Isotope (δ13C, δ15N, δ2H) diet–tissue discrimination in African grey parrot Psittacus erithacus: implications for forensic studies

Diet–tissue isotopic relationships established under controlled conditions are informative for determining the dietary sources and geographic provenance of organisms. We analysed δ¹³C, δ¹⁵N, and non-exchangeable δ²H values of captive African grey parrot Psittacus erithacus feathers grown on a fixed mixed-diet and borehole water. Diet–feather Δ¹³C and Δ¹⁵N discrimination values were +3.8?±?0.3?‰ and +6.3?±?0.7?‰ respectively; significantly greater than expected. Non-exchangeable δ²H feather values (?62.4?±?6.4?‰) were more negative than water (?26.1?±?2.5?‰) offered during feather growth. There was no positive relationship between the δ¹³C and δ¹⁵N values of the samples along each feather with the associated samples of food offered, or the feather non-exchangeable hydrogen isotope values with δ²H values of water, emphasising the complex processes involved in carbohydrate, protein, and income water routing to feather growth. Understanding the isotopic relationship between diet and feathers may provide greater clarity in the use of stable isotopes in feathers as a tool in determining origins of captive and wild-caught African grey parrots, a species that is widespread in aviculture and faces significant threats to wild populations. We suggest that these isotopic results, determined even in controlled laboratory conditions, be used with caution.     

Sauna,sweat and science II – do we sweat what we drink?

ABSTRACT

Inspired by a previous ‘Sauna, sweat and science’ study [Zech et al. Isot Environ Health Stud. 2015;51(3):439–447] and out of curiosity and enthusiasm for stable isotope and sauna research we aimed at answering the question ‘do we sweat (isotopically) what we drink’? We, therefore, pulse-labelled five test persons in a sauna experiment with beverages that were ²H-enriched at about +25,600?‰. Sweat samples were collected during six sauna rounds and the hydrogen isotope composition δ²H_sweat was determined using an isotope ratio mass spectrometer. Before pulse labelling, δ²H_sweat – reflecting by approximation body water – ranged from –32 to –22?‰. This is ～35?‰ enriched compared to usual mid-European drinking water and can be explained with hydrogen-bearing food as well as with the respiratory loss of ²H-depleted vapour. The absence of a clearly detectable ²H pulse in sweat after pulse labelling and δ²H_sweat results of ≤+250?‰ due to a fast ²H equilibration with body water are moreover a clearly negative answer to our research question also in a short-term consideration. Given that the recovery of the tracer based on an isotope mass balance calculation is clearly below 100?%, we finally answer the question ‘where did the rest of the tracer go?’     

Tracing the geographical origin of early potato tubers using stable hydrogen isotope ratios of methoxyl groups†

The application of stable isotope ratio measurements has become an extremely useful tool for tracing the provenance of food products, thus ensuring that consumers receive products which comply with their labelled specifications. Recently, it has been shown that relative stable hydrogen isotope abundances (δ²H values) of wood lignin methoxyl groups have a distinct range that reflects the δ²H values of their meteoric source water. Furthermore, it has been suggested that the isotope information stored in methoxyl groups in plant matter generally might assist with determining the place of origin of plant material. We now have measured δ²H values of methoxyl groups from natural compounds in tubers of early potatoes (Solanum tuberosum) grown in different geographical locations. Tubers of early potatoes were collected from across Europe and regions close to the Mediterranean Sea between April and July 2004. The methoxyl groups from the potatoes were found to be highly depleted in ²H, relative to both their meteoric water and bulk biomass, and a systematic shift of the δ²H values between methoxyl groups and meteoric water was observed. A constant fractionation of?161±11‰. between methoxyl groups and modelled meteoric water is shown over a transaction covering the δ²H values of meteoric water from?95‰ in Northern Sweden to+25‰ in Egypt. From this information, early potato tubers from Middle Europe can be clearly distinguished from those of Mediterranean regions and from Northern Europe. Thus, we suggest that δ²H values of methoxyl groups have the potential to become an effective tool in assisting with the constraint of the geographical origin of potato tubers and other food stuffs.     

NMR studies on hydrogen bonding and proton transfer in Mannich bases

A review of studies on the ortho Mannich bases containing various substituents in the phenyl ring on the basis of¹H,¹³C and¹⁵N nuclear magnetic resonance (NMR) spectra in various solvents over the temperature range 110–298 K is presented. Some new results are also included. The data gathered so far show that there is some critical (inversion) range of ΔpK _a (= pK _a(NH⁺) − pK _a(OH)) in which the proton transfer equilibrium appears. This inversion range is well reflected in the behaviour of secondary deuterium isotope effect in¹³C NMR spectra. A strong temperature effect on the strength of hydrogen bonding should be emphasized. The¹H chemical shift for trichloroderivative increases from 13.5 at room temperature up to 17 ppm at 130 K when the proton is equally shared between the bridging atoms (¹ J(¹H,¹⁵N) = 30–40 Hz). The potential for the proton motion in such bridges is discussed taking into account the behaviour in the ultraviolet and infrared spectra. The role of dimerization in proton transfer equilibria is shown. In addition the rotation of OH groups involved in hydrogen bond formation and nitrogen pyramidal inversion was studied by the¹H dynamical NMR spectra.     

Factors Influencing Stable Isotope Ratios in CH4 and CO2 Within Subenvironments of Freshwater Wetlands: Implications for δ-Signatures of Emissions

Abstract

Much uncertainty still exists regarding spatial and temporal variability of stable isotope ratios (¹³C/¹²C and D/H) in different CH₄-emission sources. Such variability is especially prevalent in freshwater wetlands where a range of processes can influence stable isotope compositions, resulting in variations of up to ～50‰ for δ¹³C-CH₄ and ～150‰ for δD-CH₄ values. Within a temperate-zone bog and marsh situated in southwestern Ontario, Canada, gas bubbles in pond sediments exhibit only minor seasonal and spatial variation in δ¹³C-CH₄, δD-CH₄ and δ¹³C-CO₂ values. In pond sediments, CO₂ appears to be the main source of carbon during methanogenesis either directly via CO₂ reduction or indirectly through dissimilation of autotrophic acetate. In contrast, CH₄ production occurs primarily via acetate fermentation at shallow depths in peat soils adjacent to ponds at each wetland. At greater depths within soils, σCO₂ and H₂O increasingly exert an influence on δ¹³C- and δD-CH₄ values. Secondary alteration processes (e.g., methanotrophy or diffusive transport) are unlikely to be responsible for depth-related changes in stable isotope values of CH₄. Recent models that attempt to predict δD-CH₄ values in freshwater environments from D/H ratios in local precipitation do not adequately account for such changes with depth. Subenvironments (i.e., soil-forming and open water areas) in wetlands should be considered separately with respect to stable isotope signatures in CH₄ emission models.     

Stable isotope determination of ester and ether methyl moieties in plant methoxyl groups

Plant methoxyl groups of lignin and pectin have both distinct stable hydrogen isotope (δ²H) and carbon isotope (δ¹³C) values that can be used for studying environmental processes and for investigating the origin and authenticity of biomaterials. Up to now, the reported methods have been applied only to determine isotope values of the bulk plant methoxyl pool. In this work, we have applied several methods to distinguish between stable isotope ratios of methoxyl groups of pectin and the bulk plant methoxyl pool. Our results demonstrate that by applying alkaline hydrolysis to specifically cleave off the ester methyl moiety (pectin-like), we can distinguish δ²H and δ¹³C values of the pectin methoxyl pool from the bulk methoxyl pool. No measureable isotope discrimination was observed either during sample preparation or during analytical measurement. Furthermore, using this method, no major isotope difference in either the hydrogen or carbon isotope signature of the methoxyl groups of plant pectin and bulk matter from plant species such as leaves from trees, apples, carrots and potatoes was noted. We show the methanol released during alkaline hydrolysis of plant material and subsequently treated with hydriodic acid to be an excellent procedure to measure specifically and precisely the δ¹³C and δ²H isotope values of plant pectin-like methoxyl groups. This method is particularly advantageous when plant matter with a low methoxyl content has to be analysed.     

Field-deployable measurements of free-living individuals to determine energy balance: fuel substrate usage through δ13C in breath CO2 and diet through hair δ13C and δ15N values

Carbon isotopes of breath CO₂ vary depending on diet and fuel substrate used. This study examined if exercise-induced δ¹³C-CO₂ changes in substrate utilization were distinguishable from baseline δ¹³C-CO₂ variations in a population with uncontrolled diet, and compared hair isotope values and food logs to develop an isotope model of diet. Study participants included nine women with diverse Body Mass Index (BMI), age, ancestry, exercise history, and diet. Breath samples were collected prior to and up to 12?h after a 5- or 10?K walk/run. Indirect calorimetry was measured with a smartphone-enabled mobile colorimetric device, and a field-deployable isotope analyzer measured breath δ¹³C-CO₂ values. Diet was assessed by food logs and δ¹³C, δ¹⁵N of hair samples. Post-exercise δ¹³C-CO₂ values increased by 0.54?±?1.09‰ (1 sd, n?=?9), implying enhanced carbohydrate burning, while early morning δ¹³C-CO₂ values were lower than daily averages (p?=?0.0043), indicating lipid burning during overnight fasting. Although diurnal δ¹³C-CO₂ variation (1.90?±?0.77‰) and participant baseline range (3.06‰) exceeded exercise-induced variation, temporal patterns distinguished exercise from dietary isotope effects. Hair δ¹³C and δ¹⁵N values were consistent with a new dietary isotope model. Notwithstanding the small number of participants, this study introduces a novel combination of techniques to directly monitor energy balance in free-living individuals.     

Isotopic characteristics of meteoric waters in the Belgrade region

Abstract

The stable isotope composition of hydrogen (δ²H) and oxygen (δ¹⁸O) in monthly precipitation and river water (Sava River and Danube) samples in the Belgrade area gathered between 1992 and 2005 are determined. The local meteoric water line δ²H=7.8 (±0.2) δ¹⁸O+7.3(±1.6) (r ²=0.98, n=60, σ=0.52) for the whole period of observation is close to the global meteoric water line. The amount-weighted mean δ²H and δ¹⁸O values of precipitation were?65±27 ‰ and?9.4±3.4 ‰, respectively. Good correlation between δ¹⁸O values (r>rsim0.67) and ambient temperature and relative humidity was obtained. Stream-water data ranged from?94 to?60 ‰ for δ²H and from?11.0 to ～5.7 ‰ for δ¹⁸O with highly statistically significant difference (p>0.01) between the Sava River and the Danube. In addition, the isotopic compositions of local precipitation and adjacent river water at monitoring sites were compared. Obtained data will give an opportunity to improve the knowledge of mixing stream water and local groundwater, and assessment of potential groundwater risks and pressures in the Belgrade basin.     

Nitrogen stable isotopes reveal age-dependent dietary shift in the Japanese scallop Mizuhopecten yessoensis

Ontogenetic niche shifts in diet are a consequence of changes in body size or resource partitioning between age classes. To better resolve the feeding patterns of the Japanese scallop Mizuhopecten yessoensis, we examined the relative importance of age and size in the diet of this species using stable isotope ratios of carbon (δ¹³C) and nitrogen (δ¹⁵N) from 2006 to 2009. Contribution of food sources was quantified using an isotope mixing model by comparing the muscle tissue isotope ratios to those of suspended particulate organic matter (SPOM) and their zooplankton prey (e.g. micro- and meso-zooplankton). Unlike the δ¹³C values, which remained constant with age and size, muscle δ¹⁵N values were more positively correlated with age accounting for 69?% of variations than size with only 46?%. Increasing ¹⁵N values with age suggested that shifts in diet from SPOM to micro- and meso-zooplankton occurred during ontogeny in M. yessoensis. Results of the isotope mixing model indicated that SPOM contribution to scallop’s diet decreased from 68 to 8?% while those of zooplankton increased from 15 to 50?% with increasing age. This study concludes that age-related dietary shift explains the enrichment of ¹⁵N, as a result of predation on zooplankton by M. yessoensis.     

First real-time measurement of the evolving 2H/1H ratio during water evaporation from plant leaves

We have studied the temporal behaviour of the deuterium isotope ratio of water vapour emerging from a freshly cut plant leaf placed in a dry nitrogen atmosphere. The leaf material was placed directly inside the sample gas cell of the stable isotope ratio infrared spectrometer. At the reduced pressure (～40 mbar) inside the cell, the appearance of water evaporating from the leaf is easily probed by the spectrometer, as well as the evolving isotope ratios, with a precision of about 1 ‰. The demonstration experiment we describe measures the ²H/¹H isotope ratio only, but the experiment can be easily extended to include the ¹⁸O/¹⁶O and ¹⁷O/¹⁶O isotope ratios. Plant leaf water isotope ratios provide important information towards quantification of the different components in the ecosystem water and carbon dioxide exchange.     

Stable Isotope Ratios and Uric Acid Preservation in Termites Belonging to Three Feeding Habits in Thailand

Abstract

Nitrogen and carbon stable isotope ratios and uric acid concentrations in termites sampled from a dry evergreen forest in Thailand, were determined across three kinds of feeding habits. Feeding habits of Microcerotermes crassus, which is an abundant woodfeeder, and Dicuspiditermes makhamensis, a common soil-feeding termite, were confirmed by isotopic signatures. Lichen feeding termites (Hospitalitermes birmanicus, H. bicolor and H. ataramensis) were characterized by low δ¹⁵N values, suggesting that they assimilated nitrogen deposited from the atmosphere. There was also a significant difference in uric acid concentrations between termites representing different feeding habits. No significant relationships were found between uric acid concentrations and δ¹⁵N or δ¹³C in Hospitalitermes. However, δ¹⁵N values were correlated with C/N ratios in H. birmanicus, except in one colony of H. ataramensis. δ¹³C values in both species were negatively correlated with C/N ratios.     

Helicobacter Pylori in Vivo Diagnosis after Food Intake: Results of Simultaneous [15N]Urea Urine and [13C]Acetate Breath Tests

Abstract

The protocols for ¹³C and ¹⁵N H. pylori tests stipulate that the diagnostic agent should be taken on an empty stomach. It is presumed that food intake prior to the tests leads to less reliable test results due to a prolongation of the gastric residence time of the diagnostic agent urea. This might allow the bacteria to split a higher proportion of urea, resulting in an increased number of false positives. 12 probands received 150mg [¹⁵N]urea and 75 mg sodium [¹³C]acetate in 75 ml orange juice as a test drink. [¹⁵N]Urea served as an agent to diagnose gastric H. pylori colonization. The ¹⁵N tests were evaluated using a urine sample of the second hour after test start. [¹³C]Acetate served as a marker of the gastric emptying of water-soluble food including the urea under the influence of food intake. Breath air samples were taken to calculate the gastric emptying half life (EHL) and the apparent resorption time (RT) of the urea. The double tests were carried out four times within four weeks using identical test protocols but different     

Record of Nile seasonality in Nubian neonates

The oxygen isotope compositions of bones (n?=?11) and teeth (n?=?20) from 12 Sudanese individuals buried on Sai Island (Nubia) were analysed to investigate the registration of the evolution of the Nile environment from 3700 to 500 years BP and the potential effects of ontogeny on the oxygen isotope ratios. The isotopic compositions were converted into the composition of drinking water, ultimately originating from the Nile. δ¹⁸O values decrease during ontogeny; this is mainly related to breastfeeding and physiology. Those of neonates present very large variations. Neonates have a very high bone turnover and are thus able to record seasonal δ¹⁸O variations of the Nile waters. These variations followed a pattern very similar to the present one. Nile δ¹⁸O values increased from 1.4 to 4.4?‰ (Vienna Standard Mean Ocean Water) from the Classic Kerma (～3500 BP) through the Christian period (～1000 BP), traducing a progressive drying of Northeast Africa.     

Three-dimensional triple-resonance NMR Spectroscopy of isotopically enriched proteins. 1990

Four new and complementary three-dimensional triple-resonance experiments are described for obtaining complete backbone ¹H, ¹³C, and ¹⁵N resonance assignments of proteins uniformly enriched with ¹³C and ¹⁵N. The new methods all rely on ¹H detection and use multiple magnetization transfers through well-resolved one-bond J couplings. Therefore, the 3D experiments are sensitive and permit relatively rapid recording of 3D spectra (l–2 days) for protein concentrations on the order of 1 mM. One experiment (HNCO) correlates the amide ¹H and ¹⁵N shifts with the ¹³C shift of the carbonyl resonance of the preceding amino acid. A second experiment (HNCA) correlates the intraresidue amide ¹H and ¹⁵N shifts with the Cα chemical shift. This experiment often also provides a weak correlation between the amide NH and ¹⁵N resonances of one amino acid and the Ca resonance of the preceding amino acid. A third experiment (HCACO) correlates the Hα and Cα shifts with the intraresidue carbonyl shift. Finally, a 3D relay experiment, HCA(CO)N, correlates Ha and Cal resonances of one residue with the ¹⁵N frequency of the succeeding residue. The principles of these experiments are described in terms of the operator formalism. To optimize spectral resolution, special attention is paid to removal of undesired J splittings in the 3D spectra. Technical details regarding the implementation of these triple-resonance experiments on a commercial spectrometer are also provided. The experiments are demonstrated for the protein calmodulin (16.7 kDa).     

H/D isotope effect for solubility of hydrogen in doped perovskites

A novel method for studying the H/D isotope separation effect in the “nonstoichiometric oxide-gas” system is suggested. The theoretical scheme of this method is represented for doped perovskites of the ABO₃ family, but this approach is also applicable to other compounds where the solubility of hydrogen is determined by the content of oxygen vacancies. The suggested approach is used to determine the H/D isotope effect in hydrogen solubility from the experimental data for proton-conducting oxides. The measurements have been taken using the nuclear microanalysis of fine-grain BaZr_0.9Y_0.1O_{3 − y} powders. The method is based on the study of the products of the ²H(d, p)³H nuclear reaction. The observed pronounced isotope effect is in quantitative agreement with our theoretical predictions.     

AgF desalination procedure for the routine determination of oxygen and hydrogen isotopic composition of saline waters and brines

The routine methods for stable oxygen and hydrogen isotope analysis of water involve water–CO₂ gas equilibration and water reduction on hot metal (e.g. Zn, Cr, U) and subsequent mass spectrometric analysis of the evolved gases of CO₂ and H₂ for ¹⁸O/¹⁶O and ²H/¹H ratios, respectively. Precise determination of the isotopic composition of water in brines with application of these standard methods is still problematic and technically often impossible due to detrimental influence of dissolved salts. The new method of brine desalination presented in this study overcomes the problem of the isotope salt effects encountered during the application of the routine techniques for the determination of the isotopic composition of high saline waters. The procedure combines two technical steps: (i) the chemical precipitation of Mg and Ca ions as insoluble non-hydroscopic fluorides, and (ii) the vacuum distillation of water from solution–precipitate mixture. The application of simple vacuum distillation allows full extraction of water and dehydration of remaining salts in a temperature range from 300 to 350?°C without hydrogen and oxygen isotope fractionation. The precision and accuracy of δ¹⁸O and δ²H determination of saline waters and brines with prior application of AgF desalination procedure is comparable with that usually obtained for fresh waters.     

Feeding and Growth of Apple Snail Pomacea lineata in the Pantanal Wetland,Brazil—a Stable Isotope Approach

Abstract

Apple snails Pomacea lineata (SPIX 1827) are widespread in the tropical regions of Brazil as well as in the Pantanal wetland of Mato Grosso in the western part of the country. They have a key position in the Pantanal food web and serve as food for many animals e.g. fishes, birds, and caimans. However, little is known about their feeding preferences and growth rates. Stable isotopes have been used successfully on numerous studies as food source indicator. Therefore, the δ¹⁵N and δ¹³C values of snails from 0.45 to 3.03cm in length, which were collected in the rainy season from March through May, were analyzed. Snails signatures revealed ambiguous evidence for food preferences. δ¹⁵N and δ¹³C values ranged between ?2.8 and 12.4‰ and between ?24.2 and ?16.4‰, respectively. This range of values mirrors the highly variable isotope values of possible food sources comprising C₃ and C₄ macrophytes. To test whether all common food sources were similarly assimilated, feeding experiments with different diets were conducted. Snail eggs were reared in tanks and offered different but single plants. Snails fed different diets and δ¹³C values of the food were reflected in the animal tissue. Growth varied considerably in experiments with different diets indicating the preference for certain food sources. Also, the fractionation of nitrogen isotopes between food and animal varied from 0.1 to 17.0‰. The results are explained by different feeding habits, and it is supposed that animals fed either on the plant itself or on bacteria mats growing in the tanks. In an additional experiment juvenile snails were offered one single food with a distinctive C₄ grass signature. These snails did not grow detectably, but nevertheless isotope signatures approached to values of the diet.     

Food Web Implications of δ13C and δ15N Variability over 370 km of the Regulated Colorado River USA

Abstract

Dual stable isotope analysis in the regulated Colorado River through Grand Canyon National Park, USA, revealed a food web that varied spatially through this arid biome. Down-river enrichment of δ¹³C data was detected across three trophic levels resulting in shifted food webs. Humpack chub δ¹³C and δ¹⁵N values from muscle plugs and fin clips did not differ significantly. Humpback chub and rainbow trout trophic position is positively correlated with standard length indicating an increase in piscivory by larger fishes. Recovery of the aquatic community from impoundment by Glen Canyon Dam and collecting refinements for stable isotope analysis within large rivers are discussed.     

Is the ‘canine surrogacy approach’ (CSA) still valid for dogs and humans in market-oriented and subsistence-oriented communities in Brazil?

Based on the assumptions that human food is available for dogs and isotope diet–tissue differences are similar in dogs and humans, the ‘canine surrogacy approach’ (CSA) has been used to infer patterns of ancient populations. The goal of this study was to test the CSA in urban (Brasília and Piracicaba) and in rural (Ubatuba and Maraã) areas. The hair C and N isotope ratios of modern dogs were compared with those of human fingernails from different regions of Brazil. Our CSA results showed a correlation between dog and human isotopes values: in rural areas δ¹⁵N of humans and dogs was not statistically different; contrarily, in urban centres, δ¹⁵N of humans was approximately 1?‰ higher (p?<?0.01) than δ¹⁵N of dogs; humans had lower δ¹³C values (p?<?0.01) than dogs in Brasília, Piracicaba and Ubatuba. In Maraã, there was not any significant difference between dogs and humans. We concluded that CSA is still valid as a first approach in modern societies. However, isotopic differences found suggest that in modern societies processed dog food is increasingly disconnecting human and dog, jeopardising the use of CSA in the future if the trend of increasing processed dog food consumption continues to occur.     

Diet–consumer nitrogen isotope fractionation for prolonged fasting arthropods

Nitrogen acquisition for cellular metabolism during diapause is a primary concern for herbivorous arthropods. Analyses of naturally occurring stable isotopes of nitrogen help elucidate the mechanism. Relevant articles have cited (58 times up to mid-June 2011) anomalously elevated δ¹⁵N (per mil deviation of ¹⁵N/¹⁴N, relative to atmospheric nitrogen=0 ‰) values (diet–consumer nitrogen isotope fractionation; up to 12 ‰) for a prolonged fasting raspberry beetle (Byturus tomentosus Degeer (Coleoptera: Byturidae)), which feeds on red raspberries (Rubus idaeus: δ¹⁵N=～+2 ‰). Biologists have hypothesised that extensive recycling of amino acid nitrogen is responsible for the prolonged fasting. Since this hypothesis was proposed in 1995, scientists have integrated biochemical and molecular knowledge to support the mechanism of prolonged diapausing of animals. To test the validity of the recycling hypothesis, we analysed tissue nitrogen isotope ratios for four Japanese arthropods: the shield bug Parastrachia japonensis Scott (Hemiptera: Cydnidae), the burrower bug Canthophorus niveimarginatus Scott (Hemiptera: Cydnidae), leaf beetle Gastrophysa atrocyanea Motschulsky (Coleoptera: Chrysomelidae) and the Japanese oak silkworm Antheraea yamamai (Lepidoptera: Saturniidae), all of which fast for more than 6 months as part of their life-history strategy. Resulting diet–consumer nitrogen isotope discrimination during fasting ranged from 0 to 7‰, as in many commonly known terrestrial arthropods. We conclude that prolonged fasting of arthropods does not always result in anomalous diet–consumer nitrogen isotope fractionation, since the recycling process is closed or nearly closed with respect to nitrogen isotopes.