NOTE: Received extension to 3/19/2013 per NSSC (Ed., 5/8/2012)

NOTE: Received extension to 5/19/2012 per C. Guidry/JSC and NSSC [Ed. 3/2/2011]

The resorption of bone when astronauts are exposed to microgravity is a major challenge for NASA's plans for human exploration of the Moon and Mars. Our proposed technique would be immediately valuable in ground-based studies of countermeasure strategies, accelerating the pace of discovery of countermeasures to bone loss. In the long run, flight-qualified versions of mass spectrometric or other systems for Ca isotope characterization could accompany astronauts on long-duration missions.

Precise measurements of the calcium isotope composition in blood or urine provide information about bone mineral balance because the isotopic composition of calcium in human soft tissues is naturally affected by the relative rates of bone formation and resorption. Specifically, lighter calcium isotopes are preferentially incorporated into bone during formation. Because of the short residence time of calcium in soft tissues, calcium isotope ratios should change rapidly in response to changes in bone gain or loss. These changes, while small, can be measured by multiple collector inductively coupled plasma mass spectrometry (MC-ICP-MS) or thermal ionization mass spectrometry (TIMS).

The research expanded on a pilot study in which we measured calcium isotopes in a small suite of urine samples from a bed rest study. The expanded study involved a larger number of bed rest subjects, measurements of blood and dietary samples as well as urine, and a much denser sample schedule, including sub-daily urine and blood sampling during critical early and late phases of the bed rest and post bed rest periods. This research allowed us to answer critical questions unresolved by the pilot study. Most importantly, we were able to determine how long after the onset of bed rest bone loss can be detected with Ca isotopes, and to begin to quantify the relationship between changes in Ca isotope ratios and changes in bone mineral balance (BMB).

Prior to the start of the research described in this report, a relationship between BMB and soft tissue Ca isotope composition had been hypothesized (Skulan and DePaolo, 1999) and confirmed in a pilot study using archived urine samples from a previous 17-week bed rest study (Skulan et al., 2007--see below). These preliminary studies raised several questions that were addressed in the NASA-funded research described here:

1. How soon after the start of bed rest does a bone loss signal appear in urine and blood? In the pilot study, the bone loss signal in urine was apparent at 28 days after the start of bed rest, which was the first bed rest sample point. Because the residence time of Ca in soft tissues is short (hours to days) we predicted that the bone loss signal should appear far earlier than this.

2. What factors other than BMB can affect soft tissue Ca isotope composition? In particular, how does the renal Ca isotope fractionation observed in earlier studies affect the ability of soft tissue Ca isotope composition to track BMB?

3. Can soft tissue Ca isotope composition be quantitatively related to BMB?

The centerpiece of our research was a suite of blood, urine, and diet samples collected in a 30 day, 12 subject bed rest study. Samples were analyzed for Ca isotope composition the University of Arizona’s W.M. Keck Foundation Laboratory for Environmental Biogeochemistry. With that aid of a mathematical model, data generated by the study allowed us to answer each of the three initial questions.

How soon after the start of bed rest does a bone loss signal appear in urine and blood? All-subject average urinary Ca isotopes begin to show bone loss on the third day of bed rest. This signal persists throughout the bed rest and recovery periods and becomes statistically significant by day 9 of bed rest. Ca isotopes in urine reflect the onset of negative BMB at least ten times faster than radiological techniques like DEXA, which can detect changes in bone mass that require several months of bed rest to produce.

What factors other than BMB can affect soft tissue Ca isotope composition? Several factors other than BMB possibly could affect soft tissue Ca isotope composition. Extraneous factors like changes in dietary Ca isotope composition and differences in geographic and dietary history between subjects can be adequately controlled for under the strict conditions of bed rest studies by normalizing each subjects Ca isotope composition to his or her individual average pre-bed rest value. The former factor—changes in dietary d44/42Ca—is harder to control for, but based on our data does not seem to obscure the bone loss signal under the carefully controlled experimental conditions of bed rest, in which all subjects are fed the same menu on the same ten day rotation. The mass of Ca in soft tissues is large enough to dampen meal-to-meal variations in d44/42Ca of absorbed Ca. This damping effect is enhanced in 24-hour pooled urine samples, which for that reason are ideal for Ca isotope analysis.

Internal, physiological processes other than bone remodeling possibly also could affect soft tissue Ca isotope composition. Two processes in particular—the production of bile salts in the liver and excretion of Ca by the kidney—involve a mass of Ca large enough to potentially affect the Ca isotope composition of the whole body. We have investigated the effects of hepatic and renal Ca isotope fractionation using the quantitative model developed and calibrated to results of this and previous research on Ca isotope fractionation. Based on results of this model, we conclude that neither hepatic nor renal fractionation interfere with the interpretation of Ca isotope data from bed rest studies in terms of changes in BMB.

Can soft tissue Ca isotope composition be quantitatively related to BMB? The mathematical model combined with data collected in this and previous studies allow us to estimate the average rate of bone loss during the 30 day bed rest period at ˜0.4%/month. This agrees well with rates of bone loss measured by DEXA in longer bed rest studies, which are about 1% over three months. Future plans

Research on the Ca isotope technique is proceeding on three fronts.

1. Validation of the Ca isotope technique in spaceflight, using urine and blood and other biological samples collected from crew members on the International Space Station.

2. Development of a flight-ready instrument for measuring Ca isotopes in spaceflight.

3. Development of Earth-based clinical applications of the Ca isotope technique. The technique holds particular promise in the detection and monitoring of skeletal involvement in cancer. In particular, we are exploring potential applications to the early detection of skeletal involvement in multiple myeloma and other cancers.

NOTE: Received extension to 5/19/2012 per C. Guidry/JSC and NSSC [Ed. 3/2/2011]

The resorption of bone when astronauts are exposed to microgravity is a major challenge for NASA's plans for human exploration of the Moon and Mars. Our proposed technique would be immediately valuable in ground-based studies of countermeasure strategies, accelerating the pace of discovery of countermeasures to bone loss. In the long run, flight-qualified versions of mass spectrometric or other systems for Ca isotope characterization could accompany astronauts on long-duration missions.

Precise measurements of the calcium isotope composition in blood or urine provide information about bone mineral balance because the isotopic composition of calcium in human soft tissues is naturally affected by the relative rates of bone formation and resorption. Specifically, lighter calcium isotopes are preferentially incorporated into bone during formation. Because of the short residence time of calcium in soft tissues, calcium isotope ratios should change rapidly in response to changes bone gain or loss. These changes, while small, can be measured by multiple collector inductively coupled plasma mass spectrometry (MC-ICP-MS) or thermal ionization mass spectrometry (TIMS).

The proposal team recently demonstrated the promise of this method in a published pilot study in which we measured calcium isotopes in a small suite of urine samples from a bed rest study. Here, we propose an expanded examination of bed rest samples, involving a larger number of subjects, measurements of blood and dietary samples as well as urine, and daily or even sub-daily sampling. This research would address critical questions unresolved by the pilot study.

1. Most of the blood and food samples collected during the 30 day bed rest study have been purified and are ready for analysis. As discussed in last year’s progress report, sample purification is the most time consuming step in Ca isotope analysis of biological samples. New chemical techniques needed to be devised in order to reduce Sr and K concentrations to the extremely low levels required for mass spectrometric analysis. These techniques were developed in the earlier project years, and published in Summer, 2011 (Morgan et al., 2011). They are also included in a patent claim being pursued by Arizona State University.

2. Largely through the efforts of Steven Romaniello, a PhD student in Dr. Anbar’s lab, we have made significant advances in the theoretical understanding of Ca isotope fractionation in the human body, and how this fractionation affects the urinary Ca isotope response to changes in bone mineral balance. Mr. Romaniello has developed a mathematical model that includes the important variables controlling the Ca isotope composition of soft tissue and urine, including the isotope composition of diet, intestinal Ca absorption, bone remodeling rate, and Ca isotope fractionation during bone formation.

Importantly, the mathematical model takes into account renal and hepatic Ca isotope fractionation. Until now, renal Ca isotope fractionation has been the major outstanding issue in biological Ca isotope research, and a potential problem for the application of Ca isotope analysis to the measure of bone mineral balance. It now is clear that renal fractionation can be adequately accounted for in the model. Moreover, given the likely value of the renal Ca isotope effect, which can be inferred from our own and others’ work on humans and animal models, renal fractionation actually increases the sensitivity of Ca isotopes in urine to changes in bone mineral balance.

Once the renal isotope effect has been measured with sufficient precision through the analysis of the remaining blood and food samples, the mathematical model will allow us to use changes in urinary Ca isotope composition to make truly quantitative calculations of bone mineral balance.

3. Results of our research have been communicated at several major international meetings, including the annual meeting of the Endocrine Society, the American Geophysical Union, and the annual Goldschmidt conference. We have made plans future collaboration with European researchers who have been independently investigating the relationship between urinary Ca isotopes and bone mineral balance.

4. Results of the analysis of approximately 200 urine samples, together with our mathematical model and a detailed discussion of the renal isotope effect, are described in our paper, Rapidly assessing changes in bone mineral balance using natural stable calcium isotopes, which currently is in revision at PNAS. This paper is the most exhaustive treatment to date of Ca isotopes in humans. It demonstrates that Ca isotopes in urine respond to changes in BMB within ten days of the start of bed rest, and the feasibility of using urinary Ca isotopes to quantitatively measure changes in BMB.

Future plans: Over the next year we will finish the analysis of blood and diet samples collected during the bed rest study, and use these new data to refine our mathematical model. In particular, we will have multiple simultaneous measures of the Ca isotope composition of blood and urine from the same subjects. These data will allow us to quantify the renal fractionation factor, and shed light on the exact mechanism of renal Ca isotope fractionation. We anticipate writing at least two major papers presenting our results.

NOTE: Received extension to 5/19/2012 per C. Guidry/JSC and NSSC [Ed. 3/2/2011]

The resorption of bone when astronauts are exposed to microgravity is a major challenge for NASA's plans for human exploration of the Moon and Mars. Our proposed technique would be immediately valuable in ground-based studies of countermeasure strategies, accelerating the pace of discovery of countermeasures to bone loss. In the long run, flight-qualified versions of mass spectrometric or other systems for Ca isotope characterization could accompany astronauts on long-duration missions.

Precise measurements of the calcium isotope composition in blood or urine provide information about bone mineral balance because the isotopic composition of calcium in human soft tissues is naturally affected by the relative rates of bone formation and resorption. Specifically, lighter calcium isotopes are preferentially incorporated into bone during formation. Because of the short residence time of calcium in soft tissues, calcium isotope ratios should change rapidly in response to changes bone gain or loss. These changes, while small, can be measured by multiple collector inductively coupled plasma mass spectrometry (MC-ICP-MS) or thermal ionization mass spectrometry (TIMS).

The proposal team recently demonstrated the promise of this method in a published pilot study in which we measured calcium isotopes in a small suite of urine samples from a bed rest study. Here, we propose an expanded examination of bed rest samples, involving a larger number of subjects, measurements of blood and dietary samples as well as urine, and daily or even sub-daily sampling. This research would address critical questions unresolved by the pilot study.

Although the bulk of blood and urine samples have not yet been analyzed, Ca isotopic measurements have been made of a full 44 day (30 days of bed rest plus one week preceding and following bed rest) time series of urine samples from all 12 subjects. These data confirm the central premise of our research, that Ca isotope ratios in urine change in response to changes in bone mineral balance (BMB). More specifically, we conclude that

1. Urine Ca becomes isotopically lighter 5 to 7 days after the start of bed rest. This change in Ca isotope composition is highly statistically significant, and probably reflects the initiation of negative BMB in response to bed rest. N-telepeptide (NTx), a biochemical marker of bone resorption, also increases sharply at 5 to 7 days; bone specific alkaline phosphatase (BSAP), a marker of bone formation, does not change.

2. Background variation, as measured by day-to-day changes in urinary Ca isotope composition of 24 hour urine pools for each subject, in subjects prior to the start of bed rest, is only about half as large as the change that that we attribute to the onset of bone loss. This demonstrates that under conditions of bed rest the Ca isotopic signal of bone loss is not swamped by background ‘noise’ from factors other than bone loss that might affect the Ca isotope composition of urine.

3. Void-by-void urine sampling was conducted on all subjects before or after the bed rest period. This high density sampling was used to detect possible diurnal changes in urinary Ca isotope composition, and to document the magnitude of intra-subject background variation in isotope composition. Only a fraction of these samples have been analyzed, but initial data show no evidence of a diurnal cycle, and indicate that variation in urinary Ca isotope composition over the course of a day is no greater than the day to day variation between 24 hour pooled urine samples.

4. Using published estimates of the magnitude of change in Ca isotope composition occurring during bone mineral formation we estimate that the change in urinary Ca isotope composition observed during bed rest reflects a bone mineral loss rate of about 6%/year. This estimate agrees well with radiometric measurements of bone loss in bed rest.

On the basis of the preceding we conclude bone loss typically observed in spaceflight should be easily resolvable using the Ca isotope technique. The success of countermeasures to bone loss also should be reflected in urinary Ca isotope composition.

Over the next year we will complete the analysis of samples from the 30 day bed rest study. These additional analyses will allow us to determine how well urine tracks changes in Ca isotope composition, to measure the effect of changes in dietary Ca isotope composition on that of urine and blood, and to better constrain the magnitude of background variation in the Ca isotope compositions of urine and blood.

The resorption of bone when astronauts are exposed to microgravity is a major challenge for NASA's plans for human exploration of the Moon and Mars. Our proposed technique would be immediately valuable in ground-based studies of countermeasure strategies, accelerating the pace of discovery of countermeasures to bone loss. In the long run, flight-qualified versions of mass spectrometric or other systems for Ca isotope characterization could accompany astronauts on long-duration missions.

Precise measurements of the calcium isotope composition in blood or urine provide information about bone mineral balance because the isotopic composition of calcium in human soft tissues is naturally affected by the relative rates of bone formation and resorption. Specifically, lighter calcium isotopes are preferentially incorporated into bone during formation. Because of the short residence time of calcium in soft tissues, calcium isotope ratios should change rapidly in response to changes bone gain or loss. These changes, while small, can be measured by multiple collector inductively coupled plasma mass spectrometry (MC-ICP-MS) or thermal ionization mass spectrometry (TIMS).

The proposal team recently demonstrated the promise of this method in a published pilot study in which we measured calcium isotopes in a small suite of urine samples from a bed rest study. Here, we propose an expanded examination of bed rest samples, involving a larger number of subjects, measurements of blood and dietary samples as well as urine, and daily or even sub-daily sampling. This research would address critical questions unresolved by the pilot study.

1. The sampling originally intended for year 1 was postponed to the end of year 2 because of unforeseen events at the bed rest facility. During year 2 we developed a sampling protocol that has been incorporated into a 12 subject, 30-day bed rest study. The 30-day bed rest study began October 2009. Samples from two patients in this study arrived at ASU in January 2010 and are currently being processed for Ca isotope analysis. The results from these initial samples are expected by mid April 2010. We expect all remaining patient samples to be delivered to us by the end of 2010. As we are analyzing the samples as they arrive, we expect the bulk of the analyses to be completed by April 2011.

This sampling protocol will generate approximately 1750 urine, blood and food samples, and is by far the most detailed investigation of Ca isotopes ever undertaken in any field. It will provide the data needed to address the most important questions in order for Ca isotopic analysis to become an effective tool for measuring bone mineral balance in space flight. The core of the sampling protocol focuses on two periods of intensive sampling, which consists of per void sampling of urine and sub daily sampling of blood during times when bone mineral balance can be expected to change rapidly. These rapid changes should occur during the first week of bed rest and the first week of recovery. Data from these samples will allow us to determine how quickly changes in skeletal loading produce detectable changes in urinary and blood Ca isotope composition, and to begin to estimate how these changes vary between individuals. The data from these intensive sampling sections will allow us to resolve diurnal changes and other variations in urinary and blood Ca isotope composition. By measuring the isotopic composition of all dietary items containing significant Ca and obtaining the detailed records of what each subject ate, we will be able to reconstruct the isotopic composition of each subjects’ dietary Ca on a meal by meal basis. This will allow us to disentangle the dietary signal in urinary and blood Ca isotope composition from the endogenous physiological signal that reflects changes in bone mineral balance.

The importance of intensive sampling is highlighted by new data from a study that our lab conducted (in collaboration with the University of Wisconsin National Primate Research Center) in parallel with our bed rest study. This new study investigated the ability of Ca isotopes to detect bone loss caused by estrogen depletion in female rhesus macaques. Although not a formal part of our NASA project, the rhesus study provided us with urine and blood samples that allowed us to perfect sample preparation and purification techniques for use on human samples. Our pilot study of Ca isotopes in bed rest (Skulan et al, 2007) demonstrated that changes in bone mineral balance induce changes in urinary Ca isotope composition by the fourth week of bed rest, which was the earliest bed rest sample point in that study. The results of the rhesus study indicate that changes in Ca isotope composition can occur very rapidly, within one day of a bone loss stimulating event and that these changes most likely reflect changes in bone mineral balance.

2. During year 2 we moved MC-ICP-MS analysis of biological samples from a theoretical possibility to a practical technique.

As explained in our original documents, application of Ca isotopes to human samples requires a high-throughput analytical techniques. High-throughput is required because the changes being monitored happen rapidly, requiring the analysis of large numbers of samples in order to determine optimum sampling strategies for application to space flight. Previously, analyses of Ca isotopes has been done primarily using thermal ionization mass spectrometry (TIMS). TIMS analyses are slow, labor-intensive and cannot easily be automated. We are instead using multiple collector inductively coupled plasma mass spectrometry (MC-ICP-MS). This method intrinsically allows 5 – 10 times higher sample throughput than TIMS. It also offers the potential for an automated process for Ca.

A key stumbling block to the use of MC-ICP-MS for such analyses of biological samples are interferences arising from chemical impurities in the samples. This is much more or a problem for MC-ICP-MS than for TIMS because, unlike TIMS, MC-ICP-MS simultaneously and completely analyzes all elements present in a sample. Early in year 2 analysis of rhesus urine and blood samples demonstrated that published techniques for purifying geological samples (the only kind of samples previously analyzed) for MC-ICP-MS analysis were not sufficient for biological samples, which continued to have unacceptably large interferences after purification.

Over the course year 2 we identified the sources of these interferences and developed methods of removing them. We now are able to process and analyze samples from the bed rest study as they arrive, and are on target to finish these analyses in year 3.

The resorption of bone when astronauts are exposed to microgravity is a major challenge for NASA's plans for human exploration of the Moon and Mars. Our proposed technique would be immediately valuable in ground-based studies of countermeasure strategies, accelerating the pace of discovery of countermeasures to bone loss. In the long run, flight-qualified versions of mass spectrometric or other systems for Ca isotope characterization could accompany astronauts on long-duration missions.

Precise measurements of the calcium isotope composition in blood or urine provide information about bone mineral balance because the isotopic composition of calcium in human soft tissues is naturally affected by the relative rates of bone formation and resorption. Specifically, lighter calcium isotopes are preferentially incorporated into bone during formation. Because of the short residence time of calcium in soft tissues, calcium isotope ratios should change rapidly in response to changes bone gain or loss. These changes, while small, can be measured by multiple collector inductively coupled plasma mass spectrometry (MC-ICP-MS) or thermal ionization mass spectrometry (TIMS).

The proposal team recently demonstrated the promise of this method in a published pilot study in which we measured calcium isotopes in a small suite of urine samples from a bed rest study. Here, we propose an expanded examination of bed rest samples, involving a larger number of subjects, measurements of blood and dietary samples as well as urine, and daily or even sub-daily sampling. This research would address critical questions unresolved by the pilot study.

The resorption of bone when astronauts are exposed to microgravity is a major challenge for NASA's plans for human exploration of the Moon and Mars. Our proposed technique would be immediately valuable in ground-based studies of countermeasure strategies, accelerating the pace of discovery of countermeasures to bone loss. In the long run, flight-qualified versions of mass spectrometric or other systems for Ca isotope characterization could accompany astronauts on long-duration missions.

Precise measurements of the calcium isotope composition in blood or urine provide information about bone mineral balance because the isotopic composition of calcium in human soft tissues is naturally affected by the relative rates of bone formation and resorption. Specifically, lighter calcium isotopes are preferentially incorporated into bone during formation. Because of the short residence time of calcium in soft tissues, calcium isotope ratios should change rapidly in response to changes bone gain or loss. These changes, while small, can be measured by multiple collector inductively coupled plasma mass spectrometry (MC-ICP-MS) or thermal ionization mass spectrometry (TIMS).

The proposal team recently demonstrated the promise of this method in a published pilot study in which we measured calcium isotopes in a small suite of urine samples from a bed rest study. Here, we propose an expanded examination of bed rest samples, involving a larger number of subjects, measurements of blood and dietary samples as well as urine, and daily or even sub-daily sampling. This research would address critical questions unresolved by the pilot study.