Oxygen Affinity Of Hemoglobin Research Articles

Validation of a Novel Noninvasive Technology to Estimate Blood Oxygen Saturation Using Green Light: Observational Study

Background Pulse oximeters work within the red-infrared wavelengths. Therefore, these oximeters produce erratic results in dark-skinned subjects and in subjects with cold extremities. Pulse oximetry is routinely performed in patients with fever; however, an elevation in body temperature decreases the affinity of hemoglobin for oxygen, causing a drop in oxygen saturation or oxyhemoglobin concentrations. Objective We aimed to determine whether our new investigational device, the Shani device or SH1 (US Patent 11191460), detects a drop in oxygen saturation or a decrease in oxyhemoglobin concentrations. Methods An observational study (phase 1) was performed in two separate groups to validate measurements of hemoglobin and oxygen concentrations, including 39 participants recruited among current university students and staff aged 20-40 years. All volunteers completed baseline readings using the SH1 device and the commercially available Food and Drug Administration–approved pulse oximeter Masimo. SH1 uses two light-emitting diodes in which the emitted wavelengths match with absorption peaks of oxyhemoglobin (hemoglobin combined with oxygen) and deoxyhemoglobin (hemoglobin without oxygen or reduced hemoglobin). Total hemoglobin was calculated as the sum of oxyhemoglobin and deoxyhemoglobin. Subsequently, 16 subjects completed the “heat jacket study” and the others completed the “blood donation study.” Masimo was consistently used on the finger for comparison. The melanin level was accounted for using the von Luschan skin color scale (VLS) and a specifically designed algorithm. We here focus on the results of the heat jacket study, in which the subject wore a double-layered heated jacket and pair of trousers including a network of polythene tubules along with an inlet and outlet. Warm water was circulated to increase the body temperature by 0.5-0.8 °C above the baseline body temperature. We expected a slight drop in oxyhemoglobin concentrations in the heating phase at the tissue level. Results The mean age of the participants was 24.1 (SD 0.8) years. The skin tone varied from 12 to 36 on the VLS, representing a uniform distribution with one-third of the participants having fair skin, brown skin, and dark skin, respectively. Using a specific algorithm and software, the reflection ratio for oxyhemoglobin was displayed on the screen of the device along with direct hemoglobin values. The SH1 device picked up more minor changes in oxyhemoglobin levels after a change in body temperature compared to the pulse oximeter, with a maximum drop in oxyhemoglobin concentration detected of 6.5% and 2.54%, respectively. Conclusions Our new investigational device SH1 measures oxygen saturation at the tissue level by reflectance spectroscopy using green wavelengths. This device fared well regardless of skin color. This device can thus eliminate racial disparity in these key biomarker assessments. Moreover, since the light is shone on the wrist, SH1 can be readily miniaturized into a wearable device.

The differential regulation of placenta trophoblast bisphosphoglycerate mutase in fetal growth restriction: preclinical study in mice and observational histological study of human placenta.

Fetal growth restriction (FGR) is a pregnancy complication in which a newborn fails to achieve its growth potential, increasing the risk of perinatal morbidity and mortality. Chronic maternal gestational hypoxia, as well as placental insufficiency are associated with increased FGR incidence; however, the molecular mechanisms underlying FGR remain unknown. Pregnant mice were subjected to acute or chronic hypoxia (12.5% O2) resulting in reduced fetal weight. Placenta oxygen transport was assessed by blood oxygenation level dependent (BOLD) contrast magnetic resonance imaging (MRI). The placentae were analyzed via immunohistochemistry and in situ hybridization. Human placentae were selected from FGR and matched controls and analyzed by immunohistochemistry (IHC). Maternal and cord sera were analyzed by mass spectrometry. We show that murine acute and chronic gestational hypoxia recapitulates FGR phenotype and affects placental structure and morphology. Gestational hypoxia decreased labyrinth area, increased the incidence of red blood cells (RBCs) in the labyrinth while expanding the placental spiral arteries (SpA) diameter. Hypoxic placentae exhibited higher hemoglobin-oxygen affinity compared to the control. Placental abundance of Bisphosphoglycerate mutase (BPGM) was upregulated in the syncytiotrophoblast and spiral artery trophoblast cells (SpA TGCs) in the murine gestational hypoxia groups compared to the control. Hif1α levels were higher in the acute hypoxia group compared to the control. In contrast, human FGR placentae exhibited reduced BPGM levels in the syncytiotrophoblast layer compared to placentae from healthy uncomplicated pregnancies. Levels of 2,3 BPG, the product of BPGM, were lower in cord serum of human FGR placentae compared to control. Polar expression of BPGM was found in both human and mouse placentae syncytiotrophoblast, with higher expression facing the maternal circulation. Moreover, in the murine SpA TGCs expression of BPGM was concentrated exclusively in the apical cell side, in direct proximity to the maternal circulation. This study suggests a possible involvement of placental BPGM in maternal-fetal oxygen transfer, and in the pathophysiology of FGR. This work was supported by the Weizmann Krenter Foundation and the Weizmann - Ichilov (Tel Aviv Sourasky Medical Center) Collaborative Grant in Biomedical Research, by the Minerva Foundation, by the ISF KillCorona grant 3777/19.

Glutathione Non-Covalent Binding Sites on Hemoglobin and Major Glutathionylation Target betaCys93 Are Conservative among Both Hypoxia-Sensitive and Hypoxia-Tolerant Mammal Species.

Intracellular tripeptide glutathione is an important agent of cell survival under hypoxia. Glutathione covalently binds to SH groups of hemoglobin cysteine residues, protecting them from irreversible oxidation, and changes its affinity to oxygen. Reduced glutathione (GSH) can also form a noncovalent complex with hemoglobin. Previously, we showed that hemoglobin tetramer has four noncovalent binding sites of glutathione GSH molecules inside, two of which are released during hemoglobin transition to deoxy form. In this study, we characterized the conserved cysteine residues and residues of noncovalent glutathione binding sites in the sequences of a number of hypoxia-tolerant and hypoxia-sensitive mammals. The solvent accessibility of all HbA and HbB residues in oxy and deoxy forms was analyzed. The alpha subunit of all species considered was shown to have no conserved cysteines, whereas the beta subunit contains Cys93 residue, which is conserved across species and whose glutathionylation changes the affinity of hemoglobin for oxygen 5-6-fold. It was found that the key residues of noncovalent glutathione binding sites in both alpha and beta subunits are absolutely conserved in all species considered, suggesting a common mechanism of hemoglobin redox regulation for both hypoxia-sensitive and hypoxia-tolerant mammals.

Pyruvate kinase activators: targeting red cell metabolism in sickle cell disease.

Hemoglobin S (HbS) polymerization, red blood cell (RBC) sickling, chronic anemia, and vaso-occlusion are core to sickle cell disease (SCD) pathophysiology. Pyruvate kinase (PK) activators are a novel class of drugs that target RBC metabolism by reducing the buildup of the glycolytic intermediate 2,3-diphosphoglycerate (2,3-DPG) and increasing production of adenosine triphosphate (ATP). Lower 2,3-DPG level is associated with an increase in oxygen affinity and reduction in HbS polymerization, while increased RBC ATP may improve RBC membrane integrity and survival. There are currently 3 PK activators in clinical development for SCD: mitapivat (AG-348), etavopivat (FT-4202), and the second-generation molecule AG-946. Preclinical and clinical data from these 3 molecules demonstrate the ability of PK activators to lower 2,3-DPG levels and increase ATP levels in animal models and patients with SCD, as well as influence a number of potential pathways in SCD, including hemoglobin oxygen affinity, RBC sickling, RBC deformability, RBC hydration, inflammation, oxidative stress, hypercoagulability, and adhesion. Furthermore, early-phase clinical trials of mitapivat and etavopivat have demonstrated the safety and tolerability of PK activators in patients with SCD, and phase 2/3 trials for both drugs are ongoing. Additional considerations for this novel therapeutic approach include the balance between increasing hemoglobin oxygen affinity and tissue oxygen delivery, the cost and accessibility of these drugs, and the potential of multimodal therapy with existing and novel therapies targeting different disease mechanisms in SCD.

Hemoglobin Affinity to Oxygen during Coronavirus Infection: New Faces of a Known Problem

The development of coronavirus infection prevents the transport of oxygen to the tissues through the mechanism of impaired hemoglobin saturation in the damaged lung. The change in the affinity of hemoglobin for oxygen, which is the most important factor in compensating for oxygen deficiency in various pathological conditions, underlies the processes of adaptation to hypoxia. The question of the significance of the shift in the oxyhemoglobin dissociation curve in this pathology is actively discussed, in which its shift to the left in the arterial blood and its shift to the right in the venous blood are noted, which in the conditions of the development of the hypoxic syndrome reflects the a-ctivation of the body’s mechanisms to compensate for oxygen deficiency. The change in its position is not specific for this pathology, but is determined by the severity of oxygen deficiency and the imbalance of the mechanisms of the cardiorespiratory system, as well as the peculiarities of the status of patients and the analyzed blood sample.

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.

Red Blood Cell Deformability and Morphological Dynamic Sickling As a Metric to Monitor Pharmacodynamic Response to Voxelotor

The recent introduction of new therapies, such as hemoglobin (Hb) oxygen affinity modifiers, fetal Hb (HbF) inducers, and gene therapy (replacement of HbS with non-sickling Hb), that affect the Hb polymerization rate for sickle cell disease (SCD), highlight the need for new metrics to assess changes in sickling characteristics as therapeutic response. Primary outcomes in clinical trials often focus on indirect clinical improvement i.e. decrease in vaso-occlusive events (VOC), Hb increase, fatigue and pain score. There is currently a lack of direct biomarkers to assess the pharmacodynamic impact of these therapies. Two methods have been developed to assess downstream Hb polymerization, LoRRca (oxygenscan), a commercial oxygen gradient ektacytometry that measures deformability as a function of oxygen tension (pO 2), and dynamic sickling assay (DSA), an enzymatic oxygen scavenging system that directly assesses morphological sickling. In the present study, we investigate the relationship between red blood cell (RBC) deformability and kinetics of sickling morphology after a treatment with different doses of voxelotor (GBT440, Global Blood Therapeutics), the only FDA approved Hb oxygen modifier. Blood samples from eight SCD patients were collected following the IRB #041718MP2E & IRB #Pro00066220 protocols. RBCs were incubated with voxelotor at five different doses based on sample hematocrit (Hct): 25% (0.32 ± 0.06 mM), 50% (0.65 ± 0.11 mM), 75% (0.97 ± 0.17 mM), 100% (1.29 ± 0.23 mM) and 125% (1.62 ± 0.28 mM), or with an equal volume of DMSO-containing buffer (vehicle). Oxygenscan is performed (LoRRca, RR Mechatronics, The Netherlands) as per manufacturer's instructions. The following parameters are calculated: i) elongation index (EImin), measured at minimum RBC deformability, ii) point of sickling (PoS), the pO 2 at which RBC deformability decreases below 5% of EImax during deoxygenation and iii) ΔEI, the difference between EImin and EImax. DSA uses an enzymatic reaction that allows for tight control of the rate and depth of hypoxia, resulting in RBC morphological changes observed through time-lapse photography. Sickling profiles were constructed to represent: i) morphological points of sickling at 50% of induced fraction (mPoS i@50%), ii) rate of sickling at 50% of induced fraction (rate i@50%) and iii) area under the curve at 15 minutes (AUC 15). Paired t-test analysis of the LoRRca elongation curves show a significant dose response in Elmin (Vehicle: 0.16 ± 0.16; 25%: 0.20 ± 0.16, p=0.0033; 50%: 0.23 ± 0.15, p=0.0001; 75%: 0.27 ± 0.15, p=0.0004; 100%: 0.30 ± 0.14, p=0.0009; 125%: 0.35 ± 0.13, p=0.0015), ΔEl (Vehicle: 0.26 ± 0.11; 25%: 0.22 ± 0.11, p=0.0016; 50%: 0.18 ± 0.11, p=0.0003; 75%: 0.15 ± 0.08, p=0.0002; 100%: 0.11 ± 0.06, p=0.0004; 125%: 0.09 ± 0.07, p=0.0011) and PoS (Figure 1; Vehicle: 35 ± 11 mmHg; 25%: 32 ± 6, p=0.0092; 50%: 28 ± 7, p=0.0038; 75%: 25 ± 6, p<0.0001; 100%: 17 ± 5, p=0.0007; 125%: 14 ± 4, p=0.0015). These findings indicate improvements in RBC deformability under hypoxic conditions when the drug is present. Furthermore, paired t-test of DSA data showed a significant dose response with AUC 15 (Vehicle: 958 ± 104; 25%: 909 ± 105, p=0.0061; 50%: 845 ± 146, p=0.0004; 75%: 768 ± 194, p=0.0014; 100%: 672 ± 179, p<0.0001; 125%: 623 ± 192, p=0.0002), rate i@50% (Vehicle: 0.87 ± 0.26; 25%: 0.77 ± 0.30, p=0.1236; 50%: 0.61 ± 0.29, p=0.0003; 75%: 0.33 ± 0.13, p<0.0001; 100%: 0.28 ± 0.10, p<0.0001; 125%: 0.25 ± 0.09, p=0.0004) and mPoS i@50% (Figure 2; Vehicle: 4.4 ± 0.7 minutes; 25%: 4.7 ± 0.9, p=0.0260; 50%: 5.0 ± 1.2, p=0.0407; 75%: 5.9 ± 1.7, p=0.0082; 100%: 7.0 ± 1.6, p=0.0002; 125%: 7.8 ± 1.8, p=0.0004). These results indicate a delay in morphological sickling due to drug treatment. Based on the results of our study there is a dose-dependent decrease in RBC deformability and in the kinetics of morphologic sickling based of DSA following voxelotor treatment. These two methods have demonstrated a comparable ability to assess downstream therapeutic effects on delaying/inhibiting HbS polymerization. The combination of these two unique properties, RBC deformability and morphology change, can possibly provide a comprehensive approach to describe the cellular level of phenotypes, which may predict SCD severity. With further development these complementary assays have the potential to become important tools for drug development and monitoring response to SCD therapies.

Real-World Outcomes and Digitally Monitored Quality of Life in Voxelotor-Treated Patients with Sickle Cell Disease

Background Voxelotor is a haemoglobin oxygen-affinity modulator that leads to inhibited red blood cell sickling, has demonstrated reduced markers of haemolysis and anaemia improvement, and is one of a small number of novel disease-modifiers approved in the US for sickle cell disease (SCD) treatment. It has marketing authorisation in the UK and is currently undergoing reimbursement appraisal. The modelled impact of voxelotor on health-related quality of life (HRQoL)remains a key issue in current trial evidence. Therefore, the need for real-world data and understanding of patient-reported outcomes and experiences following treatment remains critical. Aims This work sought to characterise the side effects, HRQoL, and wearable-captured sleep and activity metrics of SCD patients in the UK, before and after treatment initiation. Methods Feedback from 19 SCD patients who had received voxelotor was captured through an online survey (Aug 2022-Mar 2023). 15 of these patients were also consented and enrolled within an artificial intelligence (AI) platform of over 600 SCD patients that combined genomic, medical record, real-time wearable biometric, and patient-reported outcomes data into a single platform. This included a mobile phone app for day-to-day QoL questionnaire (EQ-5D-5L) entry and an FDA-cleared smartwatch to capture sleep quality and physical activity. Convenience sampling selected data for a 17-patient (89%) subcohort with available pre- and post-voxelotor initiation EQ-5D-5L scores, either through the platform or follow-up survey snapshots. For each patient, this was analysed up to the date on which they completed the survey. EQ-5D-5L was also analysed in comparison to platform patients who had hydroxyurea (HU) in the past (n=19) or were receiving HU at the time of analysis (n=60), in addition to those with no recorded HU use (n=116). Additional analysis compared pre- and post-initiation activity levels (n=6), 4 of whom also had sleep quality data. Results The 19 surveyed patients had received voxelotor for a mean ± SD (range) of 194 ± 415 (17-1,826) days. 11 (58%) had also received HU and 1 (5%) received crizanlizumab. The mean age was 33 ± 13 (14-52) years, 12 (63%) were male, and 16 (84%) had HbSS. Side effects were reported in 8 (42%) patients, mostly diarrhea (6, 32%), stomach pains (2, 11%), and nausea/vomiting (2, 11%). Comparison of the 17-patient pre- to post-voxelotor cohort showed a statistically significant (p<0.001) increase in pooled EQ-5D-5L scores, from 0.558 ± 0.314 to 0.761 ± 0.262 (Figure 1). Post-initiation EQ-5D-5L for these 17 patients was significantly higher than for those receiving HU at 0.731 ± 0.232 (p=0.023) or who had previously received HU at 0.704 ± 0.256 (p=0.040) (Figure 2). Analysis of pre- and post-initiation wearables data saw increased deep sleep in 50% (2/4) and decreased nightly wakeups in 50% (2/4) of patients. Pooled pre- and post-initiation data showed a significant difference in deep sleep (43% vs. 47%, p=0.011). The mean number of steps and distance increased for 50% (3/6) of patients. While soft activity (Metabolic Equivalent of Task (METs): <3, e.g., sleeping, sitting quietly, slow walk) decreased for 67% (4/6) of patients, moderate activity (MET: 3-6, e.g., fast walk, cleaning, gardening) increased in 67% (4/6) and intense activity (MET: >6, e.g., running, hiking) increased in 60% (3/5). Conclusions Our data present a different lens to the voxelotor treatment experience of patients in the UK, with a side effect profile similar to that of other published studies. We demonstrated that EQ-5D-5L increased by 0.203 pre- to post-initiation, and was 0.030 and 0.057 higher than those receiving HU and those who had previously received HU, respectively, in the voxelotor-treated group. These are in line with the ranges of minimally important differences (0.030-0.098) reported for the EQ-5D-5L in other chronic diseases. As such and despite the limitations of small sample size and variability in pre- to post-initiation changes, a statistically significant improvement in HRQoL was observed after initiating voxelotor, as well as in comparison to HU treatment pathways. Similarly, pre- and post-initiation wearables data demonstrated early positive trends in voxelotor's impacts on sleep and activity using an FDA-cleared device, highlighting a need for further research into these metrics and the impact of SCD-modifying treatment.

РОЛЬ ОЗОНА В АДАПТИВНЫХ ИЗМЕНЕНИЯХ ЭРИТРОЦИТАРНОГО ЗВЕНА

The analysis of literature and our own data on the study of the mechanisms of the physiological action of ozone on the erythrocyte link of the blood was carried out. The present article discusses the effect of ozone on the oxygenbinding capacity of blood and its pro-oxidant-antioxidant balance. The effect of this gas manifests itself in different ways under conditions of changing activity of the system of gas transmitters (nitrogen monoxide and hydrogen sulfide) and at different values of the partial pressure of oxygen and carbon dioxide. The antihypoxic effect of ozone is provided with the participation of gas transmitters that realize their action through intraerythrocytic mechanisms of formation of hemoglobin affinity for oxygen, which justifies its use as a means of improving the adaptive and antihypoxic capabilities of the body.

Le globule rouge drépanocytaire : données fonctionnelles: The sickle red blood cell: functional data

Sickle cell anemia is a genetic disorder that affects hemoglobin leading to the production of an abnormal hemoglobin, called HbS. HbS has the property to polymerize under deoxygenated conditions, causing a mechanical distortion of red blood cells; a phenomenon called sickling. These sickle red blood cells are more fragile and rigid, leading to chronic hemolytic anemia and painful vaso-occlusive crises, as well as chronic vascular complications that can affect many organs. The abnormal functional properties of these sickle red blood cells are responsible for a wide range of clinical expression of the disease. HbS polymerization can be influenced by many factors, such as the hydration state of the red blood cells or the affinity of hemoglobin for oxygen. Moreover, the rheological characteristics of red blood cells, including their deformability and aggregation properties, are associated with specific clinical phenotypes. The pro-inflammatory and pro-oxidant state, as well as the repeated polymerization of HbS, accelerate the senescence of sickle red blood cells, promoting the release of microparticles and contributing to vascular dysfunction. Patients' red blood cells also have molecular characteristics that promote their adhesion to the endothelium and other circulating cells, contributing to the onset of vascular complications. Massive intravascular hemolysis, due to increased erythrocyte fragility, is also responsible for chronic vascular complications. These different alterations are privileged therapeutic targets, leading to the emergence of new specific treatments. © 2023 Société nationale française de médecine interne (SNFMI). Published by Elsevier Masson SAS. All rights reserved.

Targeted modification of furan-2-carboxaldehydes into Michael acceptor analogs yielded long-acting hemoglobin modulators with dual antisickling activities.

Sickle cell disease (SCD) is the most common genetic disorder, affecting millions of people worldwide. Aromatic aldehydes, which increase the oxygen affinity of human hemoglobin to prevent polymerization of sickle hemoglobin and inhibit red blood cell (RBC) sickling, have been the subject of keen interest for the development of effective treatment against SCD. However, the aldehyde functional group metabolic instability has severly hampered their development, except for voxelotor, which was approved in 2019 for SCD treatment. To improve the metabolic stability of aromatic aldehydes, we designed and synthesized novel molecules by incorporating Michael acceptor reactive centers into the previously clinically studied aromatic aldehyde, 5-hydroxymethylfurfural (5-HMF). Eight such derivatives, referred to as MMA compounds were synthesized and studied for their functional and biological activities. Unlike 5-HMF, which forms Schiff-base interaction with αVal1 nitrogen of hemoglobin, the MMA compounds covalently interacted with βCys93, as evidenced by reverse-phase HPLC and disulfide exchange reaction, explaining their RBC sickling inhibitory activities, which at 2 mM and 5 mM, range from 0% to 21% and 9% to 64%, respectively. Additionally, the MMA compounds showed a second mechanism of sickling inhibition (12%-41% and 13%-62% at 2 mM and 5 mM, respectively) by directly destabilizing the sickle hemoglobin polymer. In vitro studies demonstrated sustained pharmacologic activities of the compounds compared to 5-HMF. These findings hold promise for advancing SCD therapeutics.

Effects of 2,3-DPG knockout on SCD phenotype in Townes SCD model mice.

Sickle cell disease (SCD) is a severe, multisystemic hematological disorder that impacts nearly every major organ in adults. The current approved treatments for SCD directly target mutant hemoglobin or address downstream disease pathology. Several compounds targeting reduction of 2,3-DPG by activation of Pyruvate Kinase-R are currently being evaluated in SCD patients. In this study, we genetically engineered a mouse lacking 2,3-DPG on the Townes SCD mouse model background and evaluated the effects of 2,3-DPG loss on disease pathology. Animals lacking 2,3-DPG showed improvements in hematological markers and reductions in RBC sickling relative to native Townes mice, however, minimal difference in organ damage was observed in 2,3-DPG deficient mice compared to native Townes animals. When animals lacking 2,3-DPG were dosed with a compound designed to increase hemoglobin oxygen affinity, oxygen delivery related toxicity was observed.

The dependence of maximum oxygen uptake and utilization (V̇O2 max) on hemoglobin-oxygen affinity and altitude.

Oxygen transport from the lungs to peripheral tissue is dependent on the affinity of hemoglobin for oxygen. Recent experimental data have suggested that the maximum human capacity for oxygen uptake and utilization (V̇O2 max) at sea level and altitude (~3000 m) is sensitive to alterations in hemoglobin-oxygen affinity. However, the effect of such alterations on V̇O2 max at extreme altitudes remains largely unknown due to the rarity of mutations affecting hemoglobin-oxygen affinity. This work uses a mathematical model that couples pulmonary oxygen uptake with systemic oxygen utilization under conditions of high metabolic demand to investigate the effect of hemoglobin-oxygen affinity on V̇O2 max as a function of altitude. The model includes the effects of both diffusive and convective limitations on oxygen transport. Pulmonary oxygen uptake is calculated using a spatially-distributed model that accounts for the effects of hematocrit and hemoglobin-oxygen affinity. Systemic oxygen utilization is calculated assuming Michaelis-Menten kinetics. The pulmonary and systemic model components are solved iteratively to compute predicted arterial and venous oxygen levels. Values of V̇O2 max are predicted for several values of hemoglobin-oxygen affinity and hemoglobin concentration based on data from humans with hemoglobin mutations. The model predicts that increased hemoglobin-oxygen affinity leads to increased V̇O2 max at altitudes above ~4500 m.

Distribution of Oxygen Tension on Microvessels and in Tissue of Rat Brain Cortex at Severe Arterial Hypocapnia

Arterial hypocapnia (AH), induced by voluntary or forced hyperventilation of the lungs, is accompanied by a decrease in cerebral blood flow (due to an increase in the arteriole tone) and an increase in the affinity of hemoglobin for oxygen. As a result, an insufficient oxygen supply to cortical tissue take place and zones with a critically low oxygen tension (pO2) are formed in brain tissue. The distribution of pO2 to cerebral cortex during AH has not been studied enough. The aim of the work was to evaluate the effectiveness of oxygen supply to brain tissue at the level of arterial and venous microvessels at AH. To do this, the following tasks were set: 1) to study the distribution of the pO2 on the arterial and venous microvessels of the rat cerebral cortex; 2) to analyze tissue pO2 profiles near the walls of these microvessels. On anesthetized Wistar rats under conditions of forced hyperventilation (PaCO2 = 17.1 ± 0.7 mm Hg), the distribution of oxygen tension on the wall of pial and radial arterioles with a lumen diameter of 7–70 μm and on the wall of pial and ascending venules with a lumen diameter of 7–300 µm was studied. In tissue, near the wall of cortical arterioles and venules with a lumen diameter of 10–20 μm, tissue pO2 profiles were measured. Measurements of pO2 during spontaneous breathing of the animal with air served as a control. All pO2 measurements were made using platinum polarographic microelectrodes with a tip diameter of 3–5 μm. Visualization of the electrode tip and microvessels was carried out using a LUMAM-K1 microscope with epiobjectives of the contact type. This work presents for the first-time direct measurements of pO2 on the walls of arterioles and venules of the rat cerebral cortex and in tissues at different distances from the walls of these microvessels at AH. It has been shown that AH results in significant decrease in the oxygen supply to cerebral cortex, that is manifested by a significant drop of the pO2’s on venous microvessels and in tissue in the immediate vicinity of the studied microvessels. It has been shown, that the role of arterioles as a direct source of oxygen to brain tissue, is significantly reduced during arterial hypocapnia. Forced hyperventilation results in significant deterioration of oxygen supply to cerebral cortex, despite elevated pO2 values in the systemic arterial blood and in blood of systemic cerebral veins (sagittal sinus).

The Role of Asprosin in the Regulation and Mechanisms of Oxygen Transport in the Blood and the Gas Transmitter System in Men with Different Body Mass Index

The purpose of this study the role of asprosin in the regulation and mechanisms of oxygen transport in the blood and the gas transmitter system in men with different body mass index. The indicators of lipid and carbohydrate metabolism, as well as the oxygen transport function of the blood and the concentration of gas-transmitters nitrogen monoxide and hydrogen sulfide in it, were determined. A higher concentration of asprosin was found in individuals with an increased body mass index and grade I obesity. With an increased content of asprosin, there is a decrease in the main indicators of blood oxygenation and an increase in the affinity of hemoglobin for oxygen. An increase in the concentration of nitrogen monoxide and a decrease in hydrogen sulfide were revealed at a high content of asprosin, which may be important for the formation of the mechanisms of oxygen transport in the blood.

The relationship between smoking dose with oxygen saturation and cardiorespiratory endurance on young adult men

Background: Healthy lifestyle can reduce the risk of disease. One of the unhealthy lifestyle patterns is consuming cigarettes. Smoking can also reduce the affinity of oxygen for hemoglobin so that it can affect oxygen saturation levels in the blood. This study aimed to determine the relationship between smoking dose, oxygen saturation, and cardiorespiratory endurance in adult men.
 Methods: The study design was a cross-sectional study with an adult male population in the Tampaksiring Village, Bali, Indonesia, in which they qualified for the inclusion and exclusion criteria. Adult men were selected by purposive sampling. It was obtained 52 samples. The measurement of smoking dose using the Brinkman index, the measurement of oxygen saturation using oximetry, and the measurement of cardiorespiratory endurance using the Harvard step test. Data were analyzed using computer software with the Spearman rank test to determine the relationship between smoking dose with oxygen saturation and cardiorespiratory endurance in adult men.
 Results: The results showed that from 52 adult men were obtained by analysis result of the relationship between smoking dose and oxygen saturation where p= 0.000 (p<0.05) and the analysis result of the relationship between smoking dose and cardiorespiratory endurance with p= 0.000 (p<0.05).
 Conclusion: There was a significant relationship between smoking dose with oxygen saturation and cardiorespiratory endurance in adult men in Tampaksiring Village, Bali, Indonesia

Tissue Oxygenation Around Capillaries: Effects of Hematocrit and Arteriole Oxygen Condition.

Oxygen transfer in the microvasculature is a complex phenomenon that involves multiple physical and chemical processes and multiple media. Hematocrit, the volume fraction of red blood cells (RBCs) in blood, has direct influences on the blood flow as well as the oxygen supply in the microcirculation. On the one hand, a higher hematocrit means that more RBCs present in capillaries, and thus, more oxygen is available at the source end. On the other hand, the flow resistance increases with hematocrit, and therefore, the RBC motion becomes slower, which in turn reduces the influx of oxygen-rich RBCs entering capillaries. Such double roles of hematocrit have not been investigated adequately. Moreover, the oxygen-hemoglobin dissociation rate depends on the oxygen tension and hemoglobin saturation of the cytoplasm inside RBCs, and the dissociation kinetics exhibits a nonlinear fashion at different oxygen tensions. To understand how these factors and mechanisms interplay in the oxygen transport process, computational modeling and simulations are favorite since we have a good control of the system parameters and also we can access to the detailed information during the transport process. In this study, we conduct numerical simulations for the blood flow and RBC deformation along a capillary and the oxygen transfer from RBCs to the surrounding tissue. Different values for the hematocrit, arteriole oxygen tension, tissue metabolism rate and hemoglobin concentration and affinity are considered, and the simulated spatial and temporal variations of oxygen concentration are analyzed in conjunction with the nonlinear oxygen-hemoglobin reaction kinetics. Our results show that there are two competing mechanisms for the tissue oxygenation response to a hematocrit increases: the favorite effect of the higher RBC density and the negative effect of the slower RBC motion. Moreover, in the low oxygen situations with RBC oxygen tension less than 50mmHg at capillary inlet, the reduced RBC velocity effect dominates, resulting in a decrease in tissue oxygenation at higher hematocrit. On the opposite, for RBC oxygen tension higher than 50mmHg when entering the capillary, a higher hematocrit is beneficial to the tissue oxygenation. More interestingly, the pivoting arteriole oxygen tension at which the two competing mechanisms switch dominance on tissue oxygenation becomes lower for higher oxygen-hemoglobin affinity and lower hemoglobin concentration. This observation has also been analyzed based on the oxygen supply from RBCs and the oxygen-hemoglobin reaction kinetics. The results and discussions presented in this article could be helpful for a better understanding of oxygen transport in microcirculation.

The dependence of maximum oxygen uptake on hemoglobin-oxygen affinity and altitude: a computational modeling approach

Background: Hemoglobin, the primary oxygen-carrying protein in humans, provides the intermediate link between pulmonary oxygen uptake and tissue oxygen consumption. Oxygen transport is greatly influenced by the hemoglobin-oxygen affinity, which is commonly characterized by the metric P50 - defined as the oxygen tension at which 50% of hemoglobin is saturated. Humans with rare hemoglobin mutations causing a low P50 (high hemoglobin-oxygen affinity) have demonstrated remarkable preservation of exercise tolerance at high altitude conditions (~3,000m). However, the influence of a low P50 on V̇O2max at extreme altitudes (>5,500m) remains largely unexamined. To examine the dependence of V̇O2max on P50 and altitude, we developed a computational model of oxygen uptake and utilization. We hypothesized that a low P50 would result in a better maintained V̇O2max at extreme altitudes compared to conditions of normal P50 and high P50 (low hemoglobin-oxygen affinity). Methods: We created a model that couples pulmonary oxygen uptake with systemic oxygen utilization to estimate V̇O2max as a function of P50, hemoglobin concentration, and altitude. Fixed values for cardiac output and tissue oxygen demand for V̇O2max at sea level were assigned in accordance with experimental data. The pulmonary oxygen uptake model assumes a single blood compartment exposed to alveolar gas, from which the arterial oxygen tension may be estimated from venous input. Using the alveolar gas equation, we interpolated respiratory parameters from data obtained during human sojourn to the summit of Everest. The systemic oxygen utilization model uses arterial input parameters along with Michaelis-Menten kinetics to compute oxygen consumption. The Fick principle was used to determine the venous oxygen tension, which was assumed to approximate tissue oxygen tension. From these values, systemic oxygen extraction and V̇O2max were determined as a function of P50, hemoglobin concentration, and altitude. Results: We present the results for several cases of P50 (low, normal, and high) and hemoglobin concentrations as a function of altitude. For a low P50, the model demonstrated a greater arterial oxygen saturation, greater oxygen content, and lower systemic extraction at extreme altitudes compared to values determined for cases of normal and high P50. Additionally, a low P50 led to better maintenance of V̇O2max at ~8,850m (~38% decrease from sea-level V̇O2max) compared to values determined for normal P50 and high P50 (~53% and ~67% decrease from sea-level V̇O2max, respectively, P<0.05). Conclusion: This model demonstrates the importance of P50 in the determination of V̇O2max at various altitudes. At low altitudes, a low P50 does not confer an advantage in terms of oxygen utilization, likely due to diffusive oxygen limitations. However, at high and extreme altitudes, a greater convective oxygen transport associated with a low P50 likely outweighs impairments in oxygen diffusivity. This project was supported by the National Institutes of Health R-35-HL139854. This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Single-FiO2 lung modelling with machine learning: a computer simulation incorporating volumetric capnography

We investigated whether machine learning (ML) analysis of ICU monitoring data incorporating volumetric capnography measurements of mean alveolar PCO2 can partition venous admixture (VenAd) into its shunt and low V/Q components without manipulating the inspired oxygen fraction (FiO2). From a 21-compartment ventilation / perfusion (V/Q) model of pulmonary blood flow we generated blood gas and mean alveolar PCO2 data in simulated scenarios with shunt values from 7.3% to 36.5% and a range of FiO2 settings, indirect calorimetry and cardiac output measurements and acid- base and hemoglobin oxygen affinity conditions. A ‘deep learning’ ML application, trained and validated solely on single FiO2 bedside monitoring data from 14,736 scenarios, then recovered shunt values in 500 test scenarios with true shunt values ‘held back’. ML shunt estimates versus true values (n = 500) produced a linear regression model with slope = 0.987, intercept = -0.001 and R2 = 0.999. Kernel density estimate and error plots confirmed close agreement. With corresponding VenAd values calculated from the same bedside data, low V/Q flow can be reported as VenAd—shunt. ML analysis of blood gas, indirect calorimetry, volumetric capnography and cardiac output measurements can quantify pulmonary oxygenation deficits as percentage shunt flow (V/Q = 0) versus percentage low V/Q flow (V/Q > 0). High fidelity reports are possible from analysis of data collected solely at the operating FiO2.

5610417 IN-VITRO REAL-TIME OBSERVATION OF MODERATE SICKLING KINETICS IN SICKLE ERYTHROCYTES TREATED BY VOXELOTOR UNDER MONOTONIC AND CYCLIC HYPOXIA

Background: Hypoxia-induced sickle hemoglobin (HbS) polymerization in sickle red blood cells (sRBCs) is the root cause of the symptoms and complications in sickle cell disease (SCD). In microcirculation, timing is critical for sRBCs to pass through narrow capillaries before they become sickled under hypoxia and rigid enough to get trapped. Hence, the delay time in cell sickling due to HbS polymerization is likely a key parameter predictive of the SCD severity. Recent studies have also shown the delay time is shortened due to repeated hypoxia cycles. Voxelotor is a known hemoglobin oxygen-affinity modulator, which has been proven to effectively inhibit HbS polymerization and favorably modify SCD outcomes. However, there has been no direct in-situ observation of the dose-dependent anti-sickling effect of voxelotor treatment under monotonic hypoxia or multiple hypoxia cycles. Aims: 1. We will test the anti-sickling effect of voxelotor on sRBCs by measuring their dose-dependent hypoxia-induced sickling kinetics using our microfluidics assay. 2. We will further test the efficacy of voxelotor on the cumulative impairment to sRBCs from repetitive sickling cycles. Methods: Blood samples from patients with homozygous SCD were studied. Sample specimens used for this study are restricted to patients that were not in crisis and patients with transfusions 30 days or longer prior to blood draw. Details about the microfluidic device and related methods can be found elsewhere [1-3]. Single deoxygenation-reoxygenation cycle or multiple deoxygenation-reoxygenation cycles is created by switching between two gas supplies with 2% and 20% O2 at predetermined time intervals [3]. Results: Figure 1A-B shows significant decrease in sickled fraction with the increase of voxelotor modification percentage from 0% to 30%, 60% and 100%, which are consistent among patients taking hydroxyurea (HU ON) or not taking hydroxyurea (HU OFF). Secondly, we have investigated the efficacy of voxelotor (30% modification) on sickling kinetics progression after 20 repeated deoxygenation-reoxygenation cycles (each cycle includes 60s of deoxygenation and 30s of oxygenation). From the measurements from 4 patient samples, we have confirmed that the sickling kinetics of sickle cells (vehicle, 0% modification) become progressively faster with subsequent deoxygenation-oxygenation cycles, which is quantified by the reduced delay time and higher sickled fraction versus deoxygenation time. Figure 1C-D shows that the treatment of voxelotor (30% modification) has significant efficacy in mitigating the progressively accelerated sickling of sRBCs due to repeated deoxygenation-oxygenation cycles in vitro. The sickled fraction measured at t = 20s in the 20th cycle decreases from 53%±9% for vehicle samples (0% modification) to 21%±11% for voxelotor treated samples (30% modification) (p < 0.05). Similarly, the sickled fraction measured at t = 60s in the 20th cycle decreases from 64%±11% (0% modification) to 30%±16% (30% modification) (p < 0.05).Summary-Conclusion: Voxelotor significantly slows down the rate of sickling and reduces the sickled fraction of sRBCs under monotonic hypoxia with dose-dependency. Furthermore, voxelotor is effective for mitigating the cumulative impairment to sRBCs from repetitive sickling events. Funding statement: This study has been supported by Global Blood Therapeutics Inc. Y.Q. and M.D. acknowledge partial support from NIH R01HL158102.