Correlation of Oxygen‐offloading rate from Red Blood Cells with Body Mass

Abstract

The diffusion of O2 across red blood cell (RBC) membranes is critically important for the delivery of O2 to peripheral tissues. Although the traditional view had been that all O2 moves through RBC membranes by dissolving in and diffusing through membrane lipid, our recent work on murine RBCs indicates that most O2 traversing RBC membranes moves via protein channels (bioRxiv. doi:10.1101/2020.08.28.265066). In stopped-flow (SF) kinetics experiments, we use the absorbance spectrum of hemoglobin (Hb) to monitor the rate constant for O2 offloading from Hb (kHbO2). In RBCs from mice genetically deficient in both aquaporin-1 (AQP1) and the Rh complex (mainly RhAG), kHbO2 is 1/3 lower than in RBCs from wild-type (WT) mice. According to mathematical simulations, this 1/3 decrease in kHbO2 corresponds to a ~55% decrease in O2 permeability of RBC membranes (PM,O2). The combination of the double-knockout and pCMBS (nonspecific inhibitor of membrane proteins that is nonetheless excluded from the RBC interior) reduces PM,O2 by ~91%. Control experiments—hematology, RBC morphology, proteomics (abundance of individual proteins), lipidomics (abundance of individual membrane lipids)—rule out other potential explanations for our striking SF data. Our next challenge is to identify the membrane protein(s) responsible for the 91% – 55% = 36% of PM,O2 not accounted for by AQP1 and the Rh complex, as well as to establish the role of membrane lipids. Comparative physiology could be an important tool for achieving these goals. For each RBC sample, we correct the raw kHbO2 data for modest degrees of hemolysis that occur inside the SF reaction chamber. We assess this hemolysis using our novel SF assay, which is based on the release of carbonic anhydrase (CA) enzymes from RBCs. We find that, compared to RBCs from our lab-standard C57BL/6 WT mice, bovine RBCs have a significantly lower hemolysis (~2.5% vs. ~5%, N=3, p <0.01). More interestingly, we find a 71% lower CA activity in bovine RBCs compared to that in mouse RBCs (N=3, p <0.01). This observation could be important as we explore CO2 permeability in the near future. The kHbO2 is 41% lower for bovine RBCs than for RBCs from WT mice (N=3, p <0.01). Hematological analyses of our limited number of bovine samples do not yet show a significant difference from extensive murine samples in either mean corpuscular volume (MCV) or mean corpuscular hemoglobin concentration (MCHC). A further evaluation of hematology, morphology, proteomics, lipidomics, and genomics (for deduced amino-acid sequences of proteins), will provide valuable insight to the contribution of specific membrane proteins and lipids to the relatively low kHbO2 of bovine RBCs. Our comparative analysis would benefit greatly from access to other mammals with a wide range of body mass (e.g., up to elephant size), from which we would use a small amount of blood (<1 ml) in the assays described above.