NOTE: End date changed to 11/30/2016 per PI and NSSC information (Ed., 12/14/15)

References:

(1). Morgan JL, Skulan JL, Gordon GW, Romaniello SJ, Smith SM, Anbar AD (2012). Rapidly assessing changes in bone mineral balance using natural stable calcium isotopes. Proc. Natl. Acad. Sci. USA 109, 9989-9994;

(2). Skulan J, Bullen T, Anbar AD, Puzas JE, Shackelford L, LeBlanc A, Smith SM (2007). Natural calcium isotopic composition of urine as a marker of bone mineral balance. Clin. Chem. 53,1155-1158;

(3). Morgan JLL, Skulan JL, Gordon GW, Romaniello SJ, Smith SM and Anbar AD (2011). High-precision measurement of variations in calcium isotope ratios in urine by multiple collector inductively coupled plasma mass spectrometry. Anal. Chem. 83, 6956–6962.

The usefulness of this technique extends beyond measuring bone loss in spaceflight to the detection and evaluation of treatment for any disease involving disruption in BMB, including osteopenia/osteoporosis, cancer, and Paget’s disease. For example, we currently are exploring the application of the Ca isotope technique to the early detection of osteolytic lesions in multiple myeloma. The potential usefulness of Ca isotopes supports their widespread clinical application. We are exploring the possibility of using laser fluorescence, rather than conventional mass spectrometry, to build small, compact Ca isotope measurement instruments suitable to both spaceflight and clinical use.

Beyond the numerous potential clinical applications of Ca isotopes per se, our research on Ca isotopes has been a driving force behind international research into biomedical application of other isotope and elemental systems, including Fe, Zn, and Cu. Collectively, these efforts hold the promise of the development of an entirely new and powerful class of disease biomarkers.

Question 1: Is the observed pattern of decreasing d 44/42Ca values with bone loss in previous bed rest studies similar to the pattern in astronauts in spaceflight?

In our previous bed rest studies (Morgan et al., 2012), urinary d44/42Ca begins to fall on day 4 of bed rest and drops to values significantly lower than baseline (-0.2‰, p < 0.001) by day 15. Data from the ISS indicates that urinary d44/42C in Advanced Resistive Exercise (ARED) and interim Resistive Exercise Device (iRED) treatment groups decreased significantly by flight day (FD) 15 (~-0.26‰) comparable in magnitude and timing to the bed rest studies.

Question 2: Is the magnitude of bone loss calculated from Ca isotopes measurements and a mass balance model supported by the DEXA data from before and after spaceflight?

Our previously developed mass balance model indicates that a shift in Ca isotopes of -0.26‰ correlates to a loss of ~100 mg of Ca per day. This is consistent with loss rates calculated from Ca isotope data in bed rest studies. Morgan et al. (2012) report that bed rest subjects lost 0.25 ± 0.07% of whole body bone mass between days 7-30, which is consistent with previously reported rates of bone loss in bed rest (e.g., 0.19 ± 0.07%/month), and with rates of bone loss measured by DXA in the bed rest subjects.

For the ISS subjects, the magnitude of bone loss calculated from Ca isotope data is consistent within error with bone loss indicated by DXA data from before and after spaceflight. For the cohorts losing bone (ARED and iRED), rates of bone lose were ~2-4% over 120-215 days in space, consistent with loss rates calculated by the mass balance model and by DXA.

Question 3: How does the high temporal resolution Ca isotope data compare to protein biomarker data (NTX, BSAP) measured in the same samples?

Ca isotopes offer high temporal resolution comparable to protein biomarkers. Previous studies indicate that protein biomarkers and Ca isotopes respond to skeletal unloading by day 9 of bed rest. Ca isotopes offer the same high temporal resolution tracking during spaceflight that they do during bed rest: data from the ISS shows both Ca isotopes and protein biomarkers responding to skeletal unloading due to microgravity by FD 15.

Question 4: What does the Ca isotope data say about the relative effectiveness of exercise (iRED, ARED) and pharmaceutical countermeasures (bisphosphonates)?

All crewmembers underwent a 30-day adjustment period at the beginning of flight, during which they performed no resistive exercise. Treatment with ARED and iRED is therefore considered to start at FD 30. The dramatic decrease in d44/42Ca in the exercise-only groups during FD 0-30 reflects untreated bone loss due to skeletal unloading. The ARED+Alendronate group received Alendronate several months before flight. Thus, their baseline and all in-flight measurements reflect a state of inhibited bone turnover.

The differences between d44/42Ca measurements in the three treatment groups is striking: crewmembers treated with ARED+Alendronate maintained near constant d44/42Ca throughout spaceflight, while those treated with exercise alone showed a more variable response, but overall had lower d44/42Ca during spaceflight relative to pre-spaceflight baselines. This suggests that ARED+Alendronate effectively maintained BMB in all crewmembers. Direct comparison of the effectiveness of ARED+Alendronate with exercise alone is not possible: The ARED+Alendronate group was protected from bone loss throughout spaceflight, while the exercise groups were unprotected for the first 30 days of spaceflight, resulting in measurable bone loss over that period. For most crewmembers, exercise alone was not sufficient to completely restore preflight BMB before the end of the mission.

Ca isotope values from the ARED and iRED groups diverge after the start of exercise on FD 30. The differences between the groups are not statistically significant, which is unsurprising given the large inter-personal variation of baseline d44/42Ca values. However, the ARED group shows a trend of increasing d44/42Ca over time, while mean d44/42Ca of the iRED changes insignificantly (+0.17‰ for ARED vs +0.03‰ for iRED), suggesting that ARED provided better protection from bone loss than iRED. In the exercise only groups, Ca isotope data reveal considerable inter-personal variation in BMB during spaceflight, which results in large standard deviations in d44/42Ca group means. Some crewmembers in each exercise group recovered to near neutral BMB during spaceflight, while others remained in negative BMB despite exercise. Individual differences in response of BMB to exercise could be caused by differences in the type and intensity of exercise performed by crewmembers, and/or by inherent physiological differences. The relative importance of these two possible causes of inter-personal differences cannot be resolved with available evidence.

Question 5: Can the Ca isotope data be used to tailor personalized treatment plans for astronauts?

The behavior of BMB in spaceflight is highly dynamic, and the degree of skeletal response to microgravity is not universal. Ca isotopes provide information on the dynamics of BMB that cannot be gained from biochemical markers or DXA. By revealing previously inaccessible dynamics of individual bone metabolism in spaceflight, d44/42Ca monitoring may be useful for guiding individualized bone loss countermeasures.

Trends in d44/42Ca values in 24-hour pooled urine samples from 30 ISS crewmembers suggest that ARED+Alendronate effectively maintained BMB in all crewmembers, while exercise alone did not always maintain BMB. Further, the data reveal what may be an interpersonal differential response to type of exercise intervention. The range of responses underlines the importance of individualized monitoring of net BMB, and of creating individualized treatment plans.

Reference

Morgan JL, Skulan JL, Gordon GW, Romaniello SJ, Smith SM, Anbar AD (2012). Rapidly assessing changes in bone mineral balance using natural stable calcium isotopes. Proc. Natl. Acad. Sci. USA 109, 9989-9994

NOTE: End date changed to 11/30/2016 per PI and NSSC information (Ed., 12/14/15)

References:

(1). Morgan JL, Skulan JL, Gordon GW, Romaniello SJ, Smith SM, Anbar AD (2012). Rapidly assessing changes in bone mineral balance using natural stable calcium isotopes. Proc. Natl. Acad. Sci. USA 109, 9989-9994;

(2). Skulan J, Bullen T, Anbar AD, Puzas JE, Shackelford L, LeBlanc A, Smith SM (2007). Natural calcium isotopic composition of urine as a marker of bone mineral balance. Clin. Chem. 53,1155-1158;

(3). Morgan JLL, Skulan JL, Gordon GW, Romaniello SJ, Smith SM and Anbar AD (2011). High-precision measurement of variations in calcium isotope ratios in urine by multiple collector inductively coupled plasma mass spectrometry. Anal. Chem. 83, 6956–6962.

The usefulness of this technique extends beyond measuring bone loss in spaceflight to the detection and evaluation of treatment for any disease involving disruption in BMB, including osteopenia/osteoporosis, cancer, and Paget’s disease. For example, we currently are exploring the application of the Ca isotope technique to the early detection of osteolytic lesions in multiple myeloma. The potential usefulness of Ca isotopes supports their widespread clinical application. We are exploring the possibility of using laser fluorescence, rather than conventional mass spectrometry, to build small, compact Ca isotope measurement instruments suitable to both spaceflight and clinical use.

Beyond the numerous potential clinical applications of Ca isotopes per se, our research on Ca isotopes has been a driving force behind international research into biomedical application of other isotope and elemental systems, including Fe, Zn, and Cu. Collectively, these efforts hold the promise of the development of an entirely new and powerful class of disease biomarkers.

We have achieved this goal. Although not all of our analyses are completed, analysis of archived urine samples from 32 crewmembers from previous ISS missions clearly shows the same pattern of change in Ca isotope composition observed in bed rest and predicted in spaceflight. More specifically, the 44Ca:42Ca ratio in urine (expressed as d44/42Ca) typically drops to below each crewmember’s individual average pre-flight value shortly after spaceflight begins, remains low during spaceflight, and returns to pre-flight values upon return to Earth. This is the pattern expected in crewmembers transitioning to more negative BMB in microgravity, and a confirmation of our ability to detect this change using Ca isotopes.

A great deal more information can be extracted from the Ca isotope data we have gathered, a task that we have begun and will be our primary activity during the remainder of the project. Changes in Ca isotope composition are revealing details of the dynamics of BMB on previously inaccessible timescales, and offer new insights into bone biology. In particular, while on Ca isotopes show what on average crewmembers lose bone during spaceflight, our data reveal striking differences between the responses of individual crewmembers. Some of these differences are related to countermeasures. For example, crewmembers treated with both bisphosphonate and Advanced Resistive Exercise (ARED) uniformly showed not bone loss, while the response of crewmembers treated with exercise alone was more variable: most lost bone, some did not. Variations in response to treatment do not appear to be related to age, sex, or BMI (body mass index). The cause of these variations is of great interest, and we hope that further analysis will shed light on this and other questions. We do anticipate, however, that the answers to many questions will require more data from future projects.

A secondary goal of our project was to increase the speed with which samples could be processed and analyzed. This goal is, of course, a moving target. Processing speed can always be increased, but we have made substantial improvements over where we were at the start of the project. We designed and validated an automated column chromatography procedure for faster and cleaner sample purification prior to analysis, and have validated a new microwave digestion system for improved sample digestion is now 80% complete. In addition, we are in the process of developing an improved mass spectrometric analytical procedure using a 46Ca-43Ca double spike, which should improve data quality, accuracy, and precision.

Finally, we have begun exploring new technology to measure Ca isotope ratios using laser fluorescence rather than conventional mass spectrometry. Conventional mass spectrometers are not suited to either inflight or widespread clinical use. Such instruments are large, difficult or impossible to miniaturize, complex, and expensive, problems that could be overcome by using laser fluorescence. Whether a practical laser fluorescence device can be built remains to be seen, but preliminary results are promising, and efforts to build small and simple devices for measuring other isotopes (C and O) have been successful.

References:

(1). Morgan JL, Skulan JL, Gordon GW, Romaniello SJ, Smith SM, Anbar AD (2012). Rapidly assessing changes in bone mineral balance using natural stable calcium isotopes. Proc. Natl. Acad. Sci. USA 109, 9989-9994;

(2). Skulan J, Bullen T, Anbar AD, Puzas JE, Shackelford L, LeBlanc A, Smith SM (2007). Natural calcium isotopic composition of urine as a marker of bone mineral balance. Clin. Chem. 53,1155-1158;

(3). Morgan JLL, Skulan JL, Gordon GW, Romaniello SJ, Smith SM, Anbar AD (2011). High-precision measurement of variations in calcium isotope ratios in urine by multiple collector inductively coupled plasma mass spectrometry. Anal. Chem. 83, 6956–6962.