Characterization of Beef Coming from Different European Countries through Stable Isotope (H, C, N, and S) Ratio Analysis.

The need to guarantee the geographical origin of food samples has become imperative in recent years due to the increasing amount of food fraud. Stable isotope ratio analysis permits the characterization and origin control of foodstuffs, thanks to its capability to discriminate between products having different geographical origins and derived from different production systems. The Framework 6 EU-project "TRACE" generated hydrogen (2H/1H), carbon (13C/12C), nitrogen (15N/14N), and sulphur (34S/32S) isotope ratio data from 227 authentic beef samples. These samples were collected from a total of 13 sites in eight countries. The stable isotope analysis was completed by combining IRMS with a thermal conversion elemental analyzer (TC/EA) for the analysis of δ(2H) and an elemental analyzer (EA) for the determination of δ(13C), δ(15N), and δ(34S). The results show the potential of this technique to detect clustering of samples due to specific environmental conditions in the areas where the beef cattle were reared. Stable isotope measurements highlighted statistical differences between coastal and inland regions, production sites at different latitudes, regions with different geology, and different farming systems related to the diet the animals were consuming (primarily C3- or C4-based or a mixed one).

Identification of a female murder victim found in Burgenland, Austria in 1993.

In 1993, the skeletal remains of a female corpse were found in Burgenland, Austria. Initial identification of the approximately 25-35-year-old female appeared impossible, but the case was reopened 23 years later. By applying biogeochemical isotope methods to her body tissues, the geographical origin of the unknown corpse could be predicted. The results of the C, N, S, H, Sr, and Pb isotope analyses suggested that the female did not originate from Europe and most likely spent her youth in the northern Caribbean. Using these findings, the police were able to identify the woman within 2 weeks. The female came from the Dominican Republic and resided in Austria for only a short period before she was murdered. This case shows that isotope biogeochemistry investigations can provide the police with crucial information that enables unknown persons to be identified.KeypointsC-N-S-H and Sr-Pb isotope analyses were applied to human remains associated with a cold case.It was possible to determine the region of origin of the unknown deceased individual as the northern Caribbean.After 23 years, the murder victim was successfully identified.

Stable isotope relationships between apatite phosphate (δ18 O), structural carbonate (δ18 O, δ13 C), and collagen (δ2 H, δ13 C, δ15 N, δ34 S) in modern human dentine.

RATIONALE: The use of multi-isotopic analysis (δ2 H, δ13 C, δ15 N, δ18 O, and δ34 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 δ18 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 δ2 H values provide similar information to apatite δ18 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 (δ18 Ophos ); oxygen and carbon of the structural carbonate (δ18 Ocarb , δ13 Ccarb ); and hydrogen, carbon, nitrogen, and sulfur of the collagen (δ2 Hcoll, δ13 Ccoll , δ15 N, δ34 S). RESULTS: δ18 Ophos , δ18 Ocarb , and δ2 Hcoll values are highly correlated in modern human dentine. There are significant relationships of δ18 O values in the apatite fraction and δ2 H values in the collagen fraction with local δ18 O and δ2 H precipitation values, respectively. Pearson correlation coefficients indicate no direct relationship between δ15 N values and the isotope ratios of any other element. Weak relationships exist between collagen δ34 S values and δ18 Ocarb or δ18 Ophos values. CONCLUSIONS: The highly significant correlation of δ18 Ophos , δ18 Ocarb , and δ2 Hcoll values in the modern human dentine implies that measurement of δ2 H values in collagen or δ18 O values in bioapatite will provide reliable information about the climate at the person's whereabouts.

Revealing details of stays abroad by sequential stable isotope analyses along human hair strands.

Multi-element stable isotope analyses of δ13C, δ15N, δ34S and δ2H values were performed along scalp hair strands to detect isotopic changes resulting from different stays abroad. One hair strand with a hair length of more than 50 cm originated from a German woman, who frequently made long-distance travels of 1 to 4 weeks. The second hair strand with a length of 15 cm was taken from a Japanese woman who went to Germany for a period of some months. Stable isotopic influences due to the stays abroad were clearly reflected in the 5-mm segments along the proximal part of the hair strand; whereas in the more distal parts, the isotopic influences were blurred. This can be regarded as the result of the highly variable intra-individual hair growth rate of single hairs of at least ± 30% compared to the mean growth rate. Consequently, the initial isotope signal obtained by short stays abroad became rapidly attenuated in the more distal parts of the hair strand. Furthermore, decreasing sulphur content associated with higher sulphur isotope values was observed with increasing hair length. The isotope shifts along the scalp hair strand, provoked by dietary changes at new locations, appeared at such points of hair length, which correspond well with the maximum growth rate of single hairs. Consequently, the exact date for any changes coming along with isotopic shifts may be calculated by best approach considering a hair growth value of 1.4 cm per 30 days, instead of the commonly used mean monthly hair growth rate of 1.1 cm. This may be important in forensics, if detailed information about a person's recent lifetime should be figured out by segmental scalp hair analyses.

PET imaging of chemokine receptor CXCR4 in patients with primary and recurrent breast carcinoma.

BACKGROUND: CXCR4 is a chemokine receptor frequently overexpressed in invasive breast cancer that has been shown to play a major role in signaling pathways involved in metastasis. The aim of this retrospective analysis was to assess the diagnostic performance of CXCR4-directed PET imaging in patients with breast cancer using the recently introduced CXCR4-targeted PET probe 68Ga-Pentixafor. RESULTS: Thirteen patients with first diagnosis of breast cancer, four patients with recurrent disease after primary breast cancer, and one patient with axillary lymph node metastasis of unknown primary underwent CXCR4-targeted PET imaging using 68Ga-Pentixafor. Maximum standardized uptake values (SUVmax) and tumor-to-background (T/B) ratios of tumor lesions were measured and compared with pathological prognostic factors and molecular subtypes. 18F-FDG PET/CT images were available in 8/18 cases and were compared semi-quantitatively. Comparison with CXCR4 expression determined by immunohistochemistry was performed in 7/18 patients. Nine of 13 primary breast cancers were visually detectable on 68Ga-Pentixafor PET images (mean SUVmax of 3.0). The visually undetectable lesions included both cases of invasive lobular carcinoma (ILC) and two cases of invasive carcinoma of no special type (NST) without any hormone receptor and HER2 expression (triple negative). Metastases of recurrent breast cancer and unknown primary cancer were visually detectable in all five cases, exhibiting a mean SUVmax of 3.5. 18F-FDG PET demonstrated higher SUVmax in all patients compared to 68Ga-Pentixafor PET. A correlation between SUVmax obtained from 68Ga-Pentixafor PET and prognostic factors including estrogen receptor (ER), progesterone receptor (PR), human epidermal growth factor receptor 2 (HER2) status, proliferation index, tumor grade, or molecular subtypes was not observed. CONCLUSIONS: CXCR4-directed PET imaging in patients with primary and recurrent breast cancer is feasible; however, tumor detectability is significantly lower compared to 18F-FDG PET. Moreover, we did not find any correlation between aforementioned prognostic factors of breast cancer and CXCR4-targeted tracer accumulation. Based on these results in a small patient cohort, CXCR4-targeted PET imaging does not seem to be suitable as a general diagnostic tool for imaging of breast cancer. Future CXCR4 imaging studies should investigate whether this modality might be useful in more specific applications, e.g., in therapeutic approaches especially under the view of current developments in targeted immune cell and immune checkpoint inhibitory therapy.

Global spatial distributions of nitrogen and carbon stable isotope ratios of modern human hair.

RATIONALE: Natural stable carbon (δ(13)C) and nitrogen isotope ratios (δ(15)N) of humans are related to individual dietary habits and environmental and physiological factors. In forensic science the stable isotope ratios of human remains such as hair and nail are used for geographical allocation. Thus, knowledge of the global spatial distribution of human δ(13)C and δ(15)N values is an essential component in the interpretation of stable isotope analytical results. METHODS: No substantial global datasets of human stable isotope ratios are currently available, although the amount of available (published) data has increased within recent years. We have herein summarised the published data on human global δ(13)C andδ(15)N values (around 3600 samples) and added experimental values of more than 400 additional worldwide human hair and nail samples. In order to summarise isotope ratios for hair and nail samples correction factors were determined. RESULTS: The current available dataset of human stable isotope ratios is biased towards Europe and North America with only limited data for countries in Africa, Central and South America and Southeast Asia. The global spatial distribution of carbon isotopes is related to latitude and supports the fact that human δ(13)C values are dominated by the amount of C4 plants in the diet, either due to direct ingestion as plant food, or by its use as animal feed. In contrast, the global spatial distribution of human δ(15)N values is apparently not exclusively related to the amount of fish or meat ingested, but also to environmental factors that influence agricultural production. CONCLUSIONS: There are still a large proportion of countries, especially in Africa, where there are no available data for human carbon and nitrogen isotope ratios. Although the interpretation of modern human carbon isotope ratios at the global scale is quite possible, and correlates with the latitude, the potential influences of extrinsic and/or intrinsic factors on human nitrogen isotope ratios have to be taken into consideration.

Multi element (C, H, O) stable isotope analysis for the authentication of balsamic vinegars.

Balsamic vinegars are important and well-acknowledged products and have become more important all over Europe during the recent past. For their analytical control, stable isotope methods play an important role to check the authenticity of the raw materials applied. For vinegar, but not yet for balsamic vinegar analysis, stable isotope methods using hydrogen, carbon, and oxygen isotope analyses by isotope ratio mass spectrometry and (2)H-NMR have already been introduced as official methods. Nevertheless, the official procedure can be also applied for balsamic vinegars. The evaluation of the stable isotope parameters obtained for the ingredients of balsamic vinegars requires a knowledge and understanding of the natural and industrial processes on which the production of balsamic vinegars is based. Ranges and 'cut-off' values for the multi element stable isotope parameters of balsamic vinegars are described, and a suitable analytical strategy is suggested.

A stable isotope approach to assessing water loss in fruits and vegetables during storage.

Plant tissue water is the source of oxygen and hydrogen in organic biomatter. Recently, we demonstrated that the stable hydrogen isotope value (δ(2)H) of plant methoxyl groups is a very reliable and easily available archive for the δ(2)H value of this tissue water. Here we show in a model experiment that the δ(2)H values of methoxyl groups remain unchanged after water loss during storage of fruits and vegetables under controlled conditions, while δ(2)H and δ(18)O values of tissue water increase. This enhancement is plant-dependent, and the correlation differs from the meteoric water line. The δ(18)O value is better correlated to the weight decrease of the samples. Therefore, we postulate that the δ(2)H value of methoxyl groups and the δ(18)O value of tissue water are suitable parameters for checking postharvest alterations of tissue water, either addition or loss.

Change of geographical location from Germany (Bavaria) to USA (Arizona) and its effect on H-C-N-S stable isotopes in human hair.

To receive information about the duration of a person's stay abroad related to those questions in forensics, stable isotopes of H-C-N-S were analysed in beard hair samples from four young soldiers, who went from Fürstenfeldbruck (Bavaria, Germany) to Phoenix (Arizona, USA) on the same date for their pilot training over a time period of 3 months. All study subjects were almost of the same age, had similar physical constitutions and stayed at the same military bases for the whole study period. However, the results showed considerable individual variabilities. In Arizona, hair δ(13)C increased by 2.3‰ (±0.6) and δ(34)S decreased by 1.8‰ (±1.2). No remarkable shifts of hair δ(15)N and δ(2)H were observed. Significant shifts of δ(13)C or δ(34)S in the shaved beard hair samples occurred 8 or 9 days after arrival in Arizona, respectively. The time lag to receive the isotope signals in hair due to US diet correspond to the growth period that hair needs to cover the distance of 2-3 mm from its root to the surface of the skin. This implies that isotopic changes due to the consumption of food and drinks were incorporated almost immediately into the hair protein. Consequently, if connected with an isotopic change of the diet, short-term stays for only a few days might be clearly recognizable within the first millimetres of a scalp hair strand which includes the hair roots.

Provenancing of unidentified corpses by stable isotope techniques - presentation of case studies.

Stable isotope methods can be used to determine the provenance of unidentified corpses. Body tissue materials such as teeth, bone, hair and nail taken from mortal remains provide information of different time periods of an individuals' life from childhood to death. Tissues of newborns contain provenance information of different time periods during pregnancy of the child's mother. The results of stable isotope analyses of body residues of two adults and a newborn found in Germany between 2010 and 2012 are presented. To determine the geographic origin and movements of unknown individuals, stable isotopes of hydrogen, carbon, nitrogen and sulphur were analysed in hair and bone collagen samples. Amino acid composition and, as a consequence, δ2H, δ13C, δ15N, and δ34S values in human keratin and bone collagen are different. Consequently correction factors were determined to compare isotopic data of bone collagen with those of an extensive worldwide reference hair collection. The isotopic signatures in hair and in bone collagen samples were compared to geographical groups of reference hair samples by canonical discriminant analysis. The results served as the basis for providing provenance constraints for the unidentified persons as requested by the police and prosecution. Ultimately the individuals were identified; hence the isotopic provenance interpretations can be critically evaluated and are shown to be successful.

Multi-element stable isotope analysis of H, C, N and S in hair and nails of contemporary human remains.

This paper presents a comparison of the isotopic ratios of hydrogen, carbon, nitrogen and sulphur of three pairings of hair and nail tissue taken from contemporary human remains. Our aim was to examine the possibility of a direct comparison of isotopic data in hair with that of nail tissue for forensic purposes. The results indicate that stable isotope ratios of the elements were best comparable between human scalp hair longer than 3 cm and the distal end of the nails. There were no distinct variations between finger and toenails. Our isotopic data for bulk hair and nail confirmed that hair samples were slightly enriched in (13)C but depleted in (15)N compared to nail samples. Furthermore, our data reveal that δ(34)S values in nail samples were more variable than in hair samples. Direct comparison of the corresponding segments of hair and nail samples may be difficult due to individual differences especially for δ(15)N and δ(2)H. Hair may have an isotopic composition more consistent with the ingested food within a specific time than is provided by nail. It can be concluded that once a hair is formed, no further metabolic changes of the isotopic pattern should occur. Nevertheless, our data suggest that there was a change in isotope ratios particularly for δ(2)H along the hair shaft. Interpretation of the isotope data in corresponding segments of hair and nail for forensic purposes must consider particular variations, especially for chronological considerations.

Does natural weathering change the stable isotope composition (²H, ¹³C, ¹5N, ¹8O and ³4S) of cattle hair?

Stable isotope analysis of hair has found applications in many fields of science because it provides a temporally resolved, fairly stable isotopic archive of mammalian individuals. We investigated whether this hair archive is modified by natural weathering while attached to a living animal. We analyzed the tail switch hairs of one suckler cow, sampled seven times over a period of four annual summer pasture-winter stall feeding cycles. We compared relative isotope ratios (δ²H, δ¹³C, δ¹5N, δ¹8O and δ³4S) of sections of hair that grew simultaneously but were exposed to natural weathering conditions over different periods of time. Natural wear caused a loss of mass of approx. 0.13% day⁻¹, with no apparent effect of environmental conditions. Changes in δ²H, δ¹³C, δ¹5N and δ¹8O were below the detection limit, indicating that hair is a reliable archive for the isotopes of these elements. In contrast, δ³4S values increased during the grazing period by about 1‰, with exposure to UV radiation appearing to have a major influence on this result. The δ³4S values decreased during the subsequent stall period, probably due to abrasion. Seasonal variation in δ³4S may indicate alternating environments that differ in their weathering conditions.

Essential methodological improvements in the oxygen isotope ratio analysis of N-containing organic compounds.

The quantitative conversion of organically bound oxygen into CO, a prerequisite for the (18)O/(16)O analysis of organic compounds, is generally performed by high-temperature conversion in the presence of carbon at ∼1450°C. Since this high-temperature procedure demands complicated and expensive equipment, a lower temperature method that could be utilized on standard elemental analyzers was evaluated. By substituting glassy carbon with carbon black, the conversion temperature could be reduced to 1170°C. However, regardless of the temperature, N-containing compounds yielded incorrect results, despite quantitative conversion of the bound oxygen into CO. We believe that the problems were partially caused by interfering gases produced by a secondary decomposition of N- and C-containing polymers formed during the decomposition of the analyte. In order to overcome the interference, we replaced the gas chromatographic (GC) separation of CO and N(2) by reversible CO adsorption, yielding the possibility of collecting and purifying the CO more efficiently. After CO collection, the interfering gases were vented by means of a specific stream diverter, thus preventing them from entering the trap and the mass spectrometer. Simultaneously, a make-up He flow was used to purge the gas-specific trap before the desorption of the CO and its subsequent mass spectrometric analysis. Furthermore, the formation of interfering gases was reduced by the use of polyethylene as an additive for analytes with a N:O ratio greater than 1. These methodological modifications to the thermal conversion of N-containing analytes, depending on their structure or O:N ratio, led to satisfactory results and showed that it was possible to optimize the conditions for their individual oxygen isotope ratio analysis, even at 1170°C. With these methodological modifications, correct and precise δ(18)O results were obtained on N-containing analytes even at 1170°C. Differences from the expected standard values were below ±1‰ with standard deviations of the analysis <0.2‰.

Isotopic and elemental data for tracing the origin of European olive oils.

H, C, and O stable isotope ratios and the elemental profile of 267 olive oils and 314 surface waters collected from 8 European sites are presented and discussed. The aim of the study was to investigate if olive oils produced in areas with different climatic and geological characteristics could be discriminated on the basis of isotopic and elemental data. The stable isotope ratios of H, C, and O of olive oils and the ratios of H and O of the relevant surface waters correlated to the climatic (mainly temperature) and geographical (mainly latitude and distance from the coast) characteristics of the provenance sites. It was possible to characterize the geological origin of the olive oils by using the content of 14 elements (Mg, K, Ca, V, Mn, Zn, Rb, Sr, Cs, La, Ce, Sm, Eu, U). By combining the 3 isotopic ratios with the 14 elements and applying a multivariate discriminant analysis, a good discrimination between olive oils from 8 European sites was achieved, with 95% of the samples correctly classified into the production site.

Multielement (H, C, N, O, S) stable isotope characteristics of lamb meat from different Italian regions.

The study focuses on the (2)H/H, (13)C/(12)C, (15)N/(14)N, (18)O/(16)O and (34)S/(32)S values of defatted dry matter (DFDM) and on the (2)H/H, (13)C/(12)C and (18)O/(16)O values of the fat fraction of meat samples from various lamb types reared in seven Italian regions, following different feeding regimes (forage, concentrate, milk). The (13)C/(12)C (r = 0.922), (2)H/H (r = 0.577) and (18)O/(16)O (r = 0.449) values of fat and DFDM are significantly correlated, the fat values being significantly lower for C and H and higher for O than for DFDM values and the differences between the two fractions not being constant for different lamb types. The feeding regime significantly affected the (13)C/(12)C, (15)N/(14)N, (18)O/(16)O and (2)H/H of fat. The DFDM (2)H/H, and (18)O/(16)O values, excluding an outlier, are significantly correlated with the corresponding values in meteoric waters, thus allowing us to trace the variability of geoclimatic factors. (15)N/(14)N is influenced by pedoclimatic conditions, whereas (34)S/(32)S is influenced by the sea spray effect and the surface geology of the provenance area. By applying stepwise linear discriminant analysis only the (2)H/H of fat was found not to be significant and 97.7% of the samples were correctly assigned to the lamb type and more than 90% cross-validated. With the feeding regime, 97.7% of the samples were both correctly assigned and cross-validated using a predictive model including (13)C/(12)C, (15)N/(14)N, (18)O/(16)O, (34)S/(32)S of DFDM and (18)O/(16)O of fat.

Assignment of unknown persons to their geographical origin by determination of stable isotopes in hair samples.

Analysing the isotope ratio of light elements in human tissue of an unknown person helps to reconstruct the life history with regard to geographical origin and/or typical food supply. In this study the isotope ratios of the bio-elements in hair samples of 111 persons from 13 different countries all over the world were measured with the aim of provenance determination. The results indicated that individuals from Costa Rica and Brazil can be differentiated from typical European individuals by delta(13)C, Australians by delta(34)S and delta(2)H in hair samples. The combination and evaluation of the data by multivariate statistical analysis considerably improved origin assignment. Investigation of hair samples from a number of individuals from one particular region (southern Germany) yielded remarkable variation of isotopic values indicating different nutritional habits. The possibilities and limitations of this method in its current state are demonstrated and discussed.

A measuring system for the fast simultaneous isotope ratio and elemental analysis of carbon, hydrogen, nitrogen and sulfur in food commodities and other biological material.

The isotope ratio of each of the light elements preserves individual information on the origin and history of organic natural compounds. Therefore, a multi-element isotope ratio analysis is the most efficient means for the origin and authenticity assignment of food, and also for the solution of various problems in ecology, archaeology and criminology. Due to the extraordinary relative abundances of the elements hydrogen, carbon, nitrogen and sulfur in some biological material and to the need for individual sample preparations for H and S, their isotope ratio determination currently requires at least three independent procedures and approximately 1 h of work. We present here a system for the integrated elemental and isotope ratio analysis of all four elements in one sample within 20 min. The system consists of an elemental analyser coupled to an isotope ratio mass spectrometer with an inlet system for four reference gases (N(2), CO(2), H(2) and SO(2)). The combustion gases are separated by reversible adsorption and determined by a thermoconductivity detector; H(2)O is reduced to H(2). The analyser is able to combust samples with up to 100 mg of organic material, sufficient to analyse samples with even unusual elemental ratios, in one run. A comparison of the isotope ratios of samples of water, fruit juices, cheese and ethanol from wine, analysed by the four-element analyser and by classical methods and systems, respectively, yielded excellent agreements. The sensitivity of the device for the isotope ratio measurement of C and N corresponds to that of other systems. It is less by a factor of four for H and by a factor of two for S, and the error ranges are identical to those of other systems.

Influence of the distillation step on the ratios of stable isotopes of ethanol in cherry brandies.

Isotope ratio mass spectrometry and site-specific natural isotope fractionation-nuclear magnetic resonance were applied to determine the overall carbon isotope ratio (delta13C) and the hydrogen isotope ratios [(D/H)I and (D/H)II] of ethanol, respectively. Ethanol was obtained by distillation of fermented cherry mash from a pot still commonly used in fruit brandy production. Analyses of distillate fractions revealed that the distillation proceeds with a fractionation of ethanol isotopologues. The inverse vapor pressure isotope effect (VPIE) observed for the carbon isotopologues is in accordance with the data reported for distillation of ethanol in spinning band columns. In contrast, the inverse VPIE for hydrogen isotopologues of ethanol observed in spinning band columns could not be confirmed. To investigate whether the observed isotope fractionations might influence the applicability of stable isotope analysis for quality and authenticity assessment of fruit brandies, the collected distillate fractions were recombined to cuts, as is common practice in commercial fruit brandy production. Taking into consideration the limits of repeatability of the method, it could be demonstrated that the isotope fractionations observed do not impair the applicability of stable isotope analysis of the carbon and hydrogen isotopes of ethanol for the authenticity assessment of cherry brandies if the cuts are placed in accordance with common distillers' practice.

Isotope characteristics of vegetables and wheat from conventional and organic production.

Multielement isotope ratio analysis was checked for its suitability as a means for the discrimination between agricultural products from integrated/conventional or organic production, respectively. Differences were mainly found for delta15N-values. Paprika and tomatoes from organic production in greenhouses showed delta15N-values above+7 per thousand, whereas corresponding products from conventional cultivation had delta15N-values near 0 per thousand. Lettuce, onions, cabbage and Chinese cabbage from field production had delta15N-values in the range of+5 to+6 and+5.5 to+7.5 per thousand, respectively (conventional and organic production); these overlapping differences do not permit a reliable discrimination. The same is true for wheat, showing average delta15N-values of+2.3+/-1.0 and+3.6+/-1.6 per thousand, respectively. The unexpected relative high 15N-enrichments of vegetables from integrated production are discussed as originating, at least partially, from 15N-enrichment in the soil by NH3 evaporation and denitrification.