Page 265 - Haematologica Vol. 110 - January 2025

COMMENT
Does hemoglobin affect measures of mitochondrial respiration in red blood cells? Comment to “Increased retention of functional mitochondria in mature sickle red blood cells is associated with increased sickling tendency, hemolysis and oxidative stress”
We are intrigued by the Connes group’s interesting study of the retention of mitochondria in mature red blood cells (RBC) in 61 patients with sickle cell anemia (SCA).1 Using the sophisticated techniques of Image Stream (Amnis, MK II) and Mitotracker Red CMXRos Dye (Invitrogen) in com- bination with flow cytometry, they reported that a signif- icant percentage of mature sickle cells in these patients retained mitochondria.1 This presence of mitochondria in human erythrocytes is a unique phenomenon, heretofore considered to be a characteristic reserved for red cells in nonmammalian vertebrates (e.g., avians, fish, amphibians, reptiles).2
However, we were particularly fascinated by one of their primary aims; specifically, “to investigate the functionality of these mitochondria”.1 Mitochondrial function was assessed with high-resolution respirometry, which according to the authors, resulted in “detectable mitochondrial oxygen con- sumption in sickle mature RBC, but not in healthy RBC.” The authors concluded that their data showed the presence of functional mitochondria in mature sickle RBC, “which could favor RBC sickling and accelerate RBC senescence, leading to increased cellular fragility and hemolysis”.1 The presence of functional mitochondria in human erythrocytes is an interesting observation with important implications. For example, the Connes group noted that the propensity of RBC to sickle when deoxygenated was greater in the SCA subgroup with a high percentage (13%) of mitochondria re- tained in mature RBC.1 These are provocative findings with great potential for both increased understanding of basic mechanisms and application to practice.
Oxygen consumption rate (JO2) is, of course, a valid indi- cator of mitochondrial function, particularly when flux is modified by agents known to inhibit or accelerate various steps in the oxidative pathway, as was done by the Connes group here.1 However, in such assessments, care must be taken to ensure the validity and accuracy of the JO2 mea- surement itself. The Connes group used intact red cells in their polarographic measurement of RBC mitochondrial JO2,1 basically following the methods of Sjövall et al.3, and Stier et al.2, with Stier being a co-author of this current study. Briefly, 100 μL of packed RBC was added to 1 mL of a potassium-based respiratory buffer (MiR05, see below), and transferred into an Oxygraph-2k high-resolution respi-
rometer (Oroboros Instruments, Innsbruck, Austria) set at 37°C and containing another 1 mL of MiR05. Subsequently, a variety of standard mitochondrial respiratory measures were made, all based on JO2. Of significant concern here is the O2 associated with hemoglobin; i.e., oxyhemoglobin (HbO2), in the red cells incubating in these respiration chambers. This store of O2 was apparently ignored in the authors’ calculation of JO2. The errors introduced by this omission are potentially quite large and variable depending on hemoglobin content, O2 binding parameters (e.g., P50), the oxygen tension (PO2) at which the analysis is being done, and the duration of a given assay. The discussion below will be restricted to a simple presentation of the overall problem. Basic concepts and details related to how the Oroboros respirometer calculates JO2 are clearly and completely described in the user’s manual.
Clark type O2 electrodes such as that used in the Oroboros instrument generate an electrical signal proportional to the oxygen tension, PO2 (mmHg), which, in turn, is proportional to the concentration of dissolved O2 (nmolO2.mL-1 or μM). As stated in Sander:4 “... the amount of dissolved gas is proportional to its partial pressure in the gas phase. The proportionality factor is called Henry’s law constant.” The value of the Henry constant (μmol.mmHg-1); i.e., solubility, depends on temperature and other factors such as ionic strength. Pure water at 37°C exposed to (and in equilibrium with) room air at 1.0 atmosphere pressure contains about 213 μM of dissolved O2 (213 nmolO2.mL-1).4 If instead, MiR05 medium is the solution of interest, the higher ionic strength reduces O2 solubility (“salting out”). In the Oroboros liter- ature this is taken into account by the “FM” factor, which is 0.92 for MiR05. Accordingly, in our example: [O2]=0.92 * 213 μM=196 μM at 1.0 atmosphere and 37°C. This value is roughly similar to the initial [O2] shown in Figure 2 in Stier et al..2 If we now multiply 196 nmol O2.mL-1 by a total medium volume of 2.0 mL, we get 392 nmol O2 in the respiratory chamber when the assay begins. Below we will compare this soluble O2 mass to the O2 mass bound to Hb.
When gas exchange with the environment is prohibited in the Oroboros, O2 consumption decreases the PO2 hence proportionally decreasing the electrode signal. The pro- gressive fall in PO2 across time (mmHg.min-1), linked by the Henry constant (μmol.mmHg-1) to the corresponding fall in
Haematologica | 110 January 2025
257