Long duration human space flight leads to loss of bone mass. As a consequence, there is a need for techniques to sensitively detect changes in the net rate of bone formation or resorption (i.e. change in "bone mineral balance") and to assess the effectiveness of countermeasures. It has been documented in bed rest experiments that measurements of the calcium (Ca) isotope composition of urine using mass spectrometry can be used to monitor rapid changes in net bone mineral balance that are not directly observable by other means. This could be extended to the International Space Station (ISS), in order to demonstrate the utility of Ca isotopes as a tool for monitoring bone mineral balance and countermeasures to bone resorption in space. This proposal built on a successful existing collaboration between researchers at Arizona State University and NASA Johnson Space Center to study and apply the Ca isotope method as a bone biomarker. The proposed project paved the way for future development of capability to measure Ca isotopes in-flight to monitor bone health during exploration-class space missions where in situ evaluation of the effectiveness of countermeasures was required to assure crew health and safety.
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Archived urine samples from ISS crewmembers were collected before, during and after flight. Aliquots of archived urine specimens from 30 ISS crewmembers were analyzed for Ca isotope composition. Urine samples were taken from 24-hr collections obtained before, during and after spaceflight. Eight crewmembers were female. Mean age at launch was 45 years. DXA measurements of all crewmembers were made less than two years prior to launch and about 30 days after return to earth. Calcium isotopes were compared to calcium excretion rate, traditional biomarkers of bone metabolisms and imaging studies to examine the correlations. Calcium isotopes were used in a model designed by the investigators to quantify the amount of bone loss undergone by the crewmembers
Crewmembers participated in one of three bone loss countermeasure regimens while in space. The ARED (advanced resistive exercise device) (n=16) and iRED (interim resistive exercise device)(N=7) groups were treated only with ARED and iRED. The bisphosphate group (n=7) was treated with both ARED and the bisphosphate Alendronate, a commonly used anti-resorptive drug. Alendronate treatment started several months before spaceflight. In- flight exercise started after an initial adjustment period of about 30 days. Each analytical run required analysis of additional quality control solutions, including a bracketing standard, two international calibration standards, a natural sample (IAPSO seawater), a calibration curve of standards of varying spike-sample ratio, and frequent blank correction. In addition, it required preparation of all standards and samples out of a single batch of tightly controlled trace metal grade molarity acid
The effect of three treatment groups – bisphosphonate, Interim Resistive Exercise Device (iRED) and Advanced Resistive Exercise Device (ARED) was compared for the relative amount of bone loss. Refrigeration was not required for any of these samples.
The results confirmed that Ca isotopes act as an accurate, near-real time monitor in space flight and allowed to address the five primary questions posed at the beginning of the study. Ca isotope data indicated that amount of bone loss correlated to intervention type. On average, exercise-only crewmembers lost bone, particularly in the first 30 days of spaceflight. Individuals who did heavier resistance exercise (using the advanced resistive exercise device, ARED) maintained BMB better than those that did lower resistance exercise (using the interim resistive exercise device, iRED). This difference became more pronounced as the length of the mission increased, highlighting the need for vigorous exercise facilities in long-term space missions for bone health.
The results of this study were consistent with data from the bed rest populations. This confirmed that variation of dietary d44/42Ca is a minor concern. Inflight d44/42Ca was significantly lower than preflight baseline in both the iRED and ARED groups, and slightly but significantly higher in the Alendronate group (Table 1) (P= 0.01). Inflight d44/42Ca measurements in the ARED and iRED groups were not significantly different overall (P=0.09). However, the two groups show distinct trends: d44/42Ca of both groups fell rapidly during the 30-day adjustment period before inflight exercise started. After flight day 30, d44/42Ca rose significantly in the ARED group (+0.17‰, P=0.02) but not in the iRED group (+0.03, P=0.81). The ARED group maintained higher d44/42Ca values than the iRED group for the remainder of spaceflight. No significant differences in d44/42Ca based on age, sex, initial BMI, or time in space were detected.
Analyses of urine samples from 30 ISS crewmembers undergoing various bone loss countermeasures showed a similar relationship between the isotopic composition of naturally occurring Ca and BMB that was observed in bed rest studies. Each individual crewmember had a baseline average Ca isotope ratio before going into space. For crewmembers whose only bone loss preventative intervention was exercise, the Ca isotope ratio typically decreased – indicating an increase in bone loss--shortly after spaceflight began, remained low during spaceflight, and returned to pre-flight values upon return to Earth.
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Gordon GW, Romaniello SR, Skulan JL, Smith SM, Zwart S, Anbar AD. "Stable Calcium Isotopes in Urine as a Biomarker of Bone Mineral Balance in Spaceflight." 2016 NASA Human Research Program Investigators’ Workshop, Galveston, TX, February 8-11, 2016.
Gordon GW, Romaniello SR, Skulan JL, Smith SM, Zwart S, Zheng W, Letcher AJ, Anbar, AD. "Natural calcium isotopes provide rapid and precise monitoring of bone mineral balance changes in microgravity." 2018 NASA Human Research Program Investigators’ Workshop, Galveston, TX, January 22-25, 2018.
Skulan J, Smith S, Anbar AD, Gordon GW, Romaniello SJ, Zwart S. "Calcium isotopes measure change in bone mineral balance during spaceflight." Presented at the 22017 NASA Human Research Program Investigators’ Workshop, Galveston, TX, January 23-26, 2017.
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