An impressive effect of the infection with SARS-Co-19 is the impairment of oxygen uptake due to lung injury. The reduced oxygen diffusion may potentially be counteracted by an increase in oxygen affinity of hemoglobin. However, hypoxia and anemia associated with COVID-19 usually decrease oxygen affinity due to a rise in [2,3-bisphosphoglycerate]. As such, COVID-19 related changes in the oxygen dissociation curve may be critical for oxygen uptake and supply, but are hard to predict. A Pubmed search lists 14 publications on oxygen affinity in COVID-19. While some investigations show no changes, three large studies found an increased affinity that was related to a good prognosis. Exact causes remain unknown. The cause of the associated anemia in COVID-19 is under discussion. Erythrocytes with structural alterations of membrane and cytoskeleton have been observed, and virus binding to Band 3 and also to ACE2 receptors in erythroblasts has been proposed. COVID-19 presentation is moderate in many subjects suffering from sickle cell disease. A possible explanation is that COVID-19 counteracts the unfavorable large right shift of the oxygen dissociation curve in these patients. Under discussion for therapy are mainly affinity-increasing drugs.

Letter to the EditorIs there a shift of the oxygen-hemoglobin dissociation curve in COVID-19?Thomas Gille, Lucile Sesé, Eric Aubourg, Jean-François Bernaudin, Jean-Paul Richalet, and Carole PlanèsThomas GilleService de Physiologie et Explorations Fonctionnelles, Hôpital Avicenne, Hôpitaux Universitaires de Paris Seine-Saint-Denis, Assistance Publique - Hôpitaux de Paris, Bobigny, FranceInserm UMR 1272 “Hypoxie et Poumon,” UFR SMBH Léonard de Vinci, Université Sorbonne Paris Nord, Bobigny, France, Lucile SeséService de Physiologie et Explorations Fonctionnelles, Hôpital Avicenne, Hôpitaux Universitaires de Paris Seine-Saint-Denis, Assistance Publique - Hôpitaux de Paris, Bobigny, FranceInserm UMR 1272 “Hypoxie et Poumon,” UFR SMBH Léonard de Vinci, Université Sorbonne Paris Nord, Bobigny, France, Eric AubourgCentre National de la Recherche Scientifique, CEA, Astroparticule et Cosmologie, Université de Paris, Paris, France, Jean-François BernaudinInserm UMR 1272 “Hypoxie et Poumon,” UFR SMBH Léonard de Vinci, Université Sorbonne Paris Nord, Bobigny, FranceFaculté de Médecine, Sorbonne Université, Paris, France, Jean-Paul RichaletInserm UMR 1272 “Hypoxie et Poumon,” UFR SMBH Léonard de Vinci, Université Sorbonne Paris Nord, Bobigny, France, and Carole PlanèsService de Physiologie et Explorations Fonctionnelles, Hôpital Avicenne, Hôpitaux Universitaires de Paris Seine-Saint-Denis, Assistance Publique - Hôpitaux de Paris, Bobigny, FranceInserm UMR 1272 “Hypoxie et Poumon,” UFR SMBH Léonard de Vinci, Université Sorbonne Paris Nord, Bobigny, FrancePublished Online:11 Jan 2022https://doi.org/10.1152/ajplung.00390.2021MoreSectionsPDF (193 KB)Download PDF ToolsExport citationAdd to favoritesGet permissionsTrack citations ShareShare onFacebookTwitterLinkedInEmailWeChat to the editor: We read with great interest the review by Böning et al. (1), which provided insightful perspectives about hemoglobin-oxygen affinity in COVID-19. We recently published a study comparing 253 blood gas samples from 100 patients with COVID-19 with 221 samples from 100 non-COVID-19 controls with homogenous age and sex ratio distribution (2). After standardization for normal conditions (body temperature = 37°C; pH = 7.4; Pco2 = 40 mmHg), we computed P50 in approximated in vivo conditions from samples with hemoglobin saturation <97%, in accordance with the manufacturer’s instructions (3). As this P50 calculation is not as accurate as that used by Böning et al. (1) and Böning and Enciso (4), we also compared standardized measured oxyhemoglobin in each sample with predicted oxyhemoglobin given by the standard oxygen-hemoglobin dissociation curve (ODC), in relation to Po2. Neither method showed any significant difference between COVID-19 and non-COVID-19 groups at any given Po2 and regardless of disease severity (28% of our patients with COVID-19 required intensive care at some point), although we were able to identify a shift of the ODC in two “positive control” groups for abnormal affinity (with carboxyhemoglobin ≥8% and with sickle cell disease).To further discuss the potential role of sex and methemoglobin pointed out by Böning et al. (1), we reanalyzed our data and found that, in the COVID-19 group, median P50 was 26.1 mmHg [25.4–27.1] in men, 26.1 mmHg [25.3 − 27.3] in women under 50 yr, and 25.7 mmHg [24.5 − 26.5] in women 50 yr or older (P = 0.21). As one would expect, our cohort only comprised 8 women aged <50 yr (extreme values: 33–48) and 22 women aged ≥50 yr (51–85). Moreover, median methemoglobin was 1.5% [1.2–1.8] in patients with COVID-19 having received hydroxychloroquine versus 1.1% [1–1.3] in the absence of hydroxychloroquine (P < 0.0001), with a maximal value of 2.5% in a critically ill 67-yr-old man. These results are in line with those recently communicated by Vogel et al. (5). However, when considering the previously reported left shift of the ODC curve (6), it may be hypothesized that it could have been overestimated by overrepresented samples from a few patients in the analyses (i.e., 3,518 samples from only 43 patients), compared with an unmatched historical cohort, or the presence of samples with a too high Po2 to accurately extrapolate P50 (7). The question about a potentially abnormal ODC in COVID-19 remains open, especially as there is conflicting data on possible impaired oxygen transport at a cellular level (8, 9).Finally, we fully agree that the measurement of 2,3-bisphosphoglycerate concentration could be of great interest in patients with COVID-19, who can present with chronic hypoxia, respiratory alkalosis, and anemia. However, in our cohort, we found no significant correlation between hemoglobin concentration and P50 value. Nevertheless, only 24% of them displayed anemia at some point: none of them exhibited obvious hemolysis; the review of blood smears and patient files by a hemobiologist revealed that inflammation was the most common cause. Of course, these results do not preclude that some patients with COVID-19 with more severe anemia, potentially related to SARS-CoV-2 infection of erythroid progenitors (10), may harbor alteration in hemoglobin-oxygen affinity.DISCLOSURESNo conflicts of interest, financial or otherwise, are declared by the authors.AUTHOR CONTRIBUTIONST.G. drafted manuscript; T.G., L.S., E.A., J.-F.B., J.-P.R., and C.P. edited and revised manuscript; T.G., L.S., E.A., J.-F.B., J.-P.R., and C.P. approved final version of manuscript.ACKNOWLEDGMENTSThe authors thank Dr. Amélie Beaugrand for proofreading the manuscript.REFERENCES1. Böning D, Kuebler WM, Bloch W. The oxygen dissociation curve of blood in COVID-19. Am J Physiol Lung Cell Mol Physiol 321: L349–L357, 2021. doi:10.1152/ajplung.00079.2021.Link | ISI | Google Scholar2. Gille T, Sesé L, Aubourg E, Fabre EE, Cymbalista F, Ratnam KC, Valeyre D, Nunes H, Richalet J-P, Planès C. The affinity of hemoglobin for oxygen is not altered during COVID-19. Front Physiol 12: 578708, 2021. doi:10.3389/fphys.2021.578708.Crossref | PubMed | ISI | Google Scholar3. Radiometer Medical ApS. ABL800 FLEX Operator’s Manual. Version 6.10. Copenhagen, Denmark: Radiometer Medical ApS, 2011.Google Scholar4. Böning D, Enciso G. Hemoglobin-oxygen affinity in anemia. Blut 54: 361–368, 1987. doi:10.1007/BF00626019.Crossref | PubMed | Google Scholar5. Vogel DJ, Formenti F, Camporota L. The increased hemoglobin oxygen affinity in COVID-19. Am J Physiol Lung Cell Mol Physiol 321: L637, 2021. doi:10.1152/ajplung.00280.2021.Link | ISI | Google Scholar6. Vogel DJ, Formenti F, Retter AJ, Vasques F, Camporota L. A left shift in the oxyhaemoglobin dissociation curve in patients with severe coronavirus disease 2019 (COVID-19). Br J Haematol 191: 390–393, 2020. doi:10.1111/bjh.17128.Crossref | PubMed | ISI | Google Scholar7. Mairbäurl H, Weber RE. Oxygen transport by hemoglobin. Compr Physiol 2: 1463–1489, 2012. doi:10.1002/cphy.c080113.Crossref | PubMed | ISI | Google Scholar8. Park KC, Donovan K, McKechnie S, Ramamurthy N, Klenerman P, Swietach P. Single-cell oxygen saturation imaging shows that gas exchange by red blood cells is not impaired in COVID-19 patients. Br J Haematol 190: e229–e232, 2020. doi:10.1111/bjh.17025.Crossref | PubMed | ISI | Google Scholar9. Thomas T, Stefanoni D, Dzieciatkowska M, Issaian A, Nemkov T, Hill RC, Francis RO, Hudson KE, Buehler PW, Zimring JC, Hod EA, Hansen KC, Spitalnik SL, D’Alessandro A. Evidence of structural protein damage and membrane lipid remodeling in red blood cells from COVID-19 patients. J Proteome Res 19: 4455–4469, 2020. doi:10.1021/acs.jproteome.0c00606.Crossref | PubMed | ISI | Google Scholar10. Huerga Encabo H, Grey W, Garcia-Albornoz M, Wood H, Ulferts R, Aramburu IV, Kulasekararaj AG, Mufti G, Papayannopoulos V, Beale R, Bonnet D. Human erythroid progenitors are directly infected by SARS-CoV-2: implications for emerging erythropoiesis in severe COVID-19 patients. Stem Cell Reports 16: 428–436, 2021.doi:10.1016/j.stemcr.2021.02.001.Crossref | PubMed | ISI | Google ScholarAUTHOR NOTESCorrespondence: T. Gille (thomas.[email protected]fr). Download PDF Previous Back to Top Next FiguresReferencesRelatedInformationRelated articlesThe oxygen dissociation curve of blood in COVID-19 10 Aug 2021American Journal of Physiology-Lung Cellular and Molecular PhysiologyReply to Gille et al. 11 Jan 2022American Journal of Physiology-Lung Cellular and Molecular PhysiologyCited ByReply to Gille et al.Dieter Böning, Wilhelm Bloch, and Wolfgang M. Kuebler11 January 2022 | American Journal of Physiology-Lung Cellular and Molecular Physiology, Vol. 322, No. 1 More from this issue > Volume 322Issue 1January 2022Pages L174-L175 Crossmark Copyright & PermissionsCopyright © 2022 the American Physiological Society.https://doi.org/10.1152/ajplung.00390.2021PubMed35015569History Received 24 September 2021 Accepted 25 September 2021 Published online 11 January 2022 Published in print 1 January 2022 Keywordsanemiahemoglobin-oxygen affinityhemolysisP50SARS-CoV-2 Metrics Downloaded 797 times

Letter to the EditorReply to Gille et al.Dieter Böning, Wilhelm Bloch, and Wolfgang M. KueblerDieter BöningInstitut für Physiologie, Campus Mitte, Charité – Universitätsmedizin Berlin, Berlin, Germany, Wilhelm BlochInstitut für Kreislaufforschung und Sportmedizin, Deutsche Sporthochschule Köln, Cologne, Germany, and Wolfgang M. KueblerInstitut für Physiologie, Campus Mitte, Charité – Universitätsmedizin Berlin, Berlin, GermanyPublished Online:11 Jan 2022https://doi.org/10.1152/ajplung.00461.2021MoreSectionsPDF (229 KB)Download PDF ToolsExport citationAdd to favoritesGet permissionsTrack citations ShareShare onFacebookTwitterLinkedInEmailWeChat to the editor: We thank Dr. Gille and collaborators for their comment (1) to our review (2). The following statements in their letter have to be discussed: 1) In contrast to the findings of Vogel et al. (3), the in vivo oxygen dissociation curve is not left-shifted in the patients of their recent study (4). 2) There is no marked increase in methemoglobin concentration in their patients as suggested by us for Vogel’s results. 3) Oxygen saturation is calculated as the sum of oxyhemoglobin (O2Hb) and carboxyhemoglobin (COHb).IN VIVO OXYGEN DISSOCIATION CURVEFour topics need to be considered, as follows.Number of MeasurementsThe number of measurements is much smaller in Gille’s than in Vogel’s investigation [19,463 from 43 patients with COVID-19 and 828 critically ill patients with acute respiratory failure (3)]. Gille et al. (4) evaluated 474 samples from 100 patients with COVID-19 and 100 subjects with other respiratory problems. In addition to the affinity difference between the patient groups, a significant decrease of P50 relative to the standard value calculated according to Severinghaus (5, 6) was found in the subjects infected with SARS-CoV-2 by Vogel et al. (3).Severity of IllnessA marked difference between the two studies is notable in terms of hemoglobin concentration: although the mean concentration is within the low normal range (median and interquartile ranges 14.0 [12.6–15.2] g/dL) in the investigation of Gille et al. (4), the patients in the article by Vogel et al. (3) are anemic (COVID-19: 8.1 ± 1.2 SD g/dL; comparison group: 9.4 ± 2.0 SD g/dL).MethodsGille et al. (4) excluded values with saturations above 97% from P50 calculations. Indeed, the variation in PO2 is large while SO2 changes very little in this part of the curve. As also the Bohr effect disappears at high saturation [described, e.g., by Garby et al. (7) and Meier et al. (8)], this approach is in any case reasonable. Severinghaus (6) even suggests using only samples between 20% and 80% SO2 for the P50 calculation. Vogel et al. (3) compare immense numbers of measurements ranging between 20% and 100% SO2 with similar mean values of ∼94% SO2 for COVID-19 and their control group of critically ill patients. Therefore, the outliers in their study are present in both groups and the detected difference should be real. Additionally, pH deviation from 7.4 and its variation are very small (7.382 ± 0.077 SD), therefore possibly false Bohr effect corrections are negligible.ResultsThere is indeed no difference in standardized P50 between patients with COVID-19 and the negative control group of patients who do not have COVID in the overall statistical evaluation presented in Table 1 in Gille’s first paper (4). But when considering Fig. 2 (mean P50 over 18 days in the hospital), there appears a tendency for lower mean values in the patients with COVID-19, especially between days 3 and 15 as compared with the control group (∼2 mmHg difference on days 8–10). The differences are not significant, but this does not prove that they do not exist. It is well possible that only the variability is too large or the number of measurements was too low. In addition, differences in the non-COVID-19 control groups (critically ill patients with acute respiratory failure, in the case of Vogel et al., vs. a mixed population of patients with infections, airway disease, interstitial lung disease, heart failure, and surgical interventions, etc., in Gille’s study) and the varying use of medians and interquartile ranges (Table 1) or mean values ± standard errors (Fig. 1) in contrast to only mean values in the article of Vogel et al. (3) makes the comparison somewhat difficult. In view of the statistical difficulties to prove absence (rather than presence) of an effect, the conclusion in the title of Gille’s first paper (1), “The affinity of hemoglobin for oxygen is not altered during COVID-19,” appears somewhat overstated, in contrast to the question mark punctuating the title of their Letter to the Editor.METHEMOGLOBINThe findings of Gille et al. (4) together with those of Vogel et al. (9) communicated in their letter to the editor indeed exclude MetHb as main factor for a left shift of the in vivo oxygen dissociation curve in COVID-19. We previously already suggested a possible influence of varying nitric oxide (NO)-binding to Hb as an alternative explanation for this finding (10). Interestingly, erythroblasts possess angiotensin-converting enzyme 2 (ACE2) receptors and are therefore potentially vulnerable to COVID-19 infection in cases of viremia (11).CALCULATION OF OXYGEN SATURATION AND P50With respect to methods, it seems puzzling that Gille et al. (4) added COHb to O2Hb for calculation of P50: “s is the combined O2 and CO saturation.” This approach will likely increase the calculated O2 saturation in both patient groups in their study and therefore reduce P50. The authors state that this was done according to Dash et al. (12), yet we were unable to find the corresponding formula in that paper. To our knowledge, this approach is also not used in the calculations by Severinghaus or other authors. Severinghaus’s curve (5, 13) is based on volumetric measurements of oxygen in the samples (therefore the COHb content was unknown) or on photometric measurements at the specific absorption wavelength for O2Hb.COHb is omitted for the calculation of P50 in many investigations according to Kirk et al. (14). They describe that the inclusion of COHb in the saturation term is used in Instrumentation Lab apparatus, but not in American Optical Company oximeters. Vogel et al. calculated P50 only for SO2 (personal communication).As the affinity for carbon monoxide (CO) is 200 times higher than for O2 (15) and influences oxygen affinity, its inclusion in the calculation of P50 appears rather unorthodox. Fortunately, the content of COHb is small in the study of Gille et al. (4) and therefore will not affect their P50 calculation very much.CONCLUSIONSThe controversial findings in the studies by Vogel and Gille and the resulting discussion highlight the need for further definite studies on this topic. In particular, additional measurements of the concentrations of key modulators of oxygen affinity in red cells such as [H+] and 2,3-bisphosphoglycerate (2,3-BPG) are required to understand the mechanisms behind a potential shift of the oxygen dissociation curve in patients with COVID-19.DISCLOSURESNo conflicts of interest, financial or otherwise, are declared by the authors.AUTHOR CONTRIBUTIONSD.B. drafted manuscript; D.B., W.B., and W.M.K. edited and revised manuscript; D.B., W.B., and W.M.K. approved final version of manuscript.REFERENCES1. Gille T, Sesé L, Aubourg E, Bernaudin JF, Richalet JP, Planès C. Is there a shift of the oxygen-hemoglobin dissociation curve in COVID-19? Am J Physiol Lung Cell Mol Physiol. In press. doi:10.1152/ajplung.00390.2021.Link | ISI | Google Scholar2. Böning D, Kuebler WM, Bloch W. The oxygen dissociation curve of blood in COVID-19. Am J Physiol Lung Cell Mol Physiol 321: L349–L357, 2021. doi:10.1152/ajplung.00079.2021.Link | ISI | Google Scholar3. Vogel DJ, Formenti F, Retter AJ, Vasques F, Camporota L. A left shift in the oxyhaemoglobin dissociation curve in patients with severe coronavirus disease 2019 (COVID-19). Br J Haematol 191: 390–393, 2020. doi:10.1111/bjh.17128. Crossref | PubMed | ISI | Google Scholar4. Gille T, Sesé L, Aubourg E, Fabre EE, Cymbalista F, Ratnam KC, Valeyre D, Nunes H, Richalet JP, Planès C. The affinity of hemoglobin for oxygen is not altered during COVID-19. Front Physiol 12: 578708, 2021. doi:10.3389/fphys.2021.578708.Crossref | PubMed | ISI | Google Scholar5. Severinghaus JW. Blood gas calculator. J Appl Physiol 21: 1108–1116, 1966. doi:10.1152/jappl.1966.21.3.1108. Link | ISI | Google Scholar6. Severinghaus JW. Simple, accurate equations for human blood O2 dissociation computations. J Appl Physiol Respir Environ Exerc Physiol 46: 599–602, 1979. doi:10.1152/jappl.1979.46.3.599. Link | ISI | Google Scholar7. Garby L, Robert M, Zaar B. Proton- and carbamino-linked oxygen affinity of normal human blood. Acta Physiol Scand 84: 482–492, 1972. doi:10.1111/j.1748-1716.1972.tb05198.x. Crossref | PubMed | Google Scholar8. Meier U, Böning D, Rubenstein HJ. Oxygenation dependent variations of the Bohr coefficient related to whole blood and erythrocyte pH. Effect of lactic and carbonic acid. Pflügers Arch 349: 203–213, 1974. doi:10.1007/BF00592448. Crossref | PubMed | ISI | Google Scholar9. Vogel DJ, Formenti F, Camporota L. The increased hemoglobin oxygen affinity in COVID-19. Am J Physiol Lung Cell Mol Physiol 321: L637, 2021. doi:10.1152/ajplung.00280.2021.Link | ISI | Google Scholar10. Böning D, Bloch W, Kuebler WM. Reply to Vogel et al. Am J Physiol Lung Cell Mol Physiol 321: L638–L639, 2021. doi:10.1152/ajplung.00327.2021.Link | ISI | Google Scholar11. Huerga Encabo H, Grey W, Garcia-Albornoz M, Wood H, Ulferts R, Aramburu IV, Kulasekararaj AG, Mufti G, Papayannopoulos V, Beale R, Bonnet D. Human erythroid progenitors are directly infected by SARS-CoV-2: implications for emerging erythropoiesis in severe COVID-19 patients. Stem Cell Reports 16: 428–436, 2021. doi:10.1016/j.stemcr.2021.02.001. Crossref | PubMed | ISI | Google Scholar12. Dash RK, Korman B, Bassingthwaighte JB. Simple accurate mathematical models of blood HbO2 and HbCO2 dissociation curves at varied physiological conditions: evaluation and comparison with other models. Eur J Appl Physiol 116: 97–113, 2016. doi:10.1007/s00421-015-3228-3. Crossref | PubMed | ISI | Google Scholar13. Severinghaus JW. Oxyhemoglobin dissociation curve correction for temperature and pH variation in human blood. J Appl Physiol 12: 485–486, 1958. doi:10.1152/jappl.1958.12.3.485. Link | ISI | Google Scholar14. Kirk BW, Raber MB, Duke KR. A simplified method for determining the P50 of blood. J Appl Physiol 38: 1140–1142, 1975. doi:10.1152/jappl.1975.38.6.1140. Link | ISI | Google Scholar15. Mairbäurl H, Weber RE. Oxygen transport by hemoglobin. Compr Physiol 2: 1463–1489, 2012. doi:10.1002/cphy.c080113. Crossref | PubMed | ISI | Google ScholarAUTHOR NOTESCorrespondence: D. Böning (dieter.[email protected]de). Download PDF Previous Back to Top FiguresReferencesRelatedInformationRelated articlesIs there a shift of the oxygen-hemoglobin dissociation curve in COVID-19? 11 Jan 2022American Journal of Physiology-Lung Cellular and Molecular Physiology More from this issue > Volume 322Issue 1January 2022Pages L176-L177 Crossmark Copyright & PermissionsCopyright © 2022 the American Physiological Society.https://doi.org/10.1152/ajplung.00461.2021PubMed35015567History Received 12 November 2021 Accepted 26 November 2021 Published online 11 January 2022 Published in print 1 January 2022 KeywordsCOHbhalf-saturation pressurehemoglobin oxygen affinityin vivo hemoglobin oxygen dissociation curve Metrics Downloaded 358 times

TO THE EDITOR: We thank Dr. Vogel and colleagues for their letter (1) in response to our recent review on the oxygen dissociation curve (ODC) in COVID-19 (2). Indeed, we initially became interested in this topic when reading the paper by Vogel et al. (3), which showed a surprising left shift of the in vivo ODC in severe disease with marked anemia. Other investigators had not detected a significant change of the ODC in COVID-19, but only measured in vitro curves in small groups of patients with less anemia. Since it was previously reported that methemoglobin (MetHb) formation may cause a left shift of the ODC (4, 5), we had contacted Dr. Vogel in January 2021 regarding respective measurements in his patients and learned that these data at that time had not been evaluated yet. In our article finally submitted on April 26, 2021, we thus proposed increased levels of MetHb as a potential cause of the ODC left shift in COVID-19 (2). Dr. Vogel’s letter to the editor (1) now clarifies, however, that MetHb is not the underlying cause for the observed effect in the study by Vogel et al. (3). This does, however, not preclude that MetHb may contribute to a left shift of the ODC in other patients with COVID-19, in particular those treated with drugs that favor MetHb formation such as chloroquine and hydrochloroquine. As the patients of Dr. Vogel were severely anemic, one would in fact even expect a right shift of the ODC due to an adaptive increase in the concentration of 2,3-bisphosphoglycerate ([2,3-BPG]). Anemia, a frequent complication in SARS-CoV-2 infection, usually causes an increase in [2,3-BPG] in most cases except those with acidosis (reviewed in our article). Astonishingly, we could detect only one publication where 2,3-BPG had been measured in patients with COVID-19 (6). The authors found an increase in a slightly anemic group of patients with COVID-19 compared with a similar group without COVID-19. We contacted the corresponding author (Dr. A. D’Alessandro), who communicated that because of methodological reasons the concentrations are only given in arbitrary units, while the actual absolute concentrations in mmol/L red cells remain unknown. That notwithstanding, it seems fair to conclude that [2,3-BPG] should be increased in patients with COVID-19 given that the concentration was higher relative to the non-COVID control group also suffering from slight anemia in the study by D’Alessandro and colleagues (6). We searched for other papers with [2,3-BPG] measurements in the past years until we were informed that the test kits are no longer produced, so that only specialized biochemistry laboratories still determine this substance quantitatively. Yet, only measurements of [2,3-BPG] will clarify with certainty whether the reported left shift of the ODC curve in COVID-19 with accompanying anemia (2) is facilitated by a lacking increase of [2,3-BPG] in these patients, or occurs despite elevated [2,3-BPG] by a so far unclear mechanism. A variety of substances that also influence oxygen affinity (e.g., ATP, Cl, La, glutathione) may play a role and should be studied, too. Finally, the role of nitric oxide (NO) should be reevaluated because its binding mechanism to Hb is similar to MetHb formation at high oxygen saturation in the lungs but changes in tissue capillaries (7).

The paper by Dominelli et al. (2020) is a very interesting publication and we follow Jerome Dempsey's suggestion (Dempsey, 2020) to discuss it. The role of haemoglobin oxygen affinity for oxygen uptake during exercise in normoxia and hypoxia has been investigated and discussed for decades. The importance of the article is to show that a left shift of the oxygen dissociation curve (ODC) of high affinity haemoglobins (HAH) may be beneficial during exercise at moderate altitude (3000 m) not only in animals but also in humans, thus confirming the case report in siblings by Hebbel et al. (1978). Moreover, the surprisingly high arterial oxygen saturation in many Ethiopians (Beall et al. 2002) suggests that high affinity may also be caused by still-unknown effects, because a large number of modified Hb molecules are not known in this population (Cheong et al. 2017). These observations are in contrast to the right shift of the (in vitro!) standard ODC by upregulation of 2,3-biphosphoglycerate concentration usually observed in moderate hypoxia. However, at extreme altitude (above 5000 m) strong hyperventilation overruns this effect, thus hindering too large a decrease of arterial saturation (Samaja et al. 2003; West et al. 2013). A specially important point is not mentioned in the paper discussed here. Oxygen delivery to the tissues is influenced by not only the position of the ODC, measured as half-saturation pressure P50, but also its shape. According to Berglund (1972) and Boyer et al. (1972) Hill's n, i.e. the slope of the ODC in a logarithmic presentation, of the high affinity Hb Malmö is much smaller (1.50–1.58) than that of normal Hb (2.86). The stabilizing effect of the marked S-form of the normal dissociation curve on at low saturation is markedly reduced and therefore the high Hb concentration is especially necessary for a sufficiently high in the tissue capillaries. What about Hill's n in Dominelli's experiments? Was it normal? This essential aspect is not considered. Interestingly, n is increased in South American highlanders (Schmidt et al. 1990), pointing to an important role of this adaptation. Measurements of the ODC have been mostly performed in vitro; this is also the case in the paper discussed here. The conditions during measurement in the Hemox-Analyzer used by Dominelli et al. are, however, even more artificial: a small volume of red cells is dispersed in a high volume of buffer solution and CO2 is absent. In any case the in vivo curve is steeper because of the lower pH (Bohr effect) and higher temperature in the tissue vessels, especially during exercise. Additionally, the Bohr effect determined in vivo is bigger than in vitro (Braumann et al. 1982), which can be partly explained as follows. The Bohr effect induced by CO2 at low saturation (the condition in tissue capillaries) is larger than for lactic acid (Meier et al. 1974). Lactic acid, however, enters the erythrocytes only slowly. But it liberates CO2 from bicarbonate in the plasma. CO2 diffuses rapidly into the red cells, therefore the in vivo Bohr effect is mainly caused by CO2 (Böning et al. 1991, 2007). Under strenuous exercise conditions, CO2 therefore accumulates in the muscle capillaries and may exceed 80 mmHg (Stringer et al. 1994). Due to this quasi-closed system, the Bohr effect strongly modulates the in vivo ODC and must therefore be considered particularly under exercise conditions. Furthermore, various papers published since the 1970s (reviewed in Böning et al. 2014) suggest that unknown factors make the in vivo curve even steeper than expected. Possible causes are concentration changes of chloride, glutathione or glutamate in the erythrocytes. Different numbers of males and females in the HAH and normal subjects groups, respectively, increase scattering, because sex differences in P50, Bohr coefficient and temperature coefficient of the ODC have been observed (Böning et al. 1978). The large scattering seems also to be the cause of a lack of significant differences between groups in weight, body mass index and peak power. Measuring method: "After deoxygenation with nitrogen, the compressed air reoxygenation curve was measured at pH 7.6 and 37°C using a laboratory-developed protocol (Winslow et al. 1977)". We emphasize again that pH 7.6 and lack of CO2 do not represent physiological conditions, especially not in the muscle capillaries. In our investigations we therefore modified the continuous Hemox-Analyzer method using a plasma-like buffer and adding CO2 to the equilibration gas (e.g. Schmidt et al. 1990). Furthermore, Winslow et al. did not use the Hemox-Analyzer in the cited paper; instead they used equilibration of undiluted blood samples in tonometers at various . A non-standardized pedalling frequency possibly explains differences in oxygen uptake between groups at low exercise intensity (Fig. 7C in Dominelli's paper). Gross efficiency markedly changes with pedal rate (Böning et al. 1984). During intense exercise in normoxia, the concentration of lactate rises as result of increased metabolism, not because of a lack of oxygen (e.g. Brooks, 2010). In acute hypoxia, however, there is an additional increase during submaximal exercise. The lactate paradox appears only after acclimatization (detailed description in West et al. 2013). The higher end-exercise lactate concentration in the HAH group is most easily explained as result of a higher relative intensity visible in the increased oxygen uptake per watt. Base excess is a measure of fixed acids like HCl or lactic acid in blood, not including the buffer Hb. Hb is only used as a measure of the buffer value of blood and is necessary for calculation of base excess. We hope that some of these questions may be answered in further discussions. Nevertheless, this paper provides new insights by using an innovative approach, which should be expanded to include more in vivo aspects. None declared. D.B. contributed to conception or design of the work; acquisition or analysis or interpretation of data for the work; drafting the work or revising it critically for important intellectual content; final approval of the version to be published; agreement to be accountable for all aspects of the work. W.S. contributed to conception or design of the work; acquisition or analysis or interpretation of data for the work; drafting the work or revising it critically for important intellectual content; final approval of the version to be published; agreement to be accountable for all aspects of the work. None.

La afinidad de la hemoglobina (Hb) por oxigeno (O2) es un factor importante que influye en el transporte de este gas, especialmente en hipoxia y en diferentes enfermedades como anemia o fibrosis quística. En la medición de la afinidad se usa la determinación de la curva de disociación Hb:O2. El método presentado para establecer la curva de disociación Hb:O2 (CDO) simplifica los protocolos normalmente utilizados, ya que elimina el requerimiento del equipo específico para equilibrar la sangre con oxígeno en niveles fijos de presión parcial (PO2). Mediante el uso de ecuaciones matemáticas es posible establecer la cinética de saturación de la hemoglobina (SO2) a valores crecientes de PO2. De igual forma, mediante el método se determinan aspectos típicos de la unión Hb: O2 como la dependencia del pH (coeficiente de Bohr) y el tipo de asociación de la proteína con su ligando mediante el diagrama de Hill. En virtud de la simplificación realizada, el método es aplicable en prácticas de laboratorio en población humana y animal, así como en la investigación de diferentes condiciones experimentales.

In Caucasians and Native Americans living at altitude, hemoglobin mass is increased in spite of erythropoietin concentrations ([Epo]) not markedly differing from sea level values. We hypothesized that a nocturnal decrease of arterial oxygen saturation (SaO(2)) causes a temporary rise of [Epo] not detected by morning measurements. SaO(2) (continuous, finger oximeter) and [Epo] (ELISA, every 4h) were determined in young highlanders (altitude 2600m) during 24h of usual daily activity. In Series I (six male, nine female students), SaO(2) fell during the night with the nadir occurring between 01:00 and 03:00; daily means (range 92.4-95.2%) were higher in females (+1.7%, P<0.01). [Epo] showed opposite changes with zenith occurring at 04:00 without a sex difference. Mean daily values (22.9 +/- 10.7SD U/L) were higher than values obtained at 08:00 (17.2 +/- 9.5 U/L, P<0.05). In Series II (seven females), only SaO(2) was measured. During follicular and luteal phases, SaO(2) variation was similar to Series I, but the rhythm was disturbed during menstruation. While daily [Epo] variations at sea level are not homogeneous, there is a diurnal variation at altitude following changes in SaO(2). Larger hypoventilation-dependent decreases of alveolar PO2 decreases during the night probably cause a stronger reduction of SaO(2) in highlanders compared to lowlanders. This variation might be enlarged by a diurnal fluctuation of Hb concentration. In spite of a lower [Hb], the higher SaO(2) in women compared to men led to a similar arterial oxygen content, likely explaining the absence of differences in [Epo] between sexes.

In modern societies there is strong belief in scientific progress, but, unfortunately, a parallel partial regress occurs because of often avoidable mistakes. Mistakes are mainly forgetting, erroneous theories, errors in experiments and manuscripts, prejudice, selected publication of "positive" results, and fraud. An example of forgetting is that methods introduced decades ago are used without knowing the underlying theories: Basic articles are no longer read or cited. This omission may cause incorrect interpretation of results. For instance, false use of actual base excess instead of standard base excess for calculation of the number of hydrogen ions leaving the muscles raised the idea that an unknown fixed acid is produced in addition to lactic acid during exercise. An erroneous theory led to the conclusion that lactate is not the anion of a strong acid but a buffer. Mistakes occur after incorrect application of a method, after exclusion of unwelcome values, during evaluation of measurements by false calculations, or during preparation of manuscripts. Co-authors, as well as reviewers, do not always carefully read papers before publication. Peer reviewers might be biased against a hypothesis or an author. A general problem is selected publication of positive results. An example of fraud in sports medicine is the presence of doped subjects in groups of investigated athletes. To reduce regress, it is important that investigators search both original and recent articles on a topic and conscientiously examine the data. All co-authors and reviewers should read the text thoroughly and inspect all tables and figures in a manuscript.

UNLABELLED:Relatively long-lasting metabolic alkalizing procedures such as bicarbonate ingestion have potential for improving performance in long-sprint to middle-distance events. Within a few minutes, hyperventilation can induce respiratory alkalosis. However, corresponding performance effects are missing or equivocal at best. PURPOSE:To test a potential performance-enhancing effect of respiratory alkalosis in a 30-s Wingate Anaerobic Test (WAnT). METHODS:10 men (mean ± SD age 26.6 ± 4.9 y, height 184.4 ± 6.1 cm, body-mass test 1 80.7 ± 7.7 kg, body-mass test 2 80.4 ± 7.2 kg, peak oxygen uptake 3.95 ± 0.43 L/min) performed 2 WAnTs, 1 with and 1 without a standardized 15-min hyperventilation program pre-WAnT in randomized order separated by 1 wk. RESULTS:Compared with the control condition, hyperventilation reduced (all P < .01) pCO2 (40.5 ± 2.8 vs 22.5 ± 1.6 mm Hg) and HCO3 - (25.5 ± 1.7 vs 22.7 ± 1.6 mmol/L) and increased (all P < .01) pH (7.41 ± 0.01 vs 7.61 ± 0.03) and actual base excess (1.4 ± 1.4 vs 3.2 ± 1.6 mmol/L) pre-WAnT with an ergogenic effect on WAnT average power (681 ± 41 vs 714 ± 44 W) and total metabolic energy (138 ± 12 vs. 144 ± 13 kJ) based on an increase in glycolytic energy (81 ± 13 vs 88 ± 13 kJ). CONCLUSION:Hyperventilation-induced respiratory alkalosis can enhance WAnT cycling sprint performance well in the magnitude of what is seen after successful bicarbonate ingestion.

In modern societies there is strong belief in scientific progress. But unfortunately a parallel partial regress occurs because of often evitable mistakes. These are mainly forgetting, erroneous theories, mistakes in experiments and manuscripts, selected publication of “positive” results and fraud. PURPOSE: In this contribution typical examples in the area of sports medicine will be presented. METHODS: Evaluation of experiments and of scientific articles. RESULTS: An example for forgetting is that initiating basic articles for methods introduced since decades are no more read. This may cause incorrect interpretation of results. For instance false use of actual base excess instead of standard base excess for calculations of the number of hydrogen ions leaving the muscles raised the idea that an unknown fixed acid is produced besides lactic acid during hard exercise. An erroneous theory led to the conclusion that lactate is not the anion of a strong acid but a buffer. Mistakes may occur after incorrect application of a method, during evaluation of measurements by false calculations or exclusion of unwelcome values (extreme values sometimes point to subjects with unexpected properties), or during preparation of manuscripts. Astonishingly coauthors as well as reviewers do not always carefully read the papers before publication. As an example for various mistakes in a partly excellent publication of 7 authors, mice had a resting heart rate of 500/min but 160/min at exhaustion. A general problem in science is selected publication of “positive” result. An example special for fraud in sports medicine is the presence of doped subjects in a group of investigated athletes which is not always checked for. CONCLUSIONS: To reduce regress it is important that investigators search not only for recent articles on a topic, that they examine conscientiously the data and that all coauthors as well as reviewers read not only the text thoroughly but revise also tables and figures in a manuscript.

In patients with cystic fibrosis lung damages cause arterial hypoxia. As a typical compensatory reaction one might expect changes in oxygen affinity of hemoglobin. Therefore position (standard half saturation pressure P50st) and slope (Hill's n) of the O2 dissociation curve as well as the Bohr coefficients (BC) for CO2 and lactic acid were determined in blood of 14 adult patients (8 males, 6 females) and 14 healthy controls (6 males, 8 females). While Hill's n amounted to approximately 2.6 in all subjects, P50st was slightly increased by 1 mmHg in both patient groups (controls male 26.7 ± 0.2, controls female 27.0 ± 0.1, patients male 27.7 ± 0.5, patients female 28.0 ± 0.3 mmHg; mean and standard error, overall p<0.01). Main cause was a rise of 1-2 µmol/g hemoglobin in erythrocytic 2,3-biphosphoglycerate concentration. One patient only, clearly identified as an outlier and with the mutation G551D, showed a reduction of both P50st (24.5 mmHg) and [2,3-biphosphoglycerate] (9.8 µmol/g hemoglobin). There were no differences in BCCO2, but small sex differences in the BC for lactic acid in the controls which were not detectable in the patients. Causes for the right shift of the O2 dissociation curve might be hypoxic stimulation of erythrocytic glycolysis and an increased red cell turnover both causing increased [2,3-biphosphoglycerate]. However, for situations with additional hypercapnia as observed in exercising patients a left shift seems to be a more favourable adaptation in cystic fibrosis. Additionally when in vivo PO2 values were corrected to the standard conditions they mostly lay left of the in vitro O2 dissociation curve in both patients and controls. This hints to unknown fugitive factors influencing oxygen affinity.