The resorption of bone when astronauts are exposed to microgravity is a major challenge for humans engaged in long-term space travel. The goal of our research is to develop a Ca isotope assay of urine, blood, or other soft tissues and soft tissue proxies that allows rapid, quantitative measures of the changes in bone mineral balance (BMB) that lead to bone loss, providing key information that other techniques, for example dual energy X-ray absorptiometry (DEXA) and biochemical markers of bone metabolism, cannot provide. Such a Ca isotope technique would facilitate quick evaluation of countermeasures to bone loss in space. In the long run, flight-qualified versions of mass spectrometric or other systems required for Ca isotope measurement could accompany astronauts on long-duration missions. The final outcome will be better monitoring of human health in space through more rapid and accurate measurement of changes in 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.