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Nutrition and Disease

Body Iron Stores and Oxidative Damage in Humans Increased during and after a 10- to 12-Day Undersea Dive^1–3,

⁴ Universities Space Research Association, Houston, TX 77058; ⁵ Enterprise Advisory Services, Inc., Houston, TX 77058; and ⁶ Human Adaptation and Countermeasures Division, National Aeronautics and Space Administration Johnson Space Center, Houston, TX 77058

^* To whom correspondence should be addressed. E-mail: sara.zwart-1{at}nasa.gov.

	ABSTRACT

TOP ABSTRACT Introduction Subjects and Methods Results Discussion LITERATURE CITED

 
The National Aeronautics and Space Administration Extreme Environment Mission Operations (NEEMO) underwater habitat is a useful analogue for spaceflight. However, the increased air pressure in the habitat exposes crewmembers to higher oxygen pressures, which increases their risk for oxidative damage to DNA, proteins, and lipids. Studies from a previous NEEMO mission suggested that DNA oxidation occurs at an increased level, similar to that in smokers and astronauts returning from space. Astronauts in space and NEEMO crewmembers also have similar changes in iron metabolism. Newly formed RBC are destroyed and body iron stores are elevated. Because excess iron can act as an oxidant and cause tissue damage, we investigated aspects of oxidative damage and tested whether toxic forms of iron were present when iron stores increased during NEEMO missions. Subjects (n = 12) participated in 10- to 12-d saturation dives, and blood and 24-h urine samples were collected twice before, twice during, and twice after the dive. During the dive, ferritin was higher (P < 0.001), transferrin was lower (P < 0.001), and transferrin receptors were lower (P < 0.01). Serum iron was higher during and immediately after the dive (P < 0.001). Total homocysteine (P < 0.001) and superoxide dismutase (SOD) (P < 0.05) activity were affected by time; homocysteine increased during the dive and SOD decreased during and after the dive. Labile plasma iron was measurable only during the dive. These data indicate that the NEEMO environment increases body iron stores and labile forms of iron, which may contribute to oxidative damage.

	Introduction

TOP ABSTRACT Introduction Subjects and Methods Results Discussion LITERATURE CITED

In an underwater-based analogue of spaceflight, the National Aeronautics and Space Administration Extreme Environment Mission Operations (NEEMO) project, crewmembers live in an underwater laboratory for days to weeks. NEEMO missions are saturation dives, meaning that the diver's tissue gases reach equilibrium with the pressure environment and therefore divers are allowed to live and work underwater for long periods. Aquarius, the unique laboratory in which the divers stay, provides a remote, confined environment similar to that found aboard the International Space Station. The habitat is located 19 m below the ocean surface, 4.8 km off the coast of Key Largo, FL. The 2.5-atm (253 kPa) pressure and 21% oxygen inside the habitat provide increased oxygen availability, similar to that on International Space Station EVA, during which spacewalkers breathe 100% oxygen at reduced pressure (0.3 atm) (5). Because of the increased air pressure in the Aquarius habitat, crewmembers are exposed to higher oxygen pressures, which increases their risk for oxidative damage to DNA, proteins, and lipids in tissues and blood (6–9).

Our laboratory performed a comprehensive nutritional status assessment of 6 crewmembers from an earlier mission, NEEMO V (10). Along with increased 8-hydroxy-2'-deoxyguanosine (8OHdG) excretion during the dive, decreased activity of RBC glutathione peroxidase (GPX) and superoxide dismutase (SOD) during and after the dive implied that oxidative stress increased. Changes in iron metabolism were also observed during this saturation dive, similar to those during and after spaceflight (4,11–13). Hemoglobin decreased and body iron storage increased. Because of the increased pressure in the habitat, and therefore greater oxygen availability, it is hypothesized that newly formed RBC are selectively hemolyzed in a process called neocytolysis (which occurs during both spaceflight and descent from high altitude) (14).

Both during spaceflight and in the ground analogue NEEMO, evidence exists that body iron stores are increased (4,10,13,15). Excess iron can act as a pro-oxidant and cause tissue damage (16,17). In this project, we investigated specific aspects of oxidative damage and sought to test whether toxic forms of iron (18,19) and circulating heme are present when body iron stores increase because of increased oxygen exposure.

	Subjects and Methods

TOP ABSTRACT Introduction Subjects and Methods Results Discussion LITERATURE CITED

 
    Environment. The NEEMO XII and XIII missions were 12-d and 10-d saturation dives, respectively. Each mission involved 6 divers living in an underwater habitat for the duration of the mission. The habitat is described in detail in (10); briefly, it is 14 m long and 4 m in diameter, is located 19 m below the ocean surface, and has an atmospheric pressure of 253 kPa (2.5 atm) with a normal air mixture. Subjects were required to undergo a 17-h decompression before resurfacing at the end of the dive. The NEEMO XII and XIII missions took place in April and August of 2007.

    Subjects. This study was approved by the Johnson Space Center (JSC) Committee for the Protection of Human Subjects. The combined crew consisted of 1 female and 11 males, all of whom were subjects in the study. The female was on the NEEMO XII mission. Subjects were trained in all procedures required for successful completion of the in-dive sample and data collections.

All subjects were required to pass a modified Air Force Class III physical examination and were required to have logged a minimum of 25 dives before they were selected to participate in the mission. Their mean body weight (± SD) before the dive was 85 ± 14 kg.

    Blood collection and processing. Sample collection days before the dive were designated as Dive–X days (D-X days), during the mission as Mission Day X (MDX), and after the dive as Return+X days (R+X days).

Blood was collected before (D-7 and D-2), during (MD 6 or 7 and MD 9 or 11), and after the dive (R+0 and R+6 or 7). During the dive, NEEMO XII blood samples were collected 1 or 2 d later in the mission than NEEMO XIII blood samples because of the length of the mission. Blood collections (14.2 mL at each session) were performed at the same time each day after an 8-h fast. Pre- and postdive (D-6, D-2, R+0, and R+6/7) blood samples were collected near the Aquarius habitat (on shore) and centrifuged for processing within 45 min of phlebotomy. Aliquots of whole blood were made before centrifugation. Serum aliquots were frozen at –20°C until they were transported to JSC in Houston. Processing of blood samples during the dive was conducted after a longer delay (see details below).

Whole blood was transported to JSC and analyzed for hemoglobin and hematocrit within 48 h of blood collection (20). Aliquots used for other tests remained frozen at –20°C until they were transported on dry ice to JSC.

    Delayed centrifugation study. Because of the possibility that altered biochemical analyte concentration during the dive could be an artifact of delayed blood processing (blood was processed 8 h after phlebotomy during the dive, but only 45 min after phlebotomy before and after the dive), a ground-based study simulating this scenario was tested. Six subjects were recruited at the JSC Human Test Subject Facility and 2 sets of blood tubes were collected. One set was centrifuged (1850 x g; 15 min) and processed within 45 min of phlebotomy and the other set was kept on ice and protected from light for 8 h before centrifugation (1850 x g; 15 min) and processing. Biochemical analytes were measured as described below.

    Urine collection and processing. Each subject's urine was collected for 24-h periods before (D-7 and D-2), during (MD 6 or 7 and MD 9 or 11), and after the dive (R + 6 or 7). Pre- and postdive urine samples were collected in individual bottles and stored cool (at 4°C or on ice packs) until they were processed (<24 h later). During the dive, the crew collected voids into either a beaker or a graduated cylinder. The volume of each void was recorded and a 50-mL aliquot was sent to the surface. All urine and blood samples collected during the dive were kept in a cooler on ice and protected from light in the habitat before (and during) ascent to the surface. The samples were also kept on ice and protected from light aboard the boat when it returned to shore 8 h after the blood draw. On shore, 24-h urine pools based on void volumes were created, pH was measured, and aliquots were prepared and frozen (–20°C) for analysis as soon as possible.

    Biochemical analyses. Most analytical determinations were performed by standard commercial techniques as described previously (2,4). Hemoglobin and hematocrit were determined using a Coulter MAXM instrument (Beckman Coulter). Serum ferritin and transferrin were analyzed with the Immulite (Diagnostics Products) and Array 360 (Beckman Coulter) instruments, respectively. Transferrin receptors were measured using a commercially available ELISA (Ramco Laboratories). Total plasma heme was determined spectrophotometrically at 400 nm after the addition of an aqueous alkaline solution that converts heme into a uniform colored form (QuantiChrome Heme Assay kit, DIHM-250, BioAssay Systems). Body iron was estimated from serum ferritin and transferrin receptor values (21).

RBC SOD, RBC GPX, and serum oxygen-radical absorbance capacity were measured spectrophotometrically using commercially available kits (Randox Laboratories; oxygen-radical absorbance capacity was analyzed on an Olympus AU400 analyzer). Plasma lipid peroxides were measured spectrophotometrically using a commercially available kit (Calbiochem Lipid Peroxidation Assay kit, EMD Biosciences). HPLC techniques (22) were used in an external commercial laboratory to determine 8OHdG in urine (ESA Laboratories). Urinary prostaglandin F₂ (PGF₂) was measured by ELISA using a commercially available kit (Oxford Biomedical Research). Oxidized and reduced glutathione (GSH) were measured spectrophotometrically using a commercially available kit (Oxford Biomedical Research). Urinary creatinine was analyzed spectrophotometrically on the NexCT clinical chemistry system (Alfa Wassermann).

Methylmalonic acid (MMA), total homocysteine, and cystathionine were determined in serum by GC-MS in an external commercial laboratory (Metabolite Laboratories). Non-transferrin–bound iron and labile plasma iron were measured in plasma using fluorometry in an external commercial laboratory as previously described (Aferrix) (23).

    Statistical analysis. Data are reported as means ± SD. A mean predive value was calculated for each variable and 2-way repeated-measures ANOVA was performed with mission and time as factors. A post hoc Bonferroni t test was performed comparing the predive value to all in- and postdive samples. In the delayed centrifugation study, we used a 1-way repeated-measures ANOVA to determine differences between 1-h and 8-h processing. Statistical analyses were performed using Sigma Stat software (SPSS). Whenever data were not normally distributed (SOD, total antioxidant capacity, cystathionine, MMA, oxidized glutathione, hemoglobin, hematocrit, and ferritin), an attempt was made to transform and normalize data (using log, 1/x, square root, etc.), but they could not be normalized. Significance was defined as P < 0.05. The relationships between total body iron and SOD and between plasma heme and urinary 8OHdG were determined by calculating the Pearson correlation coefficient for each pair.

	Results

TOP ABSTRACT Introduction Subjects and Methods Results Discussion LITERATURE CITED

 
Blood tubes from each session were visually inspected for hemolysis and samples collected during the dives exhibited more hemolysis than samples collected on land. This could have been caused by the increased pressure inside the habitat. Some tests (such as total antioxidant capacity) are affected by hemolysis and in such a case, the hemolyzed samples were not included in the means or statistical analyses.

Data for NEEMO XII and XIII individually are presented in Supplemental Tables 1 and 2 and the combined data are presented in Tables 1 and 2.

    Biochemical analyses. For both NEEMO missions, SOD activity was lower toward the end of the dive (P < 0.05) (Table 1). Lipid peroxide concentrations were greater during the dive than before the dive (P < 0.001) and were greater in NEEMO XIII subjects than in NEEMO XII subjects. Total antioxidant capacity during or after the dive did not differ from predive capacity. GPX activity was 46% and 31% higher during and immediately after the dive, respectively, for NEEMO XIII but not NEEMO XII subjects (Supplemental Table 1).

Oxidized glutathione was elevated to 600% of baseline at the end of the dive (MD 9/11) (P < 0.01), whereas GSH did not change.

MMA, a marker of vitamin B-12 status, did not change during or after the dive. Homocysteine and cystathionine, both 1-carbon metabolism intermediates, were differentially affected by the dive. Homocysteine increased early during the dive (16% elevation) and continued to remain elevated through the end of the dive (37% elevation), but postdive levels were similar to predive levels. Cystathionine, on the other hand, decreased at the end of the dive and on splash-up day (R+0) compared with before the dive but did not differ 1 wk after the dive.

    Iron metabolism. Hemoglobin and hematocrit both decreased during the dive but not consistently in the 2 NEEMO missions. Immediately after the dive, hemoglobin and hematocrit were lower in NEEMO XII than in NEEMO XIII crewmembers.

Body iron stores increased during the dive, as indicated by increased ferritin and decreased transferrin and transferrin receptors (Table 2). Total body iron, estimated from ferritin and transferrin receptors (21), increased up to 29% during and after the dive. Total body iron was negatively correlated with SOD (Fig. 1; r = –0.39; P < 0.01). Total plasma heme increased 48% during the dive (Table 2; P < 0.01) and was generally higher in NEEMO XII crewmembers than in the NEEMO XIII crew. Plasma heme was positively correlated with urinary PGF₂ (r = 0.38; P < 0.01) and there was a trend for a relationship with 8OHdG (r = 0.28; P = 0.054). Statistical analyses were not conducted for non-transferrin–bound iron and labile plasma iron, because they were not detectable in most samples; however, when these pro-oxidant forms of iron were detectable, it was always during or immediately after the dive and never before the dive. One-fourth of the subjects had detectable levels of non-transferrin–bound iron at the end of the dive period (MD 9/11) and 5 of the 12 crewmembers had detectable levels of labile plasma iron during or immediately after the dive.

View larger version (8K): [in this window] [in a new window]   FIGURE 1  Correlation between SOD activity and total body iron (TBI) in humans. All time points for each subject are plotted (5 time points per subject). Pearson r = –0.39; P < 0.01.

    Delayed centrifugation study. Oxidized glutathione was greater (P < 0.05) when blood was processed 8 h after phlebotomy than when it was processed at the nominal 45–60 min after phlebotomy. None of the other variables tested differed when processed 8 h after phlebotomy. Data for each time point are in Supplemental Table 3.

	Discussion

TOP ABSTRACT Introduction Subjects and Methods Results Discussion LITERATURE CITED

 
Oxidative damage is a health concern for astronauts and for individuals, such as divers, in environments with altered atmospheres. We have previously shown that saturation diving affects iron metabolism and creates oxidative stress in the body, and the studies reported here further evaluated these 2 systems in this environment. The changes in iron metabolism, which reflect an iron overload, and their relationship with increased oxidative damage are of broad concern because of the association of oxidative damage with chronic disease and cataracts (24–28).

A decrease in hemoglobin and hematocrit is likely caused by a process called neocytolysis, by which newly formed RBC are lysed and thus removed from circulation. This is similar to what happens to athletes who train at high altitudes and then return to sea level and is also similar to what astronauts experience during the first few weeks of spaceflight (12,13,29,30). Implications of neocytolysis include a potential for increased release of iron from the lysed RBC into a labile free form (non-transferrin–bound iron) as a redox-active form in free circulating heme or into non-labile storage forms in the body. Heme can be cytotoxic in the free form, particularly in the presence of oxidants (31,32).

We have previously shown that the NEEMO environment induces changes associated with increased iron storage (increased serum ferritin and decreased transferrin receptors) (10), but the presence of labile forms of iron has not been investigated before. In the present study, circulating plasma heme increased 50% at the beginning of the dive but was elevated only 10% by the end of the dive. Non-transferrin–bound labile iron was not detectable in most samples, but 4 of the 12 crewmembers had detectable levels during or immediately after the dive. Ferritin levels were elevated in the crewmembers during the dive, with a simultaneous decrease in transferrin receptors. In a recent study, a positive correlation was found between the ratio of serum soluble transferrin receptors to serum ferritin concentration and urinary 8OHdG excretion, suggesting that body iron contributes to excess oxidative stress even at non-iron overload concentrations (33,34). Using an equation described by Cook et al. (21), body iron was estimated and determined to be greater during the dive than before the dive. Although ferritin (an acute-phase protein) is used in the equation to estimate body iron and an acute-phase response could explain a change, the changes in transferrin receptors, transferrin, and serum iron all support a shift in body iron stores.

Free iron causes the formation of highly reactive hydroxyl radicals that damage cells' macromolecules, including DNA and proteins. During the dive in NEEMO V (10), 8OHdG was elevated, but only a trend of an increase was found in the current missions (P = 0.06). It was more difficult to get complete 24-h urine collections during NEEMO XII and XIII, because crewmembers on these missions spent much of their time during the dive on EVA. This probably explains the difference between results from these missions and NEEMO V, because urinary measures could not be expressed per day and had to be normalized to creatinine. Similar to what was observed in NEEMO V (10), SOD decreased during the dive in both NEEMO XII and XIII missions. Body iron was negatively correlated with SOD (P < 0.05), meaning that the more iron stores a subject had, the less SOD was measured. This is not surprising given that SOD is often decreased in patients with iron-overload disorders (35,36).

The reason GPX concentration increased during the dive in NEEMO XIII but not NEEMO XII crewmembers is more difficult to explain. Serum lipid peroxide concentrations of NEEMO XIII crewmembers were generally higher than those of NEEMO XII crewmembers, but the percentage increase during and after the dive was similar in the 2 groups. It was not feasible to collect diet data from these crews and it is possible that 1 group consumed more antioxidant-rich foods. Food choices were left to the discretion of each crewmember.

GSH is a major antioxidant and plays a role in the maintenance of the redox state of the cell. Upon exposure to oxidative stress, it is converted to the glutathione disulfide (GSSG) form. In the present study, there was an approximate 600% increase in GSSG levels and consequently a reduction in the GSH:GSSG ratio during the dive compared with before the dive, which suggests that oxidative stress was occurring. To make sure this was not just an artifact of the delayed centrifugation and processing of the blood sample (until 8 h after phlebotomy), a ground-based study was conducted to simulate the conditions of the delayed centrifugation. When samples were stored on ice and protected from light for 8 h before centrifugation (Supplemental Table 3), oxidized glutathione values were 50% higher than values obtained when the sample was processed within 1 h. Therefore, at least some of the 6-fold increase in oxidized glutathione observed during NEEMO missions must have been caused by the delayed processing. As expected because its concentration in the blood is tightly regulated, the levels of GSH were conserved during the dive with no observable change.

In addition to the changes in iron metabolism, one striking change was the elevated homocysteine in all crewmembers during the dive but not immediately after the dive. The results of the delayed centrifugation study showed that the homocysteine concentration was not affected by the processing schedule (centrifugation 45 min or 8 h after phlebotomy, samples on ice), suggesting that the increased homocysteine during the dive was related to the dive itself and not to the processing conditions. An increase in homocysteine suggests that a decrease might have occurred in folate, vitamin B-12, or vitamin B-6 status. Because MMA did not change during the dive, it is not likely that vitamin B-12 status changed. The decrease in cystathionine during the dive supports the likelihood that vitamin B-6 status decreased, although a decrease in folate status cannot be ruled out. RBC folate was measured in NEEMO V (10) crewmembers and was not significantly reduced, even though it tended to be lower during the dive. The lack of significance may be related to the small sample size, as we might expect folate status to be decreased. In vitro studies show that folate has radical-scavenging capacity equivalent to that of vitamin C (37,38). Folate status has been shown to be compromised in other oxidative environments, including radiotherapy, and in the presence of iron (39–41). One risk of impaired folate status is the accompanying increase in homocysteine, which itself can induce oxidative damage (42–44).

In this ground-based model of spaceflight, we have found evidence for oxidative stress. The increased oxygen exposure likely contributes oxidative damage both directly and indirectly. Indirectly, the hyperoxic environment affects RBC metabolism so that iron stores are elevated in the body. Increased iron stores were accompanied by increased serum iron, increased heme, and detectable levels of non-transferrin–bound iron, which likely contributes to further oxidative damage.

Evidence exists that iron stores increase during spaceflight, but to our knowledge heme and labile plasma iron have not been measured before. These compounds could potentiate the effects of other oxidative stressors, including ionizing radiation. Similarly, an increase in homocysteine, as observed in these NEEMO missions, could have synergistic effects in oxidative environments such as spaceflight. RBC folate has been shown to decrease after long-duration spaceflight (4–6 mo) (4) and this could lead to elevated homocysteine. The immediate clinical implications of acute elevated oxidative damage and increased body iron stores in environments like the NEEMO environment may not be obvious, but the literature is replete with examples of why these factors can be harmful in the long term to overall health, including their association with cataract formation (24,44–47). Understanding and mitigating the risk of iron excess in astronauts during space missions will be critical for ensuring crew health and individuals on Earth in high-oxygen environments must be made aware of the risk and possible consequences of iron excess in those environments.

	ACKNOWLEDGMENTS

 
We thank Kristin Hitchcox for assistance with the delayed centrifugation study and Jane Krauhs for editorial assistance. We also thank Diane DeKerlegand, YaVonne Bourbeau, Tiffany Chew, Glenda Crawford, Patricia Gillman, Amanda Messick, Barbara Rice, Ann Rogers, and Shanna Rodgers from the Nutritional Biochemistry Laboratory for assistance in sample processing and coordination.

	FOOTNOTES

 
¹ Supported by the National Aeronautics and Space Administration Human Research Program.

² Author disclosures: S. R. Zwart, G. Kala, and S. M. Smith, no conflicts of interest.

³ Supplemental Tables 1–3 are available with the online posting of this paper at jn.nutrition.org.

⁷ Abbreviations used: D–, day before dive; EVA, extravehicular activity; GPX, glutathione peroxidase; GSH, reduced form of glutathione; GSSG, glutathione disulfide; JSC, Johnson Space Center; MD, mission day; MMA, methylmalonic acid; NEEMO, NASA Extreme Environment Mission Operations; PGF₂, prostaglandin F₂; 8OHdG, 8-hydroxy-2'-deoxyguanosine; R+, days after return; SOD, superoxide dismutase.

Manuscript received 5 August 2008. Initial review completed 16 September 2008. Revision accepted 18 October 2008.

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TOP ABSTRACT Introduction Subjects and Methods Results Discussion LITERATURE CITED

 

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