STEM Today

SPACE FOOD AND NUTRITION

STEM TODAY September 2016, No.12

SPACE FOOD AND NUTRITION

STEM TODAY September 2016 , No.12

CONTENTS SPACE FOOD AND NUTRITION N15: We need to identify the most important nutritional factors for oxidative damage during spaceflight.

Iron

Editorial Editor: Mr. Abhishek Kumar Sinha Editor / Technical Advisor: Mr. Martin Cabaniss

STEM Today, September 2016, No.12

Biography Dr. Kjell N. Lindgren was selected by NASA in 2009. He spent most of his childhood abroad and returned to the U.S. to complete his education and earn a Doctorate of Medicine from the University of Colorado. He is board certified in emergency and aerospace medicine. After serving as the Deputy Crew Surgeon for STS-130 and Expedition 24, he was selected as an astronaut in June 2009 as one of 14 members of the 20th NASA astronaut class. Dr. Lindgren flew on Expedition 44/45 and logged 141 days in space.

He participated in two spacewalks and in more than a hundred different scientific experiments.

At the U.S. Air Force Academy, Dr. Lindgren was a member of the "Wings of Blue" parachute team, where he served as an instructor, a jumpmaster and a member of the academy’s intercollegiate national championship team. As a part of his masters studies at CSU, Dr. Lindgren conducted cardiovascular countermeasure research in the Space Physiology Lab at NASA Ames Research Center in Sunnyvale, California. He conducted high-altitude physiology research during medical school. Dr. Lindgren began working at Johnson Space Center in 2007. As a Wyle-University of Texas Medical Branch flight surgeon, he supported International Space Station training and operations in Star City, Russia and water survival training in the Ukraine. At the time of his selection to the astronaut corps, he was serving as the Deputy Crew Surgeon for STS-130 and Expedition 24.

STEM Today, September 2016, No.12

Biography Page Astronaut Kjell Lindgren Astronaut Kjell Lindgren floats through the Destiny lab module.

Image Credit: NASA

Cover Page Astronaut Kjell Lindgren Corrals the Supply of Fresh Fruit NASA astronaut Kjell Lindgren corrals the supply of fresh fruit that arrived August 25, 2015 on the Kounotori 5 H-II Transfer Vehicle (HTV-5.) Visiting cargo ships often carry a small cache of fresh food for crew members aboard the International Space Station.

Image Credit: NASA

Back Cover ISSpresso Machine ISS043E160227 (05/03/2015)— The new ISSpresso machine was recently installed on the International Space Station. In order to utilize the ISSpresso, a NASA standard drink bag is installed, along with a capsule containing the beverage item that the crew member wishes to drink. After the item has been brewed, the used capsule and the drink bag are removed.

Image Credit: NASA

STEM Today , September 2016

Editorial Dear Reader

STEM Today, September 2016, No.12

All young people should be prepared to think deeply and to think well so that they have the chance to become the innovators, educators, researchers, and leaders who can solve the most pressing challenges facing our world, both today and tomorrow. But, right now, not enough of our youth have access to quality STEM learning opportunities and too few students see these disciplines as springboards for their careers.

According to Marillyn Hewson, "Our children - the elementary, middle and high school students of today - make up a generation that will change our universe forever. This is the generation that will walk on Mars, explore deep space and unlock mysteries that we can’t yet imagine". "They won’t get there alone. It is our job to prepare, inspire and equip them to build the future - and that’s exactly what Generation Beyond is designed to do."

STEM Today will inspire and educate people about Spaceflight and effects of Spaceflight on Astronauts.

Editor Mr. Abhishek Kumar Sinha

Editorial Dear Reader The Science, Technology, Engineering and Math (STEM) program is designed to inspire the next generation of innovators, explorers, inventors and pioneers to pursue STEM careers.

According to President Barack Obama, "[Science] is more than a school subject, or the periodic table, or the properties of waves. It is an approach to the world, a critical way to understand and explore and engage with the world, and then have the capacity to change that world..."

STEM Today addresses the inadequate number of teachers skilled to educate in Human Spaceflight. It will prepare , inspire and educate teachers about Spaceflight. STEM Today will focus on NASA’S Human Research Roadmap. It will research on long duration spaceflight and put together latest research in Human Spaceflight in its monthly newsletter.

Editor / Technical Advisor Mr. Martin Cabaniss

STEM Today, September 2016, No.12

SPACE FOOD AND NUTRITION Gap in NASA's Human Research Roadmap

N15: We need to identify the most important nutritional factors for oxidative damage during space ight: There are numerous possible sources of oxidative stressors during space ight, including radiation, oxygen exposure, diet, or changes in metabolism. For one recent piece of evidence, data from the Nutrition SMO recently identi ed iron status as a factor that is closely related to biomarkers of oxidative damage during space ight.

Special Edition on Space Food and Nutrition

Iron is an essential trace element in the human body. Iron plays an important role in oxygen transport, electron transfer, and it serves as a cofactor in many enzyme systems. Iron is required for growth and survival of almost every organism. Both iron deficiency and iron overload are deleterious. Iron deficiency leads to the clinical irondeficiency anemia, and severe long-standing iron deficiency may also be associated with koilonychia and the Plummer-Vinson syndrome. On the other hand, iron is also a catalyst in free radical reactions, and excessive iron can be harmful to human health via the generation of reactive oxygen species. Iron overload can lead to hereditary hemochromatosis. Iron overload can also have a direct effect on the central nervous system, and has been implicated in the pathogenesis of Parkinson’s disease (PD) and Alzheimer’s disease (AD). Taken together, the maintenance of iron homeostasis is crucial for human health.

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Dietary iron is initially absorbed by apical brush border of duodenal enterocytes, then transported to basolateral surfaces, and is ultimately released into the circulation. Most of the remaining body iron is stored in hepatocytes and reticuloendothelial macrophages. Daily iron absorption or loss is relatively small, and is characterized by a limited external exchange and by efficient reutilization of iron from internal sources. For example, each day the bone marrow utilizes approximately 20 mg of iron to produce new erythrocytes, but only 1-2 mg of iron normally enters the body through the intestines. Therefore, the mononuclear phagocyte system (MPS) plays a major role in the recycling of iron by degrading the hemoglobin of senescent erythrocytes to meet the demand for continued erythropoiesis.

The MPS is composed of monocytes, macrophages, and their precursor cells. Macrophages are abundant in the liver, intestine, bone marrow, spleen, and kidney. The recycling of iron from senescent erythrocytes is mainly carried out by macrophages in the spleen, liver, and bone marrow. While the MPS is important in maintaining iron homeostasis, the molecular mechanisms underlying iron homeostasis in the MPS are poorly understood.

Recently, new proteins involved in iron metabolism have been discovered. The elucidation of their functions is beginning to reveal the process of iron metabolism in the MPS at the molecular level.

Iron acquisition by the MPS Erythrophagocytosis After migrating from the blood to various tissues, monocytes differentiate into macrophages. Macrophages engulf aged erythrocytes, degrade them with the help of hydrolytic enzymes, and then catabolize their hemoglobin in the phagolysosome yielding free heme. It is assumed that heme crosses the phagolysosomal membrane either by diffusion or by a specific transporter. Heme oxygenase (HO), localized in the endoplasmic reticulum, catabolizes heme to produce biliverdin, carbon monoxide, and Fe2+ . There are three isoforms of HO in mammals. The expression of HO-1 is inducible; HO-2 is constitutively expressed; HO-3 is nearly devoid of catalytic capability.

Although HO-2 is predominant in most organs, the expression of HO-1 is five times greater than HO-2 in the rat spleen. It is inferred that there is an abundance of erythrocyte phagocytosing MPS cells in spleen, and abundant expression of HO-1 induced to catabolize the heme liberated from hemoglobin.

Receptor-mediated iron uptake Uptake of hemoglobin It is found that 10-20% of normal erythrocyte destruction occurs intravascularly, resulting in the release of hemoglobin within the bloodstream. This hemoglobin is rapidly bound by haptoglobin, which is then cleared from the circulation by parenchymal cells of the liver. CD163,as a hemoglobin scavenger receptor, is expressed exclusively in monocytes and macrophages. The hemoglobin-haptoglobin complex (Hb-Hp complex) interacts with CD163, and then undergoes endocytosis and degradation to release heme. Finally, Fe2+ is released from hemoglobin by the action of HO-1.

Heme-carrier protein 1 (HCP1) is an intestinal heme transporter, which is able to transport heme-bound iron from the gut lumen into duodenal epithelial cells. HCP1 is highly hydrophobic and contains nine predicted transmembrane domains. HCP1 is mainly expressed in the duodenum, but is also found in other tissues, such as liver and kidney. In the cultured cells over-expressing HCP1, there is a twofold increase in heme uptake. In contrast, incubation of duodenal tissue with anti-HCP1 antibodies causes significant inhibition of heme uptake.

HCP1-dependent heme uptake is both temperature-dependent and saturable, indiindicating a carrier-mediated process. HCP1 is also expressed in human macrophages. Within early endosomes, HCP-1 colocalizes with endocytosed Hb-Hp complexes, which are taken up via the CD163 receptor scavenger pathway. Therefore, heme may reach the endoplasmic reticulum from the phagolysosomal membrane via HCP1.

When the hemoglobin-binding capacity of haptoglobin is exceeded following increased intravascular hemolysis, free hemoglobin appears in the plasma. The free hemoglobin is oxidized to methemoglobin, dissociates to free heme, and forms complex with the plasma glycoprotein hemopexin (Hpx). Hepatocytes can acquire heme-

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Special Edition on Space Food and Nutrition hemopexin complexes from the circulation by a receptor-mediated uptake mechanism. Hemopexin receptors have also been found on human monocyte cell lines, and thus it can be inferred that the MPS is able to acquire heme via hemopexin under abnormal circumstances.

Uptake of transferrin-bound iron Macrophages can utilize a transferrin-dependent mechanism to acquire iron via the cell surface receptor known as transferrin receptor 1 (TfR1). TfR1 is a dimer of 90 kDa subunits. At the cell surface, transferrin binds to TfR1 and is internalized by receptor-mediated endocytosis. Iron dissociates from transferrin within acidified endosomes and is transferred across the endosomal membrane. TfR1 expression is greatly increased when cultured monocytes are induced to differentiate into macrophages. Transferrin uptake has been demonstrated in macrophages from mice,rats, and humans. However, significant iron uptake by the MPS has not been shown after injection of radiolabeled transferrin-bound iron in humans. Therefore, TfR1-dependent iron uptake may not be the important route in the human MPS. Further study is needed to clarify the details of this mechanism.

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Recently, it is found that the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is localized on human and murine macrophage cell surface. The level of iron in culture medium can regulate the expression of GAPDH. Furthermore, GAPDH can interact with transferrin and the GAPDH-transferrin complex is subsequently internalized into early endosomes. However, the possibility that GAPDH is a novel transferrin receptor that mediates Tf (Fe2+ ) absorption needs further confirmation.

Iron stores in the MPS In MPS cells, some of the iron released from the catabolism of heme is destined for storage in ferritin, a cytosolic protein comprises of 24 subunits consisting of two types, H and L. During the first 2 h following erythrophagocytosis of senescent red blood cells, the level of HO-1 mRNA increases, whereas H- and L-ferritin mRNA levels remain unchanged. Four hours later, HO-1 mRNA decreases progressively in parallel with an increase in Hferritin mRNA levels. Twenty hours after erythrophagocytosis, the level of HO-1 mRNA returns to initial levels, while the expression of H-ferritin mRNA remains high. On the other hand, the level of L-ferritin mRNA does not change significantly throughout this entire period.

Iron is essential for cell survival as well as for the proliferation and maturation of developing erythroid precursors(EP) into hemoglobin-containing red blood cells. The main route of erythroid iron uptake is via a transferrindependent mechanism involving TfR1 on the cell surface. In transferrin-free conditions, ferritin synthesized and secreted by macrophages can serve as an iron source, partially replacing transferrin and supporting EP development. Furthermore, it has been reported that iron loading can lead to increased synthesis and secretion of ferritin in macrophages and that this ferritin accumulation is associated with atherosclerotic processes.

Iron release by the MPS It has been found that during the first few hours after erythrophagocytosis, most of the extracted heme iron is rapidly released by macrophages into the plasma. The rest of the iron is stored in ferritin and is slowly released over several days or weeks. However, the mechanism of iron release by the MPS was poorly understood until ferroportin 1 (FPN1) was independently identified by three groups. FPN1, also known as SLC40A1, iron-regulated transporter 1 (IREG1), or metal transporter protein 1 (MTP1), is the first characterized cellular iron exporter, and it possesses 9 or 10 predicted transmembrane regions. The tissue distribution of FPN1 is ubiquitous, and most abundant in reticuloendothelial macrophages of the liver, spleen, and bone marrow.

Many reports have demonstrated that FPN1 functions as an iron exporter in various cell types. The expression of FPN1 in macrophages increases after erythrophagocytosis. Macrophages over-expressing FPN1 release 70% or more 59 Fe than control cells after erythrophagocytosis. In macrophages, FPN1 is predominantly localized in intracellular vesicular compartments. However, FPN1 expression is strongly enhanced at the plasma membrane of macrophages following iron treatment or erythrophagocytosis. These all suggest that FPN1 may specifically mediate the export of non-heme iron during erythrocyte-iron recycling.

There was a remarkable increase in ferroportin mRNA levels during the first 4 h post-erythrophagocytosis.

However, 4 h later a sharp decrease in ferroportin mRNA levels was observed. Twenty hours later, the level of ferroportin mRNA was lower than that in control cells. Therefore, in the early phase of erythrophagocytosis, most of the extracted heme iron was rapidly returned to the plasma (few hours), which may protect the macrophages against iron-mediated oxidative damage. In later phases, part of the iron was stored and slowly released from macrophages (several days and weeks) in order to prevent excessive iron release into the plasma.

In macrophages, FPN1 exports from the cytosol into the circulation, and this process is facilitated by a protein called ceruloplasmin (Cp). Ceruloplasmin is a copper protein with a potent ferroxidase activity that converts

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Special Edition on Space Food and Nutrition Fe2+ to Fe3+ in the presence of molecular oxygen. Ceruloplasmin can promote the binding of iron to apotransferrin and stimulate iron release from macrophages. Recently, authors discovered that FPN1 is rapidly internalized and degraded in the absence of Cp. However, FPN1 can still be maintained on the cell surface in the absence of Cp following depletion of extracellular iron by iron chelators. Mutation of lysine 253 of FPN1 to alanine prevents ubiquitination and maintains FPN1-iron complex on cell surface in the absence of Cp. Therefore, it is inferred that in the absence of multicopper oxidases iron remains bound to FPN1. FPN1 with bound iron is recognized by a ubiquitin ligase, which ubiquitylates FPN1 on lysine 253 and induces the degradation of FPN1. Curiously, Cp−/− mice do not develop iron-deficiency anemia. Therefore, it was inferred that there are other sources of ferroxidase capable of mobilizing iron from storage sites. Recent studies on the sex-linked anemia (sla) mouse have identified a Cp homologue, hephaestin.

STEM Today, September 2016, No.12

Although hephaestin is also a copper protein with a potent ferroxidase activity, it is an integral membrane protein with a single transmembrane domain. Hephaestin plays a critical role in the export of iron from the duodenum to the portal circulation. It is expressed abundantly in the small intestine, heart, and brain. Although hephaestin mRNA has been detected in the spleen, more work is needed to determine its potential involvement in iron export from the MPS. Regulation of iron metabolism by hepcidin in the MPS What factors can regulate DMT1 and FPN1 expression? The recently identified hepatic peptide hepcidin (also known as HAMP, liver-expressed antimicrobial peptide, LEAP-1) has been proposed to act as the principal ironregulatory hormone responsible for the maintenance of iron homeostasis. The main function of hepcidin is to regulate the absorption of dietary iron from the intestine and in the release of hemoglobin-derived iron in macrophages.

Hepcidin is homeostatically regulated by plasma iron levels, and macrophages, which serve as a storage depot for excess iron, respond rapidly to changes in hepcidin levels. The regulation of iron metabolism by hepcidin in the MPS is mainly mediated by redistribution of FPN1 from the plasma membrane. It has been reported that radiolabeled hepcidin accumulates in FPN1-rich organs such as liver, spleen, and proximal duodenum. The expression of FPN1 in J774 cells is decreased dramatically after incubation with hepcidin, while the 59 Fe release from macrophages is significantly reduced after erythrophagocytosis.

In contrast, the expression of FPN1 is enhanced in peritoneal exudate macrophages following rHuEpo injection injection, while hepatic hepcidin mRNA levels decrease significantly. Moreover, FPN1 expression is up-regulated in the liver (predominantly in Kupffer cells) in hepcidin-deficient mice.

Hepcidin decreases the functional activity of FPN1 by directly binding to it and causing it to be internalized from the plasma membrane and degraded, thereby blocking cellular iron efflux. Further study showed that internalization of FPN1 from the plasma membrane is mediated by tyrosine phosphorylation following the binding of hepcidin, since mutation of both tyrosines (Y302 and Y303) prevented hepcidinmediated FPN1 internalization. Following internalization FPN1 is dephosphorylated, and subsequently ubiquitinated and degradated in the late endosome/lysosome.

Alveolar macrophages can express low levels of hepcidin mRNA, and that hepcidin mRNA levels are increased following exposure to LPS. Another study also showed that macrophages stimulated with mycobacteria and IFN-γ expressed high levels of hepcidin mRNA and peptide.

Furthermore, confocal microscopy analysis showed that hepcidin was localized to the mycobacteria-containing phagosomes and directly exerted its antimicrobial activity. Hepcidin mRNA expression induced by mycobacteria and IFN-γ was inhibited by iron loading, and iron chelation increased hepcidin mRNA expression. Interestingly, IL-1 and IL-6, which are important in hepcidin augmentation in hepatocytes, had no effect on hepcidin expression in alveolar macrophages.

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Special Edition on Space Food and Nutrition

When the body has an excess of iron, it uses specific molecules to transport and store the iron. It is observed that an increase in body iron stores in astronauts early during spaceflight, and iron stores return to preflight levels in most crew members by the end of the flight. There are several potential causes for this increase astronauts experience when they begin their time in orbit.

First - the food system contains more iron than desired, on average about three times the recommended dietary allowance. Many food items on the space station are commercially available, and common items found on grocery store shelves (like bread and cereal) are fortified with iron.

Second - iron stores increase in response to a decrease in red blood cell mass. That decrease is a normal physiological change of spaceflight.

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Iron is an essential element involved in oxygen transport, oxidative phosphorylation in carbohydrate and lipid metabolism, and electron transport in cytochromes and cytochrome oxidase. Adequate iron is crucial for meeting the needs of organs and tissues, but excess iron is detrimental to cells and can cause oxidative damage. The body achieves iron balance through regulation of absorption by enterocytes in the intestine and regulation of iron export from cells. Once iron is absorbed into the enterocyte, it can be bound to ferritin and stored. Serum ferritin has been shown to be a sensitive indicator of iron stores. Ferritin is exponentially correlated with storage iron, as determined by quantitative phlebotomy in patients with iron overload.

Toxic amounts of iron may lead to tissue damage or cancer. High iron intakes have also been related to gastrointestinal distress. The toxic potential of iron derives from its ability to exist in 2 oxidative states (ferrous and ferric forms). Iron serves as a catalyst in redox reactions; however, when these reactions are not properly modulated by antioxidants or iron-binding proteins, cellular damage can occur. Adaptation of iron metabolism in humans typically allows the maintenance of normal body iron concentrations in spite of disparate physiological requirements and dietary supply. Body iron, about 4 g in the adult human, is determined by physiological iron demands, dietary supply, and adaptation. Dietary iron is a function of both content and bioavailability of total food iron; bioavailability is lower in nonheme than in heme iron sources. Dietary factors that inhibit iron absorption include tea, coffee, bran, calcium, phosphate, egg yolk, polyphenols, and certain forms of dietary fiber. Conversely, meat, fish, poultry, and ascorbic acid will enhance the bioavailability of non-heme iron.

R

Food System contains more iron than desired

The dietary iron content is very high in the International Space Station(ISS) food system, largely because many of the commercial food items in the ISS menu are fortified with iron. The mean (ÂąSD) iron content of the standard 16-d ISS menu is 20 Âą 6 mg/d, and individual crew members have had intakes in excess of 47 mg/d for some weeks during long-duration missions.

Shuttle Menus have more Sodium and Iron than required The Shuttle menus do tend to have more sodium and iron than required. This can be partially attributed to the use of many high-sodium commercial products and to the use of commercial bread and cereal products, which are enriched with iron. The requirement for an all-shelf-stable food system also increases sodium content, since sodium is often used in shelf-stable products to improve flavor and help with preservation [Smith et.al.(2009)].

Nutrient Requirements for ISS missions with Iron and Sodium excess The nutrient requirements for ISS missions of up to 360 days are shown in Table 1. With a few exceptions (most notably vitamin D insufficiency, and iron and sodium excess),the actual menus meet these requirements[Smith et.al.(2009)].

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Special Edition on Space Food and Nutrition

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Special Edition on Space Food and Nutrition In 1999, all ISS International Partners-the Canadian Space Agency, the European Space Agency, the Japanese Space Agency (NASDA, later JAXA), the Russian Space Agency, and the U.S. space agency, NASA–held discussions to review ISS nutrient requirements. Additional reviews have been conducted since then, but a formal, signed agreement for updated nutritional requirements has yet to be established.

Dietary Intake and Requirements The current documented space flight requirement for dietary intake of iron is 8 to 10 mg/d for men and women.

In the U.S., the RDA for men 19 to 70 years is 8 mg/d, and for women aged 19 to 50 years it is 18 mg/d, dropping to 8 mg/d in women over 50. Historical space flight iron requirements for missions of 12 to 30 days were less than 10 mg/d, matching the RDA at the time.

Dietary iron provided by the space food system has always exceeded the requirement, and intakes have often been much higher (intakes as high as 20 to 25 mg iron per day have been observed, and the menu provides an average of over 22 mg/d, Table 1). This gives reason for concern because of the potential for elevated tissue iron to cause deleterious effects, including oxidative damage. This is a rare gender-based effect: women, who are at increased risk of iron deficiency on Earth, may actually be protected against iron overload during flight.

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Iron status in men and women before, during, and after long-duration space flight Subjects (n =23; 16 men and 7 women) were astronauts on ISS expeditions 14-27 (missions of 50-247 day in duration; mean ± SD duration: 157 ± 46), which were flown between 2006 and 2011. The male and female subjects were 48 ± 4 and 45 ± 4 year of age and weighed 85 ± 9 and 62 ± 4 kg, respectively, at the time of launch.

Results As determined with a food-frequency questionnaire completed once weekly, the overall iron intakes during the missions were 18 ± 5 and 16 ± 2 mg/d for the men and women, respectively.

Effect of space flight on biomarkers of iron status, acute phase proteins, and hematology A significant interaction was found between sex and space flight effects on serum ferritin. Serum ferritin was higher early during space flight (FD15: P < 0.0004; FD30: P = 0.005; Table 1) than it was before flight, and the increase was greater among women. Only on FD15 did ferritin remain significantly higher than it was before flight after adjustment for multiple comparisons. Body iron estimates (total body iron) followed the same pattern as ferritin. The iron-transporting protein transferrin was lower at all time points during space flight and on landing day, but concentrations were significantly higher in women than in men before and during flight. After adjustment for multiple comparisons, transferrin values on FD30, 60, and 120 remained significantly greater than preflight values. The transferrin index, which is an estimate of transferrin saturation, increased significantly during flight; at times during flight, it was >1 µmol Fe/µmol transferrin, which indicated iron overload.

The concentration of transferrin receptors was lower during flight on FD15, 30, 60, and 180 (only the value on FD30 remained significant after the post hoc adjustment) and on landing day. A significant interaction between space flight and sex occurred for heme (P = 0.005), in that women had a slightly lower heme concentration on landing day and men had a higher concentration, but these differences did not remain significant after the post hoc adjustment. When the data were analyzed with the statistical outliers in the model, the P value increased to 0.090.

No effects of sex or space flight on hepcidin, an important regulator of iron homeostasis, were detected. Ceruloplasmin, which is an acute phase protein, was significantly higher in women (P < 0.0004), but space flight had no effect on ceruloplasmin in either sex. CRP, an indicator of inflammation, was generally lower during flight in both sexes (P < 0.0004), but no significant differences remained on specific days compared with L-10 after the post hoc adjustment. Prealbumin, a negative acute phase protein, was significantly higher in men than in women (P < 0.0004). Prealbumin tended to increase during flight (P = 0.006), but after post hoc testing these values were not significantly different from the L-10 value.

Both hemoglobin and hematocrit were lower on landing day and 30 d after landing than on L-45, but only the decrease in hematocrit remained significant after adjustment for multiple comparisons. Hemoglobin was higher in men before and after flight (P < 0.0004), but mean corpuscular (red blood cell) hemoglobin did not change in response to space flight (Table 1).

Mean corpuscular volume and mean corpuscular hemoglobin concentration were significantly lower on landing day (P = 0.0006 and P = 0.007, respectively). These changes were not significant after post hoc analyses. Red blood cell distribution width was also lower on landing day and 30 d after landing (P < 0.0004).

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Special Edition on Space Food and Nutrition

Correlation of iron-status markers with markers of oxidative damage and bone loss Ferritin was negatively correlated with superoxide dismutase (r =-0.32, P = 0.008; Table 2), which meant that a higher ferritin concentration was associated with a lower superoxide dismutase activity. Ferritin was positively correlated with urinary 8OHdG (r = 0.54, P < 0.0004), PGF2Îą (r = 0.27, P < 0.0004), helical peptide (r = 0.52, P < 0.0004), NTX (r = 0.42, P < 0.0004), and calcium (r = 0.42, P , 0.0004). The percentage changes in ferritin and 8OHdG have remarkably similar patterns (Figure 1). Serum iron was positively correlated with total lipid peroxides (r = 0.37, P = 0.003), urinary 8OHdG (r = 0.21, P = 0.010), and PGF2a (r = 0.16, P = 0.040). The transferrin index was positively correlated with plasma lipid peroxides (P < 0.0004) and urinary 8OHdG (P < 0.0004), PGF2Îą (P = 0.002), helical peptide (P = 0.002), NTX (P < 0.001), and calcium (P < 0.0004).

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Special Edition on Space Food and Nutrition

STEM Today, September 2016, No.12

The AUC for the increase in ferritin during space flight was generated to quantify how much and how long ferritin was elevated during flight for each subject, and this was compared with changes in measures of BMD (Table 3). Negative correlations occurred between the AUC for ferritin and the percentage change from preflight in the BMD of hip (P = 0.031), trochanter (P = 0.006), hip neck (P = 0.044), and pelvis (P = 0.049). The higher the ferritin AUC, the more negative the percentage change from before flight, regardless of sex or type of exercise used during flight. Total BMD and lumbar spine BMD were not correlated with ferritin AUC.

Iron stores increased early during space flight and then returned to preflight concentrations by the end of 6-month missions. Transferrin and transferrin receptors decreased later during flight, which supports the idea that mobilization of iron to storage in tissues increased. The transferrin index tended to increase early during flight (FD15), which indicated that transferrin was more saturated during flight. At several time points during flight (FD15, FD60, and FD120), the transferrin index was >1 Âľmol Fe/Âľmol transferrin, which is considered to indicate iron overload.

Overall, the data support the idea that tissue iron stores increased during space flight, but an important question remains: are transient increases in iron stores a cause for concern? Although mean ferritin concentrations during flight were not outside the normal clinical range, the increase in ferritin was associated with evidence of oxidative damage and bone resorption.

Ferritin is well known to be an acute phase protein upregulated during an inflammatory response. The increase in ferritin early in space flight could have been caused by several factors, and one that cannot be ignored is inflammation. At the start of an inflammatory response , a rise in ferritin parallels a rise in CRP, and such a rise in CRP was not observed during flight; in fact, CRP decreased. Ceruloplasmin typically increases by 30-60% during an inflammatory response, but it did not change during flight. Prealbumin is a negative acute phase protein, which tends to decrease during infection and inflammation. Space flight data indicate that prealbumin tended to increase slightly during flight. A main effect of time was seen, but no time points were different from L-10 on post hoc testing.

Finally, the soluble transferrin receptor concentration is a sensitive indicator of tissue iron availability, and several studies indicate that it is not affected by chronic disease or inflammation. In pregnant women with tissue iron deficiency, with or without infection, the circulating transferrin receptor concentration increased. During space flight, transferrin receptors decreased early and continued to decrease later, which further suggests that inflammation was not responsible for the observed increase in serum ferritin.

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Special Edition on Space Food and Nutrition

STEM Today, September 2016, No.12

In this healthy astronaut population, ferritin, serum iron, and transferrin index were associated with markers of oxidative damage to DNA (8OHdG) and lipids (lipid peroxides and urinary PGF2Îą). The change in ferritin over the course of a 6-month mission was strikingly similar to the change in urinary 8OHdG during space flight (Figure 1). Astronauts are exposed to an environment in which multiple oxidative stressors are present (radiation, changes in oxygen, and rigorous exercise). Others have documented similar associations between iron status (ferritin) and markers of oxidative damage, including malondialdehyde, urinary 8OHdG, advanced oxidation protein products, and oxidized LDL in healthy individuals in the general population.

Furthermore, in an intervention study in which iron fortification of flour was tested for 8 and 16 month, ironrelated oxidative stress increased after exposure to a relatively small increase in dietary iron. Iron can be liberated from ferritin during periods of oxidative stress or exposure to some types of radiation. The observations that serum iron and transferrin index were also related to markers of oxidative damage support the involvement of iron in the types of oxidative damage represented by these markers, but we do not know whether the changes in oxidative damage are a cause or effect of the increase in ferritin.

The changes in iron stores during space flight were also related to markers of bone resorption and changes in BMD after flight. The greater the increase in ferritin during flight (or the longer it was elevated-either case would result in a higher AUC), the greater the decrease in BMD in the hip trochanter, hip neck, and pelvis after long-duration space flight. In support of this result are the correlations between ferritin or transferrin index and biochemical markers of bone resorption (helical peptide and NTX) or urinary calcium. Several other human and animal studies have also shown that iron overload is associated with bone loss by a mechanism believed to be related to oxidative stress.

Furthermore, the overall range was similar to the range for the 23 crew members (7-631 ng/mL), the data for whom are presented here. Although we know that consuming enough calories and exercising with the advanced resistive exercise device during space flight mitigate changes in BMD, it appears that the change in ferritin and overall iron stores during flight may be another important factor to consider when defining dietary intake recommendations for iron, and potentially for antioxidants, during space flight.

Another implication for changes in iron status during flight is related to immunity and infection. The virulence of an extensive list of pathogens and other microorganisms is enhanced by iron. In one study, women with the highest ferritin concentrations (>120 ng/mL) were less likely to clear human papillomavirus infection than were individuals with lower ferritin concentrations (<120 mg/mL), which suggests that elevated iron stores may increase the risk of persistent infections. This could have significant consequences for astronauts, considering that the documented dysregulation of the immune system, a known increase in bacterial pathogenicity and virulence during space flight, and the increase in iron stores could combine and lead to an overall increase in disease incidence.

The hepcidin peptide was recently characterized as one of the main regulatory hormones of iron absorption and recirculation, and it is induced during inflammatory processes and infections. An increase in circulating hepcidin is associated with a decrease in iron absorption and a decrease in release of iron from macrophages.

Circulating hepcidin did not change during or after flight, which suggests that, despite high iron intakes by astronauts, iron absorption may not have changed.

However, authors cannot rule out the potential for a change in iron absorption during flight because microgravity may affect the hepcidin response. One factor contributing to the increase in ferritin early during flight could be the high dietary intake of iron. A possible mechanism for reducing the elevated iron stores that occur early in flight is to decrease absorption until new red blood cell production and turnover normalize iron status.

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Special Edition on Space Food and Nutrition Another possibility is a shift to long-term iron storage in the liver or other tissues. This question of the mechanism responsible for the return to normal iron status cannot be answered with the current data set. Until further iron kinetics studies are conducted, it is recommended that iron intake during flight be lowered to levels closer to the current Recommended Dietary Allowance for iron on Earth.

The changes in hematocrit, mean corpuscular volume, and red blood cell distribution width observed at landing were most likely not direct effects of space flight. They were probably related to either dilution effects, in response to the replacement of lost plasma volume much faster than lost red blood cells, or to saline infusions received by many crew members soon after landing (to help mitigate orthostatic intolerance) and before collection of blood samples.

Nutritional Status Assessment in Semiclosed Environments: Ground-Based and Mir Space Flight Two types of studies were conducted, i.e., ground-based, semiclosed chamber studies and flight studies aboard the Mir space station. The semiclosed environment of each provided unique opportunities to examine the effect of a limited food system on dietary intake and nutritional status and to assess and implement means of monitoring dietary intake.

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Chamber Studies Subject:Subjects for the 60-d study were 1 woman and 3 men; subjects for the 91-d study were 2 women and 2 men. The ages of the 5 male subjects ranged from 26 to 36 y, and prechamber body mass ranged from 56.8 to 83.4 kg [body mass index (BMI) = 23.0 ± 3.4 kg/m2 , mean ± SD]. The ages of the 3 female subjects ranged from 28 to 41 y, and prechamber body mass ranged from 57.4 to 69.4 kg (BMI = 22.4 ± 3.3 kg/m2 ). All subjects were required to pass an Air Force Class III physical examination for clearance to participate in the study.

Flight Studies Subject: Two men aged 40 to 54 y with preflight body mass in the range from 70.5 to 88.6 kg participated in these studies. These ranges reflect data for all male astronauts (n = 6) who resided on Mir as part of the NASA Mir Science Program.

Chamber Studies Food systems: The food system for the 60-d study was designed to be similar to that planned for use on the International Space Station. Commercial products comparable to foods on the International Space Station Daily Menu Food List were located in local grocery stores and incorporated into a standardized menu that included fresh, frozen and thermostabilized items. Energy requirements were calculated for each subject based on the WHO equation, adjusted for moderate activity (specifically 1.7 for men, 1.6 for women).

Macronutrient contents of the standardized menu were calculated using the Daily Nutritional Requirements for Spaceflight. A 20-d cycle menu was repeated throughout each chamber test period. Although only foods from the menu were allowed, subjects were not required to eat exactly the planned menu. The menu was adjusted only when an item could not be supplied due to seasonal availability or some other reason. Food preparation equipment for this study consisted of two microwave ovens. A side-by-side refrigerator/freezer was available for food storage.

The food system for the 91-d study was developed in a similar manner, but it was designed to be similar to that planned for use on a planetary (e.g., Moon, Mars) base. Accordingly, during the 91-d study, the 20-d cycle menu consisted of a 50% vegetarian diet, defined as ≤4 servings of meat/wk. Additionally, an experimental diet was used for 10 d of the 91-d study (d 31-40). It consisted entirely of food items that could be produced in a regenerative food system.

During the 91-d study, food preparation equipment included a combination microwave/convection oven, a bread-making machine, a blender and a portable stove-top burner. A side-by-side refrigerator/ freezer was also available for food storage.

Flight Studies Food systems: The food system used on board Mir consisted of about half U.S. space foods and half Russian space foods. Because refrigeration was not available for food items, all foods were shelfstabledehydrated, thermostabilized (e.g., canned) or in natural form. Although a 6-d cycle menu was planned, actual eating patterns during flight rarely followed the scheduled menu. About once per mission, a cargo vehicle arrived with a limited number of fresh food items (e.g., fruits, vegetables). These items typically are edible for <1 wk.

Results Dietary assessment Chamber studies. Energy and protein intakes were similar for the 3 intake assessment techniques during both studies (Table 1). Week-by-week energy intake data are shown in Figure 1. Body weight did not change dur-

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ing the chamber studies (data not shown). During the 60-d study, questionnaire estimates of calcium and iron intakes were lower than the intakes determined from weighed diet records (Table 1). Subsequent analysis revealed that these differences were related to differences in nutrient content data for two foods (milk and cereal) between the nutrient databases used to analyze the weighed diet records and the FFQ. When the databases were synchronized for nutrient content of these food items, no differences were observed (data not presented). This problem was identified before the 91-d study began and was thus avoided in that study.

Sodium intake assessment yielded similar results for the three techniques during the 60-d chamber study. However, during the 91-d study, the 24-h FFQ sodium intake estimates were higher than those for the 7-d FFQ (Table 1). Water intake estimates during the 60-d study were different (P < 0.001) for all three assessment techniques.

Conversely, no differences were observed during the 91-d study.

Flight studies Energy intake estimated by the spaceflight FFQ was < 50% of predicted energy requirements for the two crew members studied (Fig. 2). These results are supported by data showing that postflight body weight was > 10%

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lower than preflight body weight (Fig. 3). Intake of other nutrients was also below recommended levels for spaceflight (Fig. 2), as would be expected from the reduced energy intake.

Biochemical assessment Chamber studies Biochemical results from the chamber studies are shown in Tables 2-4. Iron status tended to be negatively

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influenced throughout both studies, i.e., values for most hematologic variables (Table 2) tended to decrease. Serum ferritin was significantly (P < 0.05) lower at the end of the 91-d study than before the subjects entered the chamber; a similar trend was seen (P = 0.054) in the 60-d study. Folate levels, as assessed by the concentration of RBC folate did not change (P = 0.13) during the 60-d study and increased significantly during the 91-d study (Table 2). Vitamin B-6 and riboflavin markers were unchanged during the chamber studies. Serum 25hydroxyvitamin D declined steadily throughout the 91-d study; final concentrations were significantly lower than prechamber values (Table 3). A small but significant (P < 0.05) decline in serum calcium was noted at CD30, although all data obtained during the 91-d study were within clinical norms. Other indices of bone and calcium metabolism were generally unchanged in both studies (Table 3).

During the 91-d study, thiamin status, as assessed by erythrocyte stimulation of transketolase by thiamin pyrophosphate, did not change from prechamber levels. These data were not available for the 60-d study. General clinical chemistry and antioxidant-related measurements (Table 4) were relatively unchanged during the two chamber studies. For most of these variables, statistically significant differences generally were not clinically important. A very small, albeit significant decrease in serum sodium concentration occurred during the 60-d study, and serum sodium was elevated on CD40 during the 91-d study (Table 4). Serum total protein concentrations were decreased on CD30 and CD40, and returned to prechamber levels after the 91-d study. Glutathione peroxidase activity was elevated during the 91-d chamber study, but not during the 60-d study. There were no differences in serum albumin, creatinine, chloride, aspartate aminotransferase or alanine aminotransferase.

Urinary calcium and collagen crosslink excretion did not change during either of the chamber studies (data not presented).

Flight studies Biochemical results from the flight subjects are shown in Tables 2-4. The observed hematologic changes indicated a nominal response to spaceflight, with reduced hemoglobin and hematocrit, and increased serum ferritin

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Special Edition on Space Food and Nutrition (Table 2). Ferritin iron saturation was reduced after landing. Serum transferrin receptor concentrations tended to be lower after flight. No difference was observed between preflight and postflight measurements of serum calcium (either total or ionized) (Table 3). The level of vitamin D stores increased between the first and second preflight sessions, but was decreased after landing. Postflight urinary calcium and collagen crosslink excretions were higher than preflight excretions (data not presented). There was no apparent change (postflight compared with preflight) for serum albumin, creatinine, chloride, aspartate aminotransferase or alanine aminotransferase (data not presented).

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Red blood cell mass and iron metabolism are altered during spaceflight. Iron stores and tissue iron tended to increase during spaceflight, as indicated by increased serum ferritin concentrations and reduced transferrin receptors, respectively, in the present study. The ferritin iron results reported here are novel and intriguing. Ferritin iron saturation did not change in the 60-d chamber study, as expected (these analyses were not available for the 91-d chamber study). In the flight studies, however, ferritin iron saturation was reduced at landing. This suggests that the increase in ferritin observed after spaceflight may be related to an acute phase reaction and not necessarily to increased iron storage. Other indices of iron availability increased, confirming that the RBC mass reduction during flight is indeed not an iron-deficiency anemia. This phenomenon, observed in only two subjects, clearly requires further validation with additional subjects before conclusions may be drawn.

Body weight loss is a consistent finding during and after spaceflight and has been observed in both the Russian and U.S. space programs. The maintenance of body mass and composition in the ground-based chamber studies demonstrates that a food system like the one planned for the International Space Station can provide required energy and nutrients, assuming that the foods are consumed. Inadequate dietary intake is a significant concern during spaceflight and has been seen on Apollo, Shuttle and Mir missions. The two Mir crew members who participated in the flight studies reported here were found to have an energy intake <50% of predicted requirements. This decrease appeared to be the result of a generally reduced dietary intake, and not simply the lack of selection of a few high energy items. The implications of reduced food intake on essentially all nutrients are important; clearly, crew dietary intake must be maintained to ensure crew health and safety.

R

Mir Space Station Food

Excessive amounts of dietary iron in Mir Space Station Food The recommendations for both men and women to limit iron intake during spaceflight to <10 mg/d, both the U.S. and Russian space food systems currently provide excessive (>20 mg/d) amounts of dietary iron.

The FFQ data obtained during spaceflight confirmed that iron intake by crew members during flight was excessive. The involvement of iron in the formation of potentially toxic free radicals has been described, and the risks of increased iron stores in a high radiation environment are a concern for spaceflight[Smith et.al. (2001)].

R

Reduction in red blood cell mass

Detailed hematologic investigations had been conducted in support of selected flights in the Gemini Program.

Although the data collected were sparse and incomplete, certain trends were noted and are worthy of comment.

Radioisotope- derived plasma volume measurements, performed on the crew of Gemini 4, yielded calculated red cell mass deficits of about 12 percent after four days in orbit. Based on this observation, direct measurements of red cell mass were performed on the crew of Gemini 5 using a 51 Cr tag. The data derived from this study showed a 20 percent decrease in red cell mass following eight days in orbit, accompanied by an abnormally low red cell 51 Cr half-life in both pilots. These studies suggested that a hemolytic process was responsible for the observed red cell loss.

Affirmation of a hemolytic reduction in red cell mass was obtained from the crew of the 14-day Gemini 7

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Special Edition on Space Food and Nutrition mission. In this case, one pilot showed a modest decrease in red cell mass, whereas the other crewman lost 20 percent. Special hematology tests, accompanying the isotope studies, revealed that the reduction in red cell mass was associated with increases in mean corpuscular volume and osmotic fragility. Reticulocyte counts before and after the mission revealed no actual depression of bone marrow activity incident to flight; however, no reticulocytosis appeared until the fourth day after landing. These data imply that the erythropoietic mechanisms were insensitive to the red cell mass reduction which occurred over the 14-day interval. The red cell mass was recovered in both men by three weeks postflight.

Additional biochemical analyses of blood samples from returning Gemini crewmen reflected significant decreases in plasma alpha-tocopherol levels, total red cell membrane lipids, specifically the long chain of fatty acids of cephalin and lecithin, and red cell phosphofructokinase activity. All of these compounds influence red cell integrity. Indeed, the Gemini findings provided a new impetus in red cell investigation, with emphasis directed at the red cell membrane itself and not solely at intracellular enzyme systems.

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The Gemini findings formed the basis of a working hypothesis for the influence of space flight on red cell function and survival. This hypothesis, comparable to that of Jacob (1969) for microspherocyte formation, proved to be actually applicable only to the Gemini environment, but it strongly influenced both the selection and interpretation of test data of the earlier Apollo flights. As more information was collected in the later Apollo missions, it became apparent that the hemolytic damage characteristic of the Gemini flights was not the only hematologic consequence of space flight.

Hematology and Immunology Studies (AP007) Specific changes observed during the Gemini Program formed the basis for the major portion of the hematology-immunology test schedule for the Apollo program. These changes included cardiovascular deconditioning and substantial decreases in plasma volume and red blood cell mass. The hematology and immunology program conducted in support of the Apollo missions was designed to acquire specific laboratory data relative to the assessment of crew health prior to their commitment to space flight. A second, equally important objective was to detect and identify any alterations in the normal functions of the immunohematologic systems attributable to space flight exposure, and to evaluate the significance of these changes relative to manâ&#x20AC;&#x2122;s continuing participation in space flight missions.

The hematology analyses conducted in support of Apollo missions ranged from routine procedures (Table 1) intended primarily to provide basic information to the crew surgeon to more specialized tests (Table 2) designed to elucidate the effects of space flight on the normal functioning and integrity of the red blood cell. For the most part, standard laboratory techniques were employed. Some specific procedures are discussed in more detail in

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Special Edition on Space Food and Nutrition the text or are referenced where details are necessary for a more complete comprehension of the results. Blood samples were obtained by venipuncture beginning approximately 30 days prior to launch. No blood samples were acquired during the inflight phase of the missions.

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The first postflight sample was collected onboard the recovery vessel within one to three hours after splashdown, after which sampling continued for about two weeks. A typical blood sampling schedule for an Apollo mission is illustrated in Table 3. The logistics involved with the postflight quarantine of Apollo 11, 12, and 14 made it impractical to perform some of the analyses on those missions.

Routine hematological data from the Apollo missions are summarized in Table 4. Details concerning some of the results mentioned here are presented in subsequent sections of this chapter. There were no changes in RBC count or hematocrit following the flights. However, there was a modest (significant in one-third of the crewmen) elevation in the hemoglobin concentration, resulting in an increase in the calculated mean corpuscular hemoglobin (MCH) and the MCH concentration (MCHC) immediately postflight in those crewmen.

Determinations of concentration-dependent parameters were complicated during the recovery day (R+0) examination by the often inadequate laboratory facilities on the recovery vessel and by the changes in red cell mass and plasma volume which occurred during the mission. There were rapid postflight shifts in body fluid compartment sizes, which influenced particularly the plasma volume.

In contrast to the Gemini findings (Fischer et al., 1967) a slight, but statistically significant, reduction in the reticulocyte count was observed at R+0. The significance of this finding relative to changes in blood volume is discussed in the next section.

There was a postflight (R+0) leukocytosis generally associated with an absolute neutrophilia and a complicated lymphocyte response. This finding was also consistently observed during the Gemini missions. In all cases, the changes in the white blood cell count and differential were transient and reverted to normal within 24 to 48 hours after flight. The elevations in the neutrophil count were modest.

In most crewmen, elevations did not exceed the 10000 count required for the classical definition of neutrophilia.

While these changes were possibly a consequence of increased blood epinephrine and/or steroid levels associated with mission stresses, they were highly variable among individuals.

It should be noted that none of the changes observed in hematologic parameters were outside accepted normal ranges, and therefore were not indicative of significant medical events.

Measurement of red cell mass was of particular interest in the early Apollo flights because of the significant decreases observed in red cell mass during the Gemini flights. The procedures used to measure red cell mass and plasma volume have been reported previously (Fischer et al., 1967; Johnson et al., 1971). The red cell mass loss in the first two Apollo flights was negligible in five of six crewmen tested.

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Special Edition on Space Food and Nutrition This deviation from the pattern of the Gemini crewmen was attributed to a change in the Apollo spacecraft atmosphere composition at launch - from 100 percent oxygen in Gemini to a 60 percent oxygen 40 percent nitrogen mixture at 346 x 102 N/m2 (260 torr) in Apollo. Therefore, the Apollo 7 and 8 missions were characterized by an oxygen concentration of less than 100 percent during the entire flight interval, though it did approach the 95 percent level by the end of the flight.

On the Apollo 9 mission, the crew opened the spacecraft hatches to perform extravehicular activity. Even though denitrogenation began at the time of repressurization and the crew lived in 100 percent oxygen for the next five days, only a seven percent mean decrease in crew red cell mass was observed. This was a significant, but not dramatic, change.

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However, the crew was not denitrogenated before the mission in the manner of the crews in the Gemini Program. Gemini crewmen breathed 100 percent oxygen at 101 x 102 N/m2 (760 torr) for three hours before the mission, again on the launch complex for several hours before lift-off, and then proceeded with a mission in which a 100 percent oxygen, 346 x 102 N/m2 (260 torr) atmosphere was used. Thus, the Apollo 7 and 8 oxygennitrogen profiles differed considerably from those of the Gemini missions. The atmosphere profile of the Apollo 9 mission was also different, but it was somewhat similar to the Gemini type atmosphere profile during the later stages of the flight.

The results of Apollo 9 and subsequent chamber study at Brooks Air Force Base (Larkin et al., 1972) seemed to confirm the hypothesized toxic effect of oxygen on the circulating red blood cells. These data were integrated into a hypothesis that hyperoxia (even at low atmospheric pressures) can induce the loss of red cell mass by inhibition of red cell production and/or increased destruction of circulating red cells. The details of this hypothesis, which have been reported (Fischer & Kimzey, 1971; Fischer, 1971), are summarized in Figure 1.

Hyperoxia can cause peroxidation of red cell lipids (all membrane-bound), resulting in one or both of the fol-

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Special Edition on Space Food and Nutrition lowing: (1) the plasma vitamin E and vitamin A levels can be reduced by virtue of the fact that these sterols are lipid antioxidants and are consumed in this type of reaction, and (2) peroxidated lipids can physically compromise red cell membrane integrity. Lipid peroxides are very effective and efficient red cell membrane sulfhydryl group inhibitors, as is oxygen directly.

Thus, if red cell lipid peroxides were formed, inhibition of red cell membrane sulfhydryl groups would be expected. The sulfhydryl groups are essential in maintaining the integrity of passive red cell membrane cation transport. If active cation transport is poisoned by the same mechanism, one would observe osmotic swelling of red cells resulting in attainment of critical volume and lysis. Altered active and passive transmembrane cation transport may, therefore, be occurring simultaneously. If the integrity of the red cell membrane is disrupted, changes in shape and/or compliance of the membrane will result in the cellâ&#x20AC;&#x2122;s removal by the reticuloendothelial system (RES).

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On Apollo 9, the sodium-potassium flux in the red cell was measured before the mission, immediately afterward, and one day after recovery. The procedure used has been described previously (Larkin & Kimzey, 1972).

The controls showed essentially no change, but a significant reduction in the active component (as defined by ouabain inhibition) of potassium flux was observed in the oxygen-exposed flight personnel. This change would compromise the osmoregulatory capacity of the cells, making them more susceptible to osmotic hemolysis. No changes in cation flux were observed on Apollo 10, a mission with a normal oxygen/nitrogen profile.

The Apollo 9 mission was characterized by other changes consistent with the proposed hypothesis; specifically, (1) a reduction in plasma vitamin E and vitamin A levels, (2) a decreased phosphofructokinase activity, (3) a reduction in total red cell lipids, especially lecithin, and (4) abnormal red cell morphology characterized by acanthrocytoid cells, spherocytes and schistocytes (Fischer & Kimzey, 1971).

No measurements of red cell mass were made on Apollo flights 10 through 13 due to operational constraints imposed by the quarantine requirements. On Apollo 14, small but significant red cell mass losses were observed postflight. The mean decrease of 4.7 percent is greater than the changes found in Apollo 7 (-3.4 percent) and Apollo 8 (-1.4 percent), but less than the -7.2 percent after Apollo 9. The Apollo 14 data are somewhat misleading since one crewman had no loss of red cell mass during the flight.

A significant decrease in red cell mass (-10 percent) was measured after Apollo 15. The red cell loss during this mission was more than half recovered by the R+13 examination. The atmosphere to which the Apollo 15 crew was exposed was also higher in oxygen due to a more rapid than nominal leak rate early in the flight, an extended stay on the lunar surface, and extravehicular activity during the transearth coast.

On Apollo 16, as in other similar missions, there was a decreased red cell mass postflight when compared to preflight (F-15) values. If the Apollo 16 results are compared with data from previous missions, authors find that the percent changes in red cell mass of the three crewmembers (average of -14.2 percent) were greater than 15 of 16 other Apollo crewmembers.

This loss had not been recovered by R+7. When expressed as milliliters per kilogram of body weight, the red cell mass change was greater after Apollo 16 than in all previous Apollo missions. It would appear from data collected on the Apollo flights, that the crewmen judged to be in the best physical condition (based on their exercise testing performance) exhibited the greatest loss of red cell mass.

The crew of Apollo 17 showed an 11 percent decrease in red cell mass at recovery. One week later at R+8 the red cell mass was still nine percent below the control values of F-15 When the red cell mass is corrected for body weight loss, the decrease was seven percent at recovery. The changes in this crew were approximately the same as in crews of the other lunar flights.

Changes in plasma volume following space flight have been more variable, but with a general tendency to be reduced following the Apollo flights. The rapidity with which the plasma volume can equilibrate, combined with the varying length of time following recovery at which the plasma volume was measured and the less than optimal conditions for these tests on the recovery vessel, make these results somewhat less meaningful relative to the inflight condition. Nevertheless, the reduction in plasma volume after space flight might be expected based on similar studies of subjects during comparable periods of bed rest (Hyatt, 1969).

In contrast to the Gemini flights, the red cell survival (as measured by the 51 Cr half-life) was not significantly

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altered during the inflight or postflight phases of the Apollo flights.

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To summarize, Table 5 compares the percent change in red cell mass, plasma volume, and red cell survival of the crews of the Apollo and Gemini missions in which these studies were performed. The red cell mass decrease of the Apollo 7 and 8 crews was significantly less than the decrease after the lunar missions 14 through 17. The flight duration of the Apollo 7 and 8 missions was less than the average duration of the moon landings; however, it is improbable that flight duration was the reason for the difference since large red cell mass decreases were found after the shorter Gemini 5 mission.

Apollo 7 and 8 also differ from other Apollo missions in that the Lunar Module purged the Command Moduleâ&#x20AC;&#x2122;s atmosphere of nitrogen. After that maneuver, the Apollo atmosphere was equivalent to a Gemini atmosphere. Small amounts of residual nitrogen were present throughout Apollo 7, the only mission in which atmosphere composition was measured. The difference between these two types of missions was further evidence to support the concept that a nitrogen-free atmosphere was the cause of the red cell mass decreases. The red cell survival as measured by 51 Cr half-life was not shortened to the extent found in three of four Gemini crewmen, suggesting that hemolysis did not occur or was very slight. While it was not possible within the framework of the Apollo Program to test this hypothesis extensively, all of the Apollo, Gemini, and supporting ground-based studies can be ranked according to the mean red cell mass loss that was measured in the subjects (Table 6).

These data include the percent loss, the atmosphere composition, the number of subjects, and the exposure duration. What is noteworthy is that anytime a 100 percent oxygen atmosphere was used, significant red cell mass loss occurred. However, if a diluent gas was present, no significant red cell loss was observed.

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The initial hypothesis (Figure 1) predicted an intravascular hemolysis of the cells as a result of failure to maintain osmotic balance. Based upon additional data collected in support of the Apollo Program, this hypothesis may need to be modified. The consistent elevation of haptoglobin in all of the crewmen following Apollo flights is inconsistent with intravascular hemolysis. Red cell survival was not significantly shortened in the Apollo flights, and this finding does not support the concept of intravascular hemolysis.

It is possible that the alteration of red cell membrane lipids and/or sulfhydryl groups would alter the cellsâ&#x20AC;&#x2122; structural configuration leading to fragmentation of cells and their subsequent destruction by the reticuloendothelial system. Shape changes have been observed in red cells collected inflight (Kimzey et al., 1974).

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However, the lack of any change in the 51 Cr survival time suggests that the loss may not be due to red cell destruction at all, but to a reduction in the production of cells. Regardless of the exact cause of the red cell mass decrease, compensatory erythropoiesis B not evident. There are data from later flights to suggest that initiation of the recovery of red cell mass after completion of the mission may be delayed for up to two weeks (Johnson et al., 1974; 1975). In order to account for the loss seen in some of the Apollo flights, red cell production would have to be totally inhibited for the duration of the flight (assuming a normal loss of approximately one percent per day), and even this could not account for the large loss in the Gemini missions. It is obvious that the exact mechanism of this red cell mass loss has not been established; oxygen undoubtedly is a contributory agent, but is probably not the only one.

The Skylab missions differ from the Apollo missions by not having hyperoxic environment except for 2 hours of 100 percent oxygen at 1.0132 X 102 kPa (760 mm Hg) prior to launch and for a few hours during the first day when the atmosphere was similar to that of Apollo. The Skylab missions have afforded an opportunity to rule out the hyperoxic hypothesis of the red cell mass decrease while at the same time testing whether changes in red cell mass are progressive with longer periods in weightlessness.

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Table 26-II shows the red cell mass volume values obtained from the nine crewmembers and the nine control subjects. These are presented as total red cell mass (milliliters) and on a milliliters per kilogram body weight basis. The mean value of the premission red cell mass of the crewmembers was 2075 milliliters which is not different from the mean values of the controls, 2053 milliliters. The mean values of the red cell mass/kilogram body weight was 28.9 milliliters/kilogram for the crew and 27.2 milliliters/kilogram for the controls. These mean values are not different statistically. The recovery mean value of the crew, 1843 milliliters, was different from their preflight mean value and different from the controls postflight mean of 2046 milliliters (P<0.05).

The crewmembers showed a mean value decrease of 232 milliliters and the controls showed a decrease of 7 milliliters. Calculated on a milliliters/kilogram body weight basis, the crewâ&#x20AC;&#x2122;s postmission mean value was 26.6 milliliters/kilogram body weight or 2.3 milliliters less than premission while the controls did not change from the premission value of 27 milliliters/kilogram body weight.

Evidence against a hemolytic process is presented in Table 26-III where the 51 Cr red cell T

1 2

preflight and

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postmission values and the 14 C glycine red cell life span mean values are shown. There is no difference of statistical significance between the preflight and postflight crew mean values or the crew and control mean values either for the 51 Cr T 21 or the 14 C-glycine red cell mean life span.

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Table 26-IV shows the iron turnover results. The 0.32 milliliters/kilogram body weight per day for the crew is similar to the 0.30 milliliters/kilogram body weight per day for the control subjects. Statistical analysis indicates no difference between controls and crew in reapparance or turnover indicating that the rate of erythropoiesis was essentially the same for crewmembers and control subjects.

Table 26-V shows the reticulocyte counts arranged according to mission. These are shown as the number of reticulocytes per cubic milliliters of blood X 10â&#x2C6;&#x2019;3 . The reticulocyte counts were low when drawn at recovery following each mission. Postmission reticulocyte counts greater than pre-mission means were found in only one crewmember of the 28-day mission at 2 weeks, the three crewmembers of the 59-day mission at 1 week, and at 1 week or less for the crew of the 84-day mission.

These results indicate that red cell mass regeneration did not occur until 14 or more days after recovery from the shortest mission. The control subjects did not develop a change in the reticulocyte count at any time indicating that reticulocyte changes found in the crewmembers were not caused by the blood drawing schedule.

The plasma volume changes are shown in table 26-VI for the nine crewmembers of the three manned Skylab missions. The results are expressed as percent change from the premisson value. The values for the pilot of the first mission suggest that his plasma volume was artifically low when his premisson control was obtained.

Indeed, on the initial day preflight, his hematocrit was elevated and plasma proteins were higher than in later specimens. Therefore, we have assumed that his R + 67-day value is more representative of his normal level.

His data are expressed both ways.

The red cell mass results of the Skylab studies show that the crewmembers sustained a statistically significant decrease in circulating red cells. The decreases were not found among the ground based control subjects indicating that the blood drawn for the extensive metabolic studies did not cause the change. Additionally, the second red cell mass obtained from the crew prior to the 84- day mission showed that no decrease in red cell mass occurred prior to launch indicating that premisson preparations did not cause the change.

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Iron turnover immediately post recovery was normal. The depressed reticulocyte counts at recovery indicate inhibited reticulocyte release or accelerated loss of reticular material. The lowering of reticulocyte counts was greatest for the crew of the shortest mission and least for the crew of the longest mission. This suggests the 84-day crew may already have been in the recovery or replacement phase of red cell mass prior to their return from weightlessness. The red cell mass mean decrease found after the 28-day Skylab mission was greater than the mean results obtained from the Apollo crewmembers while the mean decrease found after the longest Skylab mission was less. The etiology of the red cell mass drop and lowered reticulocyte counts at recovery is unknown. The red cell mass is the most stable of the various blood constituents. Sudden drops in red cell mass are possible due to hemorrhaging or hemolysis. Gradual decreases are produced by inhibition of bone marrow activity, ineffective erythropoiesis or chronic hemorrhage. There was no clinical evidence of hemorrhage among the crews and haptoglobin levels have tended to be normal or elevated rather than suppressed indicating that intravascular hemolysis did not occur in Apollo or Skylab crews.

Iron reappearance data gave no clinical evidence to suggest ineffective erythropoiesis. The low reticulocyte counts are additional evidence against ineffective erythropoiesis. An age dependent loss of red cells is a possibility and would not be seen in the survival curves obtained if red cells greater than 30 days of age were sequestrated and destroyed selectively during the first few mission days. Loss of cells older than 30 days would not affect the results since the older cell did not contain the 14 C-glycine. Premature loss of older cells without intravascular hemolysis suggests red cell surface and shape changes. This was actually found since all crewmembers showed an increase in abnormal red cell shapes including crenated erythrocytes in the scanning electron microscope evaluation of their blood samples taken at the end of the flight. Hyperoxia through lipid peroxidation could cause the red cell shape changes. This may have been the cause of the red cell mass decreases found in Gemini and Apollo crewmembers. It could not explain the red cell mass decrease noted in the Skylab

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Special Edition on Space Food and Nutrition crewmembers. Therefore, other aspects of the environment must have caused this change. A possible but unproven explanation of this combination of abnormally shaped red cells and decreased red cell mass among the Skylab astronauts would be a change in splenic function during the mission. Hypersplenism could start early during the mission when the blood volume was relatively too large perhaps associated with the increased portal pressure and/or decreased portal flow. This would be consistent with the 2 or 3 days of nausea and loss of appetite reported by susceptible crewmembers.

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The crewmembersâ&#x20AC;&#x2122; reticulocyte counts were low at recovery indicating increased splenic removal of reticulum or decreased bone marrow production rates. A vitamin E deficiency is one cause of early reticulum loss, but inhibited bone marrow is more likely because the red cell mass stayed low. Bone marrow function would not increase to replace the lost red cells if oxygen delivery to the kidneys was maintained. Either hyperoxia or hyperphosphatemia could cause this by shifting the oxygen disassociation curve to the right. In this way net oxygen delivery to the tissues is increased making a lowered red cell mass adequate for tissue oxygen.

This mechanism helps account for the Skylab results since in-flight blood specimens showed higher phosphorus levels. The red cell mass decrease associated with space flight is not followed by a decrease in hemoglobin concentration since plasma volume decreases occur at the same time. The kidneys use both changes in hemoglobin concentration and oxygen delivery to modulate erythropoietin release. Thus, the decreased red cell masses of the Skylab crewmembers might not be followed by compensatory increases in erythropoietin until plasma volume increased.

Without increased erythropoietin, bone marrow activity would not increase and should appear inhibited until a new equilibrium is reached. Both hyperphosphatemia and the decreased plasma volume seem to explain the low reticulocyte counts found at recovery. At recovery iron turnover was normal indicating a possible rebound in bone marrow activity. The rapid expansion in plasma volume during that time could account for the normal iron turnover. The mean percent decrease in plasma volume of the crewmembers after Skylab 2 (28 days) was less than Skylab 3 (59 days) and Skylab 4 (84 days) but still greater than the Apollo results (-5 percent mean) indicating that the plasma volume does not return to normal even after prolonged spaceflight.

Control of red blood cell mass in Shuttle flight The effect of spaceflight on red blood cell mass (RBCM), plasma volume (PV), erythron iron turnover, serum erythropoietin, and red blood cell (RBC) production and survival and indexes were determined for six astronauts on two shuttle missions, 9 and 14 days in duration, respectively. PV decreased within the first day. RBCM decreased because of destruction of RBCs either newly released or scheduled to be released from the bone marrow. Older RBCs survived normally. On return to Earth, plasma volume increased, hemoglobin concentration and RBC count declined, and serum erythropoietin increased. Authors propose that entry into microgravity results in acute plethora as a result of a decrease in vascular space. PV decreases, causing an increase in hemoglobin concentration that effects a decrease in erythropoietin or other growth factors or cytokines. The RBCM decreases by destruction of recently formed RBCs to a level appropriate for the microgravity environment. Return to Earth results sequentially in acute hypovolemia as vascular space dependent on gravity is refilled, an increase in plasma volume, a decrease in hemoglobin concentration (anemia), and an increase in serum erythropoietin.

Main Medical Results of extended flights on space station MIR in 1986-1990 Blood alterations during a long stay in microgravity typically include development of transient erythrocytopenia. In the flights of PC-3 and PC-4 there was a decrease in erythrocyte counts (in the physician-cosmonaut from 3.6 X 1012 /l to 3.8 X 1012 /l), anisocytosis and hypochromy. The hematocrit decreased by 4-14%. Postflight studies several hours following landing performed by Legenkov et al. revealed a decrease in reticulocytes (from 32.8 X 109 /l to 21.4 X 109 /l), erythrocytes (average count decreased from 4.91 X 1012 /l to 4.68 X 1012 /l), hemoglobin concentration (decrease from 152 to 145 g/l) and hematocrit (decrease from 45 to 42%). Further declines occurred during the first 7 days following flight as body fluids were restored and symptoms of body hypohydration abated: erythrocyte counts fell to 4.1 X 1012 /l, hemoglobin concentration to 131 g/l and hematocrit to 38.5%. In this case there was the rapid onset of reticulocytosis and counts increased to 60.8 X 109 /l, i.e. an 85% increase. Hemoglobin mass at R + 7 decreased from 738 to 589 g, i.e. fell 20% compared to preflight baseline values. The reticulocytosis associated with readaptation gradually normalized over the following 1.5-3 month postflight period. By this time there was practically complete restoration of all remaining "red blood" parameters.

The physician-cosmonaut displayed changes in erythrocyte shape during flight: target cells, ovalocytes, and spherocytes. Target cells were also found in other crewmembers of PC-3 (year-long flight) and PC-4. The target cells disappeared after a period following landing.

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Red Blood of Cosmonauts during Missions Aboard the International Space Station (ISS) The aim of the experiment was the discovery of new data on the influence of Space Mission(SM) factors on the red blood system in order to expand diagnostic and prognostic opportunities, identification of the mechanisms of the shift of hematological parameters ("space anemia"), and development of recommendations on the application of prophylactic and pharmacological preparations correcting the negative influence of the SM conditions and early period of readaptation to the earth conditions.

STEM Today, September 2016, No.12

Investigation of the studied parameters of the Russian crew members of the main expeditions (MEs) on the International Space Station (ISS) (from the 6th to the 12th) was carried out before the mission under the conditions of SF and after. A background investigation was carried out 30 days before the start; under the conditions of the mission (during a change in the crew), namely, at the beginning (days 6-10) and the final stage (days 160-190); and after the mission, on days 0, 7, and 15 of the readaptation period.

The results of the hemoglobin concentration are shown in the table. Three of the tested cosmonauts had a tendency for an increase in the hemoglobin concentration under the conditions of SM on the seventh day as a result of an increase in the hemoconcentration related to the higher dehydration of the body. The concentration of hemoglobin decreased in comparison with the background data at the final stage of SM, which was also noted

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Special Edition on Space Food and Nutrition during the period of the readaptation of cosmonauts to the conditions of the earth on the 7th and 15th days of the postmission period. There were no statistically significant changes in this parameter.

STEM Today, September 2016, No.12

The study of iron metabolism parameters has shown that the concentration of serum iron has undergone greater changes in all the cosmonauts shown in the table. The data in the table show that the level of iron in blood decreased in comparison with the background data both during SM and the readaptation period at the individual analysis of received results; this change was observed also on the 7th and 15th days of the rehabilitation period.

At the same time, statistically significant changes in this indicator were not recorded.

The study of the erythropoietin (EPO) concentration (see table) did not show distinct changes, which did not allow statistical data treatment to be carried out. In two of the three cosmonauts under the conditions of SM at the beginning of mission, the level of EPO increased, but in one of them, it did not change. The considerable increase in the EPO concentration in one of the four cosmonauts at the final stage of SM was observed, with the other parameters being held unchanged. An increase in the EPO concentration was observed on day 0 and continued until day 7. It should be emphasized that the results of the study did not show any significant decrease in the EPO level under the conditions of SM. However, despite insufficient results and their considerable individual variation, the following conclusion can be drawn. A decrease in the hemoglobin concentration under normal physiological conditions results in a decrease in the oxygen supply to the tissues and development of tissue hypoxia, which enhances the EPO production, affecting renal sensors.

This situation was observed in cosmonauts during readaptation. The reduced iron concentration during this period may be related to an enhancement of the processes of hemoglobin production. The regulation of the process of hemoglobin production in microgravity under the SM conditions, at a lower oxygen demand of the body is probably related to other mechanisms, and the decrease in the iron concentration in this case may be caused by its insufficient assimilation. The amounts of hemoglobin and erythrocytes during the readaptation period, despite the intensification of erythropoiesis, remain lower than the background values.

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Similar results have been observed upon examination of cosmonauts after a long SM on board the orbital station (OS) "Mir". Activation of erythropoiesis is a complicated process mediated by the activation of the sympathoadrenal system, erythropoietic function of macrophages of bone marrow, and simultaneous removal of old and lowquality erythrocytes, which result in an increase in the sensitivity of bone marrow to erythropoietin and stimulate erythropoiesis.

STEM Today, September 2016, No.12

The decrease in the level of erythrocytes and hemoglobin concentration can be related to the early removal of part of the morphologically changed erythrocytes from the bloodstream. Elimination of erythrocytes occurs by the activation of the macrophage system found in cosmonauts on the OS "Mir" and in experiments on antiorthostatic hypokinesia (ANOH).

A study of the cytoarchitectonics of erythrocytes is shown in Fig. 1. The study’s results have shown a statistically significant decrease in the percentage of the normal forms of erythrocytes (discocytes) on days 7-10 of SM. The tendency to normalization begins from day 7(background value, 92 ± 0.6; 86.0 ± 0.01 on days 7-10 of the SM; 82.0 ± 2.29 on days 160-190 of the SM; 84.02 ± 1.92 immediately after the SM; and 87.5 ± 1.95 and 90.0 ± 0.67 on days 7 and 15 after the SM, respectively). The decrease in the amount of discocytes under the conditions of SM occurs due to a statistically significant increase in irreversible cell forms, stomatocytes and knizocytes. It is known that the shape of the cells and their deformability are related to the intracellular metabolism and the state of the plasma membrane. The results of the investigation of erythrocyte metab olism are evidence that a considerable decrease in the ATP level (from 4.15 ± 0.07 in norm until 1.94 ± 0.56 and 2.18 ± 0.32 under the conditions of SM) (Fig. 2) occurs at the beginning and the final stage of an SM. The decrease in the concentration of this nucleotide under SM conditions has been observed in crews members numbers 1517 on OS "Mir".

Probably, this decrease is caused by changes at the membrane level, which is confirmed by the data on the presence of transformed forms of erythrocytes. All these may be related to the predominance of the population of old red blood cells in the bloodstream. The study of the lactate concentration has shown its increase at the final stage of SM, which agrees with earlier data (Fig. 2) and indicates a domination of anaerobic processes in the body. A less increased concentration of lactate is retained in the readaptation period (RP). The decrease in the concentration of reduced glutathione at the beginning of SM, an increase by the end of SM, and than a clear decrease in this parameter on day 7 of the RP (from 7.59 ± 0.53 in the normal state to 6.55 ± 0.27) was noted in a study on its concentration. Glutathione is an important cell metabolite with antioxidant properties.

It was demonstrated earlier, upon examination of crews aboard the OS "Mir" that a significant decrease in the level of reduced glutathione is correlated with an activation of lipid peroxidation (LPO), as well as with a decrease in the antioxidant system activity and in the concentration of tocopherol. The data obtained upon the investigation of lipid and phospholipid spectra of the membranes of erythrocytes shown in Fig. 3 provide evidence for the possible intensification of LPO processes under the influence of SM factors. Substantial changes in the relative concentration of free cholesterol (FC) were not found under the conditions of the mission; however, a tendency towards a decrease in the relative concentration of phospholipids (PLs) was noted, which somewhat increased the FC/PL coefficient.

The decrease in the PL percentage concentration continued during readaptation and part of the FC increased, which resulted in an increase in the coefficient FC/PL, with the peak of this increase appearing on the 7th day of the rehabilitation period on Earth (1.02 ± 0.01 in norm until 1.23 ± 0.003). The tendency to normalization was noted on the 15th day. An increase in this parameter is evidence of increased microviscosity in the membrane, which may result in the disturbance of its permeability, changes in the activities of membranebound enzymes, and a decrease in the cell deformability and, hence, blood viscosity.

A statistically significant increase in phosphatidilcholine (PC) was found during investigation of the fractional

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Special Edition on Space Food and Nutrition composition of PL on the seventh day after SM, and the percentage of phosphatidylethanolamine (PEA) free oxidizing fraction, which is a substrate for LPO processes, was lower. A statistically significant increase in the percentage lysophosphatidylcholine (LPC) concentration was found during the same period, which also indicates a possible increase in the cell membraneâ&#x20AC;&#x2122;s rigidity.

Thus, the shifts in the metabolic state of red blood cells found during SMs, the presence of destabilization of the cell membrane according to the data on lipid and phospholipid compositions, and the appearance of trans formed forms of erythrocytes along with the lack of a clear decrease in erythropoietin are evidence of the possible domination of the population of old or lowquality erythrocytes with a shortened life span, which are subject to early elimination in the bloodstream. The current data in the literature and our studies undertaken under the conditions of SM are not extensive and are characterized by considerable individual variability, which do not allow an unambiguous conclusion on the state of erythropoetic activity of the bone marrow and the mechanisms of decreasing the amount of erythro cytes and hemoglobin concentration under the influence of SM factors.

The morphological and biochemical alterations found in erythrocytes suggest disturbance of the gas transport function of the red blood. It is known that the shifts in the physicochemical properties of the plasma membrane of erythrocytes (microviscosity and perme ability) influence both the efficiency of oxygen molecule transfer into the cell and the state of membranebound hemoglobin, namely, changes in the hemoporphyrin conformation and amount of complexes with oxygen and NO.

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Effects of Iron Overload

Effects of Radiation and a High Iron Load on Bone Mineral Density Astronauts on long duration space flight missions to the moon or mars are exposed to radiation and have increase iron (Fe) stores, both of which can independently induce oxidative stress and may exacerbate bone mass loss and strength.

Authors hypothesize a high Fe diet and a fractionated gamma radiation exposure would increase oxidative stress and lower bone mass. Three mo-old, SD rats (n=32) were randomized to receive an adequate Fe diet (45 mg Fe/kg diet) or a high Fe diet (650 mg Fe/kg diet) for 4 wks and either a cumulative 3 Gy dose (fractionated 8 x 0.375 Gy) of gamma radiation (Cs-137) or sham exposure starting on day 14. Elisa kit assessed serum catalase, clinical analyzer assessed serum Fe status and ex vivo pQCT scans measured bone parameters in the proximal/midshaft tibia and femoral neck. Mechanical strength was assessed by 3-pt bending and femoral neck test.

There is a significant decrease in trabecular bone mineral density (BMD) from radiation (p<0.05) and a trend in diet (p=0.05) at the proximal tibia. There is a significant interaction in cortical BMD from the combined treatments at the midshaft tibia (p<0.05). There is a trending decrease in total BMD from diet (p=0.07) at the femoral neck.

In addition, high serum Fe was correlated to low trabecular BMD (p<0.05) and high serum catalase was correlated to low BMD at all 3 bone sites (p<0.05). There was no difference in the max load of the tibia or femoral neck. Radiation and a high iron diet increases iron status and catalase in the serum and decreases BMD[Zwart et.al.(2012)].

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Iron status and its relations with oxidative damage and bone loss Increases in stored iron and dietary intake of iron during space flight have raised concern about the risk of excess iron and oxidative damage, particularly in bone.

Subjects (n = 23; 16 men and 7 women) were astronauts on ISS expeditions 14-27 (missions of 50-247 d in duration; mean ±SD duration: 157 ± 46), which were flown between 2006 and 2011. The male and female subjects were 48 ± 4 and 45 ± 4 y of age and weighed 85 ± 9 and 62 ± 4 kg.

Serum ferritin and body iron increased early in flight, and transferrin and transferrin receptors decreased later, which indicated that early increases in body iron stores occurred through the mobilization of iron to storage tissues. Acute phase proteins indicated no evidence of an inflammatory response during flight. Serum ferritin was positively correlated with the oxidative damage markers 8-hydroxy-2’deoxyguanosine (r = 0.53, P < 0.001) and prostaglandin F2α (r = 0.26, P < 0.001), and the greater the area under the curve for ferritin during flight, the greater the decrease in bone mineral density in the total hip (P = 0.031), trochanter (P = 0.006), hip neck (P = 0.044), and pelvis (P = 0.049) after flight.

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Increased iron stores may be a risk factor for oxidative damage and bone resorption [Zwart et.al.(2013)].

Iron status and insulin resistant Inflight insulin resistance resulting from six-months spaceflight in Astronauts Authors hypotheses that stiffness of arteries located above the heart would be increased post-flight, and that blood biomarkers inflight would be consistent with changes in vascular function.

Possible sex-differences in responses were explored in four male and four female astronauts who lived on the International Space Station for six-months.

Carotid artery distensibility coefficient (P = 0.005) and SS-stiffness index (P = 0.006) reflected 17-30% increases in arterial stiffness when measured within 38 hours of return to Earth compared to pre-flight. Spaceflight by sex interaction effects were found with greater changes in β-stiffness index in women (P = 0.017), but greater changes in pulse wave transit time in men (P = 0.006). Several blood biomarkers were changed from pre-flight to inflight, including an increase in an index of insulin resistance (P < 0.001) with a spaceflight by sex term suggesting greater change in men (P = 0.034).

Spaceflight by sex interactions for renin (P = 0.016) and aldosterone (P = 0.010) indicated greater increases in women than men.

Six months spaceflight caused increased arterial stiffness. Altered hydrostatic arterial pressure gradients as well as changes in insulin resistance and other biomarkers might have contributed to alterations in arterial properties included sex-differences between male and female astronauts [Hughson et.al.(2016)].

Development of insulin resistance by astronauts during spaceflight Human spaceflight is associated with the loss of body protein. On Earth, insulin is an important factor in the regulation of muscle protein synthesis and breakdown. The objectives of this study were to determine whether insulin resistance occurs in spaceflight, and if the development of insulin resistance is related to the protein loss. The urinary C-peptide excretion rate was used as a marker for insulin secretion. The experiment was conducted before, during and after the 1991 9.5-d SLS-1 (Columbia) Space Shuttle mission. Dietary intake and urine output were monitored continuously for the four payload crewmembers from 11 d before launch to 7 d after landing for a total of 27 d.

Data were obtained on the four payload crewmembers. Results were as follows: 1) the mean inflight C-peptide excretion rates were significantly lower than either the pre- or postflight rates (p < 0.05); and 2) the inflight nitrogen balance decreased as C-peptide excretion increased [Stein et.al.(1994)].

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Iron overload and infectious diseases

STEM Today, September 2016, No.12

Iron is absorbed in the duodenum by enterocytes and transferred to the plasma. There it is bound by the highaffinity iron-binding glycoproteins - transferrin and structurally related protein lactoferrin. Liver parenchymal tissue is especially rich in transferrin receptors and stores large quantities of iron. In muscle tissue, iron is used to make myoglobin, and in bone marrow, erythrocytes use it to make hemoglobin. Circulating red blood cells normally comprise the largest iron storage pool. When they become senescent, red blood cells are engulfed by reticuloendothelial macrophages, which make their iron available for redistribution to other tissues via transferrin. Transferrin has extremely high affinity for iron. This coupled with the fact that two-thirds of the iron binding sites of the protein are normally unoccupied, essentially eliminates free iron from plasma and extracellular tissues. Although there is an abundance of iron present in body fluids, the amount of free iron is very small to sustain bacterial growth. In addition, during infections the host reduces the total amount of iron bound to serum transferrin. Withholding iron from potential pathogens is an important host defense strategy.

Moreover both transferrin and lactoferrin are bacteriostatic in vitro for a number of bacteria. Lactoferrin is a prominent component of the granules of polymorphonuclear leukocytes. The protein is released at high concentrations by the cells in areas of infection. However, despite these host mechanisms, pathogenic bacteria manage to multiply successfully in vivo to establish an infection. In contrast, a state of iron excess in hereditary hemochromatosis has different implications, since it involves preferential iron loading of the parenchymal cells and not the reticuloendothelial system, which in turn hinders the growth of many intracellular organisms including Mycobacterium tuberculosis, Salmonella typhi, and Chlamydia pneumoniae.

NOTE: A state of iron excess in astronauts is compared with hereditary hemochromatosis Acquisition of iron by microorganisms To obtain host iron, successful pathogens with a few exceptions, use one or more of the following strategies to adapt to the iron-restricted environment: (1) proteolytic cleavage of iron-binding glycoprotein, releasing iron; (2) reduction of Fe+++ complex to Fe++ complex and release of Fe++ from the glycoprotein through interaction between receptors on the bacterial cell surface and the Fe+++ glycoprotein complex, in a manner analogous to the reaction between transferrin and erythrocyte; or (3) by producing low molecular weight iron-chelating compounds known as siderophores, which are able to remove iron from Fe+++ glycoprotein complex and deliver it to the bacterial cell. The list of infectious disease agents whose virulence is enhanced by iron continues to increase (Table 1). These pathogens include bacteria (Gram-negative and Gram-positive), fungi, and viruses.

Escherichia coli has been extensively studied and found to have the ability to obtain iron via siderophores.

E. coli specifically is known to secrete enterobactin under conditions of iron restriction. Similarly Vibrio has the ability to acquire iron from its host by the production of siderophores and by degradation of heme-containing compounds by proteolytic enzymes. In contrast, Yersinia species are not able to produce their own siderophores, but possess receptors for iron-siderophore complexes on their surface, which enable them to utilize siderophores synthesized by other bacteria. Therefore when patients with iron overload are treated with chelating agents like deferoxamine (a siderophore produced by Streptomyces pilosus), Yersinia may use this exogenous siderophore to acquire iron. Listeria monocytogenes as a pathogen requires iron for growth within phagocytic cells and virulence expression. Weakened cellmediated immunity fromiron overload is also likely linked to the increased

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Special Edition on Space Food and Nutrition virulence of L. monocytogenes in patients with hemochromatosis. Various studies, including experimental studies in mice, have suggested that excess iron may enhance the growth of M. tuberculosis and worsen the outcome of human tuberculosis. The association of Tropheryma whippelii with iron overload states has also been described. Plesiomonas shigelloides has been shown to produce a betahemolysin, which is thought to play a role in iron acquisition in vivo via the lysis of erythrocytes. Gemella haemolysans, which is a member of the genus Gemella, previously included under Neisseria species, may hypothetically, like certain isolates of Neisseria, usehemin or hemoglobin as an iron source, or produce siderophores.

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Data on the ability of fungal pathogens to sequester iron in vivo are not as well described and neither is this aspect well researched. From what we do know, Rhizopus species have been recognized in patients who have undergone treatment of iron overload with deferoxamine, and iron overload in the absence of deferoxamine therapy has also been linked with zygomycosis. Interestingly diabetic ketoacidosis and zygomycosis have been associated with iron metabolism; acidosis may lead to decreased binding of iron by transferrin and hence release of iron to be used by Rhizopus species. Aspergillus fumigatus survival in human serum in vitro involves proteolytic degradation and siderophore-mediated removal of iron from transferrin.

Evidence confirming the persistence and resistance to treatment of viral hepatitis in the presence of excess iron is rapidly mounting. It is now apparent that excess iron enhances fibrogenic pathways and may act as a co-carcinogen or promoter of hepatocellular carcinoma, may worsen the clinical course of HCV infection by causing oxidant stress in nonparenchymal cells, and lead to the irreversible mitochondrial derangement associated with the onset of hepatic fibrosis. Human cytomegalovirus protein US2 interferes with the expression of the hemochromatosis gene, HFE. An increase in the cellular iron pool by downregulating HFE expression may promote the persistence of viruses in general. It has also been shown that excess iron decreases the viability of HIV-infected cells, increases p24 levels, and elevates the activity of reverse transcriptase, indicating that iron overload associated with HIV infection is detrimental to host cell responses against viral infection.

Researchers found that patients with higher levels of serum iron or ferritin were less likely to achieve spontaneous recovery after acute HBV infection.

Microbial Contamination of the Russian Manned Spacecraft

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A total of 234 species of bacteria and microscopic fungi were identified in the Mir environment. The bacterial flora consisted of 108 species, whereas the fungal flora included 126 species.Opportunistic pathogens of opportunistic infections were noted among bacteria, and pathogenic saprophytes causing mycoses and mycointoxications were observed among fungi. The greatest diversity (64 species) was demonstrated by the group of technophylic fungi that destroy polymers and corrode metals. Out of 1150 samples gathered from Mir surfaces and air to study bacterial flora, the microorganisms were found in 949 samples (82.5% of the total number of samples). Table 1 contains data on occurrence of different bacterial genera in the space station environment.

Table 2 presents data on the occurrence of various fungal genera on the orbital station. Penicillium, Aspergillus, and Cladosporium dominated by occurrence in samples of the interior and equipment surfaces as well as in air samples. On the whole, they reached 76.8 - 75.8%, 39.4 - 76.6% and 27.0 - 24.2%, respectively.

Contamination of Air Bacterial flora of air has been composed of 19 genera and 46 species. At all times, the special structure of bacteria consisted primarily of commensals of human integument, i.e. staphylococci, micrococci, and coryneform bacteria. Streptococci and enterococci were occasional. In addition, air samples were periodically analyzed for residents of natural reservoirs, first of all for spore-forming bacteria g.Bacillus and gram-negative non-fermenting bacteria. The highest occurrence was characteristic of genes Staphylococcus(53.2%), Bacillus (34.0%), and Corynebacterium (16.0%). A significant number of microbial species in air were opportunistic pathogens including Staphylococcus aureus, S. capitis, S. haemoliticus, Flavobacterium meningosepticum,Escherichia coli, Serratia marcescens, Streptococcussp., Bacillus cereus. Micromycets from air displayed large diversity. Fungi were presented by 62 species and 13 genes. The population of aeroplankton was dominated by Pencillins; Apergills were also of weight. Micromycets of Penicillium, Aspergillus and Cladosporium were the most numerous findings in air.

In Mirâ&#x20AC;&#x2122;s operational lifetime, bacterial contamination of its air remained relatively stable and did not exceed the limit (500 CFU/m3 ) in 95% of the air samples. The relatively high bacterial contamination of air, which in several instances reached 3.53 103 CFU in m3 , was observed at the site of exercise machines in the basal module, in Kvant - 2 and Kristall. The highest occurrence on structural materials was characteristic of Corynebacterium sp. and Staphylococcus epidermidis (33.2% and 30.0%, respectively).

Contamination of Surfaces The interior site samples contained potential agents of opportunistic infections of the Enterobacteriaceae family (Enterobacter, Escherichia, Proteus, Serratia, and other genera), and bacilli Staphylococcus aureus. The latter were isolated in 2.3% of the total number of samples. Bacillus sp. also exhibited high occurrence; for instance, Bacillus subtilis were found in 5.2%, and Bacillus sphaericus in 5.0% of the total number of samples. Altogether, 116 fungal species of 25 genera were isolated and identified in the period under review. The microflora of the interior and equipment was dominated by representatives of Penicillium, Aspergillus, and Cladosporium sp., among which the most frequent were Penicillium expansum (28.9% of the samples),Penicillium chrysogenum and Cladosporium cladosporioides(16.8% and 17.0% of the samples, respectively),Aspergillus sp. of group A. versicolor (15.5% of the samples), Aspergillus versicolor (10.0% of the samples),and Aspergillus niger (7.9% of the samples). The majority of the fungal species on structural materials are well-known potential biodegradors of polymers and, therefore, can be considered agents that may produce biointerference, damage to structural materials, malfunctioning and failure of various space systems and equipment. Besides, a number of isolated micromycets are known to affect human organism as etiological agents of infections and allergies .

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Special Edition on Space Food and Nutrition Abundance of bacteria and fungi in the ISS Each space agency has been continuously monitoring the abundance and diversity of bacteria and fungi in their respective modules in the ISS to better understand microbial dynamics in crewed habitats in space. NASA previously reported microbial abundance in the ISS based on the continuous monitoring and recovery of viable cells using cultivation based methods. Staphylococcus, Bacillus, and Micrococcus species have been the most frequently recovered bacterial genera from air and surface sampling from the ISS based on quarterly samples returned between August 1998 and August 2011. The most commonly isolated fungal genera from the air and surface samples during the same time period were Penicillium, Aspergillus, Cladosporium, and Hyphomycetes.

The most commonly isolated organisms from spaceflight water samples analyzed during ground identification between 2009 and 2012 were Ralstonia pickettii and Burkholderia multivorans.

STEM Today, September 2016, No.12

In the United States Operating Segment of the ISS, the Water Recovery System (WRS) is a physicochemical system comprised of a Urine Processor Assembly (UPA) and Water Processor Assembly (WPA) that is designed to recycle crew member urine and humidity condensate for reuse as potable water. Direct counts by microscopic enumeration revealed 8.4 X 104 cells mLâ&#x2C6;&#x2019;1 in the humidity condensate sample, but no colony-forming cells. In contrast, 3.3 X 105 cells mLâ&#x2C6;&#x2019;1 were detected in a surface swab of the WRS waste tank, and included colony-forming bacteria and fungi recovered after a 12-d incubation on solid agar media. Based on 18S rRNA sequencing and phenotypic characterization, a fungal biofilm raft recovered from the filter was determined to be Lecythophora mutabilis. A bacterial isolate recovered from a biofouling sample of the membrane in the WRS was identified by 16S rRNA gene sequence data as Methylobacterium radiotolerans (unpublished data).

JAXA has also been continuously performing bacterial monitoring in Kibo (docked with the ISS in March 2008) since 2009 (Research title: Microbe). Sampling was performed in September 2009 (Microbe-I), October 2010 (Microbe-II), February 2011 (Microbe-IIâ&#x20AC;&#x2122;), and October 2012 (Microbe-III). Actinobacteria and Firmicutes were frequently detected on the surface of the PC palm rest and handrail, which were touched frequently by astronauts. Most of these bacteria have been detected on human hands as part of the normal human skin microbiota; thus, bacterial cells may be transferred to the surface of Kibo via astronaut contact. Staphylococci and enterococci are a part of the normal human flora and are, thus, commensal microorganisms. However, they can also be opportunistic pathogens that cause a wide range of diseases. Therefore, the antibiotic resistance of staphylococci and enterococci isolated from the ISS was determined. In a collection of ISS isolates from sampling campaigns between 2002 and 2006, twenty-nine Staphylococcus and Enterococcus isolates were investigated for antibiotic resistance, horizontal transfer capability, and biofilm formation.

Resistance to one or more antibiotics was detected in 22 out of 29 (75.8%) strains examined. The most prevalent resistance determinants among the staphylococci were ermC, tetK, and different cat genes conferring resistance to macrolide, tetracycline, and chloramphenicol, respectively. Plasmids were present in 86.2% of the isolates; eight of the Enterococcus faecalis isolates harbored a large plasmid of approximately 130 kb, likely to be selftransmissible. Transfer genes encoding key proteins of the conjugative transfer process, such as the ATPase delivering energy for the transfer process and the coupling protein indispensable for linking the DNA transfer and replication complex (Dtr) with the so-called mating pair formation (Mpf) complex from the well-known resistance plasmids from gram-positive bacteria such as pIP501, pRE25, pSK41, pGO1 and pT181, were detected in the total DNA of 86.2% of the strains. Most pSK41-homologous transfer genes were detected in isolates belonging to coagulase-negative staphylococci.

Twenty-eight percent of the isolates contained at least one vir signature gene, virB1, encoding a muramidase locally opening the peptidoglycan in the cell wall, virB4, encoding the motor ATPase of the plasmid transfer process, and virD4, encoding the conjugative coupling protein, respectively. Through solid surface matings, it was demonstrated that several Staphylococcus spp. isolates could transfer their resistance genes to distinct Enterococcus and Staphylococcus spp. Biofilm formation was observed in 83% of the isolates. As methicillin-resistant Staphylococcus aureus (MRSA) or vancomycin-resistant Enterococcus (VRE) was not detected, the health risk associated with Enterococcus and Staphylococcus infections for the crew was assessed as low.

To monitor fungal microbiota in the ISS-Kibo, culture based methods were used for swab- and flat sheet media samples in the Microbe-I experiment. Tests on orbital samples using either sample collection method were negative. Although adhesive sheet samples were also examined by field emission-scanning electron microscopy, no microbial cells were detected. However, fungal DNAs were detected using real-time PCR and then analyzed by the clone library method. Alternaria sp. was the dominant fungal species before the launch, but the most abundant fungal species changed to Malassezia spp. on orbit. The dominant species found in ground control samples collected from the air conditioner diffuser, lab bench, door push panel, and facility surfaces in a university laboratory were Inonotus sp., Cladosporium sp., Malassezia spp., and Pezicula sp., respectively. Malassezia spp. constitute human skin microbiota and Inonotus sp., Cladosporium sp., and Pezicula sp. are often found

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STEM Today, September 2016, No.12

in natural environments such as soil and plants.

These results suggested that the fungal biota in Kibo changed from soil-borne fungi to a human origin. It was concluded in 2009 that cleanliness in Kibo was equivalent to that in an ISO Class 7 clean room environment on the ground.

In the Microbe-II experiment, 14 strains belonging to 5 fungal species grew on board Kibo and changes in the fungal community structure were observed using clone libraries (unpublished data). In the Microbe-III experiment (September to October, 2012), 10 strains belonging to 4 fungal species were cultured from surface samples, but no fungi grew from air samples collected by an air-sampling device. A major source of the microbes found on surfaces in the ISS is thought to have been the human skin microbiota. The human skin microbiota includes various types of microorganisms, such as viruses, bacteria, and fungi. The body sites involved include sebaceous or oily sites, moist sites, and dry sites. The predominant bacterial species differ at each body site. For example, Actinobacteria, including Corynebacterium and Propionibacterium species, predominate at sebaceous or oily sites, glabellar creases, and canals.

Regarding the fungal microbiota, Malassezia is the predominant group at all body sites, although a few hundred fungi exist on human skin. Therefore, the fungal microbiota differs from the bacterial microbiota. The fungal microbiota of 10 astronauts who spent time on the ISS was analyzed by quantitative PCR. Skin samples were collected from the astronauts once during the pre-flight stage, twice during the in-flight stage, and once during the post-flight stage. The astronauts stayed on the ISS for 6 months. On the ISS, the colonization levels of Malassezia on the astronauts increased, but decreased in post-flight (Fig. 3)(unpublished data).

A few hundred thousand high-quality reads were obtained by pyrosequencing, and more than 100 fungal genera were detected in the samples collected from the 10 astronauts; Cladosporium and Malassezia were predominant, followed by Aspergillus and Cryptococcus. The fungal diversity of samples collected in-flight decreased while the fungal diversity of post-flight samples increased. The fungal microbiota sampled from both environmental surfaces in the ISS and skin of crew members revealed an increase in the proportion of Malassezia species.

In cheek samples, Malassezia accounted for 95% of the overall fungal species in the in-flight samples, whereas it comprised only 50 and 60% of the pre- and post-flight samples, respectively (unpublished data). The level of microbial colonization markedly increased when access to bathing facilities was limited over a long period of time and the skin microbiota of astronauts may have changed for that reason.

From microbial abundance and their phylogenetic affiliation, the Kibo module has been microbiologically well maintained. However, increases were reported in bacterial and fungal numbers in previous space stations due to long-duration habitations, and microbial abundance in Kibo may also increase with prolonged exposure to astronauts. Continuous bacterial monitoring in Kibo is required to ensure crew safety and better understanding of microbial dynamics in space habitation environments.

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Effect of Iron overload on Cardiovascular system

In the presence of iron overload, iron, in the form of ferrous iron (Fe2+ ), enters the myocytes through the voltage-dependent L-type calcium channels. Myocardial iron uptake is much slower in comparison with hepatic uptake, and thus myocardial iron overload develops at a later stage in comparison with hepatic iron overload.

Iron deposition occurs initially in the ventricular myocardium and subsequently in the atrial myocardium and also affects the conducting system, but to a lesser extent compared with the working myocardium. The epicardial iron concentration is generally higher than the subendocardial one, but it was recently shown that there

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was no variation in iron deposition among the different LV segments in patients with severe iron overload cardiomyopathy (IOC). Iron is stored in the myocytes in the form of ferritin, hemosiderin, and labile cellular iron (free iron), the latter being the most active one. Labile iron leads to the formation of reactive oxygen species via the Fenton reaction, which converts ferrous to ferric iron with the generation of the toxic hydroxyl radical. The cellular antioxidant properties are exceeded, resulting to peroxidation of membrane lipids, cellular proteins, and nucleic acids. At the same time, increased ferrous iron transportation through the L-type calcium channels also results in derangement of cardiomyocyte calcium transportation and impaired excitation-contraction coupling, which may in turn be involved in the development of the diastolic and systolic ventricular dysfunction seen in association with iron overload.

The end result of this process is the development of a cardiomyopathy characterized mainly by LV dysfunction. It should be noted that myocardial iron overload, although it holds a key role as a triggering factor in the development of IOC, is not the only mechanism involved. Early studies in thalassemia major showed that several indices of LV function had a poor correlation with the total number of blood units transfused. Those preliminary findings implied that IOC was rather a particular type of cardiomyopathy with complex pathophysiology and not a direct effect simply of iron infiltration. This concept was later confirmed, because a number of studies showed that, apart from iron, additional immunoinflammatory and genetic factors seemed to interfere in the pathogenesis of IOC, such as myocarditis, the HLA genotype, and the apolipoprotein E genotype. More specifically, myocarditis was identified as a cause of LV failure, whereas the HLA-DQA1*0501 allele and the apolipoprotein E e4 allele were both associated with an increased prevalence of adverse LV remodeling and reduced LVEF in patients with thalassemia major.

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Besides direct myocardial injury, iron overload may also affect the heart indirectly through its effects on other organs. Thus, hepatic dysfunction, endocrinopathies (diabetes mellitus, hypothyroidism, hypoparathyroidism), and immune deficiency resulting from iron overload may contribute to the pathophysiology of IOC. The pathophysiology of IOC is summarized in Figure.

STEM Today, September 2016, No.12

Echocardiography is the main modality used in screening patients with iron-loading conditions for heart disease as part of their initial and regular follow-up evaluation. Left and right ventricular systolic and diastolic function abnormalities, and pericardial and valvular involvement, as well, may easily be detected. Impaired diastolic LV function featuring pseudonormalized or restrictive filling pattern, with or without left atrial enlargement constitute early findings. Advanced-stage disease is characterized by left and right cardiac chamber dilatation and reduced LVEF (the dilated phenotype) or, alternatively, by restrictive LV filling with left atrial and right ventricular dilatation, increased pulmonary artery pressure, and preserved LVEF (the restrictive phenotype). High cardiac output with chamber dilatation, eccentric LV hypertrophy, and normal or increased LVEF may also be seen. Although echocardiography identifies the consequences of iron on myocardial structure and function, it does not accurately predict myocardial iron content. However, it provides a simple means for the screening of asymptomatic patients and the follow-up of patients with known pathology.

CMR-derived T2* relaxation time is currently the mainstay for the quantitative assessment of cardiac iron deposition. Introduced a decade ago, this modality has revolutionized the clinical management of patients with hemoglobinopathies and other iron overload conditions, because it allows the accurate diagnosis and quantification of myocardial and hepatic iron deposition and hence the tailoring and monitoring of iron chelation therapy. Actually, it is postulated that the currently observed survival improvement in thalassemia major is partly attributable to the introduction of CMR-T2* imaging into clinical practice. The T2* relaxation time is mainly affected by iron in the form of hemosiderin and not by ferritin or labile cellular iron, but because there is a continuous reflux between the 3 forms of stored iron, the technique accurately predicts tissue iron content.

Measured in a full-thickness area of interest in the interventricular septum, T2* is highly representative of global myocardial iron.

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Iron overload in skeletal muscle

Iron injections in mice increase skeletal muscle iron content, induce oxidative stress and reduce exercise performance Iron accelerates the production of reactive oxygen species (ROS). Excessive levels of ROS are thought to accelerate skeletal muscle fatigue and contribute to the loss of skeletal muscle mass and function with age and disuse. Increased iron accumulation has been observed in these conditions and it has been proposed that iron may play a role. Patients with an iron overload disorder frequently report symptoms of weakness and fatigue which is not entirely explained by reduced cardiac function. The contribution of skeletal muscle to these symptoms is unknown.

Using a mouse model of iron overload, authors determined the extent of iron accumulation in skeletal muscle and the concentrations of the iron storage protein ferritin. The level of oxidative stress, changes in antioxidant enzymes and exercise performance were also assessed. Compared with control mice, the iron overloaded mice had elevated levels of iron in the tibialis anterior muscle and a fourfold increase in ferritin light chain. The oxidative stress product malondialdehyde was increased in the iron group compared with the control group, as was the antioxidant enzyme activity of glutathione reductase and glutathione peroxidase. The iron group performed less work on an endurance test and produced less force in a strength test. Body weight and skeletal muscle weight were lower in the iron group following the intervention. Iron loading reduced the weight of the fast-twitch extensor digitorum longus muscle more than the slow-twitch soleus muscle.

In summary, iron accumulation in skeletal muscle may play a significant role in the reduced exercise capacity seen in iron overload disorders and in ageing, and may play an underlying role in skeletal muscle atrophy[Reardon et.al.].

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Iron Overload Effects On Immune System

Acute iron-loaded model In immune systems, many reports have shown the effects of iron, some of which are complex and controversial. Iron deficiency has been reported to be associated with increased susceptibility to infection, but iron overload caused by dietary excess abnormal hemolysis or inherited disorders is also associated with heightened susceptibility to infections. On the other hand, elevated ferritin levels, primarily related to RBC transfusions, have been reported to increase the risk of acute and chronic GVHD in patients received hematopoietic cell transplantation. Although this iron-related risk of GVHD may reflect the organ damage, such as liver, kidney, and pancreas, it also may be caused by imbalance in immune systems.

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To exaggerate the effects of iron overload on immune system, authors established iron-loaded models in mice. First, as acute iron-loaded model, 10mg of iron dextran, which is even equal to 200U of RCC transfusion in human (define as 200U of iron dextran), was intraperitoneally injected into mice once a day for 18 days.

In peripheral blood, T cell and B cell populations were decreased but monocyte/macrophage and neutrophil populations were increased as compared in control mice administered with dextran only. And notably, regulatory T cell (CD4+Foxp3+; Treg) population was significantly reduced in iron loaded mice than control mice (1.40% vs 0.58%).

Next, as chronic iron-loaded model, 2U of iron dextran was injected once a week 45 times. Although significant difference was not observed in Treg population in PB, that in spleen was significantly high in iron-loaded mice as compared with that in control mice (3.2% vs 1.7%, p<0.05). From these data, authors suspected that the excess of iron reduces Treg population in some organs, thereby effects on immune function [Ezoe et.al.].

To clarify the mechanism of Treg-reduction by iron overload, authors established an intermediate iron-loaded model, in which 50U of iron dextran was injected into mice once a week for 10 weeks, and following experiments were performed in this model. As previous examinations, Treg population in spleen was reduced in iron-loaded mice (0.86% vs 0.46%). T helper 17 (Th17) cells are recently described lymphocyte subset with common precursors with Treg but with opposing actions to Treg. The difference of Th17 cells could not be detected in spleen because of its too small population, the population of Th17 was increased in small intestine of iron-loaded mice (0.85% vs 1.51%). Treg and Th17 cells can be differentiated from naive Th cells (CD4+CD62L+) with stimulation of specific cytokines (Treg; TGFβ, IFNγ, and IL4, Th17; TGFβ, IL-6, and IL-1β).

Authors analyzed the direct effects of iron on Treg and Th17 cells during the in vitro differentiation from naive T cells. Both in Treg and Th17 cell inductions, authors could not detect significant differences between cells supplemented with iron dextran and those with dextran only. They also exaggerated the reactive oxide spesies (ROS) accumulated in induced T cell using RedCC1 staining. During the induction of Treg and Th17, although H2 O2 supplement increased the ROS accumulation dose dependently, iron dextran-addition did not change the amount of ROS. Next, they analyzed the serum concentrations of cytokines in iron-loaded mice both in acute and intermediate models. IL-1β, and IL-23 levels were elevated in iron-loaded mice. These data suggested that iron overload reduces Treg cell population and increases Th17 cell population not directly, but through the cytokine secretion from environmental cells. So they evaluated the induction rates of Treg and Th17 cells from naive T cells under co-culture with monocyte/macrophage. In this condition, Treg induction rate was significantly lower in iron dextran-supplemented cells (48.1% vs 35.3%), and Th17 induction was increased (8.4% vs 18.0%). Furthermore, mRNA expressions of IL-23 and IL-β in macrophages were increased with iron dextran-supplement in in vitro culture.

These data suggest that iron overload changes the balance of Treg and Th17 cells through the proliferation

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Special Edition on Space Food and Nutrition and activation of macrophages, and thereby effects on the immunological condition of some disease, such as GVHD and autoimmune.

How do Regulatory T Cells Work?

CD4+ T cells are commonly divided into regulatory T (Treg) cells and conventional T helper (Th) cells. Th cells control adaptive immunity against pathogens and cancer by activating other effector immune cells.

Treg cells are defined as CD4+ T cells in charge of suppressing potentially deleterious activities of Th cells.

STEM Today, September 2016, No.12

The functions for Treg cells include: prevention of autoimmune diseases by maintaining self-tolerance; suppression of allergy, asthma and pathogen-induced immunopathology; feto-maternal tolerance; and oral tolerance. Identification of Treg cells remains problematic, because accumulating evidence suggests that all the presently-used Treg markers (CD25, CTLA-4, GITR, LAG-3, CD127 and Foxp3) represent general T-cell activation markers, rather than being truly Treg-specific.

Treg-cell activation is antigen-specific, which implies that suppressive activities of Treg cells are antigen-dependent. It has been proposed that Treg cells would be self-reactive, but extensive TCR repertoire analysis suggests that self-reactivity may be the exception rather than the rule. The classification of Treg cells as a separate lineage remains controversial because the ability to suppress is not an exclusive Treg property. Suppressive activities attributed to Treg cells may in reality, at least in some experimental settings, be exerted by conventional Th cell subsets, such as Th1, Th2, Th17 and T follicular (Tfh) cells.

Recent reports have also demonstrated that Foxp3+ Treg cells may differentiate in vivo into conventional effector Th cells, with or without concomitant downregulation of Foxp3 [Corthay et.al.].

Effects of Antiorthostatic Suspension on the Immune System (NCC958IIH00208) The investigators found that not all mouse strains are equally susceptible to HU, with Balb/c being most sensitive and C3H the least, indicating genetic variability in susceptibility to HU. Nevertheless, splenocyte and thymocyte numbers decreased significantly in all mouse strains after HU. This effect occurs through apoptosis, evidenced by genomic DNA fragmentation and phosphatidylserine exposure (TUNEL staining).

Interestingly, splenocyte loss required endogenous opioid-mediated Fas expression; the effect in thymus was corticosteroid-dependent. Thus, the dramatic effect of HU on lymphocyte numbers is exerted through different mechanisms in spleen and thymus.

Low-dose photon and simulated solar particle event proton effects on Foxp3+ T regulatory cells and other leukocytes Radiation is a major factor in the spaceflight environment that has carcinogenic potential. Astronauts on missions are continuously exposed to low-dose/low-dose-rate (LDR) radiation and may receive relatively high doses during a solar particle event (SPE) that consists primarily of protons. However, there are very few reports in which LDR photons were combined with protons.

In this study, C57BL/6 mice were exposed to 1.7 Gy simulated SPE (sSPE) protons over 36 h, both with and without pre-exposure to 0.01 Gray (Gy) LDR g-rays at 0.018 cGy/h. Apoptosis in skin samples was determined by immunohistochemistry immediately post-irradiation (day 0). Spleen mass relative to body mass, white blood cells (WBC), major leukocyte populations, lymphocyte subsets (T, Th, Tc, B, NK), and CD4(+)CD25(+)Foxp3+ T regulatory (Treg) cells were analyzed on days 4 and 21.

Apoptosis in skin samples was evident in all irradiated groups; the LDR+sSPE mice had the greatest expression of activated caspase-3. On day 4 post-irradiation, the sSPE and LDR+sSPE groups had significantly lower WBC counts in blood and spleen compared to non-irradiated controls (p < 0.05 vs. 0 Gy). CD4(+)CD25(+)Foxp3(+) Treg cell numbers in spleen were decreased at day 4, but proportions were increased in the sSPE and LDR+sSPE groups (p < 0.05 vs. 0 Gy). By day 21, lymphocyte counts were still low in blood from the LDR+sSPE mice, especially due to reductions in B, NK, and CD8(+) T cytotoxic cells.

The data demonstrate, for the first time, that pre-exposure to LDR photons did not protect against the adverse effects of radiation mimicking a large solar storm. The increased proportion of immunosuppressive CD4+CD25(+) Foxp3(+) Treg and persistent reduction in circulating lymphocytes may adversely impact immune defenses that include removal of sub-lethally damaged cells with carcinogenic potential, at least for a period of time postirradiation.

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