Stable carbon and oxygen isotopes in human tooth enamel: Identifying breastfeeding and weaning in prehistory

This paper investigates the utility of stable carbon and oxygen isotopes in human dental enamel to reveal patterns of breastfeeding and weaning in prehistory. Enamel preserves a record of childhood diet that can be studied in adult skeletons. Comparing different teeth, we used δ¹³C to document the introduction of solid foods to infant diets and δ¹⁸O to monitor the decline of breastfeeding. We report enamel carbonate δ¹³C and δ¹⁸O of 33 first molars, 35 premolars, and 25 third molars from 35 burials from Kaminaljuyú, an early state in the valley of Guatemala. The skeletons span from Middle Preclassic through Late Postclassic occupations, ca. 700 B.C. to 1500 A.D. Sections of enamel were removed from each tooth spanning from the cusp to the cemento-enamel junction. Stable isotope ratios were measured on CO₂ liberated by reaction of enamel with H₃PO₄ in an automated carbonate system attached to a VG Optima mass spectrometer. Within a skeleton, teeth developing at older ages are more enriched in ¹³C and more depleted in ¹⁸O than teeth developing at younger ages. Premolars average 0.5% higher in δ¹³C than first molars from the same skeleton (P = 0.0001), but third molars are not significantly enriched over premolars. The shift from first molars to premolars may be due to the shift to solid foods from lipid-rich milk. After 2 years, when premolars begin to mineralize, the δ¹³C in childhood diets did not change systematically. First molars and premolars are similar in δ¹⁸O, but third molars average 0.7% lower than first molars (P = 0.0001) and 0.5% lower than premolars (P = 0.0003). First molar and premolar δ¹⁸O is heavier, because breast milk is more enriched in ¹⁸O than is drinking water. Hence, many children continued to nurse during the period of premolar formation. Together, these results indicate that Kaminaljuyú children had begun to eat solid maize foods before the age of 2 years but continued to drink breast milk until much later. Am J Phys Anthropol 106:1–18, 1998. © 1998 Wiley-Liss, Inc.     

Insights from the inside: histological analysis of abnormal enamel microstructure associated with hypoplastic enamel defects in human teeth

We studied the abnormalities in enamel microstructure associated with enamel hypoplasia in human teeth from the early medieval (5th-7th century AD) cemetery of Barbing, Germany, using light and scanning electron microscopy. The main aim of the study was to test the hypothesis that by analyzing the microstructure of fully formed enamel it is possible to reconstruct the reaction pattern of secretory ameloblasts to stress events leading to enamel hypoplasia. From the histological findings, a sequence of increasing impairment of secretory ameloblast function involving three thresholds was deduced. Surpassing of each of these thresholds is assumed to result in characteristic changes in enamel microstructure attributable to specific functional/morphological alterations of secretory ameloblasts. Based on our results we propose a model identifying the principal factors influencing the reaction of secretory ameloblasts to stress. The present study demonstrates that by including microscopic analysis in the study of enamel hypoplasia, it is possible to obtain a more complete picture of the formation of these developmental defects than is possible by inspection of crown surface features alone, and to draw more substantiated conclusions about the possible nature of developmental defects of enamel.     

Stable isotopes in fossil hominin tooth enamel suggest a fundamental dietary shift in the Pliocene

Accumulating isotopic evidence from fossil hominin tooth enamel has provided unexpected insights into early hominin dietary ecology. Among the South African australopiths, these data demonstrate significant contributions to the diet of carbon originally fixed by C₄ photosynthesis, consisting of C₄ tropical/savannah grasses and certain sedges, and/or animals eating C₄ foods. Moreover, high-resolution analysis of tooth enamel reveals strong intra-tooth variability in many cases, suggesting seasonal-scale dietary shifts. This pattern is quite unlike that seen in any great apes, even ‘savannah’ chimpanzees. The overall proportions of C₄ input persisted for well over a million years, even while environments shifted from relatively closed (ca 3 Ma) to open conditions after ca 1.8 Ma. Data from East Africa suggest a more extreme scenario, where results for Paranthropus boisei indicate a diet dominated (approx. 80%) by C₄ plants, in spite of indications from their powerful ‘nutcracker’ morphology for diets of hard objects. We argue that such evidence for engagement with C₄ food resources may mark a fundamental transition in the evolution of hominin lineages, and that the pattern had antecedents prior to the emergence of Australopithecus africanus. Since new isotopic evidence from Aramis suggests that it was not present in Ardipithecus ramidus at 4.4 Ma, we suggest that the origins lie in the period between 3 and 4 Myr ago.     

Differences in feeding behaviour among Chironomus species revealed by measurements of sulphur stable isotopes and cadmium in larvae

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Microstructural defects and enamel hypoplasia in teeth of Toxodon Owen, 1837 from the Pleistocene of Southern Brazil

Enamel hypoplasia is characterized by reduction in the enamel thickness, resulting from a disruption of ameloblast activity due to systemic physiological stress. The euhypsodont teeth of Toxodon, a notoungulate from the Pleistocene of South America, often exhibit signs of enamel hypoplasia, in the form of continuous grooves or a series of pits where the enamel is thinner than in normal areas. These defects alternate with areas of normal enamel, and sometimes more than one form of enamel hypoplasia is present on the same tooth. This study analysed teeth of Toxodon from the Pleistocene Touro Passo Formation and the coastal plain of State of Rio Grande do Sul, Southern Brazil. Six types of enamel hypoplasia were observed. Upper teeth present mainly superficial grooves on the buccal surface, and the defects are less severe than those observed in the lower teeth. In the lower incisors, deep grooves with mesiodistal rows of pits were observed, showing clearly cyclical changes, which to a lesser degree, exist in all teeth. These changes are likely related to the continuous growth of euhypsodont teeth. Seven specimens were analysed under scanning electron microscopy and optical microscopy, which showed the occurrence of microstructural changes associated with the macroscopic enamel defects. Enamel underlying in the vicinity of hypoplastic defects was aprismatic and associated with prominent pathologic striae. These pathological findings might indicate that toxodonts were exposed to some stressing conditions or that their teeth were more easily abraded due to a change in diet items, related to shifting climatic conditions.     

Reconstructing infant weaning histories at Roman period Kellis, Egypt using stable isotope analysis of dentition

Studies of infant feeding and weaning patterns in past populations that rely on a cross-sectional approach must make the assumption that no infant mortality bias exists. Previous investigations of infant weaning patterns at the Dakhleh Oasis, Egypt, relied on cross-sectional isotope data. In this study, we re-examine this weaning pattern, using a simulated longitudinal approach, which does not require any assumptions regarding potential infant mortality biases. This involves examining the dental isotopic signatures of individuals who survived the weaning process. Stable isotope signatures from juveniles and adults (102 individuals, 297 teeth) were examined to reconstruct the weaning history of those that survived the weaning process. Both deciduous and permanent teeth were sampled. Homogenized enamel and dentin samples were isolated from each tooth and analyzed for delta(13)C(ap) and delta(18)O(ap) from the enamel and delta(15)N(coll) and delta(13)C(coll) from dentin collagen. We investigate differences between in utero versus postbirth, preweaning versus postweaning, and juvenile versus adult stable isotope values as reflected in the dentition. A random permutation procedure was used to test for statistically significant differences in stable isotope values between tooth types. Statistically significant differences were observed in all stable isotopes between permanent and deciduous teeth, and between early and later forming permanent teeth in delta(13)C(ap) and delta(15)N(coll) isotopes. These results indicate dietary change between in utero and postbirth, and changes occurring during the weaning period. These results provide a more comprehensive picture of infant weaning practices at Kellis and provide further support that complete weaning occurred by 3 years of age.     

Brief communication: diachronic investigation of linear enamel hypoplasia in prehistoric skeletal samples from Trentino,Italy

Linear enamel hypoplasia was scored on Neolithic, Copper Age, and Early Bronze Age samples from the Trentino region, Italy, in order to compare the extent of growth disruption in different biocultural subsistence systems (foragers with little agriculture, to agriculturists and agropastoralists). The Early Bronze Age sample shows a higher frequency of enamel defects and an earlier chronological onset than the early Neolithic sample. The higher frequency of defects in the Bronze Age sample could be linked to less diversified nutrition and, because of increased sedentism, a higher risk of disease.     

Using stable isotopes to trace long-distance dispersal in birds and other taxa

The study of long‐distance dispersal (LDD) in animals may be advanced by recent applications of stable isotope analyses designed to track migratory organisms and to link populations throughout their annual cycle. This approach depends on there being enough isotopic difference in tissues among potential source populations such that individuals can be unequivocally assigned to their source. The isotopic mapping of such populations will be feasible only for species occurring in relatively few disjunct populations. However, the identification of isotopic outliers within known populations will be an extremely useful first step in the forensic application of stable isotopes to identify dispersal in general, and LDD in particular. The use of deuterium isotope analysis (δD) of tissues that can be assigned to its source (e.g. feather moult or its hair growth location) has provided a recent breakthrough in our ability to associate individuals with geographical origins at continental scales. The combination of this stable isotope with others and the ultimate combination of a variety of techniques, including the measurement of trace elements and molecular genetics markers, will undoubtedly improve resolution. The isotopic cataloguing of known history (i.e. philopatric) individuals within populations will be an important step in applying isotope techniques to evaluating LDD in any species. For aquatic insects, the isotopic marking of large numbers of individuals is possible through isotopic enrichment of local food webs using labelled compounds.     

Characterization of riparian species and stream detritus using multiple stable isotopes

Multiple stable isotopes were used to determine the effectiveness of distinguishing among several dominant riparian species and aquatic macrophytes both spatially (three sites) and temporally (three seasons) along an 8-km reach of a blackwater stream. The differences in isotopic composition were used to assess contributions of various organic matter sources to the detrital pool of the stream. Samples of riparian and aquatic macrophyte vegetation and detritus were collected at three times to represent early leaf-out (April), mid-summer (August), and just prior to abscission (October). Each sample was analyzed for stable isotopes of carbon ¹³C, nitrogen ¹⁵N, and sulfur ³⁴S Within a site and sampling date, ¹³C-values were significantly different among certain riparian species and detritus samples. Species differences persisted between seasons. ³⁴S values were the most variable of the three elements examined although they remained fairly constant through time within each species and site. The results suggest that temporal changes in isotopic compositions of riparian species and aquatic macrophytes are site-specific. Discriminant analysis dissimilarity plots (based on all three isotopes) demonstrated that the contribution of species to the detrital pool depended on the site and season. At the upper site, detritus was isotopically most similar to Quercus laurifolia and Sparganium americanum in April, and the aquatic macrophytes (S. americanum and Potamogeton spp.) in August and October. At the middle site, detritus was most similar to Carpinus caroliniana and Q. nigra in April but no single source was similar to detritus in August or October. At the lower site, detritus was most similar to Taxodium distichum for all three seasons.     

Sulphur stable isotopes can distinguish trophic dependence on sediments and plankton in boreal lakes

1. Stable isotopes of carbon are useful for differentiating between freshwater food chains based on planktonic algae or benthic algae, but are reported to be of limited use for identifying food chains based on sedimentary detritus. Because data from marine systems suggest that stable isotopes of sulphur (δ³⁴S values) have potential in this regard, we tested their utility in freshwater lakes.
2. We found that sulphate in the water column of four boreal lakes was enriched in ³⁴S compared to the sulphur in bulk sediments from these lakes. Furthermore, within a given lake, insects known to feed on sediment (directly or via predation) had δ³⁴S values similar to those of sediment, whereas planktonic and benthic invertebrates known to feed on suspended particles had δ³⁴S values similar to those of sulphate in the water column.
3. Using the stable S isotope values of invertebrates that obtain their S from either the sediment or the water column as end members in a two-source mixing model, we show that two fish species obtain their food from both planktonic and sedimentary sources. Furthermore, model results suggest that, as expected, the more benthic-feeding fish species obtains more of its S from the sediment compartment than does the species that feeds in the water-column.
4. Our results suggest that measurements of stable sulphur isotopes provide a means of distinguishing between members of food chains that are based in the water column from those based on sedimentary detritus. As such, they would be a useful complement to stable C isotopes that are used to distinguish between food chains based on planktonic or benthic algae.     

Patterns of enamel hypoplasia in two medieval populations from Nubia's Batn el Hajar

Analysis of enamel hypoplasia frequencies for two medieval populations representing the earliest and latest Christian periods of ancient Nubia reveals important diachronic shifts in childhood stress. The mean frequency for hypoplastic bands among the early Christians is 4.2, while the late Christian sample has a mean frequency of 3.7. In addition, the earlier Christians show a prolongation of hypoplastic occurrences through childhood corresponding to a prolonged period of intensified childhood mortality. The modal time interval between hypoplastic occurrences is also shorter for the early Christian children. A comparison of hypoplasia frequencies by sex also reveals a pattern of considerable interest. Females show both lower frequencies of hypoplasias as well as a delay in onset. The diachronic differences are consistent with other indications from paleopathology and paleodemography that childhood stress decreased in later Christian times. The sex differences suggest that during the infancy and early childhood females were more resilient than their male counterparts.     

The use of stable isotopes in the study of human pathophysiology

The growing prevalence of metabolic diseases including fatty liver disease and Type 2 diabetes has increased the emphasis on understanding metabolism at the mechanistic level and how it is perturbed in disease. Metabolomics is a continually expanding field that seeks to measure metabolites in biological systems during a physiological stimulus or a genetic alteration. Typically, metabolomics studies provide total pool sizes of metabolites rather than dynamic flux measurements. More recently there has been a resurgence in approaches that use stable isotopes (e.g. ²H and ¹³C) for the unambiguous tracking of individual atoms through compartmentalised metabolic networks in humans to determine underlying mechanisms. This is known as metabolic flux analysis and enables the capture of a dynamic picture of the metabolome and its interactions with the genome and proteome. In this review, we describe current approaches using stable isotope labelling in the field of metabolomics and provide examples of studies that led to an improved understanding of glucose, fatty acid and amino acid metabolism in humans, particularly in relation to metabolic disease. Examples include the use of stable isotopes of glucose to study tumour bioenergetics as well as brain metabolism during traumatic brain injury. Lipid tracers have also been used to measure non-esterified fatty acid production whilst amino acid tracers have been used to study the rate of protein digestion on whole body postprandial protein metabolism. In addition, we illustrate the use of stable isotopes for measuring flux in human physiology by providing examples of breath tests to measure insulin resistance and gastric emptying rates.     

Passage kinetics of concentrates in dairy cows measured with carbon stable isotopes

Fractional passage rates form a fundamental element within modern feed evaluation systems for ruminants, but knowledge on feed-specific fractional passage is largely lacking. Commonly applied tracer techniques based on externally applied markers, such as chromium-mordanted neutral detergent fibre (Cr-NDF), have been criticised for behaving differently to feed particles. This study describes the use of the carbon stable isotope ratio (¹³C : ¹²C) as an internal digesta marker to quantify the fractional passage rate of concentrates through the digestive tract of dairy cows. In a crossover study, five dairy cows were fed low (24.6%) and high (52.6%) levels of concentrates (dry matter (DM) basis) and received a pulse-dosed Cr-NDF and ¹³C isotopes. The latter was administered orally by exchanging part of the dietary concentrates of low ¹³C natural abundance with a pulse dose of maize bran-based concentrates of high ¹³C natural abundance. Fractional passage rates from the rumen (K₁) and from the large intestine (K₂) were determined from faecal marker concentrations of Cr-NDF and of ¹³C in the DM (¹³C-DM), NDF (¹³C-NDF) and neutral detergent soluble (¹³C-NDS). No differences in K₁ estimates were found for the two concentrate levels fed but significant differences between markers (P<0.001) were observed. Faecal Cr-NDF excretions gave lower K₁ estimates (0.037–0.039/h) than ¹³C-DM (0.054–0.056/h) and ¹³C-NDF (0.061–0.063/h). The ¹³C-NDS was calculated by the difference of ¹³C in the DM and NDF, and K₁ values (0.039–0.043/h) were comparable to Cr-NDF. Total mean retention time was considerably higher for Cr-NDF (40.9–42.0 h) as compared to ¹³C-DM and ¹³C-NDF (32.0–33.5 h; P<0.001). The accuracy of the curve fits for Cr-NDF and ¹³C-DM and ¹³C-NDF was overall good (mean prediction error of 9.9–13.9%). Fractional passage rate of Cr-NDF was comparable to studies where this marker was assumed to represent the fractional passage of roughages. However, K₁ estimates based on the ¹³C : ¹²C ratio varied considerably from studies based on external markers. Our results suggest that the use of ¹³C isotopes as digesta passage markers can provide feed component-specific K₁ estimates for concentrates and provides new insight into passage kinetics of NDF from technologically treated compound feed.     

Corticosterone and stable isotopes in feathers predict egg size in Atlantic Puffins Fratercula arctica

Examining factors that operate outside the breeding season may provide new insights into life‐history traits such as egg size, in which individual variation has not been fully explained. We measured corticosterone (CORT) levels and δ¹⁵N values (trophic level) in feathers grown several months before egg‐laying to test the prediction that a female's physiological state and feeding behaviour prior to the breeding season can influence egg mass in Atlantic Puffins Fratercula arctica. As predicted, egg mass increased with both CORT and δ¹⁵N values in feathers, suggesting that the ability of female Puffins to meet the nutritional costs of egg production is related to CORT promoting increased foraging effort during moult and to consumption of a higher trophic‐level diet.     

Influence of individual predisposition, maternal experience and lactation environment on the responses of pigs to weaning at two different ages

This study assessed the effect of predisposition to perform harmful social behaviour, maternal rearing environment, and lactation environment on the responses of pigs to weaning at 3 or 5 weeks of age. Predisposed and non-predisposed gilts were selected as dams for this study at 7 weeks of age. Selection was based on behaviour in a “tail chew” test and performance of harmful social behaviour towards penmates. The gilts were mated at puberty with boars of a similar predisposition, and farrowed at approximately 44 weeks of age. Half of the gilts of each predisposition were reared from the time of selection until farrowing in barren environments, and half in enriched environments. During lactation, gilts and litters were either housed in a similar environment to that which gilts had experienced during rearing, or in a different environment (i.e. in terms of being barren or enriched). Litters from each treatment group were weaned at either 3 weeks of age (early weaning), or 5 weeks of age. After weaning, piglets were regrouped and housed in slatted pens without access to substrates. Non-predisposition to perform harmful social behaviour was associated with reduced growth during the post-weaning period (P < 0.01), and increased belly nosing behaviour in response to early weaning (P < 0.05). These effects were not mitigated by maternal experience or lactation environment factors, and it is concluded that this type of selection may not be commercially viable. Rearing dams in barren rather than enriched environments led to reduced welfare in offspring. This was reflected in increased adrenocortical reactivity during the lactation period (P < 0.01), and increased belly nosing behaviour in response to early weaning (P < 0.05). The effect of barren maternal rearing environments on belly nosing behaviour by offspring was eliminated when pigs were housed in enriched lactation environments (P < 0.01). Enrichment during the lactation period also led to improved growth rates in the post-weaning period (P < 0.01). It is suggested that this effect was due to an enhanced ability to cope with the weaning process. Overall, the results show that both genetic and early environmental factors are important determinants of the responses of pigs to weaning. Adverse effects of barren maternal rearing environments may be overcome by housing pigs in enriched lactation environments.     

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 men's blood compared to women's 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.     

Cryptic niche differentiation in West African savannah termites as indicated by stable isotopes

1. Termites consume a wide range of plant material at different stages of decomposition and, accordingly, have been classified into ‘feeding groups’. African savannah ecosystems harbour many termite species, yet most belong to Termitidae. Especially striking is the co‐occurrence of up to eight Macrotermitinae and five Trinervitermes species, each having apparently identical trophic niches, i.e. fungus‐growers and grass‐feeders, respectively. 2. This study examined stable isotope signatures of ¹⁵N and ¹³C of West African savannah termites to test the validity of existing feeding group concepts and whether there is fine‐scaled niche differentiation of species within feeding groups. Despite a phylogenetic signal that species from the same subfamily and congenerics have correlated isotope signatures, evidence of niche differentiation was found. 3. Interestingly, species with similar δ¹⁵N values generally differed in δ¹³C values, and vice versa. The dominant mound‐building fungus‐grower Macrotermes bellicosus had the lowest δ¹⁵N values among all fungus‐growers, indicating that it occupies a different trophic niche. 4. This fine‐scaled differentiation along the trophic niche axis can help to explain the coexistence of so many apparently identical termite species.     

Trophic level delineation and resource partitioning in a South African afromontane forest bird community using carbon and nitrogen stable isotopes

Southern African forests are naturally fragmented yet hold a disproportionately high number of bird species. Carbon and nitrogen stable isotopes were measured in feathers from birds captured at Woodbush (n = 27 species), a large afromontane forest in the eastern escarpment of Limpopo province, South Africa. The δ¹³C signatures of a range of forest plants were measured to categorise the food base. Most plants sampled, including two of five grass species, had δ¹³C signatures typical of a C₃ photosynthetic pathway (?29.5 ± 1.9‰). Three grass species had a C₄ signature (?12.0 ± 0.6‰). Most bird species had δ¹³C values representing a predominantly C₃‐based diet (?24.8‰ to ?20.7‰). δ¹⁵N values were as expected, with higher levels of enrichment associated with a greater proportion of dietary animal matter. The cohesive isotopic niche defining most species (n = 22), where the ranges for δ¹³C and δ¹⁵N were 2.4‰ and 3.4‰, respectively, highlight the difficulties in understanding diets of birds in a predominantly C₃‐based ecosystem using carbon and nitrogen stable isotopes. However, variation in isotopic values between and within species provides insight into possible niche width and the use of resources by different birds within a forest environment.