Calcium isotope fractionation between aqueous compounds relevant to low-temperature geochemistry, biology and medicine.

Stable Ca isotopes are fractionated between bones, urine and blood of animals and between soils, roots and leaves of plants by >1000 ppm for the 44Ca/40Ca ratio. These isotopic variations have important implications to understand Ca transport and fluxes in living organisms; however, the mechanisms of isotopic fractionation are unclear. Here we present ab initio calculations for the isotopic fractionation between various aqueous species of Ca and show that this fractionation can be up to 3000 ppm. We show that the Ca isotopic fractionation between soil solutions and plant roots can be explained by the difference of isotopic fractionation between the different first shell hydration degree of Ca2+ and that the isotopic fractionation between roots and leaves is controlled by the precipitation of Ca-oxalates. The isotopic fractionation between blood and urine is due to the complexation of heavy Ca with citrate and oxalates in urine. Calculations are presented for additional Ca species that may be useful to interpret future Ca isotopic measurements.

Can serum isotope levels accurately measure intestinal calcium absorption compared to gold-standard methods?

BACKGROUND: Low fractional calcium absorption (FCA) contributes to osteoporosis but is not measured clinically, as the gold-standard method requires administration of two calcium tracers and a subsequent 24-h urine collection. We evaluated alternate methods to measure FCA, compared to the gold standard method. METHODS: We administered two stable calcium isotope tracers (~8 mg oral (44)Ca and ~3 mg intravenous (42)Ca) with breakfast to 20 fasting post-menopausal women (Cohort 1) 59 ± 7 years old with vitamin D insufficiency. We measured subsequent calcium isotope concentrations in 24-h urine samples and serum collected 1, 3 and 5 h post tracer administration during an inpatient research stay. We assessed the candidate serum estimates in a second cohort of 9 women with similar characteristics. Methods of measuring FCA were compared using correlation coefficients and Bland-Altman tests. RESULTS: FCA estimated from a 3-h serum sample correlated highest with the levels from the 24-h urine collection (ρ 0.78, p < 0.001), but explained only 58 % of the variance in FCA. The total variance explained by 3-h estimates improved to 61 % with incorporation of glomerular filtration rate (GFR). FCA estimates from the 3-h serum measurement were assessed in a second group of nine women (Cohort 2) 60 ± 7 years old. In this cohort, however, FCA estimated by 3-h serum isotope levels did not correlate with gold-standard FCA measurements, whether determined with (ρ 0.02, p = 0.97) or without GFR values (ρ 0.03, p = 0.93). By contrast, FCA in Cohort 2 correlated best with 5-h serum isotope levels (ρ 0.75, p = 0.02). CONCLUSIONS: We conclude that serum isotope levels correlate with true fractional calcium absorption, but do not reliably estimate FCA when analyzed using Bland-Altman tests, compared to gold-standard methods. TRIAL REGISTRATION: ClinicalTrials.gov.Identifier: NCT00933244.

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.

A simple single serum method to measure fractional calcium absorption using dual stable isotopes.

The dual stable isotope method with a timed 24-h urine collection is the gold standard approach to measure fractional calcium absorption. However, the need to collect urine for 24 h makes this technique time-consuming and laborious. Our study sought to determine whether a dual isotope method using a single serum sample obtained 4 h after administration of the initial isotope provides a useful approach to measure fractional calcium absorption. Following a metabolic diet with a fixed calcium intake of 30 mmol/day for 10 days, nineteen healthy subjects age 54-74 were given a test meal with an oral isotope ((44)Ca) followed 2 h later by an intravenous isotope ((42)Ca). Once the oral isotope was administered, urine was collected for 24 h, and a serum sample was obtained after 4 h. The ratio of the oral to intravenous isotopes was measured in the urine and serum by mass spectroscopy. Fractional calcium absorption was 16.2 ± 7.7% by the 4-h single serum method versus 18.5 ± 7.5% by the 24-h urine method. There was a small mean difference between the urine and serum methods of 2.33% with a confidence interval -3.97 to 8.60%. The two methods showed a strong linear association (r = 0.912, p<0.001). Use of dual stable isotopes with a 4-h single serum method gives fractional calcium absorption values that are 12.5% lower than with the 24-h urine method; however, it rank orders subjects accurately thus making it a useful alternative method in clinical research applications.

Evaluation of an inexpensive calcium absorption index in healthy older men and women.

OBJECTIVE: Calcium absorption is an important determinant of calcium retention and bone metabolism. However, most methods of measuring calcium absorption, including the well-established dual stable isotope method, are costly and cumbersome to implement. We evaluated whether an oral calcium tolerance test (OCTT), which involves measuring calcium excretion in a fasting 2-h urine collection and two 2-h collections following an oral calcium dose, may be a useful index of calcium absorption in older adults consuming a fixed calcium intake of 30 mmol/day. DESIGN: After a 10-day metabolic diet containing 30 mmol/day of calcium, subjects had calcium absorption measured using the dual stable isotope method and the OCTT. PARTICIPANTS: Eleven healthy subjects aged 54-74 years. MEASUREMENTS: Fractional calcium absorption (FCA), calcium excretion in a fasting 2-h urine collection and two 2-h collections in response to a 10-mmol calcium dose (total intake 30 mmol/day). RESULTS: Calcium excretion from several combinations of the urine collections was examined in relation to FCA. The most predictive of FCA was calcium excretion 4 h following the calcium dose. This measure was significantly correlated with FCA (r = 0.735, P = 0.010), fitting 54% of the variability in FCA. CONCLUSION: Urinary calcium excretion during the 4 h after a 10-mmol calcium dose is a useful index of calcium absorption among older adults consuming recommended calcium intakes. This test is inexpensive, easy to implement and potentially useful in large clinical studies.

Isotopic analysis of calcium in blood plasma and bone from mouse samples by multiple collector-ICP-mass spectrometry.

The biological processing of Ca produces significant stable isotope fractionation. The level of isotopic fractionation can provide key information about the variation in dietary consumption or Ca metabolism. To investigate this, we measured the 43Ca/42Ca and 44Ca/42Ca ratios for bone and blood plasma samples collected from mice of various ages using multiple collector-ICP-mass spectrometry (MC-ICP-MS). The 44Ca/42Ca ratio in bones was significantly (0.44-0.84 per thousand) lower than the corresponding ratios in the diet, suggesting that Ca was isotopically fractionated during Ca metabolism for bone formation. The resulting 44Ca/42Ca ratios for blood plasma showed almost identical, or slightly higher, values (0.03-0.2 per thousand) than found in a corresponding diet. This indicates that a significant amount of Ca in the blood plasma was from dietary sources. Unlike that discovered for Fe, there were no significant differences in the measured 44Ca/42Ca ratios between female and male specimens (for either bone or blood plasma samples). Similarity, the 44Ca/42Ca ratios suggests that there were no significant differences in Ca dietary consumption or Ca metabolism between female and male specimens. In contrast, the 44Ca/42Ca ratios of blood plasma from mother mice during the lactation period were significantly higher than those for all other adult specimens. This suggests that Ca supplied to infants through lactation was isotopically lighter, and the preferential supply of isotopically lighter Ca resulted in isotopically heavier Ca in blood plasma of mother mice during the lactation period. The data obtained here clearly demonstrate that the Ca isotopic ratio has a potential to become a new tool for evaluating changes in dietary consumption, or Ca metabolism of animals.

Inductively coupled plasma mass spectrometric analysis of calcium isotopes in human serum: a low-sample-volume acid-equilibration method.

BACKGROUND: Analytical methods for measuring the calcium isotope distribution in enriched human serum samples that use low blood volumes, simple preparation methods, and rapid analysis are important in clinical studies of calcium kinetics. Previously, sample preparation by oxalate precipitation typically required 500 micro L of serum. This method was time-consuming, and the blood volume required was limiting in circumstances when only a small amount of serum could be obtained. METHODS: Serum was collected from humans who were administered (42)Ca, and 20 micro L of serum was mixed with 2 mL of 0.22-0.67 mol/L HNO(3) at room temperature for between 1 min and 16 h. The (42)Ca/(43)Ca ratio in the supernatant was measured by a magnetic sector inductively coupled plasma mass spectrometer (ICP-MS). Calcium isotope ratios from these equilibration solutions were compared with data from oxalate-precipitated serum samples to determine the optimum equilibrium time and the effect of acid concentration on equilibrium. RESULTS: Various amounts of aggregated particles developed in different acid-serum mixtures. These affected the time required for isotope equilibration in the mixture. The shortest equilibrium time needed for the calcium isotopes varied from 1 to 6 h for samples acidified with 0.22-0.45 mol/L HNO(3). Data obtained from these solutions were consistent with data from oxalate-precipitated calcium. The precision of (42)Ca/(43)Ca ratio measurements was better than 0.5%. CONCLUSIONS: We have developed a simple, rapid sample preparation technique for ICP-MS analysis in which 20 micro L of serum can be used for accurate measurement of the calcium isotope distribution in a sample with good precision and a rapid analysis time.

Calcium absorption measured by stable calcium isotopes ((42)Ca & (44)Ca) among Northern Chinese adolescents with low vitamin D status.

An adequate calcium intake and vitamin-D status is important for bone mineralization in adolescents. In Northern China, calcium intake and plasma vitamin-D level of adolescents is low due to low consumption of dairy foods and inadequate sunshine exposure. True fractional calcium absorption (TFCA) in Chinese adolescents has never been performed. This study aims to evaluate nutritional adaptation namely, TFCA and urinary calcium excretion among Chinese adolescents in northern China.

The predictive value of perioperative calcium levels after thyroid/parathyroid surgery.

BACKGROUND: This work was done to determine whether the slope of change between preoperative and early postoperative (2 hr and 8 hr) ionized calcium levels (ciCa) could predict significant postoperative hypocalcemia. METHODS: We did a retrospective chart review of 79 patients undergoing thyroid (n = 56) or parathyroid surgery (n = 23) where overall parathyroid function was felt to be at risk. Results/Conclusions A positive slope between the preoperative ciCa and any postoperative ciCa or the first two postoperative ciCa's predicted normocalcemia in 100% of patients. For the thyroid group, the difference in the slope of the change from preoperative ciCa to first (<2 hours) postoperative ciCa (-15.940%/hours vs -9.375%/hours, p =.082) did not differentiate patients who developed hypocalcemia (n = 11) and those who remained normocalcemic (n = 45). By contrast the slope of change from preoperative ciCa to the second postoperative (8 hr) ciCa (-1.671%/ hour vs -0.849%/hours, p =.006) and first to second postoperative ciCa (-1.022%/hour vs -0.473%/hour, p =.001) did. For the parathyroid group, none of the differences in similar comparisons between the normocalcemic (n = 20) and hypocalcemic patients (n = 3) approached statistical significance. The relatively small number of patients who became hypocalcemic in this study precluded definition of an absolute slope of calcium level change above which normocalcemia can be assured.