Developing δ15N and δ13C isoscapes using whole blood from Magellanic penguins, Spheniscus magellanicus.

RATIONALE: Understanding the migration of marine animals is hindered by the limitations of traditional tracking methods. It is therefore crucial to develop alternative methods. Stable isotope-based tracking has proven useful for this task, although it requires detailed isoscapes in the focal area. Here, we present predator-based isoscapes of the coastal zone of the Patagonian Shelf Large Marine Ecosystem (PSLME), which offers a novel tool for geolocation. METHODS: Whole-blood samples from breeding Magellanic penguins nesting at 11 colonies were used to create δ15N and δ13C isoscapes. Isotopic values were assigned to random positions inside their corresponding foraging area. Spatial analysis and data interpolation resulted in δ15N and δ13C isoscapes for the coastal zone of the PSLME, which were validated through cross-validation. RESULTS: The isoscapes mean standard error ranged from 0.05 to 0.41 for δ15N and from 0.07 to 0.3 for δ13C, similar to the error range of the mass spectrometer used for measuring isotope ratios. Predictive surfaces reflected the latitudinal trends, with δ13C and δ15N values increasing northwards. δ13C values showed a strong latitudinal gradient, while δ15N values had two distinct domains, with higher values in the north. The error surface indicated the highest certainty within 130 km from the shore and within the reported Magellanic penguin foraging areas. CONCLUSIONS: Both isoscapes revealed strong spatial variation. The δ13C isoscape showed a latitudinal gradient, consistent with patterns in other oceans. The δ15N isoscape clearly separated northern and southern colonies, likely influenced by nitrogen sources. The error obtained fell within the measurement error ranges, adding credibility to the models.

A Single-Carbon Stable Isotope Ratio Model Prediction Equation Can Estimate Self-Reported Added Sugars Intake in an Adult Population Living in Southwest Virginia.

The δ13C value of blood is a novel proposed biomarker of added sugars (AS) intake. AS prediction equations using either a single- (δ13C) or dual-isotope model (δ13C and δ15N) were previously developed in an adult population with high AS intake living in southwest Virginia (reference group). The purpose of this investigation was to test the δ13C single- and δ13C and δ15N dual-isotope prediction equations for AS intake in adults with a lower mean AS intake and different demographic characteristics (test group). The blood samples for the reference (n = 257 for single-isotope, n = 115 for dual-isotope) and test groups (n = 56) were analyzed for δ13C and δ15N values using natural abundance stable isotope mass spectrometry and were compared to reported dietary AS intake. When the δ13C single-isotope equation was applied to the test group, predicted AS intake was not significantly different from reported AS intake (mean difference ± standard error = -3.6 ± 5.5 g, Z = -0.55, p = 0.51). When testing the dual-isotope equation, predicted AS was different from reported AS intake (mean difference ± SEM = 13.0 ± 5.4 g, Z = -2.95, p = 0.003). δ13C value was able to predict AS intake using a blood sample within this population subset. The single-isotope prediction equation may be an alternative method to assess AS intake and is more objective, cost-feasible, and efficient than traditional dietary assessment methods. However, more research is needed to assess this biomarker with rigorous study designs such as controlled feeding.

Validation of dried blood spot sampling for determining trophic positions of Arctic char using nitrogen stable isotope analyses of amino acids.

RATIONALE: Dried blood spots (DBSs) are gaining popularity for biomarker analyses in ecological research due to their advantages for use in field-based research and in remote settings; however, many DBS biomarkers remain unvalidated. We validated the application of compound-specific stable nitrogen isotope analyses of amino acids (CSIA-AAs) to field-prepared DBSs for determining trophic positions of wild-caught Arctic char (Salvelinus alpinus). METHODS: Whole blood and muscle from Arctic char were collected, and DBSs were created in the field. We measured the stable nitrogen isotope ratios (expressed as δ15 N values) of the amino acids glutamic acid (Glu) and phenylalanine (Phe) isolated from Arctic char samples using CSIA-AAs. We then compared amino acid δ15 N values from DBSs and the other sample types (whole blood and muscle) from the same specimens. We calculated and compared trophic position estimates generated from whole blood, DBSs, and muscle. RESULTS: The δ15 N values of Glu and Phe, as well as trophic position estimates from DBSs, were highly correlated with δ15 N values and estimates from both whole blood and muscle. The DBS amino acid δ15 N values and trophic position estimates agreed well with those from whole blood. Although mean differences between amino acid δ15 N values from DBSs and muscle were noted, the offsets were small and resulted in a 0.2 mean difference between trophic position estimates for DBSs and muscle. CONCLUSIONS: We demonstrate that the application of CSIA-AAs to field-prepared DBSs of Arctic char generates similar trophic position estimates to those from whole blood and muscle. We suggest that DBSs could be developed as a minimally invasive sampling technique to study feeding ecology of wild fish and perhaps other organisms of interest.

Effect of Organic Food Intake on Nitrogen Stable Isotopes.

Food choices affect the isotopic composition of the body with each food item leaving its distinct isotopic imprint. The common view is that the natural abundance of the stable isotopes of nitrogen (expressed as δ15N) is higher in animals than in plants that constitute our contemporary diets. Higher δ15N is thus increasingly viewed as a biomarker for meat and fish intake. Here we show that organic compared to conventional farming increases plant δ15N to an extent that can appreciably impact the performance of δ15N as a biomarker. The error that can arise when organic plants are consumed was modelled for the entire range of proportions of plant versus animal protein intake, and accounting for various intakes of organic and conventionally grown crops. This mass balance model allows the interpretation of differences in δ15N in light of organic food consumption. Our approach shows that the relationship between δ15N and meat and fish intake is highly contextual and susceptible to variation at the population, community or group level. We recommend that fertilization practices and organic plant consumption must not be overlooked when using δ15N as a biomarker for meat and fish intake or to assess compliance to nutritional interventions.

Blood 15N:13C Enrichment Ratios Are Proportional to the Ingested Quantity of Protein with the Dual-Tracer Approach for Determining Amino Acid Bioavailability in Humans.

BACKGROUND: Assessment of amino acid bioavailability is of key importance for the evaluation of protein quality; however, measuring ileal digestibility of dietary proteins in humans is challenging. Therefore, a less-invasive dual stable isotope tracer approach was developed. OBJECTIVE: We aimed to test the assumption that the 15N:13C enrichment ratio in the blood increases proportionally to the quantity ingested by applying different quantities of 15N test protein. METHODS: In a crossover design, 10 healthy adults were given a semi-liquid mixed meal containing 25 g (low protein) or 50 g (high protein) of 15N-labeled milk protein concentrate simultaneous with 0.4 g of highly 13C-enriched spirulina. The meal was distributed over multiple small portions, frequently provided every 20 min during a period of 160 min. For several amino acids, the blood 15N- related to 13C-isotopic enrichment ratio was determined at t = 0, 30, 60, 90, 120, 180, 240, 300, and 360 min and differences between the 2 meals were compared using paired analyses. RESULTS: No differences in 13C AUC for each of the measured amino acids in serum was observed when ingesting a low- or high-protein meal, whereas 15N AUC of amino acids was ∼2 times larger on the high-protein meal (P < 0.001). Doubling the intake of 15N-labeled amino acids increased the 15N:13C ratio by a factor of 2.04 ± 0.445 for lysine and a factor between 1.8 and 2.2 for other analyzed amino acids, with only phenylalanine (2.26), methionine (2.48), and tryptophan (3.02) outside this range. CONCLUSIONS: The amino acid 15N:13C enrichment ratio in the peripheral circulation increased proportionally to the quantity of 15N-labeled milk protein ingested, especially for lysine, in healthy adults. However, when using 15N-labeled protein, correction for, e.g., α-carbon 15N atom transamination is advised for determination of bioavailability of individual amino acids. This trial was registered at www.clinicaltrials.gov as NCT02966704.

Isotopic Discrimination (δ15N, δ13C) in Captive and Wild Common Murres (Uria aalge) and Atlantic Puffins (Fratercula arctica).

Studying the diet of consumers using stable isotopes provides insight into the foraging ecology of individuals and species. To accurately reconstruct the integrated diet of animals using stable isotope values, we must quantify diet-tissue discrimination factors (DTDFs), or the way in which stable isotopes in prey are incorporated into the tissues of consumers. To quantify DTDFs, controlled experiments are needed, whereby consumers are fed a constant diet. However, relatively few controlled-diet studies have been conducted for seabirds. In this study, captive adult Atlantic puffins (Fratercula arctica) and common murres (Uria aalge) were fed a two-source diet of capelin (Mallotus villosus) and Atlantic silverside (Menidia menidia) to determine the DTDFs for the cellular component of blood and plasma for both δ15N and δ13C. The DTDFs for the cellular component (Δ15N: 2.80±0.28; Δ13C: 1.21±0.22) and plasma (Δ15N: 1.72±1.03; Δ13C: -0.18±0.56) of puffins were similar to those for the cellular component (Δ15N: 2.91±0.18; Δ13C: 1.09±0.23) and plasma (Δ15N: 2.18±0.77; Δ13C: -0.70±0.18) of murres. We reconstructed the diet of wild murres and puffins breeding on the northeastern coast of Newfoundland using previously published DTDFs and estimated DTDFs from our feeding experiment. Reconstructed dietary proportions supported a priori knowledge of diet, although outputs were sensitive to the DTDF used. Despite the similarity of our DTDFs for puffins and murres, along with the similarity of our DTDFs with those of other seabird species, our sensitivity analysis revealed considerable differences among resultant dietary contributions from mixing models, further highlighting the importance of using species- and tissue-specific DTDFs to enhance knowledge in the foraging ecology of seabirds using stable isotopes.

Isotopic variation in blood components based on their biochemistry and physiology: A comparison between sharks and fur seals.

Research using stable isotopes analysis (SIA) of carbon (δ13 C) and nitrogen (δ15 N) in blood components is lacking, because of the challenge of sample collection, processing, and storage in remote areas. There also is a paucity of information regarding the effect of tissue biochemical composition on isotopic ratios with few comparisons among taxa. We collected blood samples from shortfin mako sharks (n = 70; 2016) and Guadalupe fur seals (n = 25; 2017). All samples were centrifuged to obtain plasma from sharks and serum from the Guadalupe fur seals, and all the samples were prepared for SIA and analyzed using a Costech 4010 elemental analyzer interfaced with a Delta V Plus isotope ratio mass spectrometer. We found significant differences between plasma δ13 C values of shortfin mako sharks (-17.6 ± 0.9‰) and serum of Guadalupe fur seals (-20.3 ± 1.2‰), but we did not find any differences for δ15 N values between the two species. The differences in δ13 C values between species are probably due to the specific blood composition and to the different biochemical characteristics and different adaptations within taxa. These findings highlight the importance of further research on the influence of biochemistry features on isotopic results, in this way a more accurate assessment will be possible for this factor, separating it from the dietary influences on stable isotopic values.

Diet-tissue discrimination factors (δ15 N and δ13 C values) for blood components in Magellanic (Spheniscus magellanicus) and southern rockhopper (Eudyptes chrysocome) penguins.

RATIONALE: Analysis of the stable isotope ratios of carbon and nitrogen (δ13 C and δ15 N values) is increasingly being used to gain insight into predator trophic ecology, which requires accurate diet-tissue discrimination factors (DTDFs), or the isotopic difference between prey and predator. Accurate DTDFs must be calculated from predators consuming an isotopically constant diet over time in controlled feeding experiments, but these studies have received little attention to date, especially among seabird species. METHODS: In this study, aquarium-housed Magellanic (Spheniscus magellanicus) and southern rockhopper (Eudyptes chrysocome) penguins were fed a single-prey source diet (capelin Mallotus villosus) for eight weeks. Stable isotope ratios (δ13 C and δ15 N values) of penguin blood (cellular component and plasma) and capelin were measured using mass spectrometry and then used to calculate DTDFs for both components of penguin blood by comparison with prey values. RESULTS: The DTDFs for plasma were -0.63 ± 0.49 (mean ± SD) and -0.27 ± 0.22 for δ13 C values, and 2.60 ± 0.50 and 2.78 ± 0.22 for δ15 N values for Magellanic and southern rockhopper penguins, respectively, while the DTDFs for the cellular component were 1.22 ± 0.03 and 1.26 ± 0.03 for δ13 C values, and 2.54 ± 0.07 and 2.43 ± 0.17 for δ15 N values. CONCLUSIONS: We compare our DTDFs with published values from blood components of penguins and discuss the effects that lipid extraction, sample storage, and diet have on the DTDFs of penguin blood components. This study provides accurate DTDFs of blood components for two seabird species of conservation concern, and is one of the first to provide plasma DTDFs for penguins, which are underrepresented in the seabird literature.

Associations of plasma, RBCs, and hair carbon and nitrogen isotope ratios with fish, meat, and sugar-sweetened beverage intake in a 12-wk inpatient feeding study.

BACKGROUND: Naturally occurring carbon and nitrogen stable isotope ratios [13C/12C (CIR) and 15N/14N (NIR)] are promising dietary biomarkers. As these candidate biomarkers have long tissue residence times, long-term feeding studies are needed for their evaluation. OBJECTIVE: Our aim was to evaluate plasma, RBCs, and hair CIR and NIR as biomarkers of fish, meat, and sugar-sweetened beverage (SSB) intake in a 12-wk dietary intervention. METHODS: Thirty-two men (aged 46.2 ± 10.5 y; BMI: 27.2 ± 4.0 kg/m2) underwent a 12-wk inpatient dietary intervention at the National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK) in Phoenix, Arizona. The effects of fish, meat, and SSB intake on CIR and NIR were evaluated using a balanced factorial design, with each intake factor at 2 levels (present/absent) in a common, background diet (50% carbohydrate, 30% fat, 20% protein). Fasting blood samples were taken biweekly from baseline, and hair samples were collected at baseline and postintervention. Data were analyzed using multivariable regression. RESULTS: The postintervention CIR of plasma was elevated when diets included meat (ß = 0.89, 95% CI: 0.73,1.05) and SSBs (ß = 0.48, 95% CI: 0.32, 0.64). The postintervention NIR of plasma was elevated when diets included fish (ß = 0.85, 95% CI: 0.64, 1.05) and meat (ß = 0.61, 95% CI: 0.42, 0.8). Results were similar for RBCs and hair. Postintervention RBC CIR and NIR had strong associations with baseline, suggesting that turnover to the intervention diets was incomplete after 12 wk. Estimates of isotopic turnover rate further confirmed incomplete turnover of RBCs. CONCLUSIONS: CIR was associated with meat and SSBs, and more strongly with meat. NIR was associated with fish and meat, and more strongly with fish. Overall, CIR and NIR discriminated between dietary fish and meat, and to a lesser extent SSBs, indicating their potential utility as biomarkers of intake in US diets. Approaches to make these biomarkers more specific are needed. This trial was registered at clinicaltrials.gov as NCT01237093.

City life alters the gut microbiome and stable isotope profiling of the eastern water dragon (Intellagama lesueurii).

Urbanisation is one of the most significant threats to biodiversity, due to the rapid and large-scale environmental alterations it imposes on the natural landscape. It is, therefore, imperative that we understand the consequences of and mechanisms by which, species can respond to it. In recent years, research has shown that plasticity of the gut microbiome may be an important mechanism by which animals can adapt to environmental change, yet empirical evidence of this in wild non-model species remains sparse. Using an empirical replicated study system, we show that city life alters the gut microbiome and stable isotope profiling of a wild native non-model species - the eastern water dragon (Intellagama lesueurii) in Queensland, Australia. City dragons exhibit a more diverse gut microbiome than their native habitat counterparts and show gut microbial signatures of a high fat and plant rich diet. Additionally, we also show that city dragons have elevated levels of the Nitrogen-15 isotope in their blood suggesting that a city diet, which incorporates novel anthropogenic food sources, may also be richer in protein. These results highlight the role that gut microbial plasticity plays in an animals' response to human-altered landscapes.

Determination of Cetuximab in Plasma by Liquid Chromatography-High-Resolution Mass Spectrometry Orbitrap With a Stable Labeled 13C,15N-Cetuximab Internal Standard.

BACKGROUND: Cetuximab (CTX) is a chimeric IgG1 Kappa monoclonal antibody used to treat head and neck cancer and colorectal cancer. Previous clinical studies indicated that the pharmacokinetics of CTX influences patient survival. Thus, individualizing CTX treatment by measuring trough levels of the drug in plasma could have a major impact on clinical efficacy. METHODS: To measure these levels, a full-length stable isotope-labeled CTX standard was used in a generic, rapid, and high-throughput sample preparation protocol based on IgG capture followed by trypsin digestion, on-line solid-phase extraction cleanup, and liquid chromatography-high resolution mass spectrometry (LC-HRMS). RESULTS: The optimized method displayed good analytical performance and was linear over a range from 5 to 150 mcg/mL. The within-run and between-run imprecision of the assay were equal to or less than 10%, for 6 replicates at 3 different concentrations and for runs performed on 5 separate days. The plasma CTX concentrations in 19 patients were also determined. CONCLUSIONS: The results showed that quantification of mAb in clinical samples does not strictly require a tandem mass spectrometry system, and LC-HRMS is also relevant in this context. This first study implementing a quantitative LC-HRMS assay with a specific stable isotope-labeled mAb internal standard paves the way for more robust clinical monitoring of anticancer mAbs.

The Nitrogen Isotope Ratio Is a Biomarker of Yup'ik Traditional Food Intake and Reflects Dietary Seasonality in Segmental Hair Analyses.

BACKGROUND: The nitrogen isotope ratio (NIR) is a promising index of traditional food intake for an Alaska Native (Yup'ik) population, which can be measured in blood and hair. However, the NIR has not been calibrated to high-quality measures of Yup'ik traditional food intake. OBJECTIVES: Our primary objective was to examine associations between intakes of Yup'ik traditional food groups, including fish, marine mammals, birds, land mammals, berries, greens, and total traditional foods, and the NIR. In an exploratory analysis, we also examined whether NIR analyzed sequentially along hair could reflect dietary seasonality. METHODS: We recruited 68 participants from 2 Yup'ik communities in the Yukon Kuskokwim region of Southwest Alaska (49% female, aged 14-79 y). Participants completed 4 unscheduled 24-h food recalls over the period peak of RBC and hair synthesis preceding a specimen collection visit. The NIR was measured in RBCs ( n = 68), a proximal hair section (n = 58), and sequential segments of hair from individuals in the upper 2 quartiles of traditional food intake having hair >6 cm in length, plus 2 low subsistence participants for reference (n = 18). Diet-biomarker associations were assessed using Pearson's correlation and linear regression. RESULTS: Intakes of fish, marine mammals, berries, and greens were significantly associated with the NIR. The strongest dietary association was with total traditional food intake (R2 = 0.62), which indicated that each 1‰ increase in the RBC NIR corresponded to 8% of energy from traditional foods. Hair NIR appeared to fluctuate seasonally in some individuals, peaking in the summertime. CONCLUSIONS: Findings support the use of the RBC and hair NIR to assess total traditional food intake in a Yup'ik population. Analyses of sequential hair NIR provided evidence of seasonality in traditional food intake, although seasonal variations were modest relative to interindividual variation.

Citrulline Generation Test: What Does It Measure?

BACKGROUND: The citrulline generation test (CGT) has been proposed as a tool to determine gut function. However, the increase in plasma citrulline concentration that follows a bolus dose of alanyl-glutamine may also result from a reduction in citrulline clearance due to competition with glutamine for transport. MATERIALS AND METHODS: A swine model was developed, and stable isotope tracers were used to determine the mechanism behind the increase in plasma citrulline that follows a bolus dose of alanyl-glutamine. Plasma concentrations and enrichments were determined, and a non-steady-state model was used to calculate rates of appearance, disappearance, and conversion. RESULTS: The pig model recapitulated the increase in plasma citrulline observed in humans after a dose of alanyl-glutamine. The dipeptide was rapidly hydrolyzed to its constitutive amino acids. Both citrulline plasma concentration and citrulline rate of appearance increased by ≈45% after the bolus dose of alanyl-glutamine. The conversion of citrulline to arginine and the rate of appearance of arginine also increased. Glutamine contributed up to 25% ± 2% of the rate of appearance of citrulline. No changes in the rate of disappearance of citrulline were observed. CONCLUSION: Our results indicate that a single bolus dose of alanyl-glutamine increases plasma citrulline concentration by increasing citrulline production without any effect on citrulline disposal. Our findings strongly indicate that the CGT assesses the metabolic response of the gut and that CGT can become a useful tool to evaluate gut mass and function.

Serum Nitrogen and Carbon Stable Isotope Ratios Meet Biomarker Criteria for Fish and Animal Protein Intake in a Controlled Feeding Study of a Women's Health Initiative Cohort.

Background: Natural abundance stable isotope ratios are candidate biomarkers of dietary intake that have not been evaluated in a controlled feeding study in a US population. Objectives: Our goals were to evaluate dietary associations with serum carbon (CIR), nitrogen (NIR), and sulfur (SIR) isotope ratios in postmenopausal women, and to evaluate whether statistical models of dietary intake that include multiple isotopes and participant characteristics meet criteria for biomarker evaluation. Methods: Postmenopausal women from the Women's Health Initiative (n = 153) were provided a 2-wk controlled diet that approximated each individual's habitual food intake. Dietary intakes of animal protein, fish/seafood, red meat, poultry, egg, dairy, total sugars, added sugars, sugar-sweetened beverages (SSBs), and corn products were characterized during the feeding period with the use of the Nutrition Data System for Research (NDS-R). The CIR, NIR, and SIR were measured in sera collected from fasting women at the beginning and the end of the feeding period. Linear models based on stable isotope ratios and participant characteristics predicted dietary intake. The criterion used for biomarker evaluation was R2 ≥ 0.36, based on the study's power to detect true associations with R2 ≥ 0.50. Results: The NIR was associated with fish/seafood intake and met the criterion for biomarker evaluation (R2 = 0.40). The CIR was moderately associated with intakes of red meat and eggs, but not to the criterion for biomarker evaluation, and was not associated with intake of sugars (total, added, or SSB). A model of animal protein intake based on the NIR, CIR, and participant characteristics met the criterion for biomarker evaluation (R2 = 0.40). Otherwise, multiple isotopes did not improve models of intake, and improvements from including participant characteristics were modest. Conclusion: Serum stable isotope ratios can, with participant characteristics, meet biomarker criteria as measures of fish/seafood and animal protein intake in a sample of postmenopausal women. This trial was registered at clinicaltrials.gov as NCT00000611.

Isotopic Composition of Blood of Polar Bears (Ursus maritimus) of the Kara-Barents Sea Population.

The data on the content of carbon and nitrogen isotopes in the blood samples of polar bears obtained in the present study confirm that polar bears in the Taimyr region (and the Kara-Barents sea population in general) are partly dependent on the resources of terrestrial origin. However the "terrestrial carbon" evidently reaches bears' tissues indirectly, via marine food webs utilizing organic carbon brought into the polar basin by Siberian rivers.

Identification of sources and bioaccumulation pathways of MeHg in subantarctic penguins: a stable isotopic investigation.

Seabirds are widely used as bioindicators of mercury (Hg) contamination in marine ecosystems and the investigation of their foraging strategies is of key importance to better understand methylmercury (MeHg) exposure pathways and environmental sources within the different ecosystems. Here we report stable isotopic composition for both Hg mass-dependent (e.g. δ202Hg) and mass-independent (e.g. Δ199Hg) fractionation (proxies of Hg sources and transformations), carbon (δ13C, proxy of foraging habitat) and nitrogen (δ15N, proxy of trophic position) in blood of four species of sympatric penguins breeding at the subantarctic Crozet Islands (Southern Indian Ocean). Penguins have species-specific foraging strategies, from coastal to oceanic waters and from benthic to pelagic dives, and feed on different prey. A progressive increase to heavier Hg isotopic composition (δ202Hg and Δ199Hg, respectively) was observed from benthic (1.45 ± 0.12 and 1.41 ± 0.06‰) to epipelagic (1.93 ± 0.18 and 1.77 ± 0.13‰) penguins, indicating a benthic-pelagic gradient of MeHg sources close to Crozet Islands. The relative variations of MeHg concentration, δ202Hg and Δ199Hg with pelagic penguins feeding in Polar Front circumpolar waters (1.66 ± 0.11 and 1.54 ± 0.06‰) support that different MeHg sources occur at large scales in Southern Ocean deep waters.

Development of a multi-matrix LC-MS/MS method for urea quantitation and its application in human respiratory disease studies.

In human respiratory disease studies, liquid samples such as nasal secretion (NS), lung epithelial lining fluid (ELF), or upper airway mucosal lining fluid (MLF) are frequently collected, but their volumes often remain unknown. The lack of volume information makes it hard to estimate the actual concentration of recovered active pharmaceutical ingredient or biomarkers. Urea has been proposed to serve as a sample volume marker because it can freely diffuse through most body compartments and is less affected by disease states. Here, we report an easy and reliable LC-MS/MS method for cross-matrix measurement of urea in serum, plasma, universal transfer medium (UTM), synthetic absorptive matrix elution buffer 1 (SAMe1) and synthetic absorptive matrix elution buffer 2 (SAMe2) which are commonly sampled in human respiratory disease studies. The method uses two stable-isotope-labeled urea isotopologues, [15N2]-urea and [13C,15N2]-urea, as the surrogate analyte and the internal standard, respectively. This approach provides the best measurement consistency across different matrices. The analyte extraction was individually optimized in each matrix. Specifically in UTM, SAMe1 and SAMe2, the unique salting-out assisted liquid-liquid extraction (SALLE) not only dramatically reduces the matrix interferences but also improves the assay recovery. The use of an HILIC column largely increases the analyte retention. The typical run time is 3.6min which allows for high throughput analysis.

Blood-specific isotopic discrimination factors in the Magellanic penguin (Spheniscus magellanicus).

RATIONALE: The use of stable isotopes for ecological studies has increased exponentially in recent years. Isotopic trophic studies are based on the assumption that animals are what they eat plus a discrimination factor. The discrimination factor is affected by many variables and can be determined empirically. The Magellanic penguin is a highly abundant marine bird that plays a key role in the southern oceans. This study provides the first estimation of the Magellanic penguin blood discrimination factor for (13) C and (15) N. METHODS: A two and a half month feeding experiment was performed, in which ten captive penguins were fed their main natural prey (anchovy Engraulis anchoita). The discrimination factors were estimated by comparing anchovy δ(13) C and δ(15) N values (obtained with isotope ratio mass spectrometry using lipid-extracted and bulk anchovy muscle) with penguin blood δ(13) C and δ(15) N values. RESULTS: Penguin blood was shown to be enriched, compared with anchovies, for (13) C and (15) N. No changes were observed in the stable isotope ratios of anchovies and discrimination factors during the experiment. The overall discrimination factors were 0.93 ± 0.12 (bulk) and 0.41 ± 0.12 (lipid-free) for (13) C; and 2.81 ± 0.17 (bulk) and 2.31 ± 0.17 (lipid-free) for (15) N. CONCLUSIONS: Having an accurate discrimination factor for the studied species is key in any trophic or food web isotopic study. Comparisons of estimated diet-to-blood discrimination factors with published values of aquatic piscivore birds showed that the (13) C discrimination factor is particularly variable, and therefore ecologists should be cautious when using a surrogate value from other species. In this study, the Magellanic penguin discrimination factor of a tissue that does not require euthanasia was obtained, a fundamental input for trophic isotopic modeling of the species. Copyright © 2016 John Wiley & Sons, Ltd.

Estimating tissue-specific discrimination factors and turnover rates of stable isotopes of nitrogen and carbon in the smallnose fanskate Sympterygia bonapartii (Rajidae).

This study aimed to estimate trophic discrimination factors (TDFs) and metabolic turnover rates of nitrogen and carbon stable isotopes in blood and muscle of the smallnose fanskate Sympterygia bonapartii by feeding six adult individuals, maintained in captivity, with a constant diet for 365 days. TDFs were estimated as the difference between δ(13) C or δ(15) N values of the food and the tissues of S. bonapartii after they had reached equilibrium with their diet. The duration of the experiment was enough to reach the equilibrium condition in blood for both elements (estimated time to reach 95% of turnover: C t95%blood = 150 days, N t95%blood = 290 days), whilst turnover rates could not be estimated for muscle because of variation among samples. Estimates of Δ(13) C and Δ(15) N values in blood and muscle using all individuals were Δ(13) Cblood = 1·7‰, Δ(13) Cmuscle = 1·3‰, Δ(15) Nblood = 2·5‰ and Δ(15) Nmuscle = 1·5‰, but there was evidence of differences of c.0·4‰ in the Δ(13) C values between sexes. The present values for TDFs and turnover rates constitute the first evidence for dietary switching in batoids based on long-term controlled feeding experiments. Overall, the results showed that S. bonapartii has relatively low turnover rates and isotopic measurements would not track seasonal movements adequately. The estimated Δ(13) C values in S. bonapartii blood and muscle were similar to previous estimations for elasmobranchs and to generally accepted values in bony fishes (Δ(13) C = 1·5‰). For Δ(15) N, the results were similar to published reports for blood but smaller than reports for muscle and notably smaller than the typical values used to estimate trophic position (Δ(15) N c. 3·4‰). Thus, trophic position estimations for elasmobranchs based on typical Δ(15) N values could lead to underestimates of actual trophic positions. Finally, the evidence of differences in TDFs between sexes reveals a need for more targeted research.