Stable isotope relationships between apatite phosphate ( δ 18 O), structural carbonate ( δ 18 O, δ 13 C), and collagen ( δ 2 H, δ 13 C, δ 15 N, δ 34 S) in modern human dentine

Rationale: The use of multi-isotopic analysis ( δ 2 H, δ 13 C, δ 15 N, δ 18 O, and δ 34 S values) of modern human body tissues for provenancing of unknown individuals in forensics is increasing. Tooth dentine develops during childhood and adolescence, therefore providing geographical information from that period of life. Tooth apatite δ 18 O values are commonly used for the reconstruction of drinking water values, and H – C – N – S isotope ratios in collagen supply additional information about the composition of diet. We tested if dentine collagen δ 2 H values provide similar information to apatite δ 18 O values with a proof-of-concept study. Methods: Tooth samples were taken from modern-day individuals born in different regions of the world. Apatite and collagen were prepared from dentine. Stable isotope analyses were performed on apatite phosphate oxygen ( δ 18 O phos ); oxygen and carbon of the structural carbonate ( δ 18 O carb , δ 13 C carb ); and hydrogen, carbon, nitrogen, and sulfur of the collagen ( δ 2 H coll, δ 13 C coll , δ 15 N, δ 34 S). Results: δ 18 O phos , δ 18 O carb , and δ 2 H coll values are highly correlated in modern human dentine. There are significant relationships of δ 18 O values in the apatite fraction and δ 2 H values in the collagen fraction with local δ 18 O and δ 2 H precipitation values, respectively. Pearson correlation coefficients indicate no direct relationship between δ 15 N values and the isotope ratios of any other element. Weak relationships exist between collagen δ 34 S values and δ 18 O carb or δ 18 O phos values. Conclusions: The highly significant correlation of δ 18 O phos , δ 18 O carb , and δ 2 H coll values in the modern human dentine implies that measurement of δ 2 H values in collagen or δ 18 O values in bioapatite will provide reliable information about the climate at the person's whereabouts. other element. Weak relationships exist between collagen δ 34 S values and δ 18 O carb or δ 18 O phos values.

tissues are linked to the composition of an individual's diet and the origin of food and drink. In general, δ 13 C values show the content of C4 or C3 plants or marine versus terrestrial sources in diet, δ 15 N values show the amount of animal protein (meat, dairy products, fish), and δ 34 S values hints at any marine influences (sea products, sea-spray effect, sediments of marine origin). δ 2 H and δ 18 O values predominantly contain information about the climatic conditions at the whereabouts of individuals, as the isotopic composition of regional meteoric water is strongly linked to annual air temperature. 5 Region-specific stable isotope ratios of the elements from food and drink are integrated into body tissues of living organisms.
Stable isotope data of human teeth may provide information as to where a person spent his or her childhood and adolescence. In view of the molecular level of dentine collagen, its amino acid composition is almost similar to that of bone collagen. 6 Calculated correction factors enable a direct comparison of the stable isotope ratios of hydrogen, carbon, nitrogen and sulfur of bone collagen with those of reference hair samples collected worldwide, allowing the prediction of the geographical origin of unidentified individuals. 3 For the prediction of a person's residence during childhood and youth, the H-C-N-S stable isotope ratios of tooth collagen might be applicable in the same way.
Tooth collagen is primarily abundant in the organic fraction of dentine. Due to its bone-like structure, dentine contains 70% calcium hydroxyl apatite, 20% organic material (containing 90% collagen), and 10% water. 7 In contrast, tooth enamel contains up to 95% calcium hydroxyl apatite and has a maximum collagen content of 0.4%. 8 The final structure of primary dentine and enamel develops during ontogenesis, and neither of them is subjected to any marked conversion during a person's lifetime. 9 In addition to the stable isotopes in the organic fraction of the tooth, the 18  values in body water, and more than 50% of the oxygen in apatite is derived from water in food and drink. 10 Hydrogen and oxygen isotope ratios in body tissues are linked to the δ 18 O or δ 2 H values of body water, which mainly stem from the consumed drinks and liquid components of food. The main water source for modern humans is tap water, and the δ 18 O or δ 2 H values of local tap water are connected to the local precipitation values and the climate. 11 The relationship of δ 18 O values between apatite in enamel and drinking or tap water for humans is well established, [12][13][14] and for the δ 18 O carb and δ 18 O phos values in enamel of archaeological individuals, a strong correlation has been documented. 15 Oxygen isotope analyses on apatite from bones and teeth have been primarily used in archaeology for the reconstruction of geographical provenance and migration behaviors of individuals.
Studies on δ 18 O values in tooth apatite of contemporary humans confirm their relationship to the oxygen isotope ratios of drinking water, tap water, or precipitation. [12][13][14][16][17][18][19][20] In addition, the δ 2 H and δ 18 O values of modern human hair and nails are connected to precipitation. 3,[21][22][23][24] In human hair, 27% of the oxygen atoms and 36% of the hydrogen atoms are derived from drinking water. 25 As previously shown by the relationship between keratin δ 2 H and bone collagen δ 2 H values, 3 the same may be supposed for dentine collagen δ 2 H values. Therefore, dentine collagen δ 2 H values should be related to precipitation or drinking water δ 2 H values and consequently may serve as a proxy for the climatic conditions in which an individual grew up.
There are several studies dealing with the relationships between the δ 18 O phos , δ 18 O carb , and δ 2 H values in mammal teeth and bone samples [26][27][28] and archaeological human enamel samples, 15,29 but human dentine samples have been rarely investigated. There is one study about carbon and oxygen isotope spacing between tooth collagen and hydroxyapatite in human archaeological remains. 29  in human bone or enamel, we expect that the δ 18 O values in the dentine will be associated with the isotope ratios of the person's drinking water or the local precipitation. Our main concern was to examine whether the dentine collagen δ 2 H values can also be used as a climate proxy for the isotopic composition of local water at the individuals' whereabouts.
Furthermore, we will test if there are any direct relationships between the δ 13 C carb , δ 13 C coll , δ 15 N coll , and δ 34 S coll values in modern human dentine. As the teeth were extracted within a few days after the death of the persons, we can assume that diagenetic changes of dentine by environmental factors such as soil pH, soil hydrology, ambient temperature, or microbial degradation had not occurred. 31

| Materials
With permission of the ethic commission at the LMU Munich, 29 permanent teeth were collected anonymously from corpses for whom autopsies were ordered by prosecution authorities. After cleaning and removing adhesions mechanically, each tooth was divided in two parts, the crown and the root section, by a small saw. The whole root section, which is mainly composed of dentine, was ground by a ball mill, and tweezers removed the dental nerve.

| Preparation of collagen
Collagen was extracted from dentine following previous reports. 32,33 About 400 mg of the defatted dentine material was demineralized with 1M hydrochloric acid for 40 min at room temperature. After centrifugation and neutralization with demineralized water, the residue was gelatinized with 1mM hydrochloric acid (pH 3) for 16-18 hours at 90 C. The gelatin solution was pressure-filtered through a glass-microfiber disc and a membrane filter (cellulose nitrate, 5 μm). Afterward, the filtered solution was frozen and freeze-dried. The dry collagen was used for stable isotope measurements of hydrogen, carbon, nitrogen, and sulfur, with an isotope ratio-mass spectrometer.

| Bulk isotope analysis of collagen
Collagen δ 2 H values were measured using the comparative equilibration method. 34 Collagen samples and laboratory reference materials were stored under identical conditions for at least 3 days before analysis to enable hydrogen exchange with hydrogen from ambient air moisture. After equilibration, 150 μg of the samples, international and in-house casein reference materials, 35   with an Isoprime VisIon mass spectrometer. For H isotope analysis, a Thermo high temperature conversion unit connected with a Thermo XP plus isotope ratio mass spectrometer (Thermo Fisher, Bremen, Germany) was used. The high temperature conversion unit was equipped with a helium-flushed autosampler, the reaction column was filled with glassy carbon chips, and the temperature of the furnace was set to 1450 C. The reaction gases were passed through an absorption tube containing sodium hydroxide on support and another one containing phosphorus pentoxide. All δ values were indicated in per mil (‰) relative to international reference standards (δ 2 H VSMOW, δ 13 C VPDB , δ 15 N AIR , and δ 34 S VCDT ) as follows: δ = [(R sample /R standard ) − 1], where R is the 2 H/ 1 H, 13 C/ 12 C, 15

| Apatite preparation
To remove organic compounds, 5 mL 4% NaOCl solution was added to 100 mg pulverized dentine and thoroughly mixed. After 2 days, the NaOCl solution was replaced. On the third day, the supernatant was removed, and the pellet was rinsed with distilled water several times. The pellet was stirred with 5 mL 1M calcium acetate-acetic acid buffer overnight. After several rinses with distilled water, the apatite was dried at 40 C.

| δ 18 O and δ 13 C analyses on structural carbonate of dentine
For carbonate isotope analyses 2.0 ± 0.1 mg of the dry dental apatite (section 2.4) was weighed into borosilicate glass autosampler vials. Then, these vials were closed with a septum cap and flushed with helium for 700 seconds. CO 2 gas was produced by adding 7-9 droplets of 103% H 3 PO 4 using a syringe. The samples were equilibrated for at least 3 hours at a constant temperature (70 C) in a GasBench II on-line gas preparation and introduction system for isotope ratio mass spectrometry (Thermo Fisher). The equilibrated CO 2 was measured using a Delta V Advantage isotope ratio mass spectrometer (Thermo Fisher). The

| Dentine phosphate preparation
For the precipitation of dental phosphate as silver phosphate, the method of Joachimski et al 48

| δ 18 O analysis in dentine phosphate
A quantity of 0.92-0.94 mg of dry silver phosphate was weighed into silver capsules (4 × 6 mm) in triplicate. Prior to analysis, the samples were stored in a vacuum-drying cabinet at 60 C for 48 hours to remove water vapor. After drying, the samples were immediately  (Table 1) is from the third to the 16th year. 4 We are operating on the assumption that for each individual the residential region during the period of tooth growth is the same as the place of their birth.  Table 2).
The relationships between apatite and collagen isotope ratios were investigated using linear regression. Table 3 Table 2 show that the mean Δ carb-phos spacing is 6.6 ± 0.7‰ for pretreated and 7.2 ± 0.7‰ for untreated samples (Table 2). With respect to different climatic settings in the regions of origin, Δ carb-phos in the modern dentine samples remains nearly constant. Figure 2 shows the correlation between the δ 18 O phos or δ 18 O carb pretreated value and the δ 2 H value of each dentine sample.
The dentine collagen δ 13 C values range from −20.7‰ to −15.4‰. The δ 13 C coll and δ 13 C carb values are highly correlated ( The Δ carb-phos offsets in the modern dentine of 6.6 ± 0.7‰ or 7.2 ± 0.7‰ are much lower than the usually observed differences of  Nevertheless, because the structure of dentine is more similar to that of bone than to enamel, we also expect lower δ 18 O carb values in dentine than in enamel. Thus, this may also explain the low Δ carb-phos offsets in the modern dentine samples.

| Relationships between apatite δ 18 O, collagen δ 2 H, and local δ 18 O/δ 2 H precipitation values
One of the main concerns of our study was to examine the relationship between apatite δ 18  Particularly with regard to modern dentine, it should be proved in further studies whether it is in fact necessary to remove organic content prior to apatite carbonate or phosphate stable isotope analyses.

| Relationship among isotope ratios of nitrogen and sulfur in collagen and other elements
There was no relationship between the δ 15 N coll and δ 2 H coll values for the modern dentine samples (  65 We reasonably assume that in modern human samples, a trophic effect based on the δ 15   Our results indicate weakly correlated dentine δ 34 S coll and δ 18 O carb values. A similar pattern was recognized in mammal bones from coastal regions, where the relationship was explained as being the result of the sea spray effect. 80 However, the very weak correlation between isotope ratios from two different precursor pools in two different matrices, one organic and one inorganic, should be treated with extreme caution until such time that more data become available. value in the dentine can be used as a proxy for the climatic condition at the residence of an individual during childhood and adolescence.

| CONCLUSIONS
With regard to this issue, from the C-N-S-H stable isotope ratios in dentine collagen, sufficient information about the isotopic composition of the food and of the drinking water can be obtained.