The measurement of mitochondrial aerobic metabolism from blood cells has gained in popularity over the past decade due to its low invasiveness.1,2 In non-mammalian vertebrates, red blood cells are nucleated and possess functional mitochondria,3 which enables the assessment of mitochondrial respiration from small blood samples (as low as 20 μL of whole blood4). Recently, we have shown that human mature sickle red blood cells retain some functional mitochondria, which was associated with increased sickling tendency, hemolysis and oxidative stress.5 Willis et al.6 recently questioned the methodology used to demonstrate the functionality of the mitochondria retained in sickle red blood cells, since hemoglobin (Hb)-O2 dissociation could influence the oxygen consumption rate (JO2) measured with high-resolution respirometry.
As rightly noted by Willis et al.,6 the oxygen tension (PO2) within the in vitro chamber declines over time due to oxygen consumption by the cells. Such a decline in PO2 leads to the potential release of O2 by hemoglobin, to an extent depending mostly on: i) the change in PO2, ii) the HbO2 binding parameters, and iii) the amount of Hb in the chamber. Willis et al.6 state that because the PO2 is progressively falling, the errors will confound not only the absolute rates but also the relative differences between respiratory states (i.e., the proof of mitochondrial functionality we used in5). Such a statement assumes that O2 dissociation from Hb will be higher at lower PO2. However, using the same protocol as in a previous study on avian red blood cells7 Figure 1 shows that it would not be the case since non-mitochondrial JO2 (after antimycin A addition) does not vary over a broad range of PO2 (see also Online Supplementary Material (ESM) S1 for raw data). At low PO2, O2 release from Hb could have been anticipated as assumed by Willis et al.,6 which would have led to decreased or even negative JO2 (i.e., O2 release >O2 consumed).
Actually, the release of O2 from Hb will not directly depend on the PO2, but on the instantaneous rate of change in PO2 linked to oxygen consumption (i.e., JO2 in a given state), and on the position in terms of absolute PO2 on the Hb-O2 dissociation curve. As supposed by Willis et al.6 based on Stier et al.1 and Esperti et al.5, our measurements on both avian species and human were conducted at high PO2 (Figure 2A, see raw data in ESM S2), enabling to remain within the linear and almost flat portion of the Hb-O2 dissociation curve (Figure 2B). Extracting the data from Abdu et al. (2008)8 and Powell (2015)9 for human and birds (Figure 2B; ESM S3), respectively, enabled us to calculate the change in Hb saturation (%) linked to JO2-induced changes in PO2, and the associated release of O2 for each respiratory state (see ESM S4 and S5 for calculations). Based on these calculations, we can note that the release of O2 is relatively minor (difference between JO2 and corrected JO2 in Figure 2C, D). While absolute respiration rates are affected to a minor extent (Figure 2C, D), the relative differences between respiratory states are not, as demonstrated for instance by the similarity between OXPHOS coupling efficiencies calculated from raw versus corrected JO2 values (i.e., for Japanese quail: raw =0.731±0.013 vs. corrected =0.730±0.013; for human sickle red blood cells: raw =0.437±0.065 vs. corrected =0.424±0.061). Since O2 release is proportional to JO2 and influenced by Hb content (that does not vary between the different respiratory states), it is not surprising from our perspective that the relative differences between respiratory states are not influenced by O2 release from Hb, as long as the assay is conducted within the linear and almost flat part of the Hb-O2 dissociation curve (Figure 2B). As rightly pointed out by Willis et al.,6 issues can arise when two groups have different Hb contents and/or O2 binding kinetics. This is however unlikely to confound the results presented in Esperti et al.5 because O2 binding kinetics does not vary between healthy and sickle red blood cells in normoxic conditions8 (>92.5% saturation, PO2 >65 mm Hg), and Hb content (and thus potential O2 release) is lower in sickle cell patients than healthy individuals.10 Willis et al.6 also rightly questioned the choice of Mir05 as a respiratory medium and the lack of exogenous substrate (i.e., glucose) when assessing mitochondrial respiration of ‘intact’ red blood cells. Respiration of intact blood cells can for instance be conducted in PBS or plasma,11 but our own experience with avian blood cells shows that mitochondria loose functionality along the assay with PBS, as evidenced by a FCCP-induced respiration being lower than the endogenous respiration, which does not happen when using Mir05. Mir05 also enables to first measure the endogenous respiration and then to permeabilize the red blood cells for more detailed investigation.12 From our perspective, refraining from using exogenous substrates enables the measurement of mitochondrial respiration rates being more closely related to the in vivo physiology, where substrates are usually not at saturating levels. Using the subject’s own plasma,11 whenever possible, is likely the best way to obtain the more meaningful information about in vivo mitochondrial metabolism. Regarding the amount of red blood cells used in respirometry assays, Willis et al.6 also question the precision of pipetting packed red blood cells, which we have questioned before (see1). Counting red blood cells is likely the best approach possible (as done in4 ,1 2 ).
Figure 1.Relationship between non-mitochondrial JO2 and PO2 in Japanese quail (Coturnix japonica) red blood cells measured in vitro with high-resolution respirometry. Measurements were conducted according to the methodology described in Stier et al.,7 and the various oxygen tension (PO2) at which non-mitochondrial oxygen consumption rate (JO2) was measured were achieved by letting intact red blood cells consume more or less O2 within the chamber before adding antimycin A. N=2 biological replicates per PO2; mean ± standard deviation.
Figure 2.Relationship between PO2 and JO2 in red blood cells of Japanese quail (blue) and human with sickle cell disease (red). (A) Raw oxygen consumption rate (JO2) measurements and the associated oxygen tension (PO2) during measurement. (B) Hemoglobin (Hb)-oxygen dissociation curves, redrawn from Powell9 and Abdu et al..8 (C) Comparison of raw JO2 and JO2 corrected for O2 release by Hb for Japanese quails. (D) Comparison of raw JO2 and JO2 corrected for O2 release by Hb for human sickle red blood cells. N=8 biological replicates for each species, data from Stier et al.7 and Esperti et al.5 re-analyzed (mean ± standard error).
Around 75% of the patients included in our study5 were under hydroxyurea therapy, and all patients from the mitochondrial respiration experiments received this treatment. Hydroxyurea has recently been shown to promote erythroid differentiation by accelerating maturation processes,13 which may impact the degree of mitochondrial retention into mature red blood cells. To answer this question, a study would be needed to test the degree of mitochondrial retention into mature red blood cells and their functionality before and after hydroxyurea therapy.
In light of the excellent comment by Willis et al.,6 here are some recommendations that should be useful to ensure the best accuracy when measuring mitochondrial respiration from red blood cells: i) provide the range of PO2 at which JO2 measurements are conducted; ii) ensure that measurements are conducted at a PO2 being within a linear and almost flat part of the Hb-O2 dissociation curve; iiii) check for potential bias between experimental groups in terms of Hb content or O2 binding kinetics; iv) measure Hb in individual samples to correct JO2 if deemed necessary based on theoretical expectations (e.g., if Hb content is high and variable between samples) or if a difference exists between groups in terms of Hb content.
Footnotes
- Received August 5, 2024
- Accepted August 8, 2024
Correspondence
Disclosures
No conflicts of interest to disclose.
Funding
Acknowledgments
We are grateful to Willis et al.
References
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