Aims: Total haemoglobin mass (tot-Hb) increases during high-altitude acclimatization. Normalization of tot-Hb upon descent is thought to occur via neocytolysis, the selective destruction of newly formed erythrocytes. Because convincing experimental proof of neocytolysis is lacking, we performed a prospective study on erythrocyte survival after a stay at the Jungfraujoch Research Station (JFJRS; 3450 m). Methods: Newly formed erythrocytes of 12 male subjects (mean age 23.3 years) were age cohort labelled in normoxia (110 m) and during a 19-day high-altitude sojourn by ingestion of 13 C2-and 15 N-labelled glycine respectively. Elimination dynamics for erythrocytes produced in normoxia and at high altitude were measured by isotope ratio mass spectrometry of haem, by determining tot-Hb, reticulocyte counts, erythrocyte membrane protein 4.1a/4.1b ratio and by mathematical modelling. Results: Tot-Hb increased by 4.7% ± 2.7% at high altitude and returned to pre-altitude values within 11 days after descent. Elimination of 13 C-(normoxia) and 15 N-(high altitude) labelled erythrocytes was not different. Erythropoietin levels and counts of CD71-positive reticulocytes decreased rapidly after descent. The band 4.1a/4.1b ratio decreased at altitude and remained low for 3-4 days after descent and normalized slowly. There was no indication of haemolysis. Conclusion: We confirm a rapid normalization of tot-Hb upon descent. Based on the lack of accelerated removal of age cohorts of erythrocytes labelled at high altitude, on patterns of changes in reticulocyte counts and of the band 4.1a/4.1b ratio and on modelling, this decrease did not occur via neocytolysis, but by a reduced rate of erythropoiesis along with normal clearance of senescent erythrocytes.

Key pointsr It is generally accepted that mitochondrial volume density in human skeletal muscle diminishes with chronic high altitude exposure.r All data supporting this concept were collected during mountaineering expeditions, which are associated with the confounding effects of whole body negative energy balance.r Here we examine the effect of 28 days of exposure to 3454 m on skeletal muscle mitochondrial volume density in a setting where whole body weight, whole body composition, leg lean mass, skeletal muscle fibre area and maximal power output were preserved.r Our results demonstrate that total skeletal muscle mitochondrial volume density increases in response to high altitude exposure secondary to a preferential increase in intermyofibrillar mitochondrial populations.r This study provides direct evidence contradicting the notion that high altitude exposure diminishes skeletal muscle mitochondrial volume density, highlighting an inconsistent understanding of the role of hypoxia on skeletal muscle mitochondria.Abstract The role of hypoxia on skeletal muscle mitochondria is controversial. Studies superimposing exercise training on hypoxic exposure demonstrate an increase in skeletal muscle mitochondrial volume density (Mito VD ) over equivalent normoxic training. In contrast, reductions in both skeletal muscle mass and Mito VD have been reported following mountaineering expeditions. These observations may, however, be confounded by negative energy balance, which may obscure the results. Accordingly we sought to examine the effects of high altitude hypoxic exposure on mitochondrial characteristics, with emphasis on Mito VD , while minimizing changes in energy balance. For this purpose, skeletal muscle biopsies were obtained from nine lowlanders at sea level (Pre) and following 7 and 28 days of exposure to 3454 m. Maximal ergometer power output, whole body weight and composition, leg lean mass and skeletal muscle fibre area all remained unchanged following the altitude exposure. Transmission electron microscopy determined that intermyofibrillar (IMF) Mito VD was augmented (P = 0.028) by 11.5 ± 9.2% from Pre (5.05 ± 0.9%) to 28 Days (5.61 ± 0.04%). In contrast, there was no change in subsarcolemmal (SS) Mito VD . As a result, total Mito VD (IMF + SS) was increased (P = 0.031) from 6.20 ± 1.5% at Pre to 6.62 ± 1.4% at 28 Days (7.8 ± 9.3%). At the same time no changes in mass-specific respiratory capacities, mitochondrial protein or antioxidant content were found. This study demonstrates that skeletal muscle Mito VD may increase with 28 days acclimation to 3454 m.

The ability of red blood cells (RBCs) to transport gases, their lifespan as well as their rheological properties invariably depend on the deformability, hydration, and membrane stability of these cells, which can be measured by Laser optical rotational red cell analyser (Lorrca® Maxsis, RR Mechatronics). The osmoscan mode of Lorrca is currently used in diagnosis of rare anemias in clinical laboratories. However, a broad range of normal values for healthy subjects reduces the sensitivity of this method for diagnosis of mild disease phenotype. In this pilot study, we explored the impact of age and gender of 45 healthy donors, as well as RBC age on the Lorrca indices. Whereas gender did not affect the Lorrca indices in our study, the age donors had a profound effect on the O_hyper parameter. To study the impact of RBC age on the osmoscan parameters, we have isolated low (L)-, medium (M)-, or high (H)- density fractions enriched with young, mature, and senescent RBCs, respectively, and evaluated the influence of RBC age-related properties, such as density, morphology, and redox state, on the osmoscan indices. As before, O_hyper was the most sensitive parameter, dropping markedly with an increase in RBC density and age. Senescence was associated with a decrease in deformability (EI_max) and tolerability to low and high osmolatites (Area). L-fraction was enriched with reticulocytes and cells with high projected area and EMA staining, but also contained a small number of cells small in projected area and most likely, terminally senescent. L-fraction was on average slightly less deformable than mature cells. The cells from the L-fraction produced more oxidants and NO than all other fractions. However, RBCs from the L-fraction contained maximal levels of reduced thiols compared to other fractions. Our study suggests that reference values for O_hyper should be age-stratified, and, most probably, corrected for the average RBC age. Further multi-center study is required to validate these suggestions before implementing them into clinical practice.

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This study focuses on assessing the possible impact of changes in hemoglobin (Hb) oxygenation on the state of water in its hydration shell as it contributes to red blood cell deformability. Microwave Dielectric Spectroscopy (MDS) was used to monitor the changes in interactions between water molecules and Hb, the number of water molecules in the protein hydration shell, and the dynamics of pre-protein water in response to the transition of Hb from the tense (T) to the relaxed (R) state, and vice versa. Measurements were performed for Hb solutions of different concentrations (5 g/dl-30 g/dl) in phosphatebuffered saline buffer. Cole-Cole parameters of the main water relaxation peak in terms of interactions of water molecules (dipole-dipole/ionic dipole) during the oxygenation-deoxygenation cycle were used to analyze the obtained data. The water mobility-represented by as a function of ln -differed dramatically between the R (oxygenated) state and the T (deoxygenated) state of Hb at physiologically relevant concentrations (30 g/dl-35 g/dl or 4.5 mM-5.5 mM). At these concentrations, oxygenated hemoglobin was characterized by substantially lower mobility of water in the hydration shell, measured as an increase in relaxation time, compared to deoxyhemoglobin. This change indicated an increase in red blood cell cytosolic viscosity when cells were oxygenated and a decrease in viscosity upon deoxygenation. Information provided by MDS on the intraerythrocytic water state of intact red blood cells reflects its interaction with all of the cytosolic components, making these measurements powerful predictors of the changes in the rheological properties of red blood cells, regardless of the cause.

ZUSAMMENFASSUNGNeozytolyse bezeichnet die bevorzugte Zerstörung von jungen roten Blutzellen (Neozyten), wenn zu viele Erythrozyten vorhanden sind. Das kommt in der Raumfahrt oder beim Abstieg nach langem Höhenaufenthalt vor. Mit dem Ziel, die Existenz von Neozytolyse erstmals zu beweisen, haben wir in der Forschungsstation Jungfraujoch (3450 Hm) eine prospektive Studie durchgeführt. Neu gebildete Erythrozyten von 12 männlichen Probanden wurden durch Einnahme von nichtradioaktivem Glyzin mit seltenen Isotopen markiert: in Heidelberg (110 Hm) und während eines 19-tägigen Aufenthalts (3450 Hm). Die Eliminationsdynamik für Erythrozyten, die in Normoxie und in großer Höhe produziert wurden, unterschied sich nicht. Zusammen mit Änderungen der Retikulozytenzahlen konnte damit das Konzept der Neozytolyse nicht bestätigt werden.

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