Calcium isotope fractionation by osteoblasts and osteoclasts, across endothelial and epithelial cell barriers, and with binding to proteins.

Timely and accurate diagnosis of osteoporosis is essential for adequate therapy. Calcium isotope ratio (δ44/42Ca) determination has been suggested as a sensitive, noninvasive, and radiation-free biomarker for the diagnosis of osteoporosis, reflecting bone calcium balance. The quantitative diagnostic is based on the calculation of the δ44/42Ca difference between blood, urine, and bone. The underlying cellular processes, however, have not been studied systematically. We quantified calcium transport and δ44/42Ca fractionation during in vitro bone formation and resorption by osteoblasts and osteoclasts and across renal proximal tubular epithelial cells (HK-2), human vein umbilical endothelial cells (HUVECs), and enterocytes (Caco-2) in transwell systems and determined transepithelial electrical resistance characteristics. δ44/42Ca fractionation was furthermore quantified with calcium binding to albumin and collagen. Calcified matrix formed by osteoblasts was isotopically lighter than culture medium by -0.27 ± 0.03‰ within 5 days, while a consistent effect of activated osteoclasts on δ44/42Ca could not be demonstrated. A transient increase in δ44/42Ca in the apical compartment by 0.26‰ occured across HK-2 cells, while δ44/42Ca fractionation was small across the HUVEC barrier and absent with Caco-2 enterocytes, and with binding of calcium to albumin and collagen. In conclusion, δ44/42Ca fractionation follows similar universal principles as during inorganic mineral precipitation; osteoblast activity results in δ44/42Ca fractionation. δ44/42Ca fractionation also occurs across the proximal tubular cell barrier and needs to be considered for in vivo bone mineralization modeling. In contrast, the effect of calcium transport across endothelial and enterocyte barriers on blood δ44/42Ca should be low and is absent with physiochemical binding of calcium to proteins.

Use of Calcium Isotopic Tracers To Determine Factors That Perturb Calcium Metabolism.

Calcium plays an important role in maintaining bone health. Ensuring adequate calcium intake throughout life is essential for reaching greater peak bone mass in young adulthood and lowering osteoporotic fracture risk when aging. Calcium homeostasis involves a complex interaction between three organ systems: intestine, kidney, and bone. Metabolic balance plus kinetic studies using calcium isotopic tracers can estimate calcium metabolism parameters and pinpoint how organs and processes are perturbed by internal and external modifiers. Both modifiable factors (e.g., vitamin D supplementations and dietary bioactives) and non-modifiable factors (e.g., age, sex, and race) influence calcium utilization. Current evidence suggests that prebiotic fibers may offer an alternative approach to enhance calcium absorption through altering gut microbiota to ultimately improve bone health.

44Ca doped remineralization study on dentin by isotope microscopy.

OBJECTIVE: The dental caries is developed as a result of an alternative course of mineral gain and loss. In order to distinguish between intrinsic Ca (tooth-derived mineral) and extrinsic Ca (solution-derived mineral) uptakes, a 44Ca doped pH-cycling was performed using 44Ca (a stable calcium isotope) remineralization solution. METHODS: The natural abundance of 40Ca and 44Ca is 96.9% and 2.1%, respectively. The remineralization solution was prepared using 44Ca to contain 1.5mmol/L CaCl2 (44Ca), 0.9mmol/L KH2PO4, 130mmol/L KCl, 20mmol/L HEPES at pH 7.0. The pH-cycling was conducted on bovine root dentin daily by demineralization (pH 5.0) for 2h, incubation in 0% (control) and 0.2% NaF (900ppm fluoride) for 2h and 44Ca doped remineralization for 20h. After 14days pH-cycling, the specimens were sectioned longitudinally. On the sectioned surface, isotope imaging of 40Ca and 44Ca labeled mineral distribution was observed by a high mass-resolution stigmatic secondary ion 77 (Camera IMS 1270, Gennevilliers Cedex, France). RESULTS: Uptake of 44Ca was greater in intensity for the 0.2% fluoride group than the control, especially in the superficial lesions. The control group showed 40Ca (intrinsic) distribution in the subsurface lesions and in the superficial lesions, meanwhile the fluoride group showed 40Ca distribution limited in subsurface lesions. The total Ca (44Ca+40Ca) image revealed more homogeneously for the control than the fluoride group. SIGNIFICANCE: Since the fluoride-treated surface is more acid-resistant than intrinsic dentin, alternative minerals were dissolved from the intact intrinsic lesion in the demineralization cycle.

Calcium-43 chemical shift and electric field gradient tensor interplay: a sensitive probe of structure, polymorphism, and hydration.

Calcium is the 5th most abundant element on earth, and is found in numerous biological tissues, proteins, materials, and increasingly in catalysts. However, due to a number of unfavourable nuclear properties, such as a low magnetogyric ratio, very low natural abundance, and its nuclear electric quadrupole moment, development of solid-state (43)Ca NMR has been constrained relative to similar nuclides. In this study, 12 commonly-available calcium compounds are analyzed via(43)Ca solid-state NMR and the information which may be obtained by the measurement of both the (43)Ca electric field gradient (EFG) and chemical shift tensors (the latter of which are extremely rare with only a handful of literature examples) is discussed. Combined with density functional theory (DFT) computations, this 'tensor interplay' is, for the first time for (43)Ca, illustrated to be diagnostic in distinguishing polymorphs (e.g., calcium formate), and the degree of hydration (e.g., CaCl2·2H2O and calcium tartrate tetrahydrate). For Ca(OH)2, we outline the first example of (1)H to (43)Ca cross-polarization on a sample at natural abundance in (43)Ca. Using prior knowledge of the relationship between the isotropic calcium chemical shift and the calcium quadrupolar coupling constant (CQ) with coordination number, we postulate the coordination number in a sample of calcium levulinate dihydrate, which does not have a known crystal structure. Natural samples of CaCO3 (aragonite polymorph) are used to show that the synthetic structure is present in nature. Gauge-including projector augmented-wave (GIPAW) DFT computations using accepted crystal structures for many of these systems generally result in calculated NMR tensor parameters which are in very good agreement with the experimental observations. This combination of (43)Ca NMR measurements with GIPAW DFT ultimately allows us to establish clear correlations between various solid-state (43)Ca NMR observables and selected structural parameters, such as unit cell dimensions and average Ca-O bond distances.

Production of high intensity 48Ca for the 88-Inch Cyclotron and other updates.

Recently the Versatile ECR for NUclear Science (VENUS) ion source was engaged in a 60-day long campaign to deliver high intensity (48)Ca(11+) beam to the 88-Inch Cyclotron. As the first long term use of VENUS for multi-week heavy-element research, new methods were developed to maximize oven to target efficiency. First, the tuning parameters of VENUS for injection into the cyclotron proved to be very different than those used to tune VENUS for maximum beam output of the desired charge state immediately following its bending magnet. Second, helium with no oxygen support gas was used to maximize the efficiency. The performance of VENUS and its low temperature oven used to produce the stable requested 75 eµA of (48)Ca(11+) beam current was impressive. The consumption of (48)Ca in VENUS using the low temperature oven was checked roughly weekly, and was found to be on average 0.27 mg/h with an ionization efficiency into the 11+ charge state of 5.0%. No degradation in performance was noted over time. In addition, with the successful operation of VENUS the 88-Inch cyclotron was able to extract a record 2 pµA of (48)Ca(11+), with a VENUS output beam current of 219 eµA. The paper describes the characteristics of the VENUS tune used for maximum transport efficiency into the cyclotron as well as ongoing efforts to improve the transport efficiency from VENUS into the cyclotron. In addition, we briefly present details regarding the recent successful repair of the cryostat vacuum system.

Calcium-43 NMR studies of polymorphic transition of calcite to aragonite.

Phase transformation between calcite and aragonite is an important issue in biomineralization. To shed more light on the mechanism of this process at the molecular level, we employ solid-state (43)Ca NMR to study the phase transformation from calcite to aragonite as regulated by magnesium ions, with (43)Ca enrichment at a level of 6%. Using the gas diffusion approach, the phase of Mg-calcite is formed initially and the system subsequently transforms to aragonite as the reaction time proceeds. Our (43)Ca solid-state NMR data support the dissolution-recrystallization mechanism for the calcite to aragonite transition. We find that the (43)Ca NMR parameters of Mg-calcite are very similar to those of pure calcite. Under the high-resolution condition provided by magic-angle spinning at 4 kHz, we can monitor the variation of the (43)Ca NMR parameters of the aragonite signals for the samples obtained at different reaction times. Our data suggest that in the presence of a significant amount of Mg(2+) ions, aragonite is the most stable polymorph of calcium carbonate. The initial precipitated crystallites of aragonite have spine-like morphology, for which the (43)Ca spin-lattice relaxation data indicate that the ions in the lattice have considerable motional dynamics. As the crystallinity of aragonite improves further, the (43)Ca T(1) parameter of the aragonite phase changes considerably and becomes very similar to that obtained for pure aragonite. For the first time, the difference in crystal morphologies and crystallinity of the aragonite phase has been traced down to the subtle difference in the motional dynamics at the molecular level.

Natural-abundance 43Ca solid-state NMR spectroscopy of bone.

Structural information about the coordination environment of calcium present in bone is highly valuable in understanding the role of calcium in bone formation, biomineralization, and bone diseases like osteoporosis. While a high-resolution structural study on bone has been considered to be extremely challenging, NMR studies on model compounds and bone minerals have provided valuable insight into the structure of bone. Particularly, the recent demonstration of (43)Ca solid-state NMR experiments on model compounds is an important advance in this field. However, application of (43)Ca NMR is hampered due to the low natural-abundance and poor sensitivity of (43)Ca. In this study, we report the first demonstration of natural-abundance (43)Ca magic angle spinning (MAS) NMR experiments on bone, using powdered bovine cortical bone samples. (43)Ca NMR spectra of bovine cortical bone are analyzed by comparing to the natural-abundance (43)Ca NMR spectra of model compounds including hydroxyapatite and carbonated apatite. While (43)Ca NMR spectra of hydroxyapatite and carbonated apatite are very similar, they significantly differ from those of cortical bone. Raman spectroscopy shows that the calcium environment in bone is more similar to carbonated apatite than hydroxyapatite. A close analysis of (43)Ca NMR spectra reveals that the chemical shift frequencies of cortical bone and 10% carbonated apatite are similar but the quadrupole coupling constant of cortical bone is larger than that measured for model compounds. In addition, our results suggest that an increase in the carbonate concentration decreases the observed (43)Ca chemical shift frequency. A comparison of experimentally obtained (43)Ca MAS spectra with simulations reveal a 3:4 mol ratio of Ca-I/Ca-II sites in carbonated apatite and a 2.3:3 mol ratio for hydroxyapatite. 2D triple-quantum (43)Ca MAS experiments performed on a mixture of carbonated apatite and the bone protein osteocalcin reveal the presence of protein-bound and free calcium sites, which is in agreement with a model developed from X-ray crystal structure of the protein.

Mass dependence of calcium isotope fractionations in crown-ether resin chromatography.

Benzo 18-crown-6-ether resin was synthesised by the phenol condensation polymerisation process in porous silica beads, of which particle diameter was ca 60micro Calcium adsorption chromatography was performed with the synthesised resin packed in a glass column. The effluent was sampled in fractions, and the isotopic abundance ratios of (42)Ca, (43)Ca, (44)Ca, and (48)Ca against (40)Ca were measured by a thermo-ionisation mass spectrometer. The enrichment of heavier calcium isotopes was observed at the front boundary of calcium adsorption chromatogram. The mass dependence of mutual separation of calcium isotopes was analysed by using the three-isotope-plots method. The slopes of three-isotope-plots indicate the relative values of mutual separation coefficients for concerned isotopic pairs. The results have shown the normal mass dependence; isotope fractionation is proportional to the reduced mass difference, (M - M')/MM', where M and M' are masses of heavy and light isotope, respectively. The mass dependence clarifies that the isotope fractionations are originated from molecular vibration. The observed separation coefficient epsilon is 3.1x10(-3) for the pair of (40)Ca and (48)Ca. Productivity of enriched (48)Ca by crown-ether-resin was discussed as the function of the separation coefficient and the height equivalent to the theoretical plate.

Calcium binding environments probed by (43)Ca NMR spectroscopy.

Calcium is an important component of materials, metalloproteins, minerals, glasses, and small inorganic and organic complexes. However, NMR spectroscopy of the quadrupolar (43)Ca nuclide remains difficult primarily due to its low natural abundance and low resonance frequency. In this Perspective, experimental challenges and recent successes in the field are highlighted, with a focus on solid-state (43)Ca NMR spectroscopy. Solution (43)Ca NMR studies of calcium-binding biomolecules are also presented. The structural insights afforded from quadrupolar and chemical shift parameters are examined. For example: isotropic chemical shifts have been shown to correlate with the mean Ca-O distance and also with calcium coordination number; quadrupolar coupling constants and chemical shift tensor spans have been shown to be useful probes of polymorphism; and, distance measurements involving (43)Ca have been recently demonstrated. Lastly, challenges and opportunities for the future are considered.

Two-dimensional (43)Ca-(1)H correlation solid-state NMR spectroscopy.

Calcium-43 (nuclear spin, S=7/2) is an NMR insensitive low-gamma quadrupolar nucleus and up until recently only one-dimensional solid-state (43)Ca NMR spectra have been reported. Through-space correlation experiments are challenging between spin-12 and low-gamma quadrupolar nuclei because of the intrinsically weak dipolar interaction and the often-low natural abundance of the quadrupolar nucleus. Rotary-resonance recoupling (R(3)) has recently been used to re-introduce hetero-nuclear dipolar interactions for sensitive high-gamma quadrupolar nuclei, but has not yet been applied in the case of low-gamma half-integer quadrupolar nuclei. Here an effective and robust 2D (1)H-(43)Ca NMR correlation experiment combining the R(3) dipole-recoupling scheme with 2D HMQC is presented. It is demonstrated that the weak (43)Ca-(1)H dipolar coupling in hydroxyapatite and oxy-hydroxyapatite can be readily re-introduced and that this recoupling scheme is more efficient than conventional cross-polarization transfer. Moreover, three (43)Ca-(1)H dipolar coupled calcium environments are clearly resolved in the structurally unknown oxy-hydroxyapatite. This local information is not readily available from other techniques such as powder XRD and high resolution electron microscopy. R(3)-HMQC is also a desirable experiment because the set-up is simple and it can be applied using conventional multi-resonance probes.

Calcium-43 chemical shift tensors as probes of calcium binding environments. Insight into the structure of the vaterite CaCO3 polymorph by 43Ca solid-state NMR spectroscopy.

Natural-abundance (43)Ca solid-state NMR spectroscopy at 21.1 T and gauge-including projector-augmented-wave (GIPAW) DFT calculations are developed as tools to provide insight into calcium binding environments, with special emphasis on the calcium chemical shift (CS) tensor. The first complete analysis of a (43)Ca solid-state NMR spectrum, including the relative orientation of the CS and electric field gradient (EFG) tensors, is reported for calcite. GIPAW calculations of the (43)Ca CS and EFG tensors for a series of small molecules are shown to reproduce experimental trends; for example, the trend in available solid-state chemical shifts is reproduced with a correlation coefficient of 0.983. The results strongly suggest the utility of the calcium CS tensor as a novel probe of calcium binding environments in a range of calcium-containing materials. For example, for three polymorphs of CaCO3 the CS tensor span ranges from 8 to 70 ppm and the symmetry around calcium is manifested differently in the CS tensor as compared with the EFG tensor. The advantages of characterizing the CS tensor are particularly evident in very high magnetic fields where the effect of calcium CS anisotropy is augmented in hertz while the effect of second-order quadrupolar broadening is often obscured for (43)Ca because of its small quadrupole moment. Finally, as an application of the combined experimental-theoretical approach, the solid-state structure of the vaterite polymorph of calcium carbonate is probed and we conclude that the hexagonal P6(3)/mmc space group provides a better representation of the structure than does the orthorhombic Pbnm space group, thereby demonstrating the utility of (43)Ca solid-state NMR as a complementary tool to X-ray crystallographic methods.

Optimization and application of ICPMS with dynamic reaction cell for precise determination of 44Ca/40Ca isotope ratios.

An inductively coupled plasma mass spectrometer with dynamic reaction cell (ICP-DRC-MS) was optimized for determining (44)Ca/(40)Ca isotope ratios in aqueous solutions with respect to (i) repeatability, (ii) robustness, and (iii) stability. Ammonia as reaction gas allowed both the removal of (40)Ar+ interference on (40)Ca+ and collisional damping of ion density fluctuations of an ion beam extracted from an ICP. The effect of laboratory conditions as well as ICP-DRC-MS parameters such a nebulizer gas flow rate, rf power, lens potential, dwell time, or DRC parameters on precision and mass bias was studied. Precision (calculated using the "unbiased" or "n - 1" method) of a single isotope ratio measurement of a 60 ng g(-1) calcium solution (analysis time of 6 min) is routinely achievable in the range of 0.03-0.05%, which corresponded to the standard error of the mean value (n = 6) of 0.012-0.020%. These experimentally observed RSDs were close to theoretical precision values given by counting statistics. Accuracy of measured isotope ratios was assessed by comparative measurements of the same samples by ICP-DRC-MS and thermal ionization mass spectrometry (TIMS) by using isotope dilution with a (43)Ca-(48)Ca double spike. The analysis time in both cases was 1 h per analysis (10 blocks, each 6 min). The delta(44)Ca values measured by TIMS and ICP-DRC-MS with double-spike calibration in two samples (Ca ICP standard solution and digested NIST 1486 bone meal) coincided within the obtained precision. Although the applied isotope dilution with (43)Ca-(48)Ca double-spike compensates for time-dependent deviations of mass bias and allows achieving accurate results, this approach makes it necessary to measure an additional isotope pair, reducing the overall analysis time per isotope or increasing the total analysis time. Further development of external calibration by using a bracketing method would allow a wider use of ICP-DRC-MS for routine calcium isotopic measurements, but it still requires particular software or hardware improvements aimed at reliable control of environmental effects, which might influence signal stability in ICP-DRC-MS and serve as potential uncertainty sources in isotope ratio measurements.

Behavior of bacteria in the inductively coupled plasma: atomization and production of atomic ions for mass spectrometry.

The combination of perfusion chromatography (PC) and inductively coupled plasma mass spectrometry (ICPMS) is a fast, general way to monitor metal incorporation into bacteria. U+ signals from U incorporated intrinsically in Bacillus subtilis (B. subtilis) are measured with 4 ms time resolution to investigate the behavior of individual cells in the ICP. When intact B. subtilis cells are introduced directly into the ICP, occasional U+ spikes are observed. The positive U+ spikes suggest that bacteria behave more like solid particles than wet droplets in the ICP, compared to previous studies of such transient effects in the ICP. Drying the bacterial aerosol does not eliminate the spikes. Lysing the bacteria by sonication increases the U+ response by 30% compared to that from the untreated sample. PC results from a 10 ppb U standard, partially lysed and fully lysed bacteria samples show that the intracellular U-bound species are released by sonication and are small in size. The atomization-ionization efficiencies for different elements (U, Ca, and Mg) from cells differ somewhat. Reducing the aerosol gas flow rate by 0.1 L min-1 improves the relative U+ response for unlysed bacteria to 85% of that for lysed cells, although the absolute U+ signal is attenuated greatly.

Mg and Ca isotope fractionation during CaCO3 biomineralisation.

The natural variation of Mg and Ca stable isotopes of carbonates has been determined in carbonate skeletons of perforate foraminifera and reef coral together with Mg/Ca ratios to assess the influence of biomineralisation processes. The results for coral aragonite suggest its formation, in terms of stable isotope behaviour, approximates to inorganic precipitation from a seawater reservoir. In contrast, results for foraminifera calcite suggest a marked biological control on Mg isotope ratios presumably related to its low Mg content compared with seawater. The bearing of these observations on the use of Mg and Ca isotopes as proxies in paleoceanography is considered.