Normal Hemoglobin Concentrations in Obstructive Sleep Apnea and Associated Neocytolysis-Mediated Hemolysis and Inflammation Mediated Suppression of Expected Elevated Hemoglobin
Authors: Song Jihyun , Sundar Krishna M , Horvathova Monika , Tashi Tsewang , Lundby Carsten , Ganz Tomas , Prchal Josef T. 
Published: November 2019

Obstructive sleep apnea (OSA) is characterized by chronic intermittent hypoxia (CIH) caused by recurrent episodes of upper airway obstruction during sleep. Chronic hypoxia increases red blood cell volume (RBCV) and hemoglobin concentration (Hb) in order to improve tissue oxygen delivery. However, the prevalence of increased Hb in OSA was only 1.7% (9 out of 527 patients) in our prior study (Gangaraju et al Blood 2016 128:2444). The reasons for this relative lack of discernable erythrocytosis is not yet fully understood. Further, the studies of possible changes in RBC and plasma volume in OSA are lacking.
We collected blood samples from OSA patients before and after 3 months of continuous positive airway pressure (CPAP) treatment. We estimated erythropoietic activity by measuring erythropoietin (EPO) and reticulocyte count. EPO levels and reticulocytes were higher in OSA patients compared to controls; CPAP treatment normalized these levels. Time spent below sPO2 89% correlated with EPO levels (r=0.3434 and p=0.0403) indicating that episodes of hypoxia augmented erythropoiesis. However, Hb remained normal, leading us to hypothesize that augmented erythropoietic stimulus in OSA is counterbalanced by either 1) increase of hemolysis or 2) erythropoietic resistance to EPO. Hemolysis was detected in some but not all OSA patients by end-tidal carbon monoxide (ETCO), a product of heme catabolism; however, the instrument's limited sensitivity precluded detection of mild hemolysis. We previously showed that hypoxia-increased RBCV is transiently overcorrected upon rapid return to normoxia by preferential destruction of young red cells -neocytolysis. Neocytolysis is caused by excessive accumulation of reactive oxygen species (ROS) from increased retention of mitochondria in reticulocytes and decreased catalase in young RBCs (1). In our OSA subjects, we found that mitochondrial mass and mitochondrial ROS were higher in reticulocytes. Further, ROS were higher in B- cells, T-cells, monocytes, and granulocytes compared to controls. After CPAP treatment, these values normalized and catalase mRNA and enzyme activity also increased. These results support neocytolysis as one of the factors that prevent the increase of Hb in OSA.
OSA also induces inflammation, a factor known to inhibit erythropoiesis, in part by induction of hepcidin which inhibits release of iron from macrophages. IL-6 and TNF transcript levels in granulocytes were higher in OSA compared to controls and normalized after CPAP treatment. Hepcidin levels were higher in OSA compared to controls. Hepcidin levels were correlated with inflammatory markers, IL-6 levels were inversely correlated with EPO levels, and iron and transferrin saturation levels were lower in OSA, inversely correlating with elevated hepcidin.
The normal Hb does not rule out the presence of polycythemia, as increased RBCV could be offset by concomitant increase in plasma volume resulting in a normal Hb concentration. This was recently demonstrated in Tibetan Sherpas living in hypoxic high altitude who have normal Hb but increased RBCV and plasma volume (PV) (2). One can determine RBCV and PV by inhaling known amount of CO and measuring increments of carboxyhemoglobin together with Hb and hematocrit. We recently obtained the required instrumentation (OpCO, DETALO INSTRUMENTS, Denmark), and are currently correlating our clinical and biochemical measurement with OSA subjects' RBCV and PV to either confirm presence of hypoxia-induced polycythemia or conclusively rule it out.
We conclude that the absence of elevated Hb in OSA is attributable not only to neocytolysis but also to the suppression of erythropoiesis by inflammation.
1) Song J, Yoon D, Christensen RD., Horvathova M,Thiagarajan P, and Prchal JT. HIF mediated increased ROS from reduced mitophagy and decreased catalase causes neocytolysis. J Mol. Med., (Berl). 2015 Aug., PMID: 26017143.
2) Stembridge M et all. The overlooked significance of plasma volume for successful adaptation to high altitude in Sherpa and Andean natives. Proc Natl Acad Sci U S A.2019 Jul 29. [Epub ahead of print].

Disclosures
Lundby: Detalo: Other: CEO and founder. Ganz:Intrinsic LifeSciences: Consultancy, Equity Ownership.