absorption spectrum, particularly in the characteristic Soret band (414 nm). We extensively validated the method by testing human blood samples containing normal Hb and Hb variants. We show that rapid Hb-O2 dissociation can be used to screen for and detect Hb-O2 affinity disorders and to evaluate the function and efficacy of Hb-modifying therapies. The ubiquity of optical absorption spectrophotometers positions this approach as an accessible, rapid, and accurate Hb-O2 dissociation measurement method for basic research and clinical use. We anticipate this method's broad adoption will democratize the diagnosis and prognosis of Hb disorders, such as sickle cell disease. Further, this method has the potential to transform the research and development of new targeted and genome-editing-based therapies that aim to modify or improve Hb-O2 affinity.

Functional characterization of compound heterozygosity Hb S/Hb Deer Lodge in Brazil

IntroductionThe Hb Deer Lodge (β2 His>Arg; HBB:c.8A>G) is a structural hemoglobin variant described in some populations around the world, characterized by increased oxygen affinity, but does not confer clinical symptoms to its carriers. The coinheritance of the Hb Deer Lodge with the most common hemoglobin variant, Hb S, has been reported only once; however, functional data were not described. Here we show a case of the Hb S and Hb Deer Lodge carrier in heterozygosity. MethodsThe Hb S and Hb Deer Lodge association was identified by High-Performance Liquid Chromatography (HPLC), reverse phase HPLC and the β globin gene sequencing. The functional characterization of this interaction was obtained using the O2 dissociation curve, determination of the cooperativity between the globin chains and the Bohr effect in the presence and absence of organic phosphates. ResultsWhen the Hb S and Hb Deer Lodge were associated, there was a decrease in cooperativity, no significant changes in oxygen affinity and no significant Bohr effect changes. ConclusionDespite these genetic variations, the carrier showed no hematological alterations and no clinical symptoms, possibly due to the high oxygen affinity of the Hb Deer Lodge, which interferes with the Hb S polymerization.

Loss of Oxygen Transport Potency As a Function of RBC Storage Duration

BACKROUND: Red blood cell (RBC) oxygen (O 2) capture/release is regulated via homeostatic modulation of hemoglobin oxygen (Hb-O 2) affinity in a manner that stabilizes O 2 delivery in the setting of reduced O 2 availability (i.e., hypoxia, anemia) or increased O 2 consumption (i.e., exercise, stress, disease). This is achieved via production of allosteric effectors (i.e., 2,3 DPG, ATP) and adjustment of the RBC biochemical milieu (i.e., pH, anion concentration) in response to regional stimuli including temperature and carbon dioxide (CO 2) abundance, encountered during circulatory transit (aka Bohr effect). Blood storage is known to affect Hb-O 2 affinity, because the environment to which the RBCs are exposed affects cellular metabolism, influencing [allosteric effector], pH, and [anion]. In addition, key proteins may either become depleted or impaired during storage, including anion exchange-1 (AE-1 or Band 3) and carbonic anhydrase, the two proteins that enable the Bohr effect. We therefore explored the novel hypothesis that RBC oxygen transport potency is diminished as a function of blood storage duration, due to loss of the Bohr effect. METHODS: Fresh (healthy control) or stored RBCs units (leuko-reduced in AS-1; Impact Life Davenport IA, USA), were analyzed weekly for up to 42 days. Hb-O 2 affinity was determined from the oxygen dissociation curve (ODC), which describes the relationship between the partial pressure of oxygen (pO 2) and the fraction of oxyhemoglobin (FHbO 2). ODCs were constructed at 2 fixed pH values (HEMOX analyzer, TSC Scientific Corp, PA, USA) or 2 fixed CO 2 levels (thin film rotating tonometer; Meon, Graz, Austria, in conjunction with a gas blender; Oxystreamer, Biospherix, Parish, NY, USA, with samples measured by arterial blood gas machine; ABL90Flex, Radiometer, Brea, CA, USA). Analysis included (1) standard p50 determination (i.e., pO 2 at which Hb is 50% saturated with O 2), (2) delta p50 between ODCs at fixed pH 7.6 and 7.2 - HEMOX, or ODCs at fixed pCO 2, eucarbia ~40 mmHg and hypercarbia ~70 mmHg - tonometer), (3) Area between the two curves measured (ABC), as described above. Additional analyses were performed from ODC data, including comparison of blood O 2 content per gram Hb (with SO 2 converted to blood O 2 content assuming 1-gram Hb binds 1.34 ml O 2), and blood O 2 capacitance (βO2), which quantifies the amount of O 2 unloaded for a given arteriovenous pO 2 gradient. RESULTS: As a function of blood storage duration, we found significant (1) reduction in p50 at 2 fixed pHs (HEMOX) and 2 fixed pCO 2s (tonometer) (2) reduction in delta p50 between ODCs at fixed pH 7.6 and 7.2 (HEMOX), or fixed pCO 2; eucarbia ~40 mmHg and hypercarbia ~70 mmHg (tonometer), i.e., significantly reduced Bohr effect (3) reduction in the area between ODC curves at different pH or CO 2 levels (ABC), a more comprehensive representation of the Bohr effect, across the full range of pO 2s encountered during circulatory transit (rather than the single point p50 measurement), (4) reduction in blood oxygen content per gram Hb, and (5) reduction in βO2. SUMMARY: We demonstrate that blood storage significantly impairs the Bohr effect. Consequently, compared to fresh RBCs, the oxygen transport potency of stored RBCs in AS-1 within one week is ~50-60% that of fresh RBCs and by the time the unit is outdated, is down to ~35%. Consequently, 2.86 the number of RBCs from an expiring stored transfusion unit are required to move the same amount of O 2 as a similar number of RBCs in fresh blood.

RBC eNOS Transduces Regional O2 Gradients to Orchestrate RBC O2 Delivery Phenotype during Circulatory Transit

BACKGROUND: The role of endogenous endothelial NO synthase (eNOS) in mature RBCs is poorly understood. We have previously demonstrated functionally O2-dependent eNOS migration in RBCs, mediating (via s-nitrosylation) glycolytic metabolon assembly/disassembly during circulation in support of antioxidant systems during stress. We have further explored eNOS migration and now propose the following mechanism along with its functional significance. We (and others) suggest that hemoglobin (Hb) conformational change resulting from deoxygenation leads to its docking at the cytoplasmic domain of Band 3 (cdB3). We propose that (1) cdB3 consequently undergoes a conformational change that is transduced by Piezo1 (a mechanosensitive ion channel located in the RBC membrane); (2) Piezo1 activation results in calcium entry into the RBC, which (3) aids in the disassembly of the RBC cytoskeleton, (4) facilitates eNOS migration to the membrane (where calmodulin is mainly localized - thereby bringing these two important proteins together), and (5) binds to calmodulin, activating eNOS to generate nitric oxide (NO) - which then influences RBC phenotype that facilitates O 2 delivery, specifically related to RBC deformability and regulation of Hb~O 2 release, via enhanced Bohr effect. METHODS: Heparinized human, eNOS (-/-), or wt murine (C57BL/6J) red blood cells (RBCs) were imaged (Nikon) for the analysis of cytoskeletal protein arrangement and eNOS migration. RBCs were subjected to oxygenation/deoxygenation, with/without pre-treatment with the Piezo1 agonist Yoda1, or a membrane tension buffer grammostola mechanotoxin #4 (GSMTx4; decoupling Piezo1), or carbon monoxide (CO) to maintain R-state Hb conformation upon RBC deoxygenation. RBCs were fixed (paraformaldehyde 4%/glutaraldehyde 0.08%), permeabilized (Triton X100 amount 0.1%) and incubated with primary antibodies (spectrin, actin, protein 4.1, or eNOS), then with the secondary antibody prior to visualization. Interaction between eNOS and cytoskeleton proteins, as well as calmodulin, was determined using a proximity ligation assay (Duolink). Intracellular RBC calcium levels were assessed in oxygenated/deoxygenated RBCs utilizing an environmentally controlled fluorescent plate reader, and the calcium probe Fluo 3. NO production in RBCs was assessed using DAF-FM. To assess the functional significance of this mechanism, Hb-O 2 affinity was determined from murine blood (eNOS (-/-) or wt). Oxygen dissociation curves (ODCs), were constructed at 2 fixed CO 2 levels (thin film rotating tonometer; in conjunction with a gas blender; with samples measured by arterial blood gas machine).Bohr effect was assessed by determining the area between the ODCs (ABC) at the 2 fixed CO2 levels (~40mmHg and ~70mmHg). RESULTS: We observed several significant findings related to red RBC deoxygenation. Upon RBC deoxygenation, there is an increase in calcium levels indicated by the Fluo3 signal, which is Piezo1 dependent. Cytoskeleton proteins, such as Actin, Spectrin, and protein 4.1, undergo disassembly upon RBC deoxygenation. This disassembly process is contingent on both Piezo1-mediated calcium flux and the conformational changes in hemoglobin (Hb) (and not pO 2). We found that eNOS is associated with 4.1 throughout the oxy/deoxy cycle and that eNOS exhibits the same migratory pattern as cytoskeleton proteins to the RBC membrane, where calmodulin is located, irrespective of the RBC's oxygenation state. Additionally, when eNOS localizes in the membrane, it exhibits interaction with calmodulin. The generation of nitric oxide (NO) by eNOS, as indicated by the DAF-FM signal, increases in a manner dependent on both calcium, Piezo1 and Hb conformational transition. The functional consequence of this mechanism appears to be related to O 2 delivery phenotype during circulatory transit, with cell deformability being affected in addition to an inhibition of the Bohr effect in eNOS (-/-) compared to wt mice (2006 ± 61 vs 1892 ± 69 ABC in WT (n = 3) vs eNOS (-/-) (n = 4), respectively; p = 0.0747). SUMMARY: Upon circulatory transit, RBC deoxygenation triggers the activation of eNOS. This activated eNOS is associated with Piezo1-mediated calcium flux, which is facilitated by calmodulin. These events are coordinated with the migration of eNOS within the red blood cell between the cytosol and the cell membrane. The migration of eNOS is influenced by cytoskeleton proteins.

A coupled model for the dynamics of gas exchanges in the human lung with Haldane and Bohr’s effects

We propose an integrated dynamical model for oxygen and carbon dioxide transfer from the lung into the blood, coupled with a lumped mechanical model for the ventilation process, for healthy patients as well as in pathological cases. In particular, we take into account the nonlinear interaction between oxygen and carbon dioxide in the blood volume, referred to as the Bohr and Haldane effects. We also propose a definition of the physiological dead space volume (the lung volume that does not contribute to gas exchange) which depends on the pathological state and the breathing scenario. This coupled ventilation-gas diffusion model is driven by the sole action of the respiratory muscles. We analyse its sensitivity with respect to characteristic parameters: the resistance of the bronchial tree, the elastance of the lung tissue and the oxygen and carbon dioxide diffusion coefficients of the alveolo-capillary membrane. Idealized pathological situations are also numerically investigated. We obtain realistic qualitative tendencies, which represent a first step towards classification of the pathological behaviours with respect to the considered input parameters.

A novel mechanism for high-altitude adaptation in hemoglobin of black-spotted frog (Pelophylax nigromaculatus)

Understanding how animals living in highland adapt to extreme conditions is critical to evolutionary biology. In contrast to birds and mammals, little information was available on the adaptation mechanisms for O2 transport in high-altitude ectothermic vertebrates. Here we report for the first time on hematological parameters, amino acid sequences of α and β chains of hemoglobin (Hb), O2 affinity of purified hemoglobins (Hbs) and their sensitivities to anion allosteric effector (H+, Cl−, ATP) and temperature in the high-altitude (2,292 m) black-spotted frogs (Pelophylax nigromaculatus) from the Qinghai-Tibet plateau (QTP) compared with the low-altitude (135 m) population. Our results showed that high-altitude black-spotted frogs exhibit significantly increased relative lung mass, hematocrit, and hemoglobin concentration, but significantly decreased body mass and erythrocyte volume, which could improve the blood O2 carrying capability. Compared with the low-altitude population, the purified Hbs of high-altitude black-spotted frogs possessed significantly higher intrinsic Hb-O2 affinity, similar low anion allosteric effector sensitivities, Bohr effects and temperature sensitivities. The elevated Hb-O2 affinity of highland frogs could maximize the O2 extraction from the lungs. Molecular dynamics simulations showed that the Gln123Glu substitution on α2 chain in highland frogs could form a hydrogen bond with 127Lys on α2 chain, resulting in the elimination of a hydrogen bond between 127Lys on α2 chain and 141Arg on α1 chain. This could weaken the interaction between two semirigid dimers (α1β1 and α2β2) and then lead to the high intrinsic Hb-O2 affinity in high-altitude black-spotted frogs.

Sphingomyelin and Phosphatidylserine Derived Compounds Found into RBCs of Two Unrelated Individuals with Idiopathic Erythrocytosis with Altered Hemoglobin (Hb) Function Properties and No Hemoglobinopathies - Preliminary Results

Sphingomyelin and phosphatidylserine derived compounds were found into Red Blood Cells (RBCs) of two unrelated individuals with idiopathic erythrocytosis, whom were referred to the Laboratory of Hemoglobinopathies - State University of Campinas (State of São Paulo, Brazil) to investigate the presence of hemoglobin (Hb) variants with high oxygen affinity. Both unrelated individuals were 5-year-old age Caucasian Brazilian boys with the following counts: RBC (x 10/6mm3) 6.15 and 7.69, Hb (g/dL) 20.1 and 20, Htc (%) 54 and 58.7, respectively. Most common causes of erythrocytosis, including polycythemia vera (PV) and other secondary erythrocytosis; JAK2, VHL and EPOR mutational screen were previously excluded in the Centro Infantil Boldrini (Campinas, State of São Paulo, Brazil), where the children are followed. No hemoglobin or globin variants were detected by standard methods: alkaline electrophoresis in cellulose acetate (pH 8.9), acidic electrophoresis in agar gel (pH 5.8), cation-exchange HPLC (Variant II, Bio-Rad) and RP-HPLC (Alliance, Waters). Direct genome sequencing of HBA and HBB performed in automated sequencer (ABI PRISM™ 377 DNA Automated Sequencer, Applied BioSystems) after DNA amplification obtained from peripheral leukocytes (QIAamp DNA Blood, Qiagen) did not reveal any mutation. Familial study also did not show any alteration in blood count, hemoglobin or even globin gene mutation in their relatives. Deletional and non-deletional alpha thalassemia was also excluded by Multiplex-gap-PCR and restriction enzyme reaction (Hph and NcoI). Despite of normal results for Hb characterization, functional tests performed with Hb purified by exclusion (Sephadex G-25, Sigma-Aldrich) and ion exchange chromatography (Amberlite MB-3, Sigma-Aldrich) in Hepes buffer indicated some alteration. O2 affinity was evaluated by determining P50 with stripped lysates ([Hb]=70 μM/Heme) in pHs of 6.5, 7.0, 7.5, 8.0 and 8.5, for Bohr effect calculations. Heme-heme cooperativity was determined by Hill coefficient (n) and the activity of allosteric effector in the Hb-O2 ligation was also tested, using inositol hexaphosphate ([IHP]=1mM), a mimetic compound of the intraerythrocytic 2,3-BPG. P50 was slightly decreased (both patients, stripped Hb samples) at extreme acidic and alkaline pH (pH 6.5 and 8.5) leading to altered Bohr effect, in comparison to healthy and erythrocytosis control (caused by cardiopathy, with no Hb variant). P50 was also found decreased at pH 8.5 in the presence of IHP. Heme-heme cooperativity was very decreased at pHs 6.5, 7.0, 8.0 and 8.5, in stripped state as well as in the presence of IHP. In order to investigate the unexpected results mass spectrometry of the purified intraerythrocytic content was performed. RBC lysate was separated from stroma by ultracentrifugation (14.000rpm) and purified by molecular exclusion filtration (Amicon filter - Millipore, 10KDa cutoff) and ion exchange chromatography (Amberlite MB-3, Sigma-Aldrich) for desalinization. Quadrupole Mass spectrometry (MS) with 0.8 ppm MS and MS/MS accuracy (Impact High DefinitionTM, Bruker), preceded by UPLC (Ultra Pressure Liquid Chromatography) system (Shimadzu Nexera) with C18 Polymer RP-column (Supelco apHera C18 Polymer 150 x 2 mm, 5um) was used to separate and identify and compare the compounds to control sample (secondary erythrocytosis with no Hb function alteration). No alteration in peptide, hemoglobin or heme were found. Four small compounds, two derived of sphingomyelin and two derived of phosphatidylserine were identified in both patient samples (C39H79N2O6P, C42H80NO8P, C42H82NO8P, C44H84NO8P) - Figure 1. Sphingomyelin and phosphatidylserine are RBC membrane compounds rich in PO4-, which theoretically could bind b1b2 allosteric 2,3-BPG site in Hb. There is no evidence that these molecules were effectively bound to Hb except that were found in purified Hb samples with some function alteration, particularly with decreased heme-heme cooperativity. Some evidence suggests that analog compounds could be found in higher concentrations in PV or secondary erythrocytosis. To our knowledge it is the first-time that alteration in Hb function was observed in samples with these molecules. These preliminary results should be further validated and at least 10 other similar cases should be better characterized. (Financial support: Fapesp, Faepex-Unicamp, Educorp-Unicamp). Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Local application of a transcutaneous carbon dioxide paste prevents excessive scarring and promotes muscle regeneration in a bupivacaine-induced rat model of muscle injury.

In postoperative patients with head and neck cancer, scar tissue formation may interfere with the healing process, resulting in incomplete functional recovery and a reduced quality of life. Percutaneous application of carbon dioxide (CO2 ) has been reported to improve hypoxia, stimulate angiogenesis, and promote fracture repair and muscle damage. However, gaseous CO2 cannot be applied to the head and neck regions. Previously, we developed a paste that holds non-gaseous CO2 in a carrier and can be administered transdermally. Here, we investigated whether this paste could prevent excessive scarring and promote muscle regeneration using a bupivacaine-induced rat model of muscle injury. Forty-eight Sprague Dawley rats were randomly assigned to either a control group or a CO2 group. Both groups underwent surgery to induce muscle injury, but the control group received no treatment, whereas the CO2 group received the CO2 paste daily after surgery. Then, samples of the experimental sites were taken on days 3, 7, 14, and 21 post-surgery to examine the following: (1) inflammatory (interleukin [IL]-1β, IL-6), and transforming growth factor (TGF)-β and myogenic (MyoD and myogenin) gene expression by polymerase chain reaction, (2) muscle regeneration with haematoxylin and eosin staining, and (3) MyoD and myogenin protein expression using immunohistochemical staining. Rats in the CO2 group showed higher MyoD and myogenin expression and lower IL-1β, IL-6, and TGF-β expression than the control rats. In addition, treated rats showed evidence of accelerated muscle regeneration. Our study demonstrated that the CO2 paste prevents excessive scarring and accelerates muscle regeneration. This action may be exerted through the induction of an artificial Bohr effect, which leads to the upregulation of MyoD and myogenin, and the downregulation of IL-1β, IL-6, and TGF-β. The paste is inexpensive and non-invasive. Thus, it may be the treatment of choice for patients with muscle damage.

IDF21-0639 Preliminary Results of Wound Healing in Persons with Diabetes Mellitus Using Transcutaneous Carbon Dioxyde Application

Background: Number of processes including aging, jeopardy integrity of the body as well as all organs while only defence and reparation depends on proper nutrition through all sizes of blood vessels, haemorrheology and circulation. A number of negative changes with or without chronic noncommunicable diseases are faced with appropriate circulation and characteristics of blood vessels especially legs and feet. Aim: The idea came up based on thermal baths which were used for different treatment and rehabilitation for many years. The aims were to test the positive influence of transcutaneous gas form composite carbon dioxide (CO2) on legs and feet blood vessels. The following parameters were analysed: skin temperature, Rydel Seiffer vibrametar tests, Semmes Weinsteinov monofilament test. Method: The persons with diabetes mellitus, with peripheral neuropathy or foot wound was included in the study. Beside patient history and physical examination, skin temperature, Rydel Seiffer vibrametar tests, Semmes Weinsteinov monofilament test were performed. The patient is put in a therapeutic wrap closed at the level of abdomen for 50 minutes, during 20 days. Wound toilette was performed during every visit if necessary. The detailed description of the transcutaneous CO2 therapy using a peripheral vascular system (PVR System®; Produced by Derma Art, Brezice, Slovenia) is described earlier. Results: The size of the wound decreases faster compared to reference values. Statistical analysis were test between Study and Control group of patients. Skin temperature increases (p<0,001). Vibrameter test showed better results (p<0,001), as well as monofilament tests (p<0,001). Previous published analysis showed that vasodilatation of blood vessels was achieved, as well as number of collateral circulation as an intermediate changes important to achieving clinical results. Discussion: Based on Bohr effect we recognise positive effect of CO2 influence on tissues (healing, improving nerves velocity in particular) and other surrounding tissues. Previously published data significantly proved improved changes, (90 % shorter wound healing time), although more comprehensive investigation through random clinical trials is still necessary.

Blood Flow and Respiratory Gas Exchange in the Human Placenta at Term: A Data Update.

Reliable measurements using modern techniques and consensus in experimental design have enabled the assessment of novel data sets for normal maternal and foetal respiratory physiology at term. These data sets include (a) principal factors affecting placental gas transfer, e.g., maternal blood flow through the intervillous space (IVS) (500mL/min) and foeto-placental blood flow (480mL/min), and (b) O2, CO2 and pH levels in the materno-placental and foeto-placental circulation. According to these data, the foetus is adapted to hypoxaemic hypoxia. Despite flat oxygen partial pressure (pO2) gradients between the blood of the IVS and the umbilical arteries of the foetus, adequate O2 delivery to the foetus is maintained by the higher O2 affinity of the foetal blood, high foetal haemoglobin (HbF) concentrations, the Bohr effect, the double-Bohr effect, and high foeto-placental (=umbilical) blood flow. Again, despite flat gradients, adequate CO2 removal from the foetus is maintained by a high diffusion capacity, high foeto-placental blood flow, the Haldane effect, and the double-Haldane effect. Placental respiratory gas exchange is perfusion-limited, rather than diffusion-limited, i.e., O2 uptake depends on O2 delivery.

Sepsis Induced Red Cell Dysfunction (SiRD): Physiology and Mechanisms

BACKGROUND: In sepsis, many red blood cell (RBC) defects are described: ↑ oxygen (O2) affinity, ↓ deformability, ↑ aggregation & adhesion, and dysregulated RBC-based vasoregulation. We suggest these comprise a unique class of organ (RBC) failure, which we term sepsis-induced RBC dysfunction (SiRD). We propose SiRD arises from RBC metabolic failure and depowered antioxidant systems - resulting in impaired RBC physiology that disables O2 transport. We employed a murine cecal ligation and puncture (CLP) sepsis model to characterize SiRD features and explore mechanisms of SiRD initiation.METHODS: Mice underwent sham (Gcont), mild (Gmild: D1 mortality 10%), or severe CLP (Gsev: D1 mortality 50%); N=4-8/group. D1 RBCs were collected. SiRD phenotype was characterized by analyzing: (1) O2 affinity (p50, Bohr effect), (2) deformability (LORRCA - elongation index, EI), (3) osmotic fragility (LORRCA - ΔOsm resilience), (4) aggregation (LORRCA - syllectogram) and (5) vasoregulation (organ chamber bioassay - RBC hypoxic vasodilaton). Mechanistic explanation for the SiRD phenotype was explored by evaluating: (6) Ca+ accumulation (flow cytometry), (7) caspase3 activation (flow cytometry), (8) cdB3 proteolysis (western blot), (9) RBC metabolomic analysis (13C1,2,3-glucose based MS, +/- exogenous oxidant stress), (10) nitric oxide content (photolysis:chemiluminescence - total RBC NO, FeNO, and SNO content), and (11) eryptosis:phosphatidylserine (PS) exposure (flow cytometry). SiRD physiologic impact was evaluated by (12) quantifying tissue O2 delivery impairment in vivo (HIF-1a bioluminescent mouse, IVIS).RESULTS: RBCs from septic mice demonstrated the following physiologic Δs: (1) ↑ O2 affinity (L-shifted HbO2 dissociation) at 3 pHs (7.2, 7.4, and 7.6, with ↓ Bohr effect), with ↑ p50 (Gsev vs Gcont) (pH7.2: 47.8±1.2* vs. 53.3±0.9 Torr; pH7.4: 37.2±1.1** vs. 42.9±0.7 Torr; and pH7.6: 29.0±0.6*** vs. 34.2±0.8 Torr); (2) ↓ RBC deformability - with similar EImax, shear stress (SS)1/2 was ↑ in Gsev vs Gcont (0.92 vs. 0.65 Pa); (3) ↑ osmotic fragility - min & max tolerated osmolality (Omin & Omax): Omin in Gcont, Gmild, and Gsev were 166.1±8.9, 158.8±4.5, and 159.1±2.8 mOsm/kg; and Omax were 336.0±21.5, 315.3±9.1, and 314.0±8.5 mOsm/kg, respectively; (4) ↑ aggregation - syllectogram backscatter intensity (Isc): in Gsev, Isc ↓ from 32.5±5.0 to 12.5±2.8 Au; but in Gcont, Isc only changed from 31.1±3.4 to 23.6±4.6 Au; (5) hypoxic vasodilation response ↓ in Gsev RBCs vs Gcont (8.9±4.2% vs 10.2±4.0%*, respectively). Exploration of potential mechanisms for SiRD revealed: (6) ↑ RBC Ca+ (Fluo3-based flow cytometry): ↑ mean florescence intensity (MFI) with sepsis severity (Gcont: 61.3±11.6; Gmild: 73.4±14.6, and Gsev: 105±36.0); (7) MIF of active caspase3 probe (CaspGlow-DEVD-FMK) did not change following CLP; however, after incubation with 0.1mM H2O2, MIF for Gsev ↑ significantly (Gcont: 549.3±28.8 vs. Gsev: 3123±782.2*); (8) ↑ cdB3 proteolysis was observed, Gsev > Gmild; (9) principal component and pathway analysis of metabolomic data identified sepsis severity-based constraints in the hexose monophosphate shunt pathway & linked antioxidant systems (NADPH and GSH); (10) RBC SNOHb & membrane SNO ↓ in Gsev vs Gcont (0.08 ± 0.04* & 0.04 ± 0.02* vs 0.22 ± 0.08 & 0.17 ± 0.07 SNOHb (SNO:Hb molar ratio) and membrane SNO mol/mg protein, respectively; (11) ↑ %age of eryptotic RBCs after CLP (annexin V-based flow cytometry): Gsev annexin V positive sub-population was 5.3±2.9%**, compared to Gcont 1.3±0.6% - following oxidative stress (0.1mM H2O2), annexin V positive sub- population in Gcont ↑ further to 11.5±4.1%; whilst Gsev, ↑ to 31.0±9.2%***. (12) O2 delivery impairment was quantified by imaging (IVIS) HIF-bioluminescent mice (FVB/Gt(ROSA)26Sor tm2(HIF1A/luc)kael): D1 normalized luciferase activity in Gsev was ↑ 3.4±0.8* fold vs baseline; a similar difference was observed after washed RBC exchange from Gsev → Gcont mice. (NB: * P<0.05; ** P<0.01; *** P<0.001)CONCLUSIONS: In sepsis, RBCs undergo major biochemical & physiologic alterations that impair O2 delivery, which we term SiRD. We propose SiRD arises from acquired RBC metabolic constraints, oxidative RBC injuries and impaired physiology, comprising a distinct form of organ (RBC) failure that may influence sepsis outcome. This well characterized model will allow further characterization, mechanistic study, and therapeutic exploration. DisclosuresDoctor:KaloCyte, Inc.: Equity Ownership, Research Funding; Washington University in St. Louis: Patents & Royalties; NIH: Research Funding; DoD: Research Funding; Biogen: Consultancy; Children's Discovery Institute: Research Funding.

A Pilot Randomized, Double-blind, Placebo-controlled Clinical Trial on the Efficacy and Safety of a Transdermal Gel that Delivers CO2 in the Treatment of Vulvovaginal Atrophy

This prospective study aimed to assess the efficacy of a transdermal CO2 gel in promoting regeneration in the vulvovaginal area in postmenopausal women experiencing symptoms as part of genitourinary syndrome of menopause (GSM). Transdermal carboxytherapy refers to the application of CO2 through the skin's superficial layer, which is intended to increase oxygenation (through the Bohr effect) and angiogenesis, thus helping to reduce symptoms of GSM. In this study, 20 women with moderate or severe signs or symptoms of vulvovaginal atrophy were randomized to receive either transdermal CO2 therapy (n=10) or a placebo gel (ultrasound gel, n=10). The gels (transdermal CO2 gel or the placebo) were applied by the patients at home. The gels were applied in two treatment cycles (10 days of total treatment). Each cycle involved daily applications of the gel to the vulvovaginal area, from Monday to Friday, with no application on weekends. The Female Sexual Function Index (FSFI) and Day-to-Day Impact of Vaginal Aging (DIVA) questionnaires were used to gather standardized information, and biopsy samples were obtained from the vagina and vulva for histological analysis, both on the first day of treatment and 10 days after the final application of gel. Patients who received the transdermal CO2 gel showed significant improvements on the FSFI and DIVA questionnaires and the biopsies revealed that the CO2 gel had a regenerative effect on vulvovaginal tissues.

Comparison of hematological traits and oxygenation properties of hemoglobins from highland and lowland Asiatic toad (Bufo gargarizans).

The Asiatic toad (Bufo gargarizans) belonging to the family of Bufonidae (Anura: Amphibia) is successfully residing on the Qinghai-Tibetan Plateau (QTP). To investigate whether the oxygen delivery undergoes adaptive adjustments to high-altitude environments in Asian toads inhabiting the QTP (Zoige County, 3446m), choosing low-altitude populations (Chengdu City, 500m) as control, we measured hematological traits, O2 affinities of whole blood, Hb-O2 affinities of purified Hbs, their sensitivities to temperature, and allosteric effectors (H+, Cl- and ATP). Our results showed that high-altitude Asiatic toads possessed significantly increased hemoglobin concentration, hematocrit, and red blood cell count, but significantly decreased erythrocyte volume compared with low-altitude toads. The whole blood and purified Hbs of high-altitude Asiatic toads both exhibited significantly higher O2 affinities compared with low-altitude toads. Substantially increased intrinsic Hb-O2 affinities of high-altitude Asiatic toads Hbs are likely to be the main reason for its elevated Hb-O2 affinities given the anionic cofactor sensitivities of high- and low-altitude toads were similar. The Hbs of high-altitude toads were also characterized by distinctly strong Bohr effects at the low temperature and low-temperature sensitivities. The adaptive adjustments of hematological traits could enhance the blood-O2 carrying capacity of high-altitude Asiatic toads. The increased Hb-O2 affinities could safeguard the pulmonary O2 uploading under hypoxia. The strong Bohr effects at the low temperature could help the release of O2 in metabolic tissues and cold limbs, while low-temperature sensitivity could minimize the effect of temperature fluctuation on the Hb-O2 affinity.