Oxygen Dissociation Research Articles

Data‐driven prediction of room‐temperature density for multicomponent silicate‐based glasses

AbstractDensity is one of the most commonly measured or estimated material properties, especially for glasses and melts that are of significant interest to many fields, including metallurgy, geology, materials science, and sustainable cements. Here, three types of machine learning models (i.e., random forest [RF], artificial neural network [ANN], and Gaussian process regression [GPR]) have been developed to predict the room‐temperature density of glasses in the compositional space of CaO–MgO–Al2O3–SiO2–TiO2–FeO–Fe2O3–Na2O–K2O–MnO (CMASTFNKM), based on ∼2100 data points mined from ∼140 literature studies. The results show that the RF, ANN, and GPR models give accurate predictions of glass density with R2 values, root mean square error (RMSE), and mean absolute percentage error (MAPE) of ∼0.95–0.97, ∼0.03–0.04 g/cm3 and ∼0.63%–0.83%, respectively, for the 15% testing set, which are more accurate compared with empirical density models based on ionic packing ratio (with R2 values, RMSE, and MAPE of ∼0.28–0.91, ∼0.05–0.15 g/cm3, and ∼1.40%–4.61%, respectively). Furthermore, glass density is shown to be a reliable reactivity indicator for a range of CaO–Al2O3–SiO2 (CAS) and volcanic glasses due to its strong correlation (R2 values above ∼0.90) with the average metal–oxygen dissociation energy (a structural descriptor) of these glasses. Analysis of the predicted density–composition relationships from these models (for selected compositional subspaces) suggests that the single‐layer ANN and GPR models exhibit a certain level of transferability (i.e., ability to extrapolate to compositional spaces not [or less] covered in the database) and capture some known features, including the mixed alkaline earth (or alkali) effects.

Dynamic catalysis of sub-nanometer metal clusters in oxygen dissociation

When the catalyst size decreases down to the nanometer or even sub-nanometer scale, their geometric and electronic structures are very different from the bulk materials. Especially, their structural dynamics can greatly affect catalytic performances. Herein, we report a theoretical study on oxygen dissociation on Ag19 cluster, a critical step of ethylene epoxidation, to investigate the effect of structural dynamics on the catalytic activity. Combining ab initio molecular dynamics (AIMD) and free energy calculation, we obtain the free energy profiles under different temperatures and find sudden drops in the reaction free energy and barrier at certain temperature ranges. The reaction entropy change shows a positive peak because of the different melting temperatures between initial and final states. Additionally, we further investigate the oxygen diffusion process on the Ag cluster and compare it with the Au and Cu clusters. We find that weak adsorption of oxygen on the silver cluster leads to its facile diffusion and thus reduces the melting temperature of the cluster, therefore facilitating partial oxidation under mild conditions.

REFINED DATA ON O2 DISSOCIATION RATE MEASURED BYO-ARAS BEHIND SHOCKWAVES

The formation of atomic oxygen in high-temperature gas flows affects both the thermodynamic state of the gas and the kinetics of proceeding chemical processes. During hypersonic and space flights, there is still a significant lack of understanding of the phenomena of flow around high-speed vehicles. One of the main reactions occurring in the bow shock zone is the dissociation of molecular oxygen into O atoms. Experimental studies of the kinetics of O2 dissociation were carried out by various methods; however, the O2 dissociation rate constants in modern combustion mechanisms still differ by orders of magnitude. Therefore, the clarification of these values is a very urgent task. In this work, precision measurements of the rate constant of oxygen dissociation performed by the ARAS (atomic resonance absorption spectroscopy) method in the temperature range of 2600-5000 K behind the reflected shock waves were carried out which made it possible to noticeably refine the previous data. The best fit to the present experimental data is given by the expression k = 1.34 ± 0.4 • 1014 exp(-(53620 ± 2620)/T) cm6 /(mole-s).

The oxygen dissociation curve of blood in COVID-19-An update.

An impressive effect of the infection with SARS-Co-19 is the impairment of oxygen uptake due to lung injury. The reduced oxygen diffusion may potentially be counteracted by an increase in oxygen affinity of hemoglobin. However, hypoxia and anemia associated with COVID-19 usually decrease oxygen affinity due to a rise in [2,3-bisphosphoglycerate]. As such, COVID-19 related changes in the oxygen dissociation curve may be critical for oxygen uptake and supply, but are hard to predict. A Pubmed search lists 14 publications on oxygen affinity in COVID-19. While some investigations show no changes, three large studies found an increased affinity that was related to a good prognosis. Exact causes remain unknown. The cause of the associated anemia in COVID-19 is under discussion. Erythrocytes with structural alterations of membrane and cytoskeleton have been observed, and virus binding to Band 3 and also to ACE2 receptors in erythroblasts has been proposed. COVID-19 presentation is moderate in many subjects suffering from sickle cell disease. A possible explanation is that COVID-19 counteracts the unfavorable large right shift of the oxygen dissociation curve in these patients. Under discussion for therapy are mainly affinity-increasing drugs.

Resveratrol, a New Allosteric Effector of Hemoglobin, Enhances Oxygen Supply Efficiency and Improves Adaption to Acute Severe Hypoxia

Acute altitude hypoxia represents the cause of multiple adverse consequences. Current treatments are limited by side effects. Recent studies have shown the protective effects of resveratrol (RSV), but the mechanism remains unknown. To address this, the effects of RSV on the structure and function of hemoglobin of adult (HbA) were preliminarily analyzed using surface plasmon resonance (SPR) and oxygen dissociation assays (ODA). Molecular docking was conducted to specifically analyze the binding regions between RSV and HbA. The thermal stability was characterized to further validate the authenticity and effect of binding. Changes in the oxygen supply efficiency of HbA and rat RBCs incubated with RSV were detected ex vivo. The effect of RSV on the anti-hypoxic capacity under acute hypoxic conditions in vivo was evaluated. We found that RSV binds to the heme region of HbA following a concentration gradient and affects the structural stability and rate of oxygen release of HbA. RSV enhances the oxygen supply efficiency of HbA and rat RBCs ex vivo. RSV prolongs the tolerance times of mice suffering from acute asphyxia. By enhancing the oxygen supply efficiency, it alleviates the detrimental effects of acute severe hypoxia. In conclusion, RSV binds to HbA and regulates its conformation, which enhances oxygen supply efficiency and improves adaption to acute severe hypoxia.

Investigating the electrochemical performance of Nd1-xSrxCo0.8Fe0.2O3−δ (0 ≤ x ≤ 0.85) as cathodes for intermediate temperature solid oxide fuel cells

Perovskite-type structure oxides with the nominal chemical composition Nd1-xSrxCo0.8Fe0.2O3−δ (0 ≤ x ≤ 0.85) (NSCF) were synthesized by solid-state method to investigate the effect of Sr-doping on the crystal structure and electrochemical performance in the intermediate temperature range of 600 °C–750 °C. The electrical conductivity of the sintered NSCF pellets was found to be in the range of 300–1000 S cm−1. All the NSCF compositions showed a transition from semiconducting to metallic behavior with an increase in temperature. NSCF showed reactivity with the La0.8Sr0.2Ga0.8Mg0.2O3−δ (LSGM) electrolyte. The electrochemical performance was tested by preparing the symmetrical cells with the configuration 70 wt% NSCF +30 wt% LSGM using LSGM electrolyte in the temperature range of 650–800 °C. AC-impedance results showed a decrease in polarization resistance (Rp) of the cathode with increase in Sr-doping due to increase in electrical conductivity. Among the samples studied, composite electrode of Nd0.3Sr0.7Co0.8Fe0.2O3−δ – LSGM showed the lowest area specific resistance (ASR) of 0.1 Ω cm2 at 750 °C in air. It was chosen to investigate the effect of pO2 on the electrochemical performance of the cathode to determine the rate determining step (RDS) in oxygen reduction reaction (ORR). Dissociation of molecular oxygen into oxygen atoms seems to be the RDS in the ORR.

Cobalt-Free Double Perovskite Oxide as a Promising Cathode for Solid Oxide Fuel Cells.

Double perovskite oxide PrBaFe2O5+δ is a potential cathode material for intermediate-temperature solid oxide fuel cells. To improve its electrochemical performance, the trivalent element Ga is investigated to partially replace Fe, forming PrBaFe2-xGaxO5+δ (PBFGx, x = 0.05, 0.1, and 0.15). The doping effects on physicochemical properties and electrochemical properties are analyzed regarding the phase structures, element valence states, amount of oxygen vacancies, content of oxygen species, oxygen surface exchange coefficients (kchem), electrochemical polarization resistance, and single-cell performance. Specifically, PBFG0.1 exhibits improved kchem, such as a 19% improvement from 4.09 × 10-4 to 4.86 × 10-4 cm s-1 at 750 °C, due to the increased concentration of reactive oxygen species and oxygen vacancies. Consequently, the interfacial polarization resistance is decreased by 28% from 0.057 to 0.041 Ω cm2 at 800 °C. The subreaction steps of the oxygen reduction reaction in the PBFG0.1 cathode are further investigated, which suggests that the oxygen dissociation process is greatly enhanced by doping Ga. Meanwhile, doping Ga increases the peak power density of the anode-supported single cell by 36% from 629 to 856 mW cm-2 at 800 °C. The single cell with the PBFG0.1 cathode also exhibits good stability in 100 h of long-term operation at 750 °C.

Kinetics of Oxygen Exchange and N2O Decomposition Reaction over MeOx/CeO2 (Me = Fe, Co, Ni) Catalysts.

MeOx/CeO2 (Me = Fe, Co, Ni) samples were tested in an 18O2 temperature-programmed isotope exchange and N2O decomposition (deN2O). A decrease in the rate of deN2O in the presence of oxygen evidences the competitive adsorption of N2O and O2 on the same sites. A study of isotope oxygen exchange revealed dissociative oxygen adsorption with the subsequent formation of surface oxygen species. The same species, more probably, result from N2O adsorption and the following N2 evolution to the gas phase. We supposed the same mechanism of O2 formation from surface oxygen species in both reactions, including the stages responsible for its mobility. A detailed analysis of the kinetics of isotope exchange has been performed, and the rates of one-atom (RI) and two-atom (RII) types of exchange were evaluated. The rate of the stage characterizing the mobility of surface oxygen was calculated, supposing the same two-step mechanism was relevant for both types of exchange. The effect of oxygen mobility on the kinetics of deN2O was estimated. An analysis of the possible pathways of isotope transfer from MeOx to CeOx showed that direct oxygen exchange on the Me-Ce interface makes a valuable contribution to the rate of this reaction. The principal role of the Me-Ce interface in deN2O was confirmed with independent experiments on FeOx/CeO2 samples with a different iron content.

Catalyzed oxidation of nuclear graphite by simulated fission products Sr, Eu, and I

The influence of three fission products Sr, Eu, and I on the oxidation of IG-110 nuclear graphite was studied in the temperature range of 400 to 1000 °C. Sr and Eu were introduced as chlorides, and I was introduced as NaI. The temperature dependence of both CO2 and CO production during the graphite oxidation measured with mass spectroscopy and infrared spectrometry shows that the introduction of these three compounds to graphite significantly decreases the onset temperature for the oxidation of graphite. Among the three compounds, NaI is the most active towards the oxidation reaction, characterized by a significant decrease of the onset temperature from approximately 650 to 400 °C before and after its introduction to graphite. Separate measurements of CO2 and CO concentration at varying temperatures enable the calculation of the activation energy for the formation of CO2 and CO. The activation energies for the oxidation of pure and fission product-impregnated graphite samples decrease in the following order: standard IG-110 graphite, EuCl3-impregnated IG-110, SrCl2-impregnated IG-110, and NaI-impregnated IG-110. This trend indicates that the three compounds catalyze the oxidation of graphite at temperatures relevant to the operation of high-temperature gas-cooled reactors. Furthermore, it is found that the three compounds can also affect the molar ratio of reaction products CO2 and CO, and the rates of the graphite oxidation. At temperatures higher than about 850 °C, the impregnated samples exhibit lower CO2: CO ratios than the pure graphite. Different from EuCl3 and NaI, the introduction of SrCl2 decreases the graphite oxidation rates at temperatures higher than about 770 °C. Their catalytic mechanism can be understood based on a redox cycle of the intermediate active species, promoting the dissociation of molecular oxygen and transfer to the carbon.

Surface-strain-enhanced oxygen dissociation on gold catalysts.

The excellent low-temperature oxidation performance and stability of nanogold catalysts have attracted significant interest. However, the main active source of the low-temperature oxidation of gold remains to be determined. In situ electron microscopy and mass spectrometry results show that nitrogen is oxidized, and the catalyst surface undergoes reconstruction during the process. Strain analysis of the catalyst surface and first-principles calculations show that the tensile strain of the catalyst surface affects the oxidation performance of gold catalysts by enhancing the adsorption ability and dissociation of O2. The newly formed active oxygen atoms on the gold surface act as active sites in the nitrogen oxidation reaction, significantly enhancing the oxidation ability of gold catalysts. This study provides evidence for the dissociation mechanism of oxygen on the gold surface and new design concepts for improving the oxidation activity of gold catalysts and nitrogen activation.

Triple conducting perovskite Ba0.95La0.05Fe0.8Zn0.2O3−δ as oxygen electrode for reversible protonic ceramic cells

Reversible protonic ceramic cells (R-PCCs) have drawn much attention as energy conversion devices. However, the performance of R-PCCs is limited by the lack of highly active oxygen electrodes under operating conditions. Herein, crystal structure, oxygen non-stoichiometry, and conductivity of a triple-conducting perovskite Ba0.95La0.05Fe0.8Zn0.2O3−δ (BLFZ) are investigated. High oxygen vacancy concentration of BLFZ (δ = 0.575–0.705) is observed at a temperature range of 400–700 °C, as confirmed by combining thermo-gravimetric and iodometric titration results. The electrochemical reaction process of BLFZ-BaZr0.1Ce0.7Y0.1Yb0.1O3−δ (BZCYYb) electrode for symmetrical cells is elucidated by the distribution relaxation time (DRT) analysis. Oxygen dissociation is the rate-limiting step for protonic ceramic fuel cell (PCFC). At 650 °C, a single cell with BLFZ-BZCYYb oxygen electrode demonstrates the current density of 1070 mA cm−2 at 1.5 V in protonic ceramic electrolysis cell (PCEC) mode under wet H2 and 10% H2O-air atmospheres, achieving no apparent degradation of a cycling test for 200 h in dual modes of PCFC and PCEC. This research suggests that the triple-conducting perovskite BLFZ is a promising candidate for R-PCCs electrodes.

Investigation of in Situ Co-assembled Sr(Co,Zr)O3−δ-Based Perovskite Nanocomposite Cathode for Intermediate-Temperature Solid Oxide Fuel Cells

Exploring cathode materials with fast oxygen reduction reaction (ORR) kinetics is crucial to intermediate-temperature solid oxide fuel cells (SOFCs). Herein, a highly active perovskite nanocomposite with a nominal composition of SrCo0.8Zr0.2O3−δ is developed. The two perovskite phases of cubic SrZr0.8Co0.2O3−δ (SZC, Pm3̅m, 30 wt %) and hexagonal SrCo0.9Zr0.1O3−δ (SCZ, R3̅cH, 70 wt %) can be in situ co-assembled at the nanoscale for well-connected nanoparticles (50–100 nm) and congenital compatibility. The SZC–SCZ nanocomposite can exhibit the active surface oxygen species and a matchable thermal expansion coefficient (15.3 × 10–6 K–1) with Ce0.8Gd0.2O1.9 (13.5 × 10–6 K–1) electrolyte. When applied to the anode-supported SOFC, SZC–SCZ nanocomposite cathode cell shows superior power densities of 1.48–0.41 W cm–2 at 750–550 °C to single cubic SZC cathode cell (0.24–0.06 W cm–2) and typical hexagonal SrCoO3−δ cathode cell (1.25–0.32 W cm–2). The distribution of relaxation times analysis reveals that the oxygen adsorption and dissociation process of SZC–SCZ nanocomposite cathode is significantly enhanced, of which the polarization resistances (0.07–0.22 Ω cm2) are 0.70–0.43 times those of SrCoO3−δ cathode. This benefits from the nanostructure for an extensive heterointerface and surface, and the synergistic interaction of components can contribute to enhancing oxygen catalysis. Thus, this work explores a promising SZC–SCZ nanocomposite cathode for enhancing ORR activity and cell performance at lower operating temperatures.

Boosting full-spectrum light driven surface lattice oxygen activation of ZnMn2O4 by facet engineering for highly efficient photothermal mineralization of toluene

To improve the catalytic activity of photothermal catalysts for efficient mineralization of volatile organic compounds still keeps challenge. Herein, ZnMn2O4 with controllably exposed {010} facets (ZMO-H), {11−1} facets (ZMO-R), and {1−1−1} facets (ZMO-C) were firstly synthesized and used to unveil an essential role of facet-dependent activation of surface lattice oxygen for toluene mineralization under full-spectrum light irradiation. The experimental studies and theoretical calculations together evidenced that as compared to ZMO-R and ZMO-C, ZMO-H is favorable for activating surface lattice oxygen, owing to its relatively stronger light absorption, higher surface lattice oxygen content, and lower oxygen dissociation energy. The theoretical calculations further confirmed that toluene is more easily adsorbed by ZMO-H than by ZMO-R and ZMO-C. All these advantages have substantially afforded the toluene photothermal oxidation activity of ZMO-H. This finding confirms that surface lattice oxygen activation of catalyst by facet engineering is crucial on the photothermal oxidation of toluene.

Photocatalytic Oxidation of Carbon Monoxide Using Synergy of Redox-Separated Photocatalyst and Ozone

Separating the redox centers of photocatalysts is the most promising strategy to enhance photocatalytic oxidation efficiency. Herein, I investigate a site-selective loading of Pt on facet-engineered TiO2 to achieve carbon monoxide (CO) oxidation at room temperature. Spatially loaded Pt on {101} facets of TiO2 attracts photoinduced electrons efficiently. Thereby, oxygen dissociation is facilitated on the Pt surface, which is confirmed by enhanced oxidation of CO by 2.4 times compared to the benchmark of Pt/TiO2. The remaining holes on TiO2 can be utilized for the oxidation of various gaseous pollutants. Specifically, gaseous ozone, which is present in indoor and ambient air, is converted to a hydroxyl radical by reacting with the hole; thus, the poisoned Pt surface is continuously cleaned during the CO oxidation, as confirmed by in situ diffuse reflectance infrared transform spectroscopy. While randomly loaded Pt can act as recombination center, reducing photocatalytic activity, redox-separated photocatalyst enhances charge separation, boosting CO oxidation and catalyst regeneration via simultaneous ozone decomposition.

Oxygen dissociation on the C3N monolayer: A first-principles study

The oxygen dissociation and the oxidized structure on the pristine C3N monolayer in exposure to air are the inevitably critical issues for the C3N engineering and surface functionalization yet have not been revealed in detail. Using the first-principles calculations, we have systematically investigated the possible O2 adsorption sites, various O2 dissociation pathways and the oxidized structures. It is demonstrated that the pristine C3N monolayer shows more O2 physisorption sites and exhibits stronger O2 adsorption than the pristine graphene. Among various dissociation pathways, the most preferable one is a two-step process involving an intermediate state with the chemisorbed O2 and the barrier is lower than that on the pristine graphene, indicating that the pristine C3N monolayer is more susceptible to oxidation than the pristine graphene. Furthermore, we found that the most stable oxidized structure is not produced by the most preferable dissociation pathway but generated from a direct dissociation process. These results can be generalized into a wide range of temperatures and pressures using ab initio atomistic thermodynamics. Our findings deepen the understanding of the chemical stability of 2D crystalline carbon nitrides under ambient conditions, and could provide insights into the tailoring of the surface chemical structures via doping and oxidation.

Designing Reactive Bridging O2- at the Atomic Cu-O-Fe Site for Selective NH3 Oxidation.

Surface oxidation chemistry involves the formation and breaking of metal-oxygen (M-O) bonds. Ideally, the M-O bonding strength determines the rate of oxygen absorption and dissociation. Here, we design reactive bridging O2- species within the atomic Cu-O-Fe site to accelerate such oxidation chemistry. Using in situ X-ray absorption spectroscopy at the O K-edge and density functional theory calculations, it is found that such bridging O2- has a lower antibonding orbital energy and thus weaker Cu-O/Fe-O strength. In selective NH3 oxidation, the weak Cu-O/Fe-O bond enables fast Cu redox for NH3 conversion and direct NO adsorption via Cu-O-NO to promote N-N coupling toward N2. As a result, 99% N2 selectivity at 100% conversion is achieved at 573 K, exceeding most of the reported results. This result suggests the importance to design, determine, and utilize the unique features of bridging O2- in catalysis.

Preparation and performance of a nano-honeycomb cathode for microtubular solid oxide fuel cells

The effect of a nano-honeycomb cathode on the performance of a microtubular solid oxide fuel cell is investigated. We successfully prepared nano-honeycomb cathodes with high unsealing pore porosity (∼64.6%) and high structural strength by freeze casting, which improved the adsorption and dissociation of oxygen. We added gadolinium doped ceria (GDC) nanopowder to the lanthanum strontium cobalt ferrite (LSCF) cathode material. The cell performance of the nano-cell structure of the GDC-LSCF cathode is significantly improved compared to a traditional GDC-LSCF cathode with a spongy porous structure. At 750 °C, the current density is 1450 mA cm−2 and the power density is 475 mW cm−2, which is better than that of conventional cathode structures. We discussed the effects of the honeycomb structure on the cell, including the migration of silver paste as a cathodic collector to the GDC-LSCF interface and the improvement of the activity of the oxygen electrodes.

Promotional effect of nitrogen-doped and pore structure for the direct synthesis of hydrogen peroxide from hydrogen and oxygen by Pd/C catalyst at ambient pressure

Nitrogen-doped porous carbon is potential support for directly synthesizing H2O2 from H2 and O2. Here, density functional theory (DFT) was used to study the effect of N-doped porous carbon on H2O2 directly synthesized. The theoretical calculation results showed that N-doped improved H2O2 productivity and H2 conversion by increasing the dispersion of Pd nanoparticles and the Pd0/Pd2+ ratio. However, N-doped decreased H2O2 selectivity by reducing oxygen's dissociation energies. The experimental results showed that adjusting the pore structure of N-doped porous carbon could improve the adverse effects of N-doping for H2O2 selectivity. The H2O2 productivity and selectivity of Pd/C catalyst with a macropore-mesoporous-microporous hierarchical porous structure were up to 328.4 molH2O2·kgcat-1·h−1 and 71.9 %, respectively, at ambient pressure. The macropore structure enhances the transfer and diffusion performance of the catalyst and effectively inhibits the effect of N-doping on OO bond dissociation, which improves H2O2 productivity and selectivity. This research provides a possible solution for designing a high-performance Pd/C catalyst to directly synthesize H2O2 from H2 and O2 at ambient pressure.

Pklr Variants Associated with Acute Pain in Sickle Cell Disease Influence ATP Concentrations in Red Blood Cells

Background: We previously demonstrated association of PKLR intron 2 variants with hospitalization rate for acute pain in people with sickle cell disease (SCD) and utilized allele-specific expression analysis to show that these variants influence PKLR activity (Wang et al, 2022). Pyruvate kinase (gene PKLR, protein PKR) is a rate-limiting enzyme in glycolysis. Reduced PKR activity leads to an increase in intracellular 2,3-diphosphoglycerate (2,3-DPG), and a concomitant decrease of adenosine triphosphate (ATP), factors that promote sickle hemoglobin (HbS) polymerization and red blood cell (RBC) sickling. On this basis, we explored how the "high risk" PKLR intron 2 variants affect levels of PKR protein expression, 2,3-DPG, ATP, oxygen dissociation (p50) and sickling kinetics (t50). Methods: We studied a total of 416 subjects which included 125 HbSS, 89 HbAS, and 202 ethnic-matched healthy controls (HbAA) enrolled under protocol NCT03685721 approved by the NHLBI Institutional Review Board. DNA was genome scanned using Ilumina's Infinium "MEGA" chip (1.7m markers). The results were quality controlled followed by genotype imputation based on the 1000 Genomes Project phase 3 data using the Michigan Imputation Server. Whole blood levels of ATP and 2,3-DPG were measured using LC-MS/MS with LLOQ at 50.0 μg/mL and converted to intracellular concentrations by dividing by the hematocrit (as a fraction). PKR was detected by antibody-based capture and detection using an electrochemiluminescence immunoassay (Meso Scale Discovery, Rockville, MD, USA) and the signal normalized to a control sample. A Hemox Analyzer [TCS, Scientific Corp, New Hope, PA] was used to obtain p50 values (defined as the partial pressure of oxygen at which Hb is 50% saturated with oxygen). t50 is single parameter measure of sickling kinetics (Dunkelberger et al, 2018). Single variant analyses (under additive genetic model adjusted for age and sex) were conducted in each of the Hb sub-populations for associations between the 7 PKLR variants (intron 4 - rs2071053, and intron 2 -rs8177970, rs116244351, rs114455416, rs12741350, rs3020781, and rs8177964) and the 5 parameters (PKR, 2,3-DPG, ATP, p50 and t50). False-discovery rate (FDR) adjusted p-values were calculated to account for the multiple testing issue. Results: Table 1A shows levels of the 5 parameters (ATP/HCT, 2,3-DPG/HCT, PKR, p50 and t50) in the 3 Hb genotypic groups (HbAA, HbAS and HbSS). Because patients with SCD have variable anemia, ATP and 2,3-DPG were normalized against HCT. The observed trending higher ATP / HCT and significantly elevated 2,3-DPG /HCT in HbSS compared to HbAS and to HbAA are consistent with previous reports (Poillon et al, 1990). HbSS subjects also had higher PKR protein expression, as well as right-shifted p50 values, compared to HbAS and HbAA (Table 1A), which suggest HbSS RBCs have lower specific activity of PKR. A one-way ANOVA showed that the differences in these mean values were highly significant (Table 1A). All 7 PKLR variants were associated with ATP levels in the HbAS group, of which 4 (rs8177970, rs116244351, rs114455416 and rs8177964) in intron 2 remained statistically significant after multiple testing adjustment (Table 1B). We also identified association between 3 variants (intron 2 - rs12741350, rs3020781, intron 4 - rs071053) and 2,3-DPG levels in the HbSS, as well as t50 in the HbAS group. Conclusion: ATP is essential for maintaining RBC membrane integrity and shape; reduced ATP dehydrates RBCs and promotes sickling. In a previous report, a PKLR intron 2 'high-risk" haplotype was implicated in acute sickle pain by affecting PKLR expression (Wang et al, 2022). In this follow-up study, we show that the same intron 2 "high-risk" haplotype negatively affects ATP levels when accounting for PKR expression, thus providing a biological basis for PKLR as a genetic modifier of SCD pain presentation. 3 of the 7 variants are also associated with elevated 2,3-DPG levels in HbSS as well as t50 (a single parameter that depends primarily on sickling kinetics) in HbAS. The variant associations are observed more clearly in HbAS but not in HbSS, possibly because of the much more homogeneous cell population in HbAS. We conclude that PKLR intron variants may directly contribute to the severity and frequency of acute pain episodes in SCD, thus additional studies with larger sample sizes are warranted. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Dynamic Sickling Assay: Using Enzymatically Induced Hypoxia to Assess Real-Time Sickling Scalable from Large Erythrocyte Populations to Individual Cells

Background: There is a pressing need for assays to assess both patient condition and to monitor the progress of Sickle Cell Disease (SCD) therapies. It would be beneficial for an assay to allow assessment of oxygen dissociation, hemoglobin polymerization and Red Blood Cell (RBc) sickling under both equilibrium (mimicking longer-term effects) and non-equilibrium (simulating in-vivo process) conditions. Also desirable is the CLIA-waived system suitable for use in clinical trials and ultimately for clinical management of patients on SCD therapies including those those targeting Hb oxygen affinity. Our proposed work introduces an RBC hypoxia-induced sickling assay as a clinical tool to assess SCD patient condition and to assist in development, monitoring, and optimization of SCD therapies. Methods: The system uses an enzymatic oxygen scrubbing system allowing for a wide range of deoxygenation rates and final hypoxia severity. Diluted blood samples were supplemented with Protocatechuic acid (PCA) and then with Protocatechuate 3,4-Dioxygenase (PCD) enzyme inducing hypoxia at a predetermined rate. The sample was injected into an anaerobic microfluidic chamber with the resulting Red Blood Cell (RBC) morphological changes observed through time-lapsed photography. Image analysis allows for identification and quantification of sickled RBC linked to different polymerization scenarios/mechanisms. An oxygen probe was used to track the rate of medium deoxygenation. Sickling profiles were constructed to represent a percent of sickled RBC in a total cell population measured every two seconds after the enzymatic reaction is initiated. Profiles were assessed though i) percent sickling at normoxia, ii) morphological Point of Sickling (mPoS) and iii) time delay of sickling both measured at 5% increase in sickling over the normoxia sickling baseline, iv rate of sickling, v) size of the sickling fraction and vi) Area under the Curve (AUC). The last four parameters are measured for each sickling fraction if more than one such is present. Results: In control experiments PCA/PCD system was shown to allow reliable and reproducible sample deoxygenation currently enabled in a range from seconds to hours. Comprehensive multidimensional profiles were constructed presenting fraction of sickled RBC as a function of a) time from enzymatic reaction initiation, and b) one or more of rate of oxygen consumption, end of reaction hypoxia level, or oxygen tension in the medium. Such profiling allows for fast optimization of assay parameters to target assay conditions most suitable for detection of desired effects.Significant changes in sickling profiles, with marked delay in sickling, were observed upon treatment of RBC with hemoglobin-modifying drugs like voxelotor.Sickling profiles of Sickle Trait (SCT) patients showed significant delay in sickling compared to SS-type SCD patients. The method shows the potential to differentiate SCT patients based on the risk of adverse conditions to occur due to the patient's trait status.By stopping the flow, the system allows tracking of morphological status of individual cells over the full course of medium deoxygenation. Individual cells show pronounced differences in their propensity to sickle, potentially correlated to individual cell Hb F content. Change in morphology of sickling cells from biconcave disk to crescent and then to holly leaf and granular forms had been observed upon progressively decreasing medium oxygen tension.RBC microparticle formation can be observed and tracked over time as an additional metric of erythrocyte response to developing hypoxia Conclusions: By using enzymatically induced hypoxia in a microfluidic environment, presented system allows comprehensive analysis of RBC sickling dynamics in a wide range of deoxygenation rates and conditions. It would find utility in SCD therapy development by offering a robust and high throughput method for assessing critical RBC response to therapies. With no need for cumbersome gas cylinders and low training requirements, a stand-alone system with single use disposable cartridges would also have utility in clinical trials both in the US and abroad, due to simplicity of use suitable for developing countries with limited infrastructure. When validated, the system can be adapted for Point of Care use either as complementary to other blood tests or in an independent monitoring mode.