Modulation of red blood cell oxygen affinity with a novel allosteric modifier of hemoglobin is additive to the Bohr effect

Modulation of red blood cell oxygen affinity with a novel allosteric modifier of hemoglobin is additive to the Bohr effect

Blood Cells, Molecules and Diseases 87 (2021) 102520 Contents lists available at ScienceDirect Blood Cells, Molecules and Diseases journal homepage:...

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Blood Cells, Molecules and Diseases 87 (2021) 102520

Contents lists available at ScienceDirect

Blood Cells, Molecules and Diseases journal homepage:

Short Communication

Modulation of red blood cell oxygen affinity with a novel allosteric modifier of hemoglobin is additive to the Bohr effect Brooke A. Evans a, *, Andrea K. Ansari a, Reed W. Kamyszek a, Michele Salvagno b, John Welsby c, Matthew Fuller d, Ian Welsby e a

Duke University School of Medicine, Durham, NC, United States of America Padova University Hospital, Department of Medicine Anesthesia and Resuscitation Institute DIMED, Padova, Italy Duke University, Durham, NC, United States of America d Department of Biostatistics, Duke University, Durham, NC, United States of America e Department of Anesthesiology, Duke University Medical Center, Durham, NC, United States of America b c



Editor: Mohandas Narla

Purpose: The Bohr effect describes hemoglobin’s affinity for oxygen dependent on solution pH. Within pH range 6.0–8.5, hemoglobin’s oxygen affinity decreases with decreasing pH. This results in increased oxygen delivery to metabolically active, acidic tissues and improved oxygen uptake in basic regions including lung tissue. MyoInositol tripyrophosphate (ITPP) translocates the erythrocyte membrane and allosterically modifies hemoglo­ bin (Hb). We tested the hypothesis that ITPP does not abrogate the Bohr effect. Methods: Experiments were conducted to determine the effect of increasing concentrations of ITPP on P50 with varying pH. We incubated 10 mL red blood cells at 37 ◦ C for 1 h with ITPP concentrations from 0 to 240 mM. The Clark oxygen electrode (Hemox-Analyzer; TCS Scientific, New Hope, PA) determined oxygen affinity of each sample, in triplicate, using buffers pH 6.8, 7.4, and 7.6. A mixed linear regression model with fixed effects for ITPP concentration and pH was used. Results: Increasing ITPP concentration and decreasing pH increased P50 (p < 0.0001 for ITPP concentration, p < 0.0001 for pH). ITPP modulated increased P50 in normal pH (7.4) and acidic condition pH (6.8); with no effect at alkaline pH (7.6). Conclusion: The Bohr effect is conserved, with ITPP augmenting the decreased oxygen affinity seen with tissue acidosis, while not affecting oxygen affinity in conditions similar to a pulmonary microenvironment.

Keywords: ITPP P50 Hemoglobin affinity Bohr effect

1. Introduction Myo-inositol tripyrophosphate (ITPP), a synthetic derivative of myoinositol hexakisphosphate, is an allosteric effector of hemoglobin that readily accumulates in erythrocytes and shifts the P50 curve to the right. The P50 (the oxygen tension corresponding to 50% saturation at a pH (7.4), pCO2 (40 mmHg), and temperature (37 ◦ C) is commonly used to characterize the oxygen affinity of hemoglobin under standard con­ ditions (see above) and thus quantifies the non-acute influences [1]. A shift of the oxygen dissociation curve to the right, that is, to a higher P50 (normal range 24–28 mmHg (3.2–3.7 kPa)) potentially increasing oxy­ gen release to tissues in vivo [2]. The ability to shift P50 in human blood with ITPP could have value as a pharmaceutical additive to transfusable RBCs or as a drug to

improve exercise capacity in heart failure, as seen in animal models [3]. However, the failure of ITPP to maintain a physiological P50 right-shift in the setting of tissue acidosis would limit its physiological benefit and needs to be measured prior to clinical trials in this setting. Therefore, we tested the hypothesis that ITPP produces a dose dependent increase in P50 that persists in acidotic conditions and is additive to the P50 shift produced by the Bohr Effect. 2. Methods The study was granted a waiver of IRB approval by the Duke Uni­ versity Institutional Review Board as no donor data were collected.

* Corresponding author at: Duke University Medical Center, Durham, NC 27710, United States of America. E-mail address: [email protected] (B.A. Evans). Received 7 September 2020; Received in revised form 12 November 2020; Accepted 12 November 2020 Available online 19 November 2020 1079-9796/© 2020 Elsevier Inc. All rights reserved.

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Blood Cells, Molecules and Diseases 87 (2021) 102520

2.1. Blood products

version 3.5.0 ( and SAS, version 9.4 (SAS Institute; Cary, NC).

Leuko reduced donor RBC units (n = 10) were acquired from Duke University Hospital Blood Bank. Each unit was further aliquoted into 5 mL samples. There was a total of 110 samples examined. There were 34 samples at pH 6.8, 35 samples at pH 7.4, 41 samples at pH 7.6.

3. Results The overall effects of ITPP concentration, pH, and interactions were all significant (p < 0.0001 for ITPP concentration, p < 0.0001 for pH, p = 0.0260 for interaction), after the mixed effects linear regression model that was fit with fixed effects for pH and ITPP concentration was modified to include an interaction term, as slopes appeared to be different under pH conditions (Fig. 1). The slope in each pH group represents the change in P50 associated with an increase in ITPP concentration of 10 mM. An increase in ITPP concentration by 10 mM results in a P50 increase of 1.2[0.6,1.8] mmHg in the pH 7.4 group (p < 0.0001), 1.0[0.4,1.6] mmHg in the pH 6.8 group (p = 0.0011). There was no significant relationship observed between ITPP concentration and P50 at a pH of 7.6(p = 0.3589).

2.2. Effect of ITPP concentration on P50 values The first aim of this study was to determine the effect of the con­ centration of ITPP on the P50 value in RBC’s. The concentrations of ITPP were determined from previous literature [2]. The concentrations of ITPP chosen were 0, 30, 60, 90, 120, and 240 millimolar ITPP. For the incubation with Hb, solutions of ITPP and RBC were mixed in the ratio 1:1 vol/vol, either at the same molarities or up to 240 mM of ITPP and measured for P50 shifts immediately. ITPP solutions were incubated at 37 ◦ C for 60 min. The ODC were recorded using the Hemox Analyzer (TCS Scientific, New Hope, PA), as described in Appendix A.

4. Discussion

2.3. Effect of pH on P50 values

In this in-vitro study, we confirmed our hypothesis that ITPP pro­ duces a dose-dependent increase in P50 that persists in acidotic condi­ tions and is additive to the P50 shift produced by the Bohr Effect. This novel determination of the maintenance of the Bohr Effect during ITPP treatment is an essential step prior to translating the use this allosteric modifier of hemoglobin into the clinical setting. Importantly, the effect of ITPP was insignificant in alkalotic environments. This is relevant because cyanosis can be seen with high P50 hemoglobin variants [4,5]. ITPP does not increase P50 in alkalotic conditions should reduce any tendency to impair loading oxygen onto hemoglobin in the lungs [6]. To translate observations of ITPP related P50 increases in vivo, there have been limited animal studies which have observed right shifts in the ODC. Biolo and colleagues also demonstrated that ITPP administration can increase oxygen delivery and exercise capacity in a mouse model, an observation supported by increased activity in a mouse model featuring high P50 Hb variants [3,7]. In normal mice, intraperitoneal adminis­ tration of ITPP (0.5–3 g/kg) caused a dose-related P50 increase of over 30% [3,7]. This study dosed both normal mice and a genetically modi­ fied mouse heart failure model then determined that ITPP increased P50

The second aim of this study was to determine whether the Bohr effect is conserved when using ITPP. Preliminary experiments were conducted to determine the effect of increasing concentrations of ITPP on P50 under normal, alkalotic (hyperventilating lung) and acidic (stressed muscle) conditions. To simulate the Bohr effect, 3 different buffers were used to simulate these different pH environments (pH = 6.8,7.4 and 7.6) using 0.5 M HEPES buffers stored at 4 ◦ C. The buffers were substituted for the 20uL using the standard protocol for the Hemox Analyzer as described in Appendix A. 2.4. Statistical analysis A mixed effects linear regression model was fit for P50, with fixed effects for ITPP concentration, pH, and an interaction term between these two effects, as well as a random effect for sample. The random effect was included to account for repeated measurements generated by replicated runs at each ITPP concentration. Significant values are defined as p < 0.05. All statistical analysis was conducted using R,

Fig. 1. In the absence of ITPP, there is a clear trend of decreasing P50 with increasing pH. At all 3 pH values, P50 increased with higher ITPP concentration. This trend is preserved with increasing ITPP concentration, however the difference between the 6.8 and 7.4 pH groups gets smaller at higher concentrations. Increasing ITPP concentration has little effect on P50 at pH 7.6. 2

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and, more importantly, also increased maximal exercise capacity in normal mice and mice with severe heart failure. Our novel findings in human blood and this animal model support evaluating whether ITPP administration will improve exercise capacity in humans with or without heart failure or promote adequate oxygen delivery when cardiac output or hemoglobin concentration are limited [8].

generated an oxygen dissociation curve (ODC) from which the P50 and Hill coefficient were derived [9]. Each sample was run in triplicate. The Hemox Analyzer determines the ODC by exposing 50 μL of blood or hemolysate to an increasing partial pressure of oxygen and deoxyge­ nating it with nitrogen gas [9]. A Clark oxygen electrode detects the change in oxygen tension, which is recorded on the x-axis of an x-y recorder. The resulting increase in oxyhemoglobin saturation were measured as described above and displayed on the y-axis. For all experiments, blood samples were incubated at 37 ◦ C for 1 h. Fifty uL of whole blood were diluted in 5 uL of 4-(2-hydroxyethyl)-1piperazineethanesulfonic acid (HEPES) buffer [9]. The sample-buffer is drawn into a cuvette and the temperature of the mixture is equilibrated and brought to 37 ◦ C; the sample is then oxygenated to 100% with air. After adjustment of the pO2 value the sample is deoxygenated with ni­ trogen. The P50 value is extrapolated on the x-axis as the point at which O2 saturation is 50%. The time requires for a complete recording is approximately 30 min [9]. Control samples were run in the morning before any data collection.

5. Conclusion The Bohr effect is conserved after the addition of ITPP, suggesting that ITPP may supplement physiological compensatory mechanisms to optimize tissue oxygenation and supporting the need for clinical research using ITPP in patients with tissue acidosis, such as those with cardiac failure, shock or anemia. CRediT authorship contribution statement Brooke Evans: Conceptualization, Methodology, Experimentation, Original Draft Preparation Andrea Ansari Conceptualization, Data curation, Writing- Original draft preparation. Reed Kamyszek: Original Draft Preparation Michele Salvagno Original Draft preparation, Sta­ tistical Graph/Chart creation John Welsby: Data curation, Investigation Matthew Fuller: Statistical Analysis, guidance on sample size Ian Welsby Original conceptualization, draft reviewing and editing.

References [1] J.A. Myburgh, R.K. Webb, L.I.G. Worthley, The P50 is reduced in critically ill patients, Intensive Care Med. 17 (6) (1991) 355–358, BF01716196. [2] Fylaktakidou KC, Lehn Jm Fau - Greferath R, Greferath R Fau - Nicolau C, Nicolau C. Inositol tripyrophosphate: a new membrane permeant allosteric effector of haemoglobin. (0960-894X (Print)). [3] Biolo A, Greferath R Fau - Siwik DA, Siwik Da Fau - Qin F, et al. Enhanced exercise capacity in mice with severe heart failure treated with an allosteric effector of hemoglobin, myo-inositol trispyrophosphate. (1091–6490 (Electronic)). [4] Y. Nagayama, M. Yoshida, T. Kohyama, K. Matsui, Hemoglobin Kansas as a rare cause of cyanosis: a case report and review of the literature, Internal medicine (Tokyo, Japan) 56 (2) (2017) 207–209, internalmedicine.56.7349. [5] Luo HY, Irving I Fau - Prior J, Prior J Fau - Lim E, et al. Hemoglobin Titusville, a low oxygen affinity variant hemoglobin, in a family of Northern European background. (0361–8609 (Print)). [6] Reissmann Kr Fau - Ruth WE, Ruth We Fau - Nomura T, Nomura T. A human hemoglobin with lowered oxygen affinity and impaired heme-heme interactions. (0021–9738 (Print)). [7] Shirasawa T, Izumizaki M Fau - Suzuki Y-i, Suzuki Y Fau - Ishihara A, et al. Oxygen affinity of hemoglobin regulates O2 consumption, metabolism, and physical activity. (0021–9258 (Print)). [8] Srinivasan AJ, Kausch K, Inglut C, et al. Estimation of Achievable Oxygen Consumption Following Transfusion With Rejuvenated Red Blood Cells. (1532–9488 (Electronic)). [9] Guarnone R, Centenara E Fau - Barosi G, Barosi G. Performance characteristics of Hemox-Analyzer for assessment of the hemoglobin dissociation curve. (0390–6078 (Print)).

Declaration of competing interest Funded by Zimmer Biomet. Appendix A Oxygen Affinity Measurement Oxygen affinity of each sample was measured by automated tonometry using a Clark oxygen electrode (Hemox Analyzer, TCS Sci­ entific, New Hope, PA), according to manufacturer’s instructions. Whole blood or RBC (50 μL) was mixed with 5 mL of buffer, 20 μL of 25% bovine serum albumin, and 10 μL of antifoaming agent. Each sample included blood, ITPP, additive A, antifoaming agent and 5 mL of buffer. The mixture was introduced into the cuvette of the Hemox Analyzer and was exposed to variation in oxygen tension (automated tonometry) while changes in oxyhemoglobin saturation were measured by dualwavelength spectrophotometry at 560 nm and 576 nm. This procedure