Page 266 - Haematologica Vol. 110 - January 2025

COMMENT
[O2] (μM or nmol O2.mL-1), along with the medium volume (mL), allows the calculation of JO2 (nmol O2.min-1). Such calculations have been routinely made and reported in a vast literature, which began rapidly expanding shortly after Leland Clark developed his O2 electrode with a cellophane covering in the 1950s.5
However, the presence of an O2 buffering molecule such as Hb in the respiratory chamber complicates the above straightforward and linear relationships in two ways: i) the Hb mass available to bind O2 in each assay must be determined, and ii) the HbO2 binding parameters must be evaluated. Obviously, accounting for the utilization of this O2 pool will involve a non-linear relationship between the electrode signal and O2 concentration, and thereby JO2. We can approximate the Hb-bound O2 by assuming a normal mean corpuscular hemoglobin concentration of 34 g.dL-1 (similar to the SCA patients in Figure 6C1 if the 10-fold unit error is overlooked), and a Hb molecular weight of 67,000 g.mol-1. With these values, the 100 μL of RBC in the assay contains a Hb mass of 0.507 μmol. At the onset of the assay, assuming 1.0 atmosphere barometric pressure, 0.2095 mole fraction of O2 in dry air, and a 37°C assay temperature (thus 47.1 mmHg water vapor pressure at the air:water interface), gives a medium (MiR05) PO2=(760-47.1)x0.2095=149 mmHg at the start of the assay. Thus, the Hb will initially be almost fully saturated, holding 4 O2.Hb-1x0.507 μmol Hb=2,030 nmol O2, which is 5.2 times greater than the soluble O2 in the medium (see above). As JO2 commences early in the assay the medium PO2 will fall, drawing predominantly from the soluble pool. With time and ongoing JO2, however, the continuous fall in PO2 will elicit significant O2 dissociation from Hb, which will dampen changes in PO2 and result in an underestimation of the true JO2 (if O2 bound to Hb is ignored). Because in a given assay the PO2 is progressively falling, the errors will confound not only the absolute rates but also the relative differences between what Stier et al.2 call “routine, oxphos, leak, ETS, and non-mito” O2 consumption. In a comparative study, like that of Esperti et al.1, such errors may be amplified when the two RBC groups have different Hb contents and/ or O2 binding kinetics. In any case, standard polarographic procedures used to measure JO2 by red blood cells must be viewed with extreme caution.
In practice, the mitochondrial respiration measurements may be largely made at high partial pressures of O2 where the oxyhemoglobin dissociation curve is quite flat.6 However, a decrease of even 0.5% in percent saturation would release ≈5.2 nmol of O2, an amount that would be about one-third of the decline in O2 content occurring during “endogenous” mitochondrial function in king penguin RBC; see Figure 2 in Stier et al.2 Of course, the further the PO2 declines during the respiratory measurements, the closer the approach to a steeper portion of the HbO2 dissociation curve. According- ly, we propose that hemoglobin must be accounted for in quantitative measures of mitochondrial respiration in RBC.
We also noted that RBC were suspended in respiratory buffer MiR05 (0.5 mM EGTA, 3 mM MgCl2, 60 mM K-lacto- bionate, 20 mM taurine, 10 mM KH2PO4, 20 mM Hepes, 110 mM sucrose, free fatty acid bovine serum albumin [1 g.L-1], pH 7.1), a solution commonly used for studies of mi- tochondria, which are intracellular organelles.1 Accordingly, we wondered i) whether a respiratory solution more closely resembling plasma might be more appropriate for RBC, and ii) whether glucose should be provided as a fuel.
Further, for quantitative mitochondrial respiratory analyses, it is important to be certain about the volume of RBC used in the assays. In this context, how an exact volume of RBC is assayed is unclear. If packed RBC are transferred, air displacement pipetting is error-laden, and if packed cells are resuspended, the amount of supernatant retained with prior centrifugation is uncertain.
Finally, we saw no mention of any pharmacotherapy for the SCA patients. Would any such treatments have potential ramifications for either the presence or function of mito- chondria in the RBC of this group?
Once again, we acknowledge and appreciate the research of Esperti et al.,1 but we also believe that their methods for mitochondrial respirometry deserve a candid discussion.
Authors
Wayne T. Willis,1 Alexander C. Berry2 and L. Bruce Gladden2
1Department of Medicine, University of Arizona, Tucson, AZ and 2School of Kinesiology, Auburn University, Auburn, AL, USA
Correspondence:
L.B. GLADDEN - gladdlb@auburn.edu
https://doi.org/10.3324/haematol.2024.286135
Received: June 20, 2024. Accepted: July 24, 2024. Early view: August 1, 2024.
©2025 Ferrata Storti Foundation Published under a CC BY-NC license
Disclosures
No conflicts of interest to disclose.
Contributions
LBG conceived the idea. WTW, ACB, and LBG reviewed the literature, and discussed the basic principles. WTW and LBG performed the calculations. LBG wrote the first draft. WTW, ACB, and LBG reviewed and revised all subsequent drafts. WTW, ACB, and LBG read and approved the final version of the manuscript.
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