Evidence for the translocation of fixed N in the N2-fixing lichen Stereocaulon vesuvianum.

The fruticose lichen Stereocaulon vesuvianum is among the most abundant and widespread lichens in upland Britain. It typically produces cephalodia (nodules) that contain the cyanobacterium Stigonema, which can fix atmospheric nitrogen. However, over much of England, Wales, and southern Scotland S. vesuvianum no longer produces cephalodia and does not fix nitrogen, a morphological change linked to elevated atmospheric nitrogen deposition. This provided a unique opportunity to compare the 15N natural abundance signatures in N2-fixing and non-N2-fixing lichen populations, keeping in mind that fixed nitrogen has a 15N content close to that of atmospheric N2 while, in comparison, several components of atmospheric combined N (e.g. nitrate and ammonium in precipitation) tend to be 15N depleted. We found that in N2-fixing samples, there was a steep gradient in 15N relative abundance in the terminal 15 mm of thallus branches (pseudopodetia), changing from 15N depleted tissues at 10-15 mm below the tips to values close to that of atmospheric N2 at the apices while in non-N2-fixing samples thallus branches were uniformly 15N depleted. The 15N gradient in N2-fixing material could not be explained by the presence of cephalodia since these are more abundant towards branch bases. The data provide the first evidence in lichens of translocation of recently fixed N to sink regions of active growth and production of asexual reproductive propagules, bringing lichens into line with N source-sink relationships in N2-fixing plant symbioses.

Growth pattern and turnover of carbon and nitrogen measured by stable isotope ratios in the hair of sika deer (Cervus nippon).

RATIONALE: Hair is known to preserve diet history and other physiological information during its growth period and is often used in chemical analyses. However, the growth patterns and turnover of hair vary according to the animal species or habitat, so understanding these patterns in the target animal is necessary for interpreting the results of hair analyses. In this study, we aimed to elucidate the growth pattern and dietary information of winter coat hair in captive sika deer (Cervus nippon). METHODS: Experiments involving hair-staining and shaving were conducted to elucidate the growth pattern of sika deer hair. A diet-switching experiment was conducted to ascertain what dietary information is reflected in the carbon and nitrogen stable isotope values of the deer winter coat. Hair samples collected from each body site (head, shoulder, back, and hip) were analyzed using an elemental analyser interfaced with an isotope-ratio mass spectrometer. RESULTS: The winter coat grows from early September to early November, and then stops after that. During the growth period of the winter coat, the hair of the shoulder and back grew at a constant rate. The carbon and nitrogen stable isotope values of hair reflected the deer's feeding history during hair growth, but there seemed to be a time lag in the hip hair. CONCLUSIONS: The results suggest that the guard hair of the shoulder is suitable for hair analysis in sika deer. The obtained information on growth patterns and isotopic change of hair from captive sika deer according to diet be useful for interpreting the results of future analyses using hair samples of wild deer.

Mercury and stable nitrogen isotope ratios in the hair of bearded seals (Erignathus barbatus nauticus) from the Sea of Okhotsk.

The mercury pollution status in the northwestern Sea of Okhotsk remains largely unexplored. In this study, hair samples were collected from 40 bearded seals harvested between August and October 2021 in the region. Total mercury (THg) concentrations in the samples exhibited a wide range from 137 to 1885 ng/g (median: 407 ng/g). While no significant differences in THg concentrations were found between male and female seals, distinctions were observed between young and potentially mature seals. Stable nitrogen isotope analysis indicated that juveniles and mature adults did not differ, although sample sizes were limiting. The higher THg concentrations in juveniles were attributed to variations in the seals' diets and/or variations in foraging locations during the juvenile stage which likely contribute to THg differences due to greater seasonal migration to offshore habitats. Notably, THg levels in bearded seals from the northwestern Sea of Okhotsk were lower in comparison to other pinniped species in the North Pacific. These findings, representing the first dataset for this pinniped species in the Russian segment of its habitat, contribute insights into mercury exposure in the Sea of Okhotsk mammalian population.

Use of stable isotope ratio analysis to investigate the biology and clinical significance of seal parasites.

Stranded seals are often infected with a broad range of parasites, although whether they are the cause of significant morbidity or an incidental finding is usually unclear. In this study we used stable isotope ratio analysis, a method frequently used to investigate food webs, to explore the extent to which common seal parasites feed directly on host tissue and fluids or absorb host-derived metabolites, which in turn may give an indication of their potential impact on the host's health. The trophic discrimination factor Δ15N for the nasal mite, Halarachne halichoeri, was mostly positive, ranging between −0.015 and 3.2‰ (n = 6), while for the Acanthocephalan worm, Corynosoma strumosum and the anisakid nematode, Pseudoterranova decipiens, Δ15N ranged between −4.2 and −2.0‰ (n = 7), and between −6.7 and −0.8‰ (n = 5) respectively. In the case of the lungworm, Otostrongylus circumlitus, Δ15N measured between −5.6 and 0‰ for worms collected in the stomach (n = 5), between −1.1 and 0.2‰ for worms collected from the heart (n = 3), between −0.7 and 1.9‰ for worms situated in the lungs (n = 4). Based on Δ15N, parasites could be clearly divided into those that were on a higher trophic level than their host suggesting a predator­prey-like relationship, and those that were not. It is hypothesized that Δ15N may be indicative of the clinical significance of parasite­host associations.

Dual isotope analysis reveals the COVID-19 lockdown impact on nitrate aerosol sources and formation pathways in Shanghai.

Nitrate (NO3-) is an important contributor to PM2.5 which can adversely affect the environment and human health. A noticeable decrease in NOx concentrations has been reported due to the lockdown measures implemented to curb the spread of Corona Virus Disease 2019 (COVID-19). However, questions remain, regarding the nonlinear relationship between NOx and NO3-. Here, we collected PM2.5 samples in two periods, before and during the lockdown of COVID-19 in Shanghai. Dual isotopes (δ18O-NO3- and δ15N-NO3-) of NO3- were measured to investigate the formation pathways and potential sources of NO3-. The results showed that the concentration of NO3- decreased significantly during the lockdown period compared to the period before the lockdown. Additionally, the hydroxyl pathway was the dominant contributor to NO3- production during the lockdown period, while N2O5 hydrolyses dominated the formation of NO3- before the lockdown. This change is largely attributable to alterations in the oxidative potential of the environment. In comparison to the period preceding the lockdown, the relative contributions of each NOx source remained largely unchanged throughout the lockdown periods. Nevertheless, the concentration of NO3- contributed by each NOx source exhibited a notable decline, particularly the mobile sources and coal combustion. Furthermore, the reduction extent of NO3- due to the lockdown period was also greater than the reduction during the Clean Air Actions (2013-2017). Our findings provide evidence that the COVID-19 lockdown led to a decrease in NO3- concentration due to changes in the formation pathway and reductions in NOx emissions from various sources.

Cross-Polarization of Insensitive Nuclei from Water Protons for Detection of Protein-Ligand Binding.

Hyperpolarization derived from water protons enhances the NMR signal of 15N nuclei in a small molecule, enabling the sensitive detection of a protein-ligand interaction. The water hyperpolarized by dissolution dynamic nuclear polarization (D-DNP) acts as a universal signal enhancement agent. The 15N signal of benzamidine was increased by 1480-fold through continuous polarization transfer by J-coupling-mediated cross-polarization (J-CP) via the exchangeable protons. The signal enhancement factor favorably compares to factors of 110- or 17-fold using non-CP-based polarization transfer mechanisms. The hyperpolarization enabled detection of the binding of benzamidine to the target protein trypsin with a single-scan measurement of 15N R2 relaxation. J-CP provides an efficient polarization mechanism for 15N or other low-frequency nuclei near an exchangeable proton. The hyperpolarization transfer sustained within the relaxation time limit of water protons additionally can be applied for the study of macromolecular structure and biological processes.

Nitrogen isotope variability of macroalgae from a small fishing village, Staithes Harbour, Yorkshire, UK.

Macroalgal nitrogen isotope analysis (δ15N) is a reliable method for the identification of nitrogen pollutant sources. Understanding δ15N geospatial variation within small bays and/or harbour environments can help identify point sources of nitrogen pollution. This study sampled over 300 Fucus vesiculosus and Ulva sp. specimens in September 2022 and May 2023 from Staithes Harbour, North Yorkshire, England. δ15N values for Staithes Beck were elevated when compared to sites in Staithes Harbour and the North Sea: this is attributed to sewage effluent and/or agricultural manure. Few sites within Staithes Harbour were significantly different from one another in terms of δ15N, suggesting a relatively homogenous nitrogen isotope record of the harbour. Simple harbour environments like Staithes may be relatively well mixed, and thus, sampling one harbour site may be enough to represent the entire harbour. Of course, more complex harbours may require more sample locations to ascertain point sources and mixing in the harbour.

Bivalve shells reflect 15N enrichment in a fertilizer-dominated estuary.

Agricultural nitrogen (N) contributes a dominant percentage to global N pollution in the coastal zone. Emerging research on N isotopes in bivalve shells has shown value for reconstructing historical increases in estuarine wastewater inputs. However, applications for fertilizer N are understudied. Here, we integrate the study of organic N isotopes, in concert with δ18O and δ13C, in estuarine bivalve shells to investigate spatial and long-term changes in nitrogen inputs and sources. Modern, museum-collected, and subfossil specimens of the genera Mytilus and Ostrea were profiled in a California estuary with an intensely agricultural watershed. Spatial patterns in bivalve isotopic composition reflected gradients in watershed nutrient inputs and productivity parameters. Furthermore, a comparison between modern and historical periods revealed changes in nutrient source or processing over the last 1,000 years. The N isotope values from shells offer perspective on agricultural pollution in estuaries.

Identification of the nitrate pollution in surface water of Dongshan Bay based on stable isotope technology.

Identifying and quantifying water nitrate pollution is crucial for managing aquatic environment of a bay. Dongshan Bay, a significant semi-enclosed bay in the southeastern coastal area of Fujian Province, features mangrove and coral reef ecosystems at its estuary and bay mouth, respectively. Dongshan Bay is impacted by human activities such as mariculture. We quantified and analyzed nitrate pollution status in the surface waters of Dongshan Bay by measuring physicochemical parameters, stable isotopes (δ15N-NO3-, δ18O-NO3- and δ15N-NH4+) of the surface waters, and using statistical methods including the MixSIAR isotope mixing model. The results showed that the concentrations of chlorophyll a and dissolved inorganic nitrogen in the surface waters exhibited a noticeable gradient change, decreasing from the estuary of the Zhangjiang River to the mouth of Dongshan Bay. The maximum concentrations of chlorophyll a, NH4+, NO3- and NO2- were 45.2 µg·L-1, 52.67 µmol·L-1, 379.2 µmol·L-1 and 3.93 µmol·L-1, respectively. The nitrogen and oxygen isotope values of NH4+ and NO3- in the surface waters showed significant spatial variations. According to the MixSIAR model results, nitrogen sources in the surface waters of Dongshan Bay were mainly freshwater inputs of the Zhangjiang River estuary, aquaculture wastewater, and groundwater. The freshwater input from the Zhangjiang River estuary contributed the most (25.2%), while aquaculture wastewater, groundwater and urban sewage accounted for 24.6%, 19.0%, and 15.1%, respectively. It is evident that freshwater input from the Zhangjiang River estuary is the primary source of nitrate in the surface waters of Dongshan Bay.

A long-term study of stable isotope ratios of fingernail keratin and amino acids in a mother-infant dyad.

OBJECTIVE: To evaluate the potential of compound-specific isotope analysis of amino acids (CSIA-AA) for investigating infant feeding practices, we conducted a long-term study that compared infant and maternal amino acid (AA) nitrogen isotope ratios. MATERIALS AND METHODS: Fingernail samples were collected from a single mother-infant dyad over 19 months postpartum. Carbon and nitrogen stable isotope ratios were measured in the bulk keratin of the fingernail samples. Selected samples were then hydrolyzed and derivatized for compound-specific nitrogen isotope analysis of keratin AAs. RESULTS: As in previous studies, infant bulk keratin nitrogen isotope values increased during exclusive breastfeeding and fell with the introduction of complementary foods and eventual cessation of breastfeeding. Infant trophic AAs had elevated nitrogen isotope values relative to the mother, while the source AAs were similar between the mother and infant. Proline and threonine appeared to track the presence of human milk in the infant's diet as the isotopic composition of these AAs remained offset from maternal isotope values until the cessation of breastfeeding. DISCUSSION: Although CSIA-AA is costly and labor intensive, it appears to hold potential for estimating the duration of breastfeeding, even after the introduction of complementary foods. Through the analysis of a full suite of AAs, it may also yield insights into infant physiology and AA synthesis.

Spatio-temporal Trends of Mercury and Stable Isotopes in Lower Food Web of Winam Gulf, Lake Victoria.

Components of the lower food web (mussels, Caridina and Omena) were collected from stations from Winam Gulf, Lake Victoria, Kenya in 2022 and 2023 to analyze for stable isotopes and total mercury (THg). Temporal comparisons were made with data generated for the same species in 1998. Values of δ15N in mussels and Caridina were similar (6.89‰ vs. 6.78 ± 0.13‰), while Omena occupied an elevated trophic position (9.97 ± 0.24‰) with minor shifts in δ15N over time. All species had elevated δ13C values in 2022-2023 versus 1998 supportive of enhanced eutrophication in the Gulf. THg concentrations exhibited modest spatial differences between sites (< 2.6 fold), but not between Caridina and Omena. Larger temporal differences were apparent relative to spatial patterns with THg concentrations decreasing in study species by 2.8 to 4.1-fold between years. An exposure assessment indicated that Omena, commonly found in local markets, can be consumed up to 0.74 kg/month without generating excess THg exposures.

Similarity recognition approach to identify zero-added MSG soy sauce using stable isotopes and amino acid profiles.

Seasonings such as naturally fermented soy sauce without added monosodium glutamate (MSG), are currently a growth market in China. However, fraudulent and mislabeled zero-added MSG soy sauce may cause a risk of excessive MSG intake, increasing food safety issues for consumers. This study investigates stable carbon and nitrogen isotopes and 16 amino acids in typical Chinese in-market soy sauces and uses a similarity method to establish criteria to authenticate MSG addition claims. Results reveal most zero-added MSG soy sauces had lower δ13C values (-25.2 ‰ to -17.7 ‰) and glutamic acid concentrations (8.97 mg mL-1 to 34.76 mg mL-1), and higher δ15N values (-0.27 ‰ +0.95 ‰) and other amino acid concentrations than added-MSG labeled samples. A combined approach, using isotopes, amino acids, similarity coefficients and uncertainty values, was evaluated to rapidly and accurately identify zero-added MSG soy sauces from MSG containing counterparts.

Source identification of organic C and N in suspended particulate matter and sediments in plateau lakes as influenced by trophic status using stable isotopic signatures.

Understanding the relationship between suspended particulate matter (SPM), sediment organic C, N stable isotopes, and lake trophic state index (TSI) is essential for managing lake pollution and eutrophication. According to the δ13C, δ15N, and C/N we found that the organic C in SPM and sediment of Caohai Lake primarily originated from macrophytes, while N was sourced from chemical fertilizers, phytoplankton, and aquatic plants. Total nitrogen, total phosphorus, NO3--N, oxidation reduction potential, and Chl.a were identified as key factors influencing the sources and variations of SPM and sediment organic C and N stable isotopes in Caohai Lake, with a significant linear correlation observed between C, N stable isotopes, and TSI in sediments. To mitigate eutrophication in Caohai Lake, it is recommended that farmers apply fertilizers judiciously to minimize nutrient loss and that aquatic plants be regularly harvested to reduce N release from plant residues.

Preliminary isotopic assessment of weaning in bonobos shows evidence for extended nursing, sibling competition and invested first-time mothers.

Although considered a hallmark in early ontogeny, weaning from breastmilk is difficult to monitor in wild primates and weaning ages remain unknown for wild bonobos (Pan Paniscus). Here, we calculated inter-birth intervals from demographic data and measured the isotopic offsets (Δ15N and Δ13C) between mother (n = 17) and offspring (n = 28) fecal sample pairs (n = 131, total n = 246) in the LuiKotale bonobos to assess nutritional weaning for the first time. We tested the effects of infant age, female parity, and sibling competition on Δ15N and Δ13C values. We found bonobo inter-birth intervals ranging from 2.2 to 7.3 years (x̄ = 4.7 ± 1.3 years) at LuiKotale. The Δ15N and Δ13C values suggested nutritional weaning on average by 6.6 and 7.0 years of age respectively, considerably exceeding weaning ages reported for chimpanzees (P. troglodytes) using the same approach. Our Δ13C data suggested that the number of offspring present affected nursing, with first-time mothers nursing more and possibly longer. The Δ15N and Δ13C values decreased with the arrival of the next sibling, suggesting sibling competition reduces milk access. Nevertheless, offspring may continue nursing 2.5-3 years after the birth of the next sibling, corresponding well with observations on low infant mortality. In conclusion, bonobo mothers provide remarkably enduring materna l support in the form of nursing concurrently to several offspring.

Automated Assignment of 15N And 13C Enrichment Levels in Doubly-Labeled Proteins.

Uniform enrichment of 15N and 13C in proteins is commonly employed for 2D heteronuclear NMR measurements of the three-dimensional protein structure. Achieving a high degree of enrichment of both elements is important for obtaining high quality data. Uniform labeling of proteins and glycoproteins expressed in higher organisms (yeast or mammalian cell lines) is more challenging than expression in Escherichia coli, a prokaryote that grows on simple, chemically defined media but does not provide appropriate eukaryotic modifications. It is difficult to achieve complete incorporation of both heavy isotopes, and quality control measures are important for quantitating the level of their enrichment. Mass spectrometry measurements of the isotopic distribution of the intact protein or its proteolytic fragments provide the means to assess the enrichment level. A mass accuracy of 1 ppm or better is shown to be required to distinguish the correct combination of 13C and 15N enrichment due to subtle shifts in peak centroids with differences in the underlying, but unresolved, isotopic fine structure. A simple computer program was developed to optimize the fitting of experimental isotope patterns to statistically derived distributions. This method can determine the isotopic abundance from isotope patterns and isotopologue masses in conventional MS data for peptides, intact proteins, and glycans. For this purpose, MATLAB's isotope simulator, isotopicdist, has been modified to permit the variation of 15N and 13C enrichment levels and to perform a two-dimensional grid search of enrichment levels of both isotopes. We also incorporated an alternate isotope simulator, js-emass, into MATLAB for use in the same fitting program. Herein we benchmark this technique on natural abundance ubiquitin and uniformly [15N,13C]-labeled ubiquitin at both the intact and peptide level, outline considerations for data quality and mass accuracy, and report several improvements we have made to the previously reported analysis of the [15N,13C]-enriched human IgG Fc domain, a glycoprotein that has been expressed in Saccharomyces cerevisiae.

Neoarchaean oxygen-based nitrogen cycle en route to the Great Oxidation Event.

The nitrogen isotopic composition of sedimentary rocks (δ15N) can trace redox-dependent biological pathways and early Earth oxygenation1,2. However, there is no substantial change in the sedimentary δ15N record across the Great Oxidation Event about 2.45 billion years ago (Ga)3, a prominent redox change. This argues for a temporal decoupling between the emergence of the first oxygen-based oxidative pathways of the nitrogen cycle and the accumulation of atmospheric oxygen after 2.45 Ga (ref. 3). The transition between both states shows strongly positive δ15N values (10-50‰) in rocks deposited between 2.8 Ga and 2.6 Ga, but their origin and spatial extent remain uncertain4,5. Here we report strongly positive δ15N values (>30‰) in the 2.68-Gyr-old shallow to deep marine sedimentary deposit of the Serra Sul Formation6, Amazonian Craton, Brazil. Our findings are best explained by regionally variable extents of ammonium oxidation to N2 or N2O tied to a cryptic oxygen cycle, implying that oxygenic photosynthesis was operating at 2.7 Ga. Molecular oxygen production probably shifted the redox potential so that an intermediate N cycle based on ammonium oxidation developed before nitrate accumulation in surface waters. We propose to name this period, when strongly positive nitrogen isotopic compositions are superimposed on the usual range of Precambrian δ15N values, the Nitrogen Isotope Event. We suggest that it marks the earliest steps of the biogeochemical reorganizations that led to the Great Oxidation Event.

Effects of gastric bypass on the digestibility and postprandial metabolic fate of 15N dietary protein in rats.

Roux-en-Y Gastric Bypass may be associated with an alteration of protein bioavailability in relation to intestinal remodeling. Our study aimed to test this hypothesis by Roux-en-Y Gastric Bypass. Diet-induced obese rats underwent Roux-en-Y Gastric Bypass surgery (RYGB rats) while a Sham-operated control group was used. All rats received a 15N-labeled protein meal 1 or 3 months after surgery and were euthanized 6h later. Protein digestibility, 15N recovered in organs and urea pool, fractional protein synthesis rate, and intestinal morphometry were assessed. Protein digestibility was similar in all groups (94.2±0.3%). The small intestine was hypertrophied in RYGB rats 1 month after surgery, weighing 9.1±0.2g vs. 7.0±0.3g in Sham rats (P = 0.003). Villus height and crypt depth were increased in the alimentary limb and ileum of RYGB rats. However, Roux-en-Y Gastric Bypass had no impact on the fractional synthesis rate. In the gastrointestinal tract, 15N retention only differed in the ileal mucosa and was higher in RYGB rats at 1 month (0.48±0.2% vs. 0.3±0.09%, P = 0.03). 15N recovery from the liver, muscle, and skin was lower in RYGB rats at 1 month. 15N recovery from urinary and plasma urea was higher in RYGB rats at both times, resulting in increased total deamination (13.2±0.9% vs. 10.1±0.5%, P<0.01). This study showed that Roux-en-Y Gastric Bypass did not affect protein digestibility. Dietary nitrogen sequestration was transitorily and moderately diminished in several organs. This was associated with a sustained elevation of postprandial deamination after Roux-en-Y Gastric Bypass, whose mechanisms merit further studies.

Understanding natural isotopic variations in cultured cancer cells.

RATIONALE: Natural variations in the abundance of the stable isotopes of nitrogen (δ15N) and carbon (δ13C) offer valuable insights into metabolic fluxes. In the wake of strong interest in cancer metabolism, recent research has revealed δ15N and δ13C variations in cancerous compared to non-cancerous tissues and cell lines. However, our understanding of natural isotopic variations in cultured mammalian cells, particularly in relation to metabolism, remains limited. This study aims to start addressing this gap using metabolic modulations in cells cultured under controlled conditions. METHODS: Prostate cancer cells (PC3) were cultured in different conditions and their δ15N and δ13C were measured using isotope ratio mass spectrometry. Isotopic variations during successive cell culture passages were assessed and two widely used cell culture media (RPMI and DMEM) were compared. Metabolism was modulated through glutamine deprivation and hypoxia. RESULTS: Successive cell culture passages generally resulted in reproducible δ15N and δ13C values. The impact of culture medium composition on δ15N and δ13C of the cells highlights the importance of maintaining a consistent medium composition across conditions whenever possible. Glutamine deprivation and hypoxia induced a lower δ13C in bulk cell samples, with only the former affecting δ15N. Gaps between theory and experiments were bridged and the lessons learned throughout the process are provided. CONCLUSIONS: Exposing cultured cancer cells to hypoxia allowed us to further investigate the relation between metabolic modulations and natural isotopic variations, while mitigating the confounding impact of changing culture medium composition. This study highlights the potential of natural δ13C variations for studying substrate fluxes and nutrient allocation in reproducible culture conditions. Considering cell yield and culture medium composition is pivotal to the success of this approach.

Introducing IsoMad, a compilation of isotopic datasets for Madagascar.

We present the first open-access, island-wide isotopic database (IsoMad) for modern biologically relevant materials collected on Madagascar within the past 150 years from both terrestrial and nearshore marine environments. Isotopic research on the island has increasingly helped with biological studies of endemic organisms, including evaluating foraging niches and investigating factors that affect the spatial distribution and abundance of species. The IsoMad database should facilitate future work by making it easy for researchers to access existing data (even for those who are relatively unfamiliar with the literature) and identify both research gaps and opportunities for using various isotope systems to answer research questions. We also hope that this database will encourage full data reporting in future publications.

Effects of pre-treatment, historical age, and sample characteristics on the stable isotope analyses of killer whale (Orcinus orca) bone.

RATIONALE: Stable isotope analysis of bone provides insight into animal foraging and allows for ecological reconstructions over time, however pre-treatment is required to isolate collagen. Pre-treatments typically consist of demineralization to remove inorganic components and/or lipid extraction to remove fats, but these protocols can differentially affect stable carbon (δ13C) and nitrogen (δ15N) isotope values depending on the chemicals, tissues, and/or species involved. Species-specific methodologies create a standard for comparability across studies and enhance understanding of collagen isolation from modern cetacean bone. METHODS: Elemental analyzers coupled to isotope ratio mass spectrometers were used to measure the δ13C and δ15N values of powdered killer whale (Orcinus orca) bone that was intact (control) or subjected to one of three experimental conditions: demineralized, lipid-extracted, and both demineralized and lipid-extracted. Additionally, C:N ratios were evaluated as a proxy for collagen purity. Lastly, correlations were examined between control C:N ratios vs. historical age and control C:N ratios vs. sample characteristics. RESULTS: No significant differences in the δ15N values were observed for any of the experimental protocols. However, the δ13C values were significantly increased by all three experimental protocols: demineralization, lipid extraction, and both treatments combined. The most influential protocol was both demineralization and lipid extraction. Measures of the C:N ratios were also significantly lowered by demineralization and both treatments combined, indicating the material was closer to pure collagen after the treatments. Collagen purity as indicated via C:N ratio was not correlated with historical age nor sample characteristics. CONCLUSIONS: If only the δ15N values from killer whale bone are of interest for analysis, no pre-treatment seems necessary. If the δ13C values are of interest, samples should be both demineralized and lipid-extracted. As historical age and specimen characteristics are not correlated with sample contamination, all samples can be treated equally.