Abstract
Bed rest studies provide an important tool for modeling physiological changes that occur during spaceflight. Markers of bone metabolism and nutritional status were evaluated in 12 subjects (8 men, 4 women; ages 25–49 yr) who participated in a 30-day −6° head-down-tilt diet-controlled bed rest study. Blood and urine samples were collected twice before, once a week during, and twice after bed rest. Data were analyzed using a mixed-effects linear regression with a priori contrasts comparing all days to the second week of the pre-bed rest acclimation period. During bed rest, all urinary markers of bone resorption increased ∼20% (P < 0.001), and serum parathyroid hormone decreased ∼25% (P < 0.001). Unlike longer (>60 days) bed rest studies, neither markers of oxidative damage nor iron status indexes changed over the 30 days of bed rest. Urinary oxalate excretion decreased ∼20% during bed rest (P < 0.001) and correlated inversely with urinary calcium (R = −0.18, P < 0.02). These data provide a broad overview of the biochemistry associated with short-duration bed rest studies and provide an impetus for using shorter studies to save time and costs wherever possible. For some effects related to bone biochemistry, short-duration bed rest will fulfill the scientific requirements to simulate spaceflight, but other effects (antioxidants/oxidative damage, iron status) do not manifest until subjects are in bed longer, in which case longer studies or other analogs may be needed. Regardless, maximizing research funding and opportunities will be critical to enable the next steps in space exploration.
Keywords: bed rest, bone metabolism, nutritional status, oxalate, parathyroid hormone
bed rest studies provide a vital experimental platform that allows researchers to investigate some of the effects of spaceflight without the resource limitations of spaceflight itself, including small subject pools, limited crew time, inherent difficulties of trying to collect data in microgravity, and limited (and time-delayed) sample return. These resource constraints typically drive the requirement for findings from bed rest or other ground analog studies to be available before flight studies will be considered. Although bed rest studies offer more research flexibility, they have their own set of resource constraints, including limited funding and bed rest facility space and difficulties in subject recruitment. Thus carefully controlled bed rest studies are needed to ensure that they are comparable with spaceflight, and the bed rest study design must be optimized to maximize return. Optimal study design becomes imperative when researchers attempt to model specific physiological effects of spaceflight, and one key issue is the required duration of the bed rest period. Longer studies provide a means to evaluate chronic adaptation in systems that change slowly, but these studies require additional resources, and subject interest is limited because of the greater time commitments.
Long-duration spaceflight poses many potential risks related to nutrition, including inadequate dietary intake, nutrient deficiencies from long-term exposure to a closed food system, altered nutrient metabolism and requirements, increased oxidative damage, cardiovascular changes, and bone and muscle loss (28, 32). The status of individual nutrients–vitamin D, folate, vitamin K, and vitamin E–has been shown to be altered during and after long-duration spaceflight (4–6 mo) (28, 29, 32). Changes in iron metabolism also occur during spaceflight. Red blood cell mass decreases, causing a shift in iron out of the destroyed red blood cells and into iron storage proteins such as ferritin (1, 6, 34). Circulating transferrin also decreases, suggesting that less iron is being transported (20, 29). This shift in iron metabolism, potentially in combination with increased space radiation exposure, may contribute to the increase in markers of oxidative damage. Specific findings from flight include an increase in urinary 8-hydroxy-2′-deoxyguanosine and a decrease in superoxide dismutase activity on landing day relative to before flight (29). Long-duration (>60 day) bed rest studies have shown that bed rest is associated with risks similar to those observed during long-duration spaceflight (41).
One of the most striking physiological changes that occurs during spaceflight is bone loss (4, 5, 25–27). During spaceflight, urinary markers of bone resorption increase to 100–150% of their preflight values (4, 5, 25–27). Markers of bone formation [such as bone-specific alkaline phosphatase (BSAP)] remain unchanged or slightly lower than preflight values. These effects, increased bone resorption with unchanged formation, lead to a net loss in bone mineral density (11, 14, 22, 24, 26–28). Although bone loss during flight is well documented, recent evidence from well-nourished astronauts participating in a regimen of weight-bearing exercise showed that using the Advanced Resistive Exercise Device maintained bone mineral density at preflight levels (24). This is the first report of maintenance of bone mineral density during actual spaceflight, and it appears that bone mineral density maintenance occurs despite increased bone remodeling, with increased rates of bone resorption and a trend (P < 0.06) for increased bone formation. This leaves open questions of bone quality and strength, along with the ability of exercise and nutritional protocols to be optimized (24).
Bed rest studies have shown similar changes in bone metabolism, albeit with decreased magnitude relative to flight. Bone resorption markers increase 50–75% in bed rest, whereas bone formation markers typically remain unchanged during bed rest (3, 10, 13, 21, 36, 39) in sedentary subjects. However, in a 17-wk bed rest study in which subjects completed heavy weight-bearing exercise, markers of bone formation increased significantly, despite increased bone resorption (18). The similarities in bone metabolism between bed rest and spaceflight make bed rest an ideal model for studying nutrition, exercise, or pharmacological countermeasures to the effects of spaceflight.
We present results from a 30-day bed rest study conducted using the National Aeronautics and Space Administration (NASA)-defined standardized procedures for bed rest studies (16, 33, 41), and we discuss the results of this study with those from bed rest studies of longer duration (>60 days) and actual spaceflight.
METHODS
Data are presented from a −6° head-down-tilt bed rest study, in which subjects were not exposed to exercise, pharmacological, or other countermeasures. A 13-day pre-bed rest ambulatory period was followed by 30 days of strict bed rest and then by a 14-day post-bed rest recovery period. The study methods have been previously described in detail (16, 23, 31, 33, 41, 42). All bed rest protocols were reviewed and approved by the Johnson Space Center Committee for the Protection of Human Subjects, the University of Texas Medical Branch (UTMB) Institutional Review Board, and the UTMB Institute for Translational Sciences-Clinical Research Center (ITS-CRC) Science Advisory Committee. All subjects received verbal and written explanation of the bed rest protocol and provided written, informed consent.
Subjects, samples, and analyses.
Twelve healthy subjects (8 men, 4 women) participated in the study. The mean (±SD) age of the subjects was 32 ± 8 yr. The subjects had an average height of 173 ± 7 cm, weight of 73.6 ± 10.6 kg, and body mass index of 24.5 ± 2.8 kg/m2. The study was designed to maintain each subject's weight throughout the study by adjusting caloric intake if mean daily weight exceeded 103% of the subject's weight on bed rest day 3 (allowing for fluid losses associated with the start of head-down-tilt bed rest). Given concerns about vitamin D status and bone health, subjects were given a 2,000 IU vitamin D3 supplement daily during the acclimation period, if their serum 25-hydroxyvitamin D concentration was <50 nmol/l at the time of screening (∼30 days before arrival at ITS-CRC). Subjects with a 25-vitamin D concentration > 50 nmol/l at the time of screening were not given a vitamin D3 supplement during the acclimation period. All subjects had a 25-hydroxyvitamin D concentration > 50 nmol/l at the start of bed rest, except one subject, who had a serum concentration of 40 nmol/l at pre-bed rest week 2, which increased to 49.6 nmol/l at bed rest week 1. All subjects were given an 800 IU vitamin D3 supplement daily during bed rest. The controlled diet protocol has been described previously (12).
Markers of bone metabolism were measured on all 12 subjects, and a subset (n = 7; 3 men, 4 women) participated in a broader nutritional status assessment before and after bed rest. Blood and 48-h urine samples were collected twice before bed rest (pre-bed rest week 1, pre-bed rest week 2), once per week during bed rest (bed rest weeks 1–4), and after bed rest (recovery days 5–7). On designated “bed rest day 30/recovery days 0–3”, the blood sample was collected just before reambulation and is, therefore, considered a bed rest data point, while the urine sample was a 48-h collection starting the morning of reambulation and is considered a post-bed rest data point. Samples were excluded from analysis if blood samples were hemolyzed, or if urine samples were contaminated with blood, as indicated by a Siemens Multistix.
Biochemical analyses were performed by standard commercial techniques, as described previously (17, 19, 23, 24, 30, 31, 38, 41). A complete vitamin B6 panel was performed, including serum 4-pyridoxic acid, pyridoxal 5′-phosphate, and pyridoxal; the results for pyridoxal are not presented because only one subject's values were above the limit of detection (10 nmol/l). Phylloquinone, undercarboxylated osteocalcin, and osteocalcin were measured as previously described (37), as were testosterone and related analytes (23). Some of the data presented here have been published in related reports. Specifically, testosterone (measured by liquid chromatography-tandem mass spectrometry) was previously published (23); the data are presented again here for comparison with the dehydroepiandrosterone and estradiol results from female subjects (23). N-telopeptide and BSAP results have been previously published (17); they are again presented here for comparison (and were not previously presented in the same detail as reported herein).
Statistical analysis.
All statistical analyses were performed using Stata IC software (version 12.1, Stata, College Station, TX) and setting two-tailed α to reject the null hypothesis at 0.05 with a Bonferroni adjustment for multiple comparisons. Some of our dependent variables required transformation (log, 1/sqrt, sqrt, or squared) to satisfy the statistical assumptions required of our methods. The variables requiring transformation are indicated in our table legends. Overly influential observations (i.e., statistical outliers) were excluded from analysis if the standardized residual exceeded ±1.96 units away from the mean (also identified in table legends).
Planned pairwise comparisons were made with pre-bed rest week 2 before the beginning of bed rest, when subjects should have been fully acclimated to the bed rest facility and diet vs. all bed rest and recovery observations. All dependent variables were assessed multiple times before, during, and after bed rest, resulting in a longitudinal (repeated measures) experimental design. Separate mixed-effects linear regression models (a.k.a. multilevel models) were used to evaluate the effects of bed rest on our continuously scaled outcomes relevant to bone and nutrient metabolism. For each outcome, our statistical model included dummy-coded β-coefficients comparing the data from immediately before the bed rest period (pre-bed rest week 2) with all other time periods. For most outcomes, this included one earlier pre-bed rest time period (pre-bed rest week 1), weekly sampling during bed rest (bed rest weeks 1, 2, 3, and 4), and two post-bed rest samples (bed rest day 30/recovery days 0–3 and 5–7); however, some outcomes were collected at fewer time periods. As typical with mixed-effects modeling, all models included a random intercept to accommodate the longitudinal (within-subject) experimental design. In addition to that, our models evaluating changes in aspartate transaminase also required random slope coefficients to adjust for heterogeneous treatment effects and improve model fit (8).
On completion of these statistical models, the uncorrected P values comparing our pre-bed rest observations with those of each of the other time periods for all of our dependent variables were submitted to a single Bonferroni multiple-testing correction (502 hypothesis tests), resulting in a conservative critical P value for rejection of 0.0000996 (that is, 0.05/502) instead of the more traditional 0.05. All tables report unadjusted P values, with an asterisk (*) further indicating that the statistical significance was below this Bonferroni-adjusted critical P value.
RESULTS
Calcium and bone metabolism.
Markers of bone metabolism from serum and urine are presented in Table 1. The amount of urinary calcium excreted per day increased significantly after 2 wk of bed rest and remained high throughout bed rest. The calcium excretion rate decreased to pre-bed rest levels within 5–7 days post-bed rest. Urinary calcium increased when it was normalized to urinary creatinine after 3 or more wk of bed rest, but rapidly returned to near baseline values during the recovery period (days 0–3). There were no significant changes in whole-blood ionized calcium or serum calcium.
Table 1.
Markers of bone metabolism
| Test | n | Outliers, no. | Pre-bed Rest, Week 1 | Pre-bed Rest, Week 2 | Bed Rest, Week 1 | Bed Rest, Week 2 | Bed Rest, Week 3 | Bed Rest, Week 4 | Bed Rest, Day 30ζ; Recovery, Days 0–3 | Recovery, Days 5–7 |
|---|---|---|---|---|---|---|---|---|---|---|
| Urinary calcium, mg/mg creatinine | 12 | 1 | 0.13 ± 0.05 | 0.13 ± 0.07 | 0.14 ± 0.05 | 0.14 ± 0.04a | 0.17 ± 0.06a* | 0.17 ± 0.07a* | 0.15 ± 0.05a | 0.14 ± 0.05 |
| (0.10–0.16) | (0.10–0.16) | (0.11–0.17) | (0.12–0.18) | (0.13–0.19) | (0.14–0.20) | (0.12–0.18) | (0.11–0.17) | |||
| Urinary calcium, mmol/day | 12 | 1 | 5.6 ± 1.6 | 5.5 ± 2.1 | 5.9 ± 2.1c | 6.3 ± 1.9a* | 7.3 ± 2.1a* | 7.0 ± 2.5a* | 6.3 ± 2.0a* | 5.7 ± 1.8 |
| (4.5–6.7) | (4.3–6.5) | (4.9–7.1) | (5.5–7.7) | (5.9–8.1) | (5.8–8.1) | (5.2–7.4) | (4.6–6.9) | |||
| Serum calcium, mmol/l | 12 | 2.34 ± 0.09 | 2.32 ± 0.07 | 2.35 ± 0.07 | 2.33 ± 0.08 | 2.34 ± 0.07 | 2.33 ± 0.07 | 2.38 ± 0.08b | 2.29 ± 0.07 | |
| (2.30–2.39) | (2.28–2.36) | (2.30–2.39) | (2.29–2.37) | (2.30–2.38) | (2.28–2.37) | (2.33–2.42) | (2.25–2.33) | |||
| Ionized calcium, mmol/l | 12 | 1.23 ± 0.04 | 1.23 ± 0.04 | 1.24 ± 0.02 | 1.24 ± 0.03 | 1.24 ± 0.05 | 1.25 ± 0.05 | 1.23 ± 0.04 | 1.23 ± 0.03 | |
| (1.21–1.25) | (1.21–1.25) | (1.22–1.26) | (1.21–1.26) | (1.22–1.27) | (1.23–1.27) | (1.21–1.25) | (1.21–1.25) | |||
| N-telopeptide†, nmol/day | 12 | 13 | 507 ± 204 | 512 ± 202 | 691 ± 302a* | 688 ± 310a* | 799 ± 358a* | 764 ± 316a* | 765 ± 282a* | 618 ± 262a* |
| (360–598) | (363–602) | (479–795) | (502–832) | (549–911) | (542–898) | (516–853) | (439–728) | |||
| N-telopeptide†, nmol/mmol creatinine | 12 | 13 | 32.9 ± 11.8 | 34.0 ± 12.3 | 42.6 ± 16.7a* | 42.2 ± 12.8a* | 49.8 ± 20.8a* | 50.7 ± 19.5a* | 48.4 ± 17.8a* | 40.7 ± 15.8a* |
| (24.8–38.1) | (25.7–39.4) | (31.8–48.9) | (34.1–52.5) | (36.9–56.6) | (38.2–58.6) | (35.3–54.0) | (30.6–46.9) | |||
| C-telopeptide, μg/day | 12 | 1 | 2,335 ± 1,018 | 2,426 ± 1,080 | 3,493 ± 1,640a* | 3,811 ± 2,031a* | 4,382 ± 1,969a* | 4,288 ± 1,968a* | 3,913 ± 1,723a* | 3,280 ± 1,610a* |
| (1,409–3,260) | (1,474–3,328) | (2,565–4,422) | (2,993–4,854) | (3,334–5,192) | (3,346–5,200) | (2,842–4,678) | (2,355–4,206) | |||
| C-telopeptide†, μg/mmol creatinine | 12 | 1 | 150 ± 59 | 156 ± 60 | 216 ± 80a* | 229 ± 88a* | 272 ± 112a* | 279 ± 103a* | 247 ± 86a* | 212 ± 80a* |
| (112–172) | (117–181) | (161–249) | (182–281) | (198–306) | (211–325) | (182–280) | (160–246) | |||
| H peptide, μg/day | 12 | 1 | 754 ± 355 | 762 ± 352 | 989 ± 482a* | 1,075 ± 606a* | 1,227 ± 586a* | 1,176 ± 521a* | 1,157 ± 517a* | 990 ± 450a* |
| (484–1,025) | (485–1,026) | (722–1,263) | (818–1,362) | (918–1,460) | (905–1,446) | (854–1,392) | (719–1,260) | |||
| H peptide†, μg/mmol creatinine | 12 | 1 | 49.5 ± 20.8 | 49.4 ± 19.5 | 61.4 ± 24.9a* | 66.5 ± 28.5a* | 76.8 ± 33.8a* | 77.5 ± 27.6a* | 73.4 ± 27.2a* | 65.2 ± 24.4a* |
| (36.8–57.4) | (36.8–57.3) | (46.1–71.8) | (52.0–81.2) | (54.8–85.4) | (58.7–91.5) | (54.5–84.6) | (48.9–76.2) | |||
| Pyridinium cross-links, nmol/day | 12 | 2 | 248 ± 70c | 269 ± 78 | 338 ± 96a* | 357 ± 104a* | 390 ± 103a* | 383 ± 109a* | 398 ± 100a* | 363 ± 105a* |
| (191–300) | (213–322) | (284–393) | (304–413) | (324–433) | (330–439) | (344–453) | (309–417) | |||
| Pyridinium cross-links, nmol/mmol creatinine | 12 | 2 | 16.2 ± 4.4c | 17.6 ± 4.4 | 21.4 ± 5.4a* | 22.6 ± 5.2a* | 24.7 ± 5.3a* | 25.8 ± 6.3a* | 26.2 ± 6.3a* | 24.2 ± 6.5a* |
| (13.1–19.3) | (14.5–20.7) | (18.4–24.6) | (20.0–26.2) | (21.0–27.2) | (22.8–29.0) | (23.3–29.4) | (21.1–27.3) | |||
| Deoxypyridinoline, nmol/day | 12 | 1 | 64.5 ± 16.1 | 61.7 ± 20.5 | 77.3 ± 19.6a* | 80.9 ± 16.9a* | 92.5 ± 23.6a* | 87.3 ± 20.8a* | 97.3 ± 26.5a* | 84.2 ± 22.4a* |
| (52.1–76.1) | (49.5–73.5) | (65.4–89.4) | (68.3–92.5) | (78.3–102.4) | (75.4–99.4) | (84.8–108.2) | (72.3–96.2) | |||
| Deoxypyridinoline, nmol/mmol creatinine | 12 | 1 | 4.2 ± 1.1 | 4.0 ± 0.9 | 4.9 ± 1.0a | 5.2 ± 0.9a* | 5.9 ± 1.4a* | 5.9 ± 1.3a* | 6.3 ± 1.4a* | 5.6 ± 1.4a* |
| (3.6–4.9) | (3.3–4.6) | (4.2–5.6) | (4.6–5.9) | (5.1–6.4) | (5.2–6.6) | (5.7–7.0) | (5.0–6.3) | |||
| Serum BSAP†, U/l | 12 | 4 | 24.3 ± 6.4 | 25.9 ± 9.3 | 25.8 ± 7.2 | 24.8 ± 6.6 | 24.7 ± 7.0 | 24.1 ± 6.1 | 25.4 ± 5.4 | 22.9 ± 5.4b |
| (21.3–27.9) | (21.3–27.9) | (21.9–28.6) | (21.1–27.6) | (21.0–27.4) | (20.6–26.8) | (21.8–28.4) | (19.4–25.4) | |||
| Serum alkaline phosphatase†, U/l | 12 | 61.3 ± 17.5 | 58.5 ± 15.6 | 60.2 ± 14.2 | 61.0 ± 13.2 | 61.7 ± 16.6 | 61.6 ± 11.6 | 64.1 ± 11.3b | 58.4 ± 12.8 | |
| (52.5–67.3) | (50.2–64.5) | (51.8–66.6) | (52.7–67.7) | (52.8–67.8) | (53.4–68.6) | (55.7–71.6) | (50.5–64.8) | |||
| Serum osteocalcin, ng/ml | 12 | 4 | 10.5 ± 1.5c | 12.2 ± 2.2 | 11.1 ± 1.7a | 11.0 ± 1.6b | 11.3 ± 1.8b | 11.2 ± 2.1b | 12.1 ± 1.9 | 11.4 ± 2.0c |
| (10.1–12.4) | (11.1–13.3) | (10.0–12.2) | (10.2–12.4) | (10.2–12.4) | (10.1–12.3) | (11.0–13.2) | (10.3–12.5) | |||
| Serum undercarboxylated osteocalcin, % | 12 | 1 | 37.3 ± 7.6a* | 31.9 ± 6.9 | 33.8 ± 4.1 | 33.5 ± 5.0 | 33.7 ± 4.4 | 33.1 ± 4.4 | 33.1 ± 4.8 | 30.9 ± 5.4 |
| (34.3–40.5) | (28.9–35.0) | (30.8–36.9) | (30.4–36.5) | (30.7–36.8) | (30.0–36.2) | (30.1–36.2) | (27.9–34.0) | |||
| Serum 25-hydroxyvitamin D‡, nmol/l | 12 | 52.4 ± 17.1 | 56.7 ± 10.8 | 62.8 ± 6.9c | 63.6 ± 6.9c | 67.7 ± 9.1a* | 65.6 ± 6.7b | 69.0 ± 7.4a* | 63.7 ± 5.3c | |
| (48.7–60.6) | (51.7–63.1) | (57.8–68.2) | (58.7–68.9) | (63.3–72.9) | (60.7–70.7) | (64.5–73.9) | (58.6–68.8) | |||
| Serum 1,25-dihydroxyvitamin D†, pmol/l | 12 | 93.2 ± 26.3 | 92.8 ± 18.1 | 85.8 ± 25.5 | 88.8 ± 22.5 | 78.5 ± 20.3c | 70.6 ± 13.1a | 94.6 ± 29.2 | 102.4 ± 33.7 | |
| (77.7–103.4) | (79.0–105.2) | (71.7–95.4) | (74.9–99.7) | (66.5–88.4) | (60.3–80.3) | (78.7–104.7) | (85.1–113.2) | |||
| Serum vitamin D binding protein, mg/dl | 7 | 28.2 ± 5.9 | 27.1 ± 2.6 | 31.9 ± 3.1b | 30.9 ± 3.3c | |||||
| (25.3–31.1) | (24.2–30.0) | (29.0–34.8) | (28.0–33.9) | |||||||
| Serum PTH†, pg/ml | 12 | 21.7 ± 7.1 | 19.9 ± 7.8 | 15.7 ± 4.9b | 16.5 ± 5.5b | 15.0 ± 6.0a* | 14.1 ± 4.8a* | 14.1 ± 4.18a* | 17.5 ± 4.7 | |
| (17.3–24.9) | (15.4–22.3) | (12.6–18.1) | (13.0–18.8) | (11.7–16.9) | (11.2–16.1) | (11.3–16.3) | (14.1–20.3) | |||
| Serum OPG, pmol/l | 12 | 2.9 ± 1.1 | 3.0 ± 1.2 | 2.7 ± 1.0c | 2.9 ± 1.1 | 3.0 ± 1.2 | 2.9 ± 1.2 | 3.0 ± 1.2 | 3.1 ± 1.2 | |
| (2.2–3.5) | (2.3–3.6) | (2.1–3.4) | (2.2–3.5) | (2.3–3.6) | (2.2–3.5) | (2.3–3.6) | (2.4–3.7) | |||
| Serum IGF-I, ng/ml | 12 | 387 ± 96 | 390 ± 73 | 400 ± 76 | 428 ± 72c | 421 ± 51 | 424 ± 69c | 438 ± 64b | 355 ± 95c | |
| (344–430) | (347–433) | (357–443) | (385–471) | (378–464) | (381–467) | (395–481) | (312–397) |
Values are means ± SD (95% confidence intervals are in parentheses); n, no. of subjects. No. of outliers excluded is given.
Log transformed.
Transformed by squaring.
Remained significant after adjusting for multiple comparisons.
For this data point, blood samples were collected just before reambulation and are considered a bed rest data point, while the urine samples are a 48-h collection, starting the morning of ambulation, and are considered a post-bed rest data point. BSAP, bone-specific alkaline phosphatase; PTH, parathyroid hormone; OPG, osteoprotegerin; IGF-I, insulin-like growth factor-I.
P < 0.001;
P < 0.01;
P < 0.05.
Bone resorption markers, including N-telopeptide, C-telopeptide, helical peptide, pyridinium cross-links, and deoyxypyridinoline increased during the first week of bed rest and remained high throughout bed rest and through the recovery period. For all resorption biomarkers, both the values normalized to creatinine and the excretion per day were significantly increased during bed rest and remained elevated 5–7 days after reambulation.
Markers of bone formation in general did not increase during this 30-day bed rest study. BSAP and alkaline phosphatase did not change during bed rest. Undercarboxylated osteocalcin decreased during the acclimation period while the subjects were ambulatory. Osteocalcin was variable throughout the study, and none of the results remained significant after adjusting for multiple comparisons. Over the course of the study, 25-hydroxyvitamin D increased, and it was significantly higher in bed rest week 3 and bed rest day 30; 1,25-dihydroxyvitamin D did not change over the course of the study.
Secondary markers of bone metabolism, including osteoprotegerin and insulin-like growth factor-I, did not change significantly during the study. However, parathyroid hormone (PTH) decreased significantly after 2 wk of bed rest and remained decreased until recovery days 5–7, when the concentration was not different from before bed rest.
Vitamins.
Vitamin and nutritional status are presented in Table 2. Vitamin status of subjects was not significantly altered by bed rest. Serum folate was significantly increased on recovery days 5–7. The unadjusted P value for bed rest day 30 indicated that serum folate increased during bed rest, but this value did not remain significant after it was adjusted for multiple comparisons.
Table 2.
Vitamins
| Test | n | Outliers, no. | Pre-bed Rest, Week 1 | Pre-bed Rest, Week 2 | Bed Rest, Week 1 | Bed Rest, Week 2 | Bed Rest, Week 3 | Bed Rest, Week 4 | Bed Rest, Day 30ζ; Recovery, Days 0–3 | Recovery, Days 5–7 |
|---|---|---|---|---|---|---|---|---|---|---|
| γ-Carboxyglutamic acid#, μmol/day | 12 | 1 | 41.1 ± 8.4 | 39.5 ± 8.8 | 42.6 ± 12.1 | 44.2 ± 9.1b | 42.3 ± 11.2 | 42.5 ± 8.1 | 43.0 ± 6.2 | 44.0 ± 8.5b |
| (35.6–45.6) | (34.6–44.5) | (37.1–47.4) | (38.6–49.2) | (36.0–46.1) | (36.9–47.1) | (36.5–46.4) | (38.5–48.9) | |||
| γ-Carboxyglutamic acid†, μmol/mmol creatinine | 12 | 2.7 ± 0.4 | 2.6 ± 0.5 | 2.7 ± 0.6 | 2.8 ± 0.4c | 2.6 ± 0.4 | 2.9 ± 0.5c | 2.8 ± 0.3 | 2.9 ± 0.5b | |
| (2.4–2.9) | (2.4–2.8) | (2.4–2.9) | (2.6–3.1) | (2.4–2.8) | (2.6–3.1) | (2.5–3.0) | (2.6–3.2) | |||
| Plasma phylloquinone, nmol/l | 7 | 1.7 ± 1.1c | 1.2 ± 0.4 | 0.8 ± 0.5 | 0.8 ± 0.4 | |||||
| (1.2–2.2) | (0.7–1.7) | (0.3–1.3) | (0.3–1.2) | |||||||
| Plasma 4-pyridoxic acid, nmol/l | 7 | 2 | 31.3 ± 18.9 | 34.7 ± 15.9 | 38.6 ± 18.0 | 27.8 ± 19.9 | ||||
| (17.2–45.6) | (21.4–48.0) | (25.3–51.9) | (13.7–42.1) | |||||||
| Urinary 4-pyridoxic acid†, μmol/day | 7 | 3 | 11.1 ± 5.2 | 11.0 ± 4.6 | 12.6 ± 4.4 | 13.1 ± 3.3 | 8.5 ± 2.2 | |||
| (7.9–13.1) | (7.7–12.8) | (8.8–14.6) | (9.2–15.0) | (6.4–10.5) | ||||||
| Plasma pyridoxal 5′-phosphate, nmol/l | 7 | 68.9 ± 36.5 | 59.6 ± 29.8 | 62.1 ± 21.8 | 56.0 ± 23.4 | |||||
| (47.8–90.0) | (38.5–80.7) | (41.0–83.2) | (34.9–77.1) | |||||||
| RBC aspartate transaminase, % | 7 | 46.6 ± 11.7 | 54.1 ± 16.6 | 64.4 ± 12.5c | 56.7 ± 10.7 | |||||
| (36.9–56.3) | (44.4–63.7) | (54.8–74.1) | (47.0–66.4) | |||||||
| RBC folate, ng/ml | 7 | 570 ± 221 | 554 ± 158 | 575 ± 104 | 617 ± 108 | |||||
| (455–685) | (439–669) | (460–690) | (502–732) | |||||||
| Serum folate, nmol/l | 7 | 33.4 ± 11.7 | 32.6 ± 7.8 | 39.9 ± 3.6b | 46.4 ± 5.3a* | |||||
| (27.7–39.2) | (26.9–38.3) | (34.1–45.6) | (40.7–52.1) | |||||||
| Serum homocysteine, μmol/l | 7 | 6.6 ± 1.1 | 6.2 ± 1.1 | 5.4 ± 0.6b | 5.2 ± 0.8a | |||||
| (6.0–7.3) | (5.5–6.9) | (4.7–6.1) | (4.6–5.9) | |||||||
| Serum methylmalonic acid, nmol/l | 7 | 1 | 197 ± 117 | 193 ± 96 | 182 ± 96 | 170 ± 92 | ||||
| (141–293) | (118–269) | (107–258) | (95–246) | |||||||
| Serum cystathionine, nmol/l | 7 | 99.7 ± 14.4 | 101.6 ± 17.1 | 114.4 ± 25.6 | 87.9 ± 25.9 | |||||
| (83.9–115.5) | (85.8–117.4) | (98.6–130.2) | (72.0–103.7) | |||||||
| Serum 2-methyl citric acid, nmol/l | 7 | 160 ± 24b | 133 ± 11 | 141 ± 24 | 137 ± 27 | |||||
| (143–177) | (117–150) | (124–158) | (120–153) | |||||||
| RBC glutathione reductase, % | 7 | 30.8 ± 17.3b | 22.4 ± 13.8 | 23.8 ± 11.5 | 18.4 ± 8.6 | |||||
| (21.0–40.6) | (12.7–32.2) | (14.0–33.6) | (8.7–28.2) | |||||||
| RBC transketolase, % | 7 | 6.3 ± 4.1 | 6.1 ± 3.8 | 0.3 ± 0.8a | 2.9 ± 3.3c | |||||
| (3.9–8.7) | (3.7–8.5) | (0.0–2.8) | (0.5–5.3) | |||||||
| Plasma β-carotene, μg/ml | 7 | 0.17 ± 0.09b | 0.24 ± 0.08 | 0.29 ± 0.12 | 0.28 ± 0.08 | |||||
| (0.10–0.24) | (0.17–0.31) | (0.21–0.36) | (0.21–0.35) | |||||||
| Plasma retinol, μg/ml | 7 | 0.46 ± 0.18 | 0.41 ± 0.13 | 0.44 ± 0.08 | 0.39 ± 0.07 | |||||
| (0.36–0.55) | (0.32–0.51) | (0.35–0.54) | (0.29–0.48) | |||||||
| Plasma retinol-binding protein, g/l | 7 | 0.043 ± 0.015 | 0.038 ± 0.010 | 0.040 ± 0.006 | 0.033 ± 0.005 | |||||
| (0.036–0.051) | (0.030–0.045) | (0.033–0.047) | (0.026–0.040) | |||||||
| Plasma α-tocopherol, μg/ml | 7 | 1 | 10.0 ± 1.1c | 9.1 ± 0.9 | 9.2 ± 1.6 | 9.2 ± 1.4 | ||||
| (9.1–11.0) | (8.3–10.2) | (8.3–10.2) | (8.2–10.1) | |||||||
| Plasma γ-tocopherol, μg/ml | 7 | 1 | 1.4 ± 0.4 | 1.3 ± 0.3 | 1.0 ± 0.2a | 1.1 ± 0.2 | ||||
| (1.2–1.7) | (1.1–1.5) | (0.8–1.2) | (0.9–1.3) | |||||||
| Serum vitamin E, mg/g total lipids | 7 | 2.2 ± 0.3 | 2.3 ± 0.4 | 2.3 ± 0.3 | 2.5 ± 0.4 | |||||
| (1.9–2.4) | (2.0–2.5) | (2.0–2.5) | (2.2–2.7) | |||||||
| Plasma vitamin C, μg/ml | 7 | 12.6 ± 2.0 | 12.8 ± 1.5 | 13.3 ± 2.5 | 12.4 ± 2.2 | |||||
| (11.1–14.2) | (11.3–14.3) | (11.8–14.9) | (10.9–13.9) |
Values are means ± SD (95% confidence intervals are in parentheses); n, no. of subjects. No. of outliers excluded is given. RBC, red blood cell.
Log transformed.
sqrt transformed.
Remained significant after adjusting for multiple comparisons.
For this data point, blood samples were collected just before reambulation and are considered a bed rest data point, while the urine samples are a 48-h collection, starting the morning of ambulation, and are considered a post-bed rest data point.
P < 0.001;
P < 0.01;
P < 0.05.
Blood chemistry.
Results from the blood chemistry panel are presented in Table 3. The P value for bed rest day 30 indicated that HDL cholesterol decreased by the end of bed rest. A decrease in HDL seemed to occur from pre-bed rest week 1 to week 2, but this difference was not significant after P was adjusted for multiple comparisons. Subjects' HDL cholesterol was significantly decreased on recovery days 5–7 relative to before bed rest.
Table 3.
Blood chemistry
| Test | n | Pre-bed Rest, Week 1 | Pre-bed Rest, Week 2 | Bed Rest, Week 1 | Bed Rest, Week 2 | Bed Rest, Week 3 | Bed Rest, Week 4 | Bed Rest, Day 30 | Recovery, Days 5–7 |
|---|---|---|---|---|---|---|---|---|---|
| Serum ALT, U/l | 7 | 13.0 ± 5.0 | 14.3 ± 5.7 | 18.4 ± 5.3b | 16.9 ± 4.3 | ||||
| (9.2–16.8) | (10.5–18.1) | (14.7–22.2) | (13.1–20.6) | ||||||
| Serum AST, U/l | 7 | 16.1 ± 3.0 | 16.0 ± 3.4 | 16.6 ± 2.9 | 18.9 ± 4.5c | ||||
| (13.8–18.5) | (13.7–18.3) | (13.8–19.3) | (16.0–21.7) | ||||||
| Serum ceruloplasmin†, mg/dl | 7 | 25.3 ± 4.0 | 23.9 ± 5.7 | 23.9 ± 4.1 | 23.7 ± 4.0 | ||||
| (22.0–28.6) | (20.5–26.7) | (20.7–26.9) | (20.5–26.7) | ||||||
| Serum cholesterol, mg/dl | 7 | 161.3 ± 22.8 | 157.1 ± 16.1 | 147.3 ± 20.7 | 140.0 ± 17.2b | ||||
| (146.9–175.6) | (142.8–171.5) | (132.9–161.6) | (125.6–154.4) | ||||||
| Serum HDL cholesterol, mg/dl | 7 | 59.0 ± 9.4a | 52.6 ± 9.2 | 42.9 ± 7.4a* | 42.6 ± 8.0a* | ||||
| (52.7–65.3) | (46.3–58.9) | (36.5–49.2) | (36.3–48.9) | ||||||
| Serum LDL cholesterol, mg/dl | 7 | 83.9 ± 25.5 | 83.7 ± 16.9 | 85.9 ± 20.5 | 79.4 ± 15.3 | ||||
| (69.1–98.6) | (68.9–98.5) | (71.1–100.6) | (64.6–94.2) | ||||||
| Serum triglycerides, mg/dl | 7 | 101.6 ± 37.2c | 84.4 ± 26.3 | 91.1 ± 26.0 | 69.6 ± 25.5c | ||||
| (80.0–123.2) | (62.8–106.0) | (69.5–112.8) | (48.0–91.2) | ||||||
| Serum creatinine, mg/dl | 12 | 0.93 ± 0.14 | 0.86 ± 0.13 | 0.93 ± 0.17 | 0.91 ± 0.12 | ||||
| (0.85–1.01) | (0.82–0.98) | (0.85–1.01) | (0.83–0.99) | ||||||
| Plasma fibrinogen, g/l | 7 | 2.3 ± 0.3 | 2.2 ± 0.5 | 2.5 ± 0.4 | 2.5 ± 0.4 | ||||
| (2.0–2.7) | (1.9–2.6) | (2.2–2.8) | (2.2–2.8) | ||||||
| Serum total protein, g/dl | 7 | 6.9 ± 0.6 | 6.6 ± 0.4 | 6.8 ± 0.4 | 6.4 ± 0.2 | ||||
| (6.6–7.2) | (6.2–6.9) | (6.4–7.1) | (6.1–6.7) | ||||||
| Serum albumin, g/dl | 7 | 4.0 ± 0.4b | 3.8 ± 0.2 | 3.9 ± 0.3 | 3.8 ± 0.2 | ||||
| (3.8–4.3) | (3.5–4.0) | (3.7–4.1) | (3.6–4.0) | ||||||
| Serum transthyretin, mg/dl | 7 | 29.6 ± 8.5 | 27.9 ± 5.3 | 28.6 ± 4.3 | 24.3 ± 4.1c | ||||
| (25.3–33.9) | (23.6–32.2) | (24.3–32.9) | (20.0–28.7) | ||||||
| Serum chloride, mmol/l | 7 | 102.3 ± 2.6c | 104.1 ± 3.0 | 104.1 ± 1.7 | 105.3 ± 2.0 | ||||
| (100.5–104.0) | (102.4–105.9) | (102.4–105.9) | (103.5–107.0) | ||||||
| Serum phosphorus, mmol/l | 7 | 1.43 ± 0.19 | 1.42 ± 0.10 | 1.44 ± 0.10 | 1.49 ± 0.09 | ||||
| (1.33–1.52) | (1.32–1.51) | (1.35–1.53) | (1.39–1.58) | ||||||
| Serum magnesium, mmol/l | 7 | 0.85 ± 0.06 | 0.83 ± 0.05 | 0.83 ± 0.05 | 0.88 ± 0.05b | ||||
| (0.81–0.89) | (0.79–0.86) | (0.79–0.87) | (0.84–0.91) | ||||||
| Serum potassium, mmol/l | 7 | 4.16 ± 0.32 | 4.20 ± 0.22 | 4.10 ± 0.24 | 3.87 ± 0.18b | ||||
| (3.98–4.34) | (4.02–4.38) | (3.92–4.28) | (3.69–4.05) | ||||||
| Serum sodium, mmol/l | 7 | 138.0 ± 2.0c | 140.0 ± 2.3 | 139.7 ± 1.4 | 140.0 ± 2.4 | ||||
| (136.5–139.5) | (138.5–141.5) | (138.2–141.3) | (138.5–141.5) | ||||||
| Whole blood tests (PCBA) | |||||||||
| Potassiumθ, mmol/l | 12 | 4.10 ± 0.40 | 3.98 ± 0.25 | 4.12 ± 0.37 | 4.06 ± 0.25 | 4.09 ± 0.23 | 3.99 ± 0.26 | 3.89 ± 0.18 | 3.63 ± 0.14a* |
| (3.92–4.23) | (3.82–4.12) | (3.94–4.25) | (3.90–4.21) | (3.93–4.24) | (3.83–4.13) | (3.75–4.03) | (3.50–3.76) | ||
| Sodium, mmol/l | 12 | 140.2 ± 1.3 | 140.6 ± 1.2 | 140.3 ± 1.0 | 140.7 ± 1.2 | 141.0 ± 1.4 | 140.8 ± 1.7 | 140.3 ± 1.5 | 141.5 ± 0.9c |
| (139.4–140.9) | (139.9–141.3) | (139.5–141.0) | (139.9–141.4) | (140.3–141.7) | (140.0–141.5) | (139.6–141.1) | (140.8–142.2) | ||
| Glucose, mg/dl | 12 | 90.0 ± 7.6a* | 85.4 ± 4.7 | 87.3 ± 5.9c | 88.2 ± 6.4b | 88.1 ± 4.8b | 88.2 ± 5.5b | 88.2 ± 5.2b | 86.1 ± 4.6 |
| (86.8–93.2) | (82.2–88.6) | (84.1–90.5) | (85.0–91.4) | (84.9–91.3) | (85.0–91.4) | (85.0–91.4) | (82.9–89.3) | ||
| pH | 12 | 7.34 ± 0.05 | 7.36 ± 0.03 | 7.36 ± 0.04 | 7.36 ± 0.02 | 7.34 ± 0.04 | 7.36 ± 0.03 | 7.37 ± 0.03 | 7.37 ± 0.04 |
| (7.32–7.36) | (7.34–7.38) | (7.34–7.39) | (7.34–7.38) | (7.32–7.36) | (7.34–7.38) | (7.35–7.40) | (7.35–7.39) |
Values are means ± SD (95% confidence intervals are in parentheses); n, no. of subjects. ALT, alanine transaminase; PCBA, portable clinical blood analyzer.
Log transformed.
1/sqrt transformed;
Remained significant after adjusting for multiple comparisons.
P < 0.001;
P < 0.01;
P < 0.05.
Potassium measured in whole blood was significantly decreased on recovery days 5–7, as was serum potassium, although the significance for serum potassium did not remain significant after P was adjusted for multiple comparisons.
Glucose concentration was significantly elevated when subjects entered the bed rest facility compared with the second week of acclimatization before bed rest. The unadjusted P values indicated that glucose increased during bed rest, but none of the differences remained significant after the P value was adjusted for multiple comparisons.
Urine chemistry.
Urine chemistry results are presented in Table 4. All urine data are presented both as excretion rate per day (24-h period) and normalized to creatinine. Urinary creatinine was significantly increased at the two transition periods: bed rest week 1 and recovery days 0–3.
Table 4.
Urine chemistry
| Test | n | Outliers, no. | Pre-bed Rest, Week 1 | Pre-bed Rest, Week 2 | Bed Rest, Week 1 | Bed Rest, Week 2 | Bed Rest, Week 3 | Bed Rest, Week 4 | Recovery, Days 0–3 | Recovery, Days 5–7 |
|---|---|---|---|---|---|---|---|---|---|---|
| 3-Methyl histidine, μmol/g creatinine | 7 | 137 ± 16 | 140 ± 22 | 130 ± 26 | 137 ± 7 | 128 ± 27 | ||||
| (124–150) | (125–153) | (114–141) | (125–150) | (115–141) | ||||||
| 3-Methyl histidine†, μmol/day | 7 | 226 ± 57 | 227 ± 36 | 205 ± 51c | 232 ± 40 | 208 ± 58 | ||||
| (187–261) | (187–261) | (166–232) | (186–257) | (169–236) | ||||||
| Creatinine†, mmol/day | 12 | 1 | 4.9 ± 0.9c | 4.5 ± 0.7 | 5.5 ± 0.8a* | 5.1 ± 0.8b | 5.3 ± 0.6a | 4.8 ± 0.8 | 5.3 ± 1.1a* | 5.0 ± 1.1b |
| (4.3–5.3) | (4.0–4.9) | (4.8–5.9) | (4.5–5.5) | (4.5–5.6) | (4.3–5.2) | (4.6–5.6) | (4.4–5.4) | |||
| Chloride, mmol/day | 7 | 119.8 ± 21.1 | 129.2 ± 18.5 | 107.4 ± 22.5a* | 88.7 ± 5.1a* | 111.0 ± 13.0a | ||||
| (107.2–133.7) | (117.3–143.3) | (94.0–119.9) | (69.4–98.9) | (97.3–123.2) | ||||||
| Phosphorus, mg/day | 12 | 11 | 774 ± 173 | 806 ± 165 | 835 ± 135 | 851 ± 168 | 891 ± 147c | 812 ± 175 | 697 ± 129a* | 695 ± 150a |
| (697–883) | (720–900) | (743–922) | (760–940) | (788–969) | (720–898) | (596–769) | (607–784) | |||
| Magnesium, mg/day | 12 | 9 | 129 ± 42b | 112 ± 34 | 121 ± 25 | 122 ± 21c | 123 ± 34c | 129 ± 30b | 130 ± 20b | 116 ± 28 |
| (111–144) | (96–128) | (104–136) | (107–140) | (106–139) | (112–145) | (109–140) | (100–132) | |||
| Potassium, mmol/day | 7 | 59.2 ± 15.3b | 70.7 ± 11.0 | 63.2 ± 10.7c | 60.4 ± 7.5b | 47.0 ± 8.2a* | ||||
| (51.8–66.7) | (63.2–78.4) | (54.5–69.8) | (51.4–65.6) | (39.5–54.4) | ||||||
| Sodium, mmol/day | 7 | 116 ± 21c | 130 ± 16 | 110 ± 23b | 72 ± 6a* | 116 ± 15c | ||||
| (103–128) | (118–145) | (96–122) | (57–82) | (103–129) | ||||||
| Citrate, mg/day | 12 | 1 | 654 ± 272a | 753 ± 309 | 714 ± 317 | 657 ± 219c | 679 ± 225 | 688 ± 293b | 747 ± 298 | 778 ± 331 |
| (494–814) | (600–920) | (563–884) | (541–862) | (547–868) | (520–840) | (598–916) | (618–938) | |||
| Oxalate†, mg/day | 12 | 48.3 ± 15.3 | 51.1 ± 16.7 | 43.3 ± 17.3a | 46.8 ± 19.4c | 41.1 ± 14.5a* | 41.0 ± 12.7a* | 40.0 ± 9.8a* | 38.1 ± 11.6a* | |
| (38.5–54.7) | (40.8–58.1) | (34.0–48.4) | (36.5–52.1) | (33.2–47.3) | (32.8–46.6) | (32.2–45.4) | (30.5–43.4) | |||
| Sulfate, mmol/day | 12 | 22.4 ± 3.5b | 20.6 ± 3.3 | 22.2 ± 3.3b | 22.4 ± 4.0b | 23.2 ± 3.1a* | 21.8 ± 3.3 | 22.0 ± 2.2 | 17.3 ± 2.7a* | |
| (20.6–24.2) | (18.7–22.3) | (20.5–24.1) | (20.7–24.3) | (21.2–24.8) | (19.9–23.4) | (19.9–23.3) | (15.5–19.0) | |||
| Uric acid, mg/day | 7 | 3 | 696 ± 152c | 646 ± 117 | 583 ± 136c | 711 ± 135 | 657 ± 130 | |||
| (594–789) | (547–742) | (492–689) | (590–783) | (560–754) | ||||||
| pHψ | 12 | 6.25 ± 0.31b | 6.46 ± 0.11 | 6.43 ± 0.18 | 6.44 ± 0.15 | 6.42 ± 0.21 | 6.50 ± 0.13 | 6.25 ± 0.16a | 6.36 ± 0.24 | |
| (6.19–6.44) | (6.39–6.59) | (6.37–6.57) | (6.35–6.56) | (6.36–6.56) | (6.41–6.60) | (6.15–6.39) | (6.29–6.51) | |||
| Osmolality, mosmol/kg | 7 | 260 ± 48 | 249 ± 36 | 261 ± 40 | 258 ± 49 | 240 ± 46 | ||||
| (226–293) | (211–279) | (227–295) | (221–287) | (206–274) | ||||||
| Specific gravityψ | 12 | 1.008 ± 0.001c | 1.007 ± 0.001 | 1.008 ± 0.001a | 1.008 ± 0.001c | 1.009 ± 0.001a* | 1.008 ± 0.001c | 1.008 ± 0.001 | 1.007 ± 0.001 | |
| (1.007–1.008) | (1.007–1.008) | (1.007–1.009) | (1.007–1.008) | (1.008–1.009) | (1.007–1.008) | (1.007–1.008) | (1.006–1.008) | |||
| Volume, ml/day | 12 | 3,236 ± 637 | 3,419 ± 407 | 2,946 ± 383a* | 3,163 ± 447b | 2,986 ± 478a | 3,187 ± 495c | 3,060 ± 533a | 3,135 ± 598b | |
| (2,951–3,522) | (3,156–3,731) | (2,668–3,243) | (2,832–3,416) | (2,765–3,344) | (2,883–3,457) | (2,760–3,310) | (2,849–3,420) | |||
| Creatinine clearance, ml/min | 12 | 130 ± 12 | 134 ± 20 | 130 ± 10 | 133 ± 15 | |||||
| (122–137) | (124–145) | (120–136) | (125–141) |
Values are means ± SD (95% confidence intervals are in parentheses); n, no. of subjects. No. of outliers excluded is given.
Log transformed.
Skewed log transformed.
Remained significant after adjusting for multiple comparisons.
P < 0.001;
P < 0.01;
P < 0.05.
Urinary potassium decreased on recovery days 5–7, corresponding to the significant decrease in serum potassium on the same day and a modest decrease in whole blood potassium. Other inorganic analytes, including chloride, phosphorus, and sodium, decreased on recovery days 0–3, although chloride significantly decreased before the recovery period (bed rest week 4). Urinary sulfate and specific gravity both increased significantly in bed rest week 3. Total urine volume per day decreased significantly during the first week of bed rest.
Oxalate excretion decreased significantly during bed rest and continued to be lower during the recovery period. The decrease was significant in bed rest weeks 3 and 4 and on recovery days 0–3 and recovery days 5–7. The unadjusted P values indicated that oxalate decreased as early as bed rest week 1, but this result was insignificant when the P values were adjusted for multiple comparisons.
Hematology, iron metabolism, and endocrinology.
Table 5 presents hematology, iron status, and endocrinology results. Transferrin decreased significantly on recovery days 5–7, but none of the other analytes changed.
Table 5.
Hematology, iron status, and endocrinology
| Test | n | Outliers, no. | Pre-bed Rest, Week 1 | Pre-bed Rest, Week 2 | Bed Rest, Week 1 | Bed Rest, Week 2 | Bed Rest, Week 3 | Bed Rest, Week 4 | Bed Rest, Day 30ζ, Recovery, Days 0–3 | Recovery, Days 5–7 |
|---|---|---|---|---|---|---|---|---|---|---|
| Whole blood hemoglobin, g/dl | 7 | 14.3 ± 1.8c | 13.7 ± 1.5 | 14.4 ± 1.6b | 13.1 ± 1.4c | |||||
| (13.1–15.5) | (12.5–14.9) | (13.2–15.6) | (11.9–14.3) | |||||||
| Whole blood hematocrit, % | 7 | 42.2 ± 5.0c | 40.4 ± 4.0 | 42.0 ± 4.5 | 38.2 ± 3.4b | |||||
| (39.0–45.3) | (37.2–43.6) | (38.8–45.2) | (35.0–41.4) | |||||||
| Whole blood mean corpuscular volume, fl | 7 | 92.5 ± 4.3 | 92.1 ± 4.5 | 91.8 ± 4.0 | 92.3 ± 4.4 | |||||
| (89.3–95.7) | (88.9–95.3) | (88.6–95.0) | (89.1–95.5) | |||||||
| Serum ferritin, ng/ml | 7 | 81.4 ± 58.4 | 76.6 ± 68.2 | 101.9 ± 78.9c | 92.4 ± 76.7 | |||||
| (28.8–134.0) | (24.0–129.2) | (49.2–154.5) | (39.8–145.0) | |||||||
| Serum transferrin, mg/dl | 7 | 273 ± 38b | 254 ± 23 | 233 ± 32b | 219 ± 25a* | |||||
| (251–295) | (232–276) | (211–255) | (197–242) | |||||||
| Serum transferrin receptors, μg/ml | 7 | 1 | 4.5 ± 0.6a | 3.7 ± 0.7 | 3.8 ± 0.8 | 3.4 ± 0.7 | ||||
| (3.7–4.9) | (3.2–4.3) | (3.3–4.4) | (2.9–4.0) | |||||||
| Serum testosterone, nmol/l | 8 | 20.5 ± 7.8c | 17.6 ± 6.1 | 17.6 ± 5.3 | 17.1 ± 6.7 | |||||
| (16.0–25.1) | (13.1–22.2) | (13.1–22.2) | (12.6–21.6) | |||||||
| Serum bioavailable testosterone†, nmol/l | 8 | 6.1 ± 2.2 | 5.7 ± 2.1 | 5.1 ± 1.3 | 4.6 ± 1.6 | |||||
| (4.5–7.5) | (4.1–6.9) | (3.8–6.4) | (3.3–5.6) | |||||||
| Serum free testosterone†, pmol/l | 8 | 375 ± 136 | 350 ± 126 | 315 ± 81 | 283 ± 100 | |||||
| (275–460) | (253–424) | (235–394) | (204–342) | |||||||
| Serum sex hormone binding globulin, nmol/l | 8 | 30.1 ± 10.6 | 25.9 ± 7.0 | 28.6 ± 7.8 | 30.7 ± 10.6 | |||||
| (23.7–36.4) | (19.5–32.2) | (22.3–34.9) | (24.3–37.0) | |||||||
| Serum DHEA, μg/l | 7 | 1 | 19.1 ± 7.4 | 18.9 ± 7.9 | 15.5 ± 7.4c | 17.4 ± 5.6 | ||||
| (13.7–24.4) | (14.8–25.7) | (10.2–20.9) | (12.0–22.7) | |||||||
| Serum DHEA-S, μg/ml | 7 | 2.6 ± 0.9 | 2.8 ± 0.8 | 2.6 ± 0.8 | 2.7 ± 0.9 | |||||
| (2.0–3.2) | (2.2–3.4) | (2.0–3.2) | (2.1–3.3) | |||||||
| Serum cortisol‡, μg/dl | 12 | 18.2 ± 4.8 | 18.6 ± 5.2 | 18.7 ± 3.8 | 19.2 ± 5.4 | 19.1 ± 5.0 | 19.1 ± 5.2 | 21.8 ± 5.1c | 19.0 ± 3.7 | |
| (15.8–21.4) | (16.4–21.9) | (16.0–21.6) | (17.0–22.3) | (16.7–22.2) | (16.8–22.2) | (19.8–24.5) | (16.4–21.9) | |||
| Urinary cortisol†, μg/day | 7 | 74.5 ± 25.4 | 72.3 ± 28.6 | 81.3 ± 46.8 | 81.6 ± 28.0 | 62.8 ± 29.4 | ||||
| (52.5–93.8) | (49.2–88.4) | (53.0–95.2) | (55.8–98.3) | (43.0–76.8) | ||||||
| Serum estradiol, pg/ml | 4 | 85.5 ± 34.8 | 99.5 ± 77.7 | 118.0 ± 73.2 | 68.8 ± 22.2 | 65.0 ± 27.9 | 91.8 ± 33.1 | 123.3 ± 85.1 | 107.3 ± 82.5 | |
| (26.5–144.5) | (40.5–158.5) | (59.0–177.0) | (9.7–127.8) | (6.0–124.0) | (32.7–150.8) | (64.2–182.3) | (48.2–166.3) | |||
| Serum leptin#, μg/l | 12 | 14.7 ± 10.1 | 14.5 ± 10.5 | 16.0 ± 12.5 | 15.1 ± 11.1 | 15.1 ± 10.1 | 16.0 ± 11.4 | 15.2 ± 9.9 | 12.1 ± 8.6b | |
| (7.8–19.5) | (7.5–19.0) | (8.4–20.4) | (8.0–19.7) | (8.0–19.8) | (8.5–20.6) | (8.2–20.1) | (5.9–16.4) |
Values are means ± SD (95% confidence intervals are in parentheses); n, no. of subjects. No. of outliers excluded is given.
Log transformed.
sqrt transformed.
Transformed by squaring.
Remained significant after adjusting for multiple comparisons.
For this data point, blood samples were collected just before reambulation and are considered a bed rest data point, while the urine samples are a 48-h collection, starting the morning of ambulation, and are considered a post-bed rest data point. DHEA, dehydroepiandrosterone; DHEA-S, dehydroepiandrosterone sulfate.
P < 0.001;
P < 0.01;
P < 0.05
Oxidative damage markers and antioxidants.
Markers of oxidative stress and damage are presented in Table 6. There were no significant changes in oxidative stress or markers of oxidative damage before, during, or after bed rest.
Table 6.
Markers of oxidative damage and antioxidants
| Test | n | Outliers, no. | Pre-bed Rest, Week 1 | Pre-bed Rest, Week 2 | Bed Rest, Week 1 | Bed Rest, Week 2 | Bed Rest, Week 3 | Bed Rest, Week 4 | Bed Rest, Day 30ζ, Recovery, Days 0–3 | Recovery, Days 5–7 |
|---|---|---|---|---|---|---|---|---|---|---|
| 8-Hydroxy 2′-deoxyguanosine, μg/g creatinine | 7 | 3.3 ± 1.1 | 2.8 ± 1.1 | 3.0 ± 0.9 | 2.9 ± 0.9 | 2.8 ± 0.9 | ||||
| (2.6–4.0) | (2.1–3.6) | (2.3–3.7) | (2.5–3.9) | (2.1–3.5) | ||||||
| PGF2α†, ng/day | 7 | 2,246 ± 473 | 2,182 ± 451 | 2,265 ± 626 | 2,300 ± 427 | 1,978 ± 484 | ||||
| (1,826–2,630) | (1,770–2,555) | (1,807–2,609) | (1,779–2,548) | (1,589–2,289) | ||||||
| Glutathione peroxidase, U/g hemoglobin | 7 | 59.8 ± 20.2 | 59.2 ± 16.2 | 57.8 ± 16.4 | 55.3 ± 13.6 | |||||
| (47.4–72.2) | (46.8–71.6) | (45.4–70.2) | (42.9–67.7) | |||||||
| Heme, μmol/l | 7 | 28.9 ± 10.3a | 21.3 ± 5.2 | 22.1 ± 4.4 | 18.6 ± 5.4 | |||||
| (23.9–33.9) | (16.3–26.2) | (17.2–27.1) | (13.7–23.6) | |||||||
| SOD, U/g hemoglobin | 7 | 1,481 ± 52 | 1464 ± 138 | 1,412 ± 113 | 1,377 ± 145 | |||||
| (1,394–1,568) | (1,376–1,551) | (1,325–1,500) | (1,289–1,464) | |||||||
| Total antioxidant capacity, mmol/l | 7 | 1.74 ± 0.12c | 1.69 ± 0.08 | 1.72 ± 0.11 | 1.70 ± 0.09 | |||||
| (1.67–1.82) | (1.61–1.76) | (1.64–1.79) | (1.63–1.78) | |||||||
| C-reactive protein, mg/l | 7 | 1 | 0.9 ± 0.9 | 0.9 ± 1.0 | 1.4 ± 1.6 | 1.4 ± 1.3c | ||||
| (0.0–1.8) | (0.0–1.8) | (0.4–2.3) | (0.7–2.7) | |||||||
| Lipid peroxides, μmol/l | 7 | 0.56 ± 0.11a | 0.46 ± 0.07 | 0.45 ± 0.05 | 0.46 ± 0.09 | |||||
| (0.49–0.62) | (0.39–0.52) | (0.39–0.51) | (0.40–0.52) | |||||||
| Malondialdehyde, μmol/l | 7 | 0.34 ± 0.07a | 0.27 ± 0.04 | 0.27 ± 0.04 | 0.27 ± 0.05 | |||||
| (0.30–0.38) | (0.23–0.31) | (0.23–0.31) | (0.23–0.31) |
Values are means ± SD (95% confidence intervals are in parentheses); n, no. of subjects. No. of outliers excluded is given.
Log transformed.
For this data point, blood samples were collected just before reambulation and are considered a bed rest data point, while the urine samples are a 48-h collection, starting the morning of ambulation, and are considered a post-bed rest data point. PGF2α, prostaglandin F2α; SOD, superoxide dismutase.
P < 0.001;
P < 0.05.
DISCUSSION
This study provides a comprehensive biochemical assessment of human adaptation to 30-day head-down-tilt bed rest. Given the carefully controlled nature of the study, and documentation of the standardized procedures for this and other NASA bed rest studies (16, 33, 41), the present study provides a 30-day time point to compare with longer studies of a similar nature. Studies of shorter duration permit savings of time and cost and may allow investigators to draw conclusions sooner and change direction of an overall program when countermeasures being tested do not prove viable. Beyond this general view, several specific findings are of note as well.
Calcium and bone metabolism.
In this study, urinary calcium increased after 2 wk of bed rest and remained high throughout bed rest. The magnitude of this increase in urinary calcium is similar to that observed in bed rest studies of longer (60 days, 90 days) duration (41). In the present study, calcium excretion increased 21% after 2 wk of bed rest and 28% after 4 wk of bed rest, relative to pre-bed rest values. During spaceflight, urinary calcium increased 45% on flight day 15 and 52% on flight day 30 (24). This increase in calcium excretion during spaceflight is ameliorated by flight day 120 (24), suggesting that it represents an adaptive response to spaceflight (likely associated with an increase in the amount and intensity of exercise crewmembers do over the course of the mission). Although the changes in calcium excretion rate in the present study are intriguing, urinary calcium is notoriously variable because of the prolific nature of calcium, so it is important to look at other markers of bone turnover in conjunction with calcium excretion rate.
All urinary biomarkers of bone resorption increased during the first week of bed rest and remained high throughout bed rest and the recovery period. The increase in all markers provided a consistent signal, indicating that bone was rapidly being resorbed early during bed rest, and resorption continued to be elevated even through a 5- to 7-day recovery period. Previous studies have shown that a rapid and consistent increase in markers of bone resorption occurs within the first few days of bed rest (3, 10, 13, 21, 36). Therefore, bed rest studies that evaluate countermeasures predicted to mitigate bone loss could be conducted over relatively short periods (14 days or 30 days). For 30-day bed rest studies to observe the return of bone marker concentrations to pre-bed rest levels, a longer (e.g., >1 wk) recovery may be required.
Markers of bone formation (BSAP, alkaline phosphatase, and osteocalcin) did not change during the study; this was expected, given that subjects were not performing any resistance exercise, the only countermeasure documented to increase bone formation during bed rest, when administered with or without vibration (2, 18). Undercarboxylated osteocalcin decreased during the acclimation period and remained low throughout the remainder of the study. This decrease could indicate an improvement in vitamin K status, which would decrease the percentage of undercarboxylated osteocalcin (9). The decrease in undercarboxylated osteocalcin suggests that the diet provided in the bed rest facility was higher in vitamin K than the subjects were typically used to consuming.
With respect to vitamin D, 25-hydroxyvitamin D increased over the course of the study and was significantly higher in bed rest week 3 and on bed rest day 30. These changes indicate that the 800 IU/day vitamin D supplements provided to the subjects during bed rest improved their vitamin D status, as expected with supplementation and their likely higher intake during the study than before. The subjects' 1,25-dihydroxyvitamin D did not change over the course of the study.
PTH is responsible for tightly regulating blood calcium levels. In healthy, weight-bearing subjects, an increase in PTH will lead to an increase in osteoclastogenesis and subsequent bone resorption, as well as an increase in intestinal absorption of calcium. During skeletal unloading, bone is being resorbed, which increases serum calcium (slightly); this provides negative feedback in the PTH pathway. In this study, PTH was significantly decreased after 3 wk of bed rest. There was a 25% decrease in serum PTH in bed rest week 3, and a 29% decrease in PTH in bed rest week 4 and on bed rest day 30. PTH returned to pre-bed rest levels by recovery days 5–7. These changes are similar to those seen in previous bed rest studies (41), but represent about one-half the decrease that has been observed during spaceflight (∼60% decrease), in individuals not performing intensive resistance exercise (24).
Overall, markers of bone metabolism indicated that bed rest caused an increase in bone resorption and no change in markers of bone formation.
Vitamins.
Serum folate was significantly increased on recovery days 5–7, and the unadjusted P value on bed rest day 30 indicated that serum folate increased. The increases in serum folate after bed rest suggest that subjects' dietary folate was higher when intake was controlled and a balanced diet was provided than it was when the subjects consumed their normal diet. In bed rest studies of longer duration, RBC folate has been shown to increase, also indicating that dietary intake of folate was higher at the bed rest facility (41). Although this result is not directly related to spaceflight or its impact on astronauts, it is a consistently observed phenomenon of bed rest studies. Subjects who volunteer for bed rest are not always in optimal nutritional status. A similar issue might occur in the astronaut population and should continue to be monitored before and after spaceflight (29).
Blood chemistry.
The majority of blood chemistry tests did not change during bed rest. However, a decrease in HDL cholesterol was observed at the end of bed rest and in the week following reambulation. Increases in HDL are associated with increased physical activity (7). A previous 20-day bed rest study showed that HDL decreased during bed rest due to lack of physical activity (35). A decrease in HDL has also been observed in spaceflight (15).
The decrease in potassium measured in whole blood on recovery days 5–7 was unique, as a similar decrease was not observed in magnesium or sodium.
Glucose level was significantly higher when subjects entered the bed rest facility than it was after they had been in the facility for a week. Glucose concentration was within the normal clinical range throughout the study. The change in glucose in the acclimation period may have resulted from a change in diet when subjects entered the bed rest facility.
Urine chemistry.
Urinary creatinine was significantly increased at two transition periods, bed rest week 1 and recovery days 0–3. This could be related to multiple factors, including fluid shifts, kidney function, or alterations in muscle metabolism/catabolism. The creatinine clearance data do not show any systematic evidence of changes in kidney function. Data for some urinary analytes are presented as both excretion per day and normalized to creatinine. While normalization to creatinine is often preferred (or required, given study design), these must be taken with caution, given the potential for creatinine itself to be a confounding factor.
Decreases in inorganic analytes on recovery days 0–3 were likely caused by fluid shifts during reambulation. Chlorine significantly decreased before the recovery period (during bed rest week 4). Urinary sulfate and specific gravity both increased significantly on bed rest week 3. Total urine volume per day decreased significantly during the first week of bed rest as subjects adjusted to their new position.
Oxalate decreased significantly during bed rest and into the recovery period. The decrease was significant on bed rest week 3, bed rest week 4, recovery days 0–3, and recovery days 5–7 with indication as early as bed rest week 1. There was a significant inverse correlation between calcium (mmol/day) and oxalate (mmol/day) (R = −0.18, P < 0.02). This correlation has not been noted in previous bed rest studies, which suggests it may be a transient correlation during early bed rest.
Hematology, iron metabolism, and endocrinology.
Unlike results of previous bed rest studies, no significant changes occurred in hematology, iron status, or endocrinology during the present study. A significant decrease in circulating transferrin on recovery days 5–7 suggests that less iron was being transported.
Oxidative damage markers and antioxidants.
No significant changes occurred in oxidative stress markers before, during, or after this 30-day bed rest study. This contrasts with results from long-duration (>60 days) bed rest studies in which superoxide dismutase, total antioxidant capacity, glutathione peroxidase, lipid peroxides, and 8-hydroxy-2′-deoxyguanosine all changed (41).
Conclusion.
These data provide a broad overview of the biochemistry associated with short-duration bed rest studies and provide an impetus for using shorter studies to save time and costs, allowing faster overall progression on efforts to counteract physiological changes during spaceflight. Markers of bone metabolism can be easily observed to change in short-duration bed rest studies, whereas bed rest studies of longer duration are required for study of other markers, such as changes in iron metabolism and oxidative stress. In future studies that examine countermeasures to bone loss, the bed rest duration needed will depend on the mechanism by which the countermeasure affects bone. Studies with the goal of altering bone formation may need to be longer, whereas studies in which bone resorption will be altered can be shorter. To examine alterations in iron metabolism and oxidative stress, either bed rest studies of longer duration will need to be performed, or other analogs, such as saturation diving (40), must be sought. This paper provides baseline data for relatively short-duration bed rest studies.
GRANTS
This study was funded in part by the NASA Flight Analogs Project of NASA's Human Research Program, and in part by grant 1UL1RR029876-01 from the National Center for Advancing Translational Sciences, National Institutes of Health.
DISCLOSURES
No conflicts of interest, financial or otherwise, are declared by the author(s).
AUTHOR CONTRIBUTIONS
Author contributions: J.L.L.M., S.R.Z., and R.P.-S. analyzed data; J.L.L.M., S.R.Z., and S.M.S. interpreted results of experiments; J.L.L.M., S.R.Z., and S.M.S. drafted manuscript; J.L.L.M., S.R.Z., M.H., R.P.-S., K.E., and S.M.S. edited and revised manuscript; J.L.L.M., S.R.Z., M.H., R.P.-S., K.E., and S.M.S. approved final version of manuscript; S.R.Z., M.H., and S.M.S. conception and design of research; M.H., K.E., and S.M.S. oversaw analyses.
ACKNOWLEDGMENTS
The NASA Flight Analogs Project Team was essential to the success of this project. We thank the subjects for their time and willingness to participate in this study. We thank the staff of the UTMB Institute for Translational Sciences-Clinical Research Center for assistance in conducting the study, and we thank the staff of the NASA Johnson Space Center Nutritional Biochemistry Laboratory for assistance in processing and analyzing the samples and in all aspects of carrying out this project. We thank Jane Krauhs for editorial assistance.
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