A simplified protocol for measurement of Ca isotopes in biological samples

Abstract

We describe a chemical separation protocol of calcium from biological materials for isotopic measurement by multiple collector inductively coupled plasma mass spectrometry (MC-ICPMS). The method was tested using elution profiles along with HCl and HNO 3 acids only, on human urine, sheep serum and red blood cells (RBC), seawater and herbaceous plants. It allows the elimination of all interfering species (including K, Sr, Mg) and the remaining matrix (including Fe, P, Na and S) beyond required levels. In order to further test this protocol and better understand the Ca isotopic signatures of mammalian fluids and organs, we purified and analyzed a wide range of materials from sheep, i.e. serum, RBC, muscle, liver, kidneys, enamel, bone, urine and feces. The data show a wide range of variations, expressed as d, over 1& per amu, with a precision of 0.1& or better, spanning most of the variability reported so far. Red blood cells appeared to be heavier than serum by 0.3& per amu. This isotopic difference between serum and red blood cells was not taken into account in previous studies and it provides further information on Ca isotopic cycling in organisms. The Ca isotopic compositions of organs are correlated with concentrations, bone and RBC representing the two end-members, bone being Ca rich and 44 Cadepleted and RBC Ca poor and 44 Ca-enriched. The trend is compatible with a distillation process by which Ca is extruded from cells along with a kinetic fractionation process favoring lighter Ca isotopes. Material and methods SamplesChemical purication of Ca was tested on seawater, freeze-dried human urine, sheep serum and red blood cells (RBC) and herbaceous plants. Seawater, SRM915b, bone meal NIST SRM1486, cave bear and two sheep enamel samples (CBE, E1634, E9646 respectively) were chosen to assess precision and

Assessing human weaning practices with calcium isotopes in tooth enamel

“…*Described in ref. 59. including horses, seals, mice, deer, sheep, and Göttingen minipigs (30,(42)(43)(44)(45) and is assumed to be comparable in humans (46,47). The average δ 44/42 Ca value of wisdom tooth enamel that we measured in nine adult individuals (−1.58 ± 0.26 ‰, 2 SD, n = 9) is thus in good agreement with a hypothetical average Western diet composition of −1.00‰.…”

“…1A; Welch's ANOVA, P < 0.001; Kruskal-Wallis, P < 0.001). More precisely, we observe a transition of Ca isotopic compositions toward 44 Ca-enriched values, from prenatal to postnatal development stages (Welch's t test, P < 0.001; Wilcoxon-Mann-Whitney, P < 0.001). For each measurement in a given individual, we can define a Δ 44/42 Ca value, given by the difference between the δ 44/42 Ca value of a considered spot and the average δ 44/42 Ca value of an individual's prenatal enamel.…”

“…Such a 44 Ca-depleted isotope composition in fetus enamel has several possible and likely combined causes. First, compared with the estimated −1.00‰ mean value of diet, the mother's blood has lower δ 44/42 Ca values: a compilation of available data in mammals yields a Ca isotopic shift value from diet to blood, denoted Δ blood-diet and given by the difference between δ 44/42 Ca blood and δ 44/42 Ca diet , of −0.30 ± 0.13 ‰ (1 SD, Table S3).…”

“…Enamel that initiates formation early, that is, during the second and third trimesters of in utero development (48), is characterized by a 44 Ca-depleted isotope composition compared with third molar enamel (Welch t test, P < 0.001; Wilcoxon-Mann-Whitney, P < 0.001) and has a mean δ 44/42 Ca lower than that of third molars by −0.31 ± 0.11 ‰ (Welch t test 95% confidence interval). Such a 44 Ca-depleted isotope composition in fetus enamel has several possible and likely combined causes.…”

“…Using a sequential microsampling method along the enamel growth axis, we collected more than 150 enamel microsamples from 51 deciduous teeth of 12 different modern human individuals of known dietary histories, as well as nine enamel samples from permanent third molars. We measured and reconstructed the evolution of 44 Ca/ 42 Ca ratios in enamel from in utero development to first months of postnatal development. We show that the observed variations of δ 44/42 Ca record a transition from placental nutrition to an adult-like diet and that Ca isotopes reflect the duration of the breastfeeding period experienced by each infant.…”