Vincent Balter

Box-modeling of 15N/14N in mammals

The 15N/14N signature of animal proteins is now commonly used to understand their physiology and quantify the flows of nutrient in trophic webs. These studies assume that animals are predictably 15N-enriched relative to their food, but the isotopic mechanism which accounts for this enrichment remains unknown. We developed a box model of the nitrogen isotope cycle in mammals in order to predict the 15N/14N ratios of body reservoirs as a function of time, N intake and body mass. Results of modeling show that a combination of kinetic isotope fractionation during the N transfer between amines and equilibrium fractionation related to the reversible conversion of N-amine into ammonia is required to account for the well-established ≈4‰ 15N-enrichment of body proteins relative to the diet. This isotopic enrichment observed in proteins is due to the partial recycling of 15N-enriched urea and the urinary excretion of a fraction of the strongly 15N-depleted ammonia reservoir. For a given body mass and diet δ15N, the isotopic compositions are mainly controlled by the N intake. Increase of the urea turnover combined with a decrease of the N intake lead to calculate a δ15N increase of the proteins, in agreement with the observed increase of collagen δ15N of herbivorous animals with aridity. We further show that the low δ15N collagen values of cave bears cannot be attributed to the dormancy periods as it is commonly thought, but inversely to the hyperphagia behavior. This model highlights the need for experimental investigations performed with large mammals in order to improve our understanding of natural variations of δ15N collagen.

Allometric constraints on Sr/Ca and Ba/Ca partitioning in terrestrial mammalian trophic chains

In biological systems, strontium (Sr) and barium (Ba) are two non-essential elements, in comparison to calcium (Ca) which is essential. The Sr/Ca and Ba/Ca ratios tend to decrease in biochemical pathways which include Ca as an essential element, and these processes are termed biopurification of Ca. The quantitative pathway of the biopurification of Ca in relation to Sr and Ba between two biological reservoirs (R n and Rn -1) is measured with an observed ratio (OR) expressed by the (Sr/Ca) Rn /(Sr/Ca) Rn-1 and (Ba/Ca) Rn /(Ba/Ca) Rn-1 ratios. For a mammalian organism, during the whole biopurification of Ca starting with the diet to the ultimate reservoir of Ca which is the bone, the mean values for ORSr and ORBa are 0.25 and 0.2, respectively. In this study, published Sr/Ca and Ba/Ca ratios are used for three sets of soils, plants, and bones of herbivorous and carnivorous mammals, each comprising a trophic chain, to illustrate the biopurification of Ca at the level of trophic chains. Calculated ORSr and ORBa of herbivore bones in relation to plants and of bones of carnivores in relation to bones of herbivores give ORSr=0.30±0.08 and ORBa=0.16±0.08, thus suggesting that trophic chains reflect the Sr/Ca and Ba/Ca fluxes that are prevalent at the level of a mammalian organism. The slopes of the three regression equations of log(Sr/Ca) vs. log(Ba/Ca) are similar, indicating that the process of biopurification of Ca with respect to Sr and Ba is due to biological processes and is independent of the geological settings. Modifications of the logarithmic expression of the Sr/Ca and Ba/Ca relationship allow a new formula of the biopurification process to be deduced, leading to the general equation ORBa=ORSr1.79±0.33,where the allometric coefficient is the mean of the slopes of the three regression equations. Some recent examples are used to illustrate this new analysis of predator-prey relations between mammals. This opens up new possibilities for the utilization of Ba/Ca and Sr/Ca in addition to stable isotope ratios (δ13C and δ15N) for the determination of the relative contribution of different food sources to an animal’s diet.

Evidence for dietary change but not landscape use in South African early hominins

The dichotomy between early Homo and Paranthropus is justified partly on morphology. In terms of diet, it has been suggested that early Homo was a generalist but that Paranthropus was a specialist. However, this model is challenged and the issue of the resources used by Australopithecus, the presumed common ancestor, is still unclear. Laser ablation profiles of strontium/calcium, barium/calcium and strontium isotope ratios in tooth enamel are a means to decipher intra-individual diet and habitat changes. Here we show that the home range area was of similar size for species of the three hominin genera but that the dietary breadth was much higher in Australopithecus africanus than in Paranthropus robustus and early Homo. We also confirm that P. robustus relied more on plant-based foodstuffs than early Homo. A South African scenario is emerging in which the broad ecological niche of Australopithecus became split, and was then occupied by Paranthropus and early Homo, both consuming a lower diversity of foods than Australopithecus.

Natural variations of copper and sulfur stable isotopes in blood of hepatocellular carcinoma patients

Significance In cancer, the metabolism of copper and sulfur are dysregulated, leading to deleterious side effects. These issues are commonly addressed by studying the variations of concentrations of the elements, but here we have used, for the first time to our knowledge, copper and sulfur stable isotope compositions variations, using methods widespread in Earth sciences. We show that in hepatocellular carcinomas patients, blood copper and sulfur are enriched in light isotopes compared with control subjects. These isotopic signatures are not compatible with a dietary origin, but rather reflect the massive reallocation in the body of copper immobilized within cysteine-rich proteins such as metallothioneins. We also propose that sulfur isotope compositions could serve to track sulfur originating from tumor-derived sulfides. The widespread hypoxic conditions of the tumor microenvironment can impair the metabolism of bioessential elements such as copper and sulfur, notably by changing their redox state and, as a consequence, their ability to bind specific molecules. Because competing redox state is known to drive isotopic fractionation, we have used here the stable isotope compositions of copper (65Cu/63Cu) and sulfur (34S/32S) in the blood of patients with hepatocellular carcinoma (HCC) as a tool to explore the cancer-driven copper and sulfur imbalances. We report that copper is 63Cu-enriched by ∼0.4‰ and sulfur is 32S-enriched by ∼1.5‰ in the blood of patients compared with that of control subjects. As expected, HCC patients have more copper in red blood cells and serum compared with control subjects. However, the isotopic signature of this blood extra copper burden is not in favor of a dietary origin but rather suggests a reallocation in the body of copper bound to cysteine-rich proteins such as metallothioneins. The magnitude of the sulfur isotope effect is similar in red blood cells and serum of HCC patients, implying that sulfur fractionation is systemic. The 32S-enrichment of sulfur in the blood of HCC patients is compatible with the notion that sulfur partly originates from tumor-derived sulfides. The measurement of natural variations of stable isotope compositions, using techniques developed in the field of Earth sciences, can provide new means to detect and quantify cancer metabolic changes and provide insights into underlying mechanisms.

Fe and Cu stable isotopes in archeological human bones and their relationship to sex.

Accurate sex assignment of ancient human remains usually relies on the availability of coxal bones or well-preserved DNA. Iron (Fe) and copper (Cu) stable isotope compositions ((56)Fe/(54)Fe and (65)Cu/(63)Cu, respectively) were recently measured in modern human blood, and an unexpected result was the discovery of a (56)Fe-depletion and a (65)Cu-enrichment in mens blood compared to womens blood. Bones, being pervasively irrigated by blood, are expected to retain the (56)Fe/(54)Fe and (65)Cu/(63)Cu signature of blood, which in turn is useful for determining the sex of ancient bones. Here, we report the (56)Fe/(54)Fe, (65)Cu/(63)Cu, and (66)Zn/(64)Zn ratios from a suite of well-preserved phalanxes (n = 43) belonging to individuals buried in the 17th and 18th centuries at the necropolis of Saint-Laurent de Grenoble, France, and for which the sex was independently estimated from pelvic bone morphology. The metals were purified from the bone matrix by liquid chromatography on ion exchange resin and the isotope compositions were measured by multiple-collector inductively coupled plasma mass spectrometry. The results show that, as expected from literature data on blood, male bone iron is depleted in (56)Fe and enriched in (65)Cu relative to female. No sex difference is found in the (66)Zn/(64)Zn ratios of bone. The concentration and isotopic data show no evidence of soil contamination. Four samples of five (77%) can be assigned their correct sex, a result comparable to sex assignment using Fe and Cu isotopes in blood (81%). Isotopic analysis of metals may therefore represent a valid method of sex assignment applicable to incomplete human remains.

A simplified protocol for measurement of Ca isotopes in biological samples

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 HNO3 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 δ, 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 44Ca-depleted and RBC Ca poor and 44Ca-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.

Palaeoenvironmental reconstruction of the Sarmatian (Middle Miocene) Central Paratethys based on palaeontological and geochemical analyses of foraminifera, ostracods, gastropods and rodents

Paleoenvironmental changes in the upper Middle Miocene Central Parathetys were reconstructed by using qualitative and quantitative palaeontological analyses of foraminifera and ostracods, coupled with trace elemental (Mg/Ca) and stable isotope (delta(18)O and delta(13)C) analyses of their carbonate skeletons and of gastropod sheets. Mean annual air temperatures were estimated using the oxygen isotope composition of contemporaneous rodent teeth. The studied aquatic fossils come from two boreholes in the Zsambek basin (northern central Hungary), while the terrestrial ones are from localities in NE Hungary and E Romania. In the studied Sarmatian successions, three zones could be distinguished, based on palaeontological and geochemical results. At the Badenian/Sarmatian boundary, faunal diversity decreased markedly. In the lower zone a transgressive event culminated in a seawater incursion into the semi-open basin system of the Central Paratheys. Stable bottom-water temperature (similar to 15 degrees C) and variable salinites (20-22\%) are estimated for the Early Sarmatian Sea. The faunal changes (notably a strong reduction in biodiversity) occuring at the boundary between the lower and the middle zone can be explained by a sea-leval highstand with dysoxic conditions. A relative sea-level fall is documented at the end of this middle zone. After a short regressive event, a marine connection between the Paratethys and Mediterranean was established at the beginning of the upper zone. This is indicated by an increased microfaunal diversity and the re-appearance of marine Bademan ostracods and foraminifera, which are completely absent from the older Samatian series. During the upper zone, the temperatures and salmities are estimated to have fluctuated from 15 degrees C to 21 degrees C and from 15 parts per thousand to 43 parts per thousand, respectively.

Record of climate-driven morphological changes in 376 Ma Devonian fossils

The Lower and Upper Kellwasser horizons represent two anoxic events that mark the mass extinction at the Frasnian-Famennian (F-F) boundary. Among other groups, conodont animals were severely affected, but the genus Palmatolepis survived with a complete turnover at the F-F boundary. Here the fine morphological variations of the genus Palmatolepis and the sea-surface temperature evolution are quantified in two F-F boundary sections using morphometrics and oxygen isotopic composition of apatite, respectively. In accordance with other F-F sections, the isotope records show two positive excursions of ~1‰ during the Lower and Upper Kellwasser anoxic events. The conodont shape and the oxygen isotopic composition of the genus Palmatolepis are significantly correlated within the Frasnian and Famennian Stages, suggesting a strong environmental influence on the morphology of the feeding apparatus of the conodont animal. We propose that the morphological differences are linked to changes in the trophic position of Palmatolepis: enhanced organic carbon burial, which is supported by global positive carbon isotope excursions in inorganic and organic carbon during both the Lower and Upper Kellwasser events altered the primary biomass production and thus, the subsequent nutrient supply to higher trophic levels. While the carbon and oxygen isotopic shifts are of similar amplitude during Lower and Upper Kellwasser events, the variation of the shape of Palmatolepis during the Upper Kellwasser, i.e., the F-F boundary, is more pronounced than during the Lower Kellwasser.

Precise analysis of calcium stable isotope variations in biological apatites using laser ablation MC-ICPMS

Laser ablation (LA) is potentially an interesting technique to measure natural variations (δ44/42Ca) of calcium isotopes in calcium-rich minerals because it allows spatial resolution and avoids micro-sampling and consecutive wet chemistry. We developed a matrix-match sample/standard normalization method and used an Excite 193 nm Photon Machines LA system coupled to a Neptune plus MC-ICPMS to measure δ44/42Ca variations in enamel apatite. First, high precision δ44/42Ca solution mode (SOL) analyses were performed on a series of 5 crystalline igneous apatite and 6 modern tooth enamel samples, which were micro-sampled using a MicroMill device. The δ44/42Ca isotopic values ranged evenly between −0.60 and +0.60‰ (per amu). Second, we sintered by means of a spark plasma sintering technique the bone ash SRM1400 standard and two synthetic apatites (doped or not with Sr). The Ca isotope compositions using LA were measured in the samples in the raster mode along 600 × 85 μm profiles and bracketed with the SRM1400 standard. We obtained very good agreement between SOL and LA measurements, i.e. δ44/42CaLAvs. δ44/42CaSOL slope of 0.960 ± 0.091 (2SE, R2 = 0.971) and null offset at origin (0.012 ± 0.084, 2SE). For all samples, residual values to the 1 : 1 slope were ≤0.1‰ (per amu). However, an unexplained and constant 0.13‰ offset occurred when considering the 43/42Ca ratio, suggesting an uncorrected isobaric interference on 43Ca in the LA mode. We also noticed that the doubly charged strontium (Sr) interference correction is of crucial importance for accurate matching between LA and SOL measurements. In the SOL mode, Sr is discarded by ion chromatography leading to typical 87Sr2+/44Ca+ ratios of 10−5 to 10−6. In the LA mode, this ratio can exceed 10−3. We show that the value set for the 87Sr/86Sr ratio is of importance to correct the Sr interference, and that optimized residuals to the 1 : 1 slope are obtained using a Sr correction that takes into account a mass fractionation factor for doubly charged Sr distinct from that of Ca. We found that deciduous teeth enamel is depleted of Ca heavy isotopes by about 0.35–0.40‰ (per amu) compared to wisdom teeth enamel, a shift compatible with a transition from a milk based diet to a plant and meat based diet.

Magnesium stable isotope ecology using mammal tooth enamel

Significance The diet of fossil organisms can be inferred through isotopic analysis of skeletal tissues, largely assessed using isotopic and elemental systems such as carbon isotopes (δ13C) and strontium/calcium (Sr/Ca) and barium/calcium (Ba/Ca) concentration ratios. In the case of complex dietary habits such as omnivory, these systems must be used jointly together with new proxies. Based on the expectation that fractionation of bio-essential metals occurs during metabolism, we explore the isotopic variability of magnesium (δ26Mg) in tooth enamel sampled from an assemblage of modern mammals. We demonstrate that δ26Mg increases from herbivores to higher-level consumers, discriminating most of the identified trophic steps. This, combined with δ13C, Ba/Ca, and/or δ44Ca might prove useful in paleodietary studies. Geochemical inferences on ancient diet using bone and enamel apatite rely mainly on carbon isotope ratios (δ13C) and to a lesser extent on strontium/calcium (Sr/Ca) and barium/calcium (Ba/Ca) elemental ratios. Recent developments in nontraditional stable isotopes provide an unprecedented opportunity to use additional paleodietary proxies to disentangle complex diets such as omnivory. Of particular relevance for paleodietary reconstruction are metals present in large quantity in bone and enamel apatite, providing that biologically mediated fractionation processes are constrained. Calcium isotope ratios (δ44Ca) meet these criteria but exhibit complex ecological patterning. Stable magnesium isotope ratios (δ26Mg) also meet these criteria but a comprehensive understanding of its variability awaits new isotopic data. Here, 11 extant mammal species of known ecology from a single locality in equatorial Africa were sampled for tooth enamel and, together with vegetation and feces, analyzed for δ26Mg, δ13C, Sr/Ca, and Ba/Ca ratios. The results demonstrate that δ26Mg incorporated in tooth enamel becomes heavier from strict herbivores to omnivores/faunivores. Using data from experimentally raised sheep, we suggest that this 26Mg enrichment up the trophic chain is due to a 26Mg enrichment in muscle relative to bone. Notably, it is possible to distinguish omnivores from herbivores, using δ26Mg coupled to Ba/Ca ratios. The potential effects of metabolic and dietary changes on the enamel δ26Mg composition remain to be explored but, in the future, multiproxy approaches would permit a substantial refinement of dietary behaviors or enable accurate trophic reconstruction despite specimen-limited sampling, as is often the case for fossil assemblages.