Rapidly assessing changes in bone mineral balance using natural stable calcium isotopes.

The ability to rapidly detect changes in bone mineral balance (BMB) would be of great value in the early diagnosis and evaluation of therapies for metabolic bone diseases such as osteoporosis and some cancers. However, measurements of BMB are hampered by difficulties with using biochemical markers to quantify the relative rates of bone resorption and formation and the need to wait months to years for altered BMB to produce changes in bone mineral density large enough to resolve by X-ray densitometry. We show here that, in humans, the natural abundances of Ca isotopes in urine change rapidly in response to changes in BMB. In a bed rest experiment, use of high-precision isotope ratio MS allowed the onset of bone loss to be detected in Ca isotope data after about 1 wk, long before bone mineral density has changed enough to be detectable with densitometry. The physiological basis of the relationship between Ca isotopes and BMB is sufficiently understood to allow quantitative translation of changes in Ca isotope abundances to changes in bone mineral density using a simple model. The rate of change of bone mineral density inferred from Ca isotopes is consistent with the rate observed by densitometry in long-term bed rest studies. Ca isotopic analysis provides a powerful way to monitor bone loss, potentially making it possible to diagnose metabolic bone disease and track the impact of treatments more effectively than is currently possible.

High-precision measurement of variations in calcium isotope ratios in urine by multiple collector inductively coupled plasma mass spectrometry.

We describe a new chemical separation method to isolate Ca from other matrix elements in biological samples, developed with the long-term goal of making high-precision measurement of natural stable Ca isotope variations a clinically applicable tool to assess bone mineral balance. A new two-column procedure utilizing HBr achieves the purity required to accurately and precisely measure two Ca isotope ratios ((44)Ca/(42)Ca and (44)Ca/(43)Ca) on a Neptune multiple collector inductively coupled plasma mass spectrometer (MC-ICPMS) in urine. Purification requirements for Sr, Ti, and K (Ca/Sr > 10 000; Ca/Ti > 10 000 000; and Ca/K > 10) were determined by addition of these elements to Ca standards of known isotopic composition. Accuracy was determined by (1) comparing Ca isotope results for samples and standards to published data obtained using thermal ionization mass spectrometry (TIMS), (2) adding a Ca standard of known isotopic composition to a urine sample purified of Ca, and (3) analyzing mixtures of urine samples and standards in varying proportions. The accuracy and precision of δ(44/42)Ca measurements of purified samples containing 25 µg of Ca can be determined with typical errors less than ±0.2‰ (2σ).

Using natural, stable calcium isotopes of human blood to detect and monitor changes in bone mineral balance.

We are exploring variations in the Ca isotope composition of blood and urine as a new tool for early diagnosis and monitoring of changes in bone mineral balance for patients suffering from metabolic bone disease, cancers that originate in or metastasize to bone, and for astronauts who spend time in low gravity environments. Blood samples are often collected instead of, or in addition to, urine in clinical settings, so it is useful to know if variations in the Ca isotope composition of blood carry the same information as variations in urine. We found that the Ca isotope composition of blood shifts in the same direction and to the same magnitude (~2 parts per ten thousand--pptt) as that of urine in response to skeletal unloading during bed rest. However, the Ca isotope composition of blood is lighter than that of urine by 12 ± 2 pptt. This offset between blood and urine may result from Ca isotope fractionation occurring in the kidneys. This is the first study to confirm the suspected offset between the Ca isotope composition of blood and urine in humans, to directly quantify its magnitude, and to establish that either blood or urine can be used to detect and quantify bone loss.