Measurement of 13C and 15N isotope labeling by gas chromatography/combustion/isotope ratio mass spectrometry to study amino acid fluxes in a plant-microbe symbiotic association

We have developed a method based on a double labeling with stable isotopes and gas chromatography/combustion/isotope ratio mass spectrometry (GC/C/IRMS) analyses to study amino acid exchange in a symbiotic plant-microbe association. Isotopic precision was studied for 21 standards including 15 amino acid derivatives, three N-protected amino acid methyl esters, three amines and one international standard. High correlations were observed between the δ(13)C and δ(15)N values obtained by GC/C/IRMS and those obtained by an elemental analyzer (EA) coupled to an isotope ratio mass spectrometer (R(2) = 0.9868 and 0.9992, respectively). The mean precision measured was 0.04‰ for δ(13)C and 0.28‰ for δ(15)N (n = 15). This method was applied in vivo to the symbiotic relationship between alfalfa (Medicago sativa L.) and N(2)-fixing bacteria. Plants were simultaneously labeled over 10 days with (13)C-depleted CO(2) ((12)CO(2)), which was assimilated through photosynthesis by leaves, and (15)N(2) fixed via nodules. Subsequently, the C and N isotope compositions (i.e. δ(13)C and δ(15)N) of free amino acids were analyzed in leaves and nodules by GC/C/IRMS. The method revealed the pattern of C and N exchange between leaves and nodules, highlighting that γ-aminobutanoic acid and glycine may represent an important form of C transport from leaves to the nodules. The results confirmed the validity, reliability and accuracy of the method for assessing C and N fluxes between plants and symbiotic bacteria and support the use of this technique in a broad range of metabolic and fluxomic studies.     

Mammalian DNA δ15N exhibits 40‰ intramolecular variation and is unresponsive to dietary protein level

We report the first high‐precision characterization of molecular and intramolecular δ¹⁵N of nucleosides derived from mammalian DNA. The influence of dietary protein level on brain amino acids and deoxyribonucleosides was determined to investigate whether high protein turnover would alter amino acid ¹⁵ N or ¹³ C values. Pregnant guinea pig dams were fed control diets, or high or low levels of dietary protein throughout gestation, and all pups were fed control diets. The cerebellar DNA of offspring was extracted at 2 and 120 days of life, nucleosides isolated and δ¹⁵N and δ¹³C values characterized. Mean diet δ¹⁵N was 0.45 ± 0.33‰, compared with cerebellar whole tissue and DNA δ¹⁵N = +4.1 ± 0.7‰ and ?4.5 ± 0.4‰, respectively. Cerebellar deoxythymidine (dT), deoxycytidine (dC), deoxyadenosine (dA), and deoxyguanosine (dG) δ¹⁵N were +1.4 ± 0.4, –2.1 ± 0.9, –7.2 ± 0.3, and ?10.4 ± 0.5‰, respectively. There were no changes in amino acid or deoxyribonucleoside δ¹⁵N values due to dietary protein level. Using known metabolic relationships, we developed equations to calculate the intramolecular δ¹⁵N values originating from aspartate (asp) in purines (pur) or pyrimidines (pyr), glutamine (glu), and glycine (gly) to be δ¹⁵N_ASP‐PUR, δ¹⁵N_ASP‐PYR, δ¹⁵N_GLN, and δ¹⁵N_GLY +11.9 ± 2.3‰, +7.0 ± 2.0‰, –9.1 ± 2.4‰, and ?31.8 ± 8.9‰, respectively. A subset of twelve amino acids from food and brain had mean δ¹⁵N values of 4.3 ± 3.2‰ and 13.8 ± 3.1‰, respectively, and δ¹⁵N values for gly and asp were 12.6 ± 2.2‰ and 15.2 ± 0.8‰, respectively. A separate isotope tracer study detected no significant turnover of cerebellar DNA in the first six months of life. The large negative δ¹⁵N difference between gly and cerebellar purine N at the gly (7) position implies either that there is a major isotope effect during DNA synthesis, or that in utero gly has a different isotope ratio during rapid growth and metabolism from that in adult life. Our data show that cerebellar nucleoside intramolecular δ¹⁵N values vary over more than 40‰ and are not influenced by dietary protein level or age. Copyright © 2011 John Wiley & Sons, Ltd.     

Liquid chromatography/isotope ratio mass spectrometry measurement of δ13C of amino acids in plant proteins

In archaeological studies, the isotopic enrichment values of carbon and nitrogen in bone collagen give a degree of information on dietary composition. The isotopic enrichments of individual amino acids from bone collagen and dietary protein have the potential to provide more precise information about the components of diet. A limited amount of work has been done on this, although the reliability of these studies is potentially limited by fractionation arising through hydrolysis of whole plant tissue (where reaction between amino acids and carbohydrates may occur) and, for certain amino acids, the use of derivatives (particularly trifluoroacetyl derivatives) for gas chromatography/isotope ratio mass spectrometry (GC/IRMS) analysis. The present study takes the approach of extracting the protein components of plant tissues before hydrolysis and using liquid chromatography/isotope ratio mass spectrometry (LC/IRMS), which does not require derivatisation, for measurement of the isotopic enrichment of the amino acids. The protocol developed offers a methodology for consistent measurement of the δ(13)C values of amino acids, allowing isotopic differences between the individual amino acids from different plant tissues to be identified. In particular, there are highly significant differences between leaf and seed protein amino acids (leaf minus grain) in the cases of threonine (-4.1‰), aspartic acid (+3.5‰) and serine (-3.2‰). In addition to its intended application in archaeology, the technique will be of value in the fields of plant sciences, nutrition and environmental food-web studies.     

Practical recommendations for the reduction of memory effects in compound-specific 15N/14N-ratio analysis of enriched amino acids by gas chromatography/combustion/isotope ratio mass spectrometry

Gas chromatography/combustion/isotope ratio mass spectrometry (GC-C-IRMS) is a highly sensitive approach which allows the analysis of the (13)C/(12)C and (15)N/(14)N isotope composition of amino acids in the range of natural abundance or in slightly (13)C- and (15)N-enriched samples. However, the accuracy of measurements remains a permanent challenge. Here we show the effect of the presence of slightly (15)N-enriched compounds in physiological samples on the accuracy and reproducibility of (15)N-abundances of amino acids within or between analytical runs. We spiked several individual amino acids with the respective (15)N-labelled isotopomer and measured the (15)N/(14)N ratios of other amino acids in the same sample or in the following analytical runs. Intra- and inter-run memory effects can be observed in (15)N/(14)N ratios of amino acids. Sample throughput is reduced when cleaning runs using standard mixtures are required to restore initial conditions after runs of samples with (15)N-enriched analytes. Possible reasons for the observed phenomenon and its implications for work in the lower (15)N-enrichment range (<0.5 APE) are discussed and include different aspects of gas chromatography, derivatisation, and hot catalytic metal surface effects. Results need to be interpreted with caution if complex physiological samples contain (15)N-enriched amino acids beyond 500‰ δ(15)N (~0.18 APE).     

A simple and economical method for the production of 13C,18O-labeled Fmoc-amino acids with high levels of enrichment: applications to isotope-edited IR studies of proteins

Isotope-edited IR of proteins has generated considerable interest. Double labeling with 13C and 18O with high levels of isotopic enrichment is required for residue-specific resolution. Current methods for the preparation of doubly labeled amino acids give modest 18O enrichment, limiting the utility of the approach. We report a simple and economical method for preparing 13C,18O-doubly labeled N-(9-fluorenylmethoxycarbonyl)amino acids with high levels of enrichment for residues that do not require acid-labile side-chain protecting groups.     

Symmetry-based constant-time homonuclear dipolar recoupling in solid state NMR

Constant-time dipolar recoupling pulse sequences are advantageous in structural studies by solid state nuclear magnetic resonance (NMR) with magic-angle spinning (MAS) because they yield experimental data that are relatively insensitive to radio-frequency pulse imperfections and nuclear spin relaxation processes. A new approach to the construction of constant-time homonuclear dipolar recoupling sequences is described, based on symmetry properties of the recoupled dipole-dipole interaction Hamiltonian under cyclic displacements in time with respect to the MAS sample rotation period. A specific symmetry-based pulse sequence called PITHIRDS-CT is introduced and demonstrated experimentally. (13)C NMR data for singly-(13)C-labeled amino acid powders and amyloid fibrils indicate the effectiveness of PITHIRDS-CT in measurements of intermolecular distances in solids. (15)N-detected and (13)C-detected measurements of intramolecular (15)N-(15)N distances in peptides with alpha-helical and beta-sheet structures indicate the utility of PITHIRDS-CT in studies of molecular conformations, especially measurements of backbone psi torsion angles in peptides containing uniformly (15)N- and (13)C-labeled amino acids.     

Diet discrimination factors are inversely related to δ15 N and δ13C values of food for fish under controlled conditions

A recent literature review reported negative relationships between diet discrimination factors (DDFs = X_fish – X_food; X = δ¹⁵N or δ¹³C) and the values of δ¹⁵N and δ¹³C in the food of wild organisms but there has been no laboratory‐based confirmation of these relationships to date. Laboratory reared guppies (Poecilia reticulata) fed a series of diets with a range of δ¹³C (?22.9 to ?6.6‰) and δ¹⁵N (6.5 to 1586‰) values were used to magnify diet‐tissue dynamics in order to calculate DDFs once the fish had achieved equilibrium with each of the diets. Values of DDFs range widely for δ¹⁵N (7.1 to ?849‰) and δ¹³C (1.1 to ?7.0‰) and showed a strong negative correlation with the stable isotope value in the food for δ¹⁵N (slope = ?0.59 ± 0.02, r² = 0.95) and δ¹³C (slope = ?0.56 ± 0.02, r² = 0.94). Based on these relationships, the magnitude of DDF change over environmentally relevant values of δ¹⁵N or δ¹³C would be significant and could confound the interpretation of stable isotopes in the environment. Using highly enriched experimental diets, our study adds to a growing number of studies that undermine the consistent trophic enrichment paradigm with results that demonstrate the currently poor mechanistic understanding of how DDFs arise. The results of our study highlight that the magnitude of the stable isotope values in prey must be considered when choosing DDF values. Future laboratory studies should therefore be directed at uncovering the mechanistic basis of DDFs and, like others before, we recommend the determination of diet‐dependent DDFs under laboratory conditions before modeling dietary proportions or calculating trophic positions. Copyright © 2010 John Wiley & Sons, Ltd.     

Analysis of carbon and nitrogen co-metabolism in yeast by ultrahigh-resolution mass spectrometry applying 13C- and 15N-labeled substrates simultaneously

Alternative metabolic pathways inside a cell can be deduced using stable isotopically labeled substrates. One prerequisite is accurate measurement of the labeling pattern of targeted metabolites. Experiments are generally limited to the use of single-element isotopes, mainly (13)C. Here, we demonstrate the application of direct infusion nanospray, ultrahigh-resolution Fourier transform ion cyclotron resonance-mass spectrometry (FTICR-MS) for metabolic studies using differently labeled elemental isotopes simultaneously--i.e., (13)C and (15)N--in amino acids of a total protein hydrolysate. The optimized strategy for the analysis of metabolism by a hybrid linear ion trap-FTICR-MS comprises the collection of multiple adjacent selected ion monitoring scans. By limiting both the width of the mass range and the number of ions entering the ICR cell with automated gain control, sensitive measurements of isotopologue distribution were possible without compromising mass accuracy and isotope intensity mapping. The required mass-resolving power of more than 60,000 is only achievable on a routine basis by FTICR and Orbitrap mass spectrometers. Evaluation of the method was carried out by comparison of the experimental data to the natural isotope abundances of selected amino acids and by comparison to GC/MS results obtained from a labeling experiment with (13)C-labeled glucose. The developed method was used to shed light on the complexity of the yeast Saccharomyces cerevisiae carbon-nitrogen co-metabolism by administering both (13)C-labeled glucose and (15)N-labeled alanine. The results indicate that not only glutamate but also alanine acts as an amino donor during alanine and valine synthesis. Metabolic studies using FTICR-MS can exploit new possibilities by the use of multiple-labeled elemental isotopes.     

Compound-Specific Isotope Analysis of Amino Acid Labeling with Stable Isotope Nitrogen (15N) in Higher Plants

Individual free amino acid δ¹⁵N values in plant tissue reflect the metabolic pathways involved in their biosynthesis and catabolism and could thus aid understanding of environmental stress and anthropogenic effects on plant metabolism. In this study, compound-specific nitrogen isotope analysis of amino acid by gas chromatography-combustion-isotope ratio mass spectrometry (GC-C-IRMS) was carried out to determine individual free amino acid δ¹⁵N values. High correlations were observed between the δ¹⁵N values obtained by GC-C-IRMS and elemental analyzer-isotope ratio mass spectrometry (EA-IRMS) determinations, and the mean precision measured was better than 1 ‰. Cation-exchange chromatography was employed to purify the sample, and the difference between prior to and following passage through the resin was within 1 ‰. The amino acid δ¹⁵N values of plant leave samples following incubation in ¹⁵N-nitrate at different time points were determined. A typical foliar free amino acid ¹⁵N-enrichment pattern was found, and glutamine was the most rapidly labeled amino acid; other amino acids derived from the GS-GOGAT cycle were also enriched. The pyruvate family amino acids were labeled less quickly followed by the aromatic amino acids. This study highlighted that amino acid metabolism pathways had a major effect on the δ¹⁵N values. With the known amino acid metabolism pathways and δ¹⁵N values determined by the presented method, the influence of various external factors on the metabolic cycling of amino acid can be understood well.

     

Advantages of compound‐specific stable isotope measurements over bulk measurements in studies on plant uptake of intact amino acids

Increasing interest in the ability of plants to take up amino acids has given rise to questions on the accuracy of the commonly used bulk method to measure and calculate amino acid uptake. This method uses bulk measurements of ¹³C and ¹⁵N enrichment in plant tissues after application of dual‐labelled amino acids but some authors have recommended the use of compound‐specific stable isotope (CSI) analysis of the plants' amino acids instead. However, there has never been a direct evaluation of both methods. We conducted a field study applying dual‐labelled (¹³C, ¹⁵N) amino acids (glycine, valine, tyrosine and lysine) to soil of a Plantago lanceolata monoculture. Root and shoot samples were collected 24 h after label application and the isotope composition of the plant tissues was investigated using bulk and CSI measurements. Enrichment of ¹³C in the case of CSI measurements was limited to the applied amino acids, showing that no additional ¹³C had been incorporated into the plants' amino acid pool via the uptake of tracer‐derived C‐fragments. Compared with this rather conservative indicator of amino acid uptake, the ¹³C enrichment of bulk measurements was 8, 5, 1.6 and 6 times higher for fine roots, storage roots, shoot and the whole plant, respectively. These findings show that the additional uptake of tracer‐derived C‐fragments will result in a considerable overestimation of amino acid uptake in the case of bulk measurements. We therefore highly recommend the use of CSI measurements for future amino acid uptake studies due to their higher accuracy. Copyright © 2009 John Wiley & Sons, Ltd.     

Determination of underivatized amino acid delta(13)C by liquid chromatography/isotope ratio mass spectrometry for nutritional studies: the effect of dietary non-essential amino acid profile on the isotopic signature of individual amino acids in fish

This study provides data for the effect of dietary non-essential amino acid composition on the delta(13)C values of individual amino acids in rainbow trout (Oncorhynchus mykiss) using liquid chromatography coupled to isotope ratio mass spectrometry (LC/IRMS). In this experiment, trout were reared either on a control diet or on three experimental diets, differing in the composition of non-essential/conditionally essential amino acids, for a period of 6 weeks. The control diet was a commercial trout starter feed with fish meal as the main protein source. The experimental diets contained no protein, only synthetic amino acids. Diet 1 resembled the composition of fish meal in both essential and non-essential amino acids, Diet 2 had all essential amino acids, but cysteine, glycine, proline and tyrosine were replaced by the corresponding amounts of their precursors, and in Diet 3 all non-essential amino acids were replaced by glutamate. LC/IRMS was used for the determination of delta(13)C values of individual amino acids from diets and tissues without derivatization. Diet affected the delta(13)C of individual amino acids in fish. For fish on Diets 1-3 amino acid delta(13)C values showed a similar trend: phenylalanine showed very little change from diet to body tissue. Arginine, lysine, tyrosine and proline showed strong depletion from diet to body tissue and glycine, alanine, aspartate and serine all showed variable but strong enrichment in (13)C. Improvements are necessary before all amino acid delta(13)C values can be determined; however, this study demonstrates that measuring amino acid isotopic signatures by LC/IRMS is a promising new technique for nutritional physiologists.     

Nitrogen isotopic composition in hair protein is different in liver cirrhotic patients

The stable-isotopic composition of nitrogen (delta15N) or carbon (delta13C) of body tissues depends on the isotopic composition of food sources and on shifts due to isotopic fractionation during metabolism. As little is known about the effects of pathophysiological conditions we measured delta15N and delta13C values in hair and hair amino acids of patients with cirrhosis (n = 21) and compared the results with those of healthy subjects (n = 100) randomly selected from the 1987-1988 VERA German nutrition survey population. Cirrhosis was reflected in lower hair 15N abundances (6.7 vs. 9.9 per thousand delta15N; P < 0.001) whereas hair 13C abundances did not differ from healthy subjects (-19.4 vs. -19.6 per thousand 13C). Distinct patterns of delta15N and delta13C values were measured in hair amino acids. The delta15N values of phenylalanine were significantly higher in cirrhotics (P < 0.001). With the exception of isoleucine, threonine, and proline all other measured amino acids showed lower delta15N values than healthy subjects (P < 0.001). Lower hair delta15N values were associated with cirrhotic liver disease which suggests that under this condition the altered liver amino acid metabolism affects the nitrogen isotopic composition of the amino acids used for hair protein synthesis. It remains to be determined in controlled studies whether the altered nitrogen isotopic composition directly reflects the pathophysiological condition or is related to differences in dietary protein intake from plant or animal food sources.     

Effects of temperature on isotopic enrichment in Daphnia magna: implications for aquatic food-web studies

Laboratory experiments were conducted with Daphnia magna and Hyalella sp. grown on a single food source of known isotopic composition at a range of temperatures spanning the physiological optima for each species. Daphnia raised at 26.5 degrees C were enriched in delta(13)C and delta(15)N by 3.1 and 2.8 per thousand, respectively, relative to diet. Daphnia raised at 12.8 degrees C were enriched 1.7 and 5.0 per thousand in delta(13)C and delta(15)N, respectively. Results imply a significant negative relationship between the delta(13)C and delta(15)N of primary consumers when a temperature gradient exists. Similar responses were observed for Hyalella. Results indicate a general increase in delta(13)C enrichment and decrease in delta(15)N enrichment as temperature rises. Deviations from the commonly applied isotopic enrichment values used in aquatic ecology were attributed to changes in temperature-mediated physiological rates. Field data from a variety of sources also showed a general trend toward delta(13)C enrichment with increasing temperature in marine and lacustrine zooplankton. Multivariate regression models demonstrated that, in oligotrophic and mesotrophic lakes, zooplankton delta(13)C was related to lake-specific POM delta(13)C, lake surface temperature and latitude. Temperature-dependent isotopic separation (enrichment) between predator and prey should be taken into consideration when interpreting the significance of isotopic differences within and among aquatic organisms and ecosystems, and when assigning organisms to food-web positions on the basis of observed isotope values.     

Nitrogen isotopic analyses by isotope-ratio-monitoring gas chromatography / mass spectrometry

Amino acids containing natural-abundance levels of ¹⁵N were derivatized and analyzed isotopically using a technique in which individual compounds are separated by gas chromatography, combusted on-line, and the product stream sent directly to an isotope-ratio mass spectrometer. For samples of N₂ gas, standard deviations of ratio measurement were better than 0.1‰ (Units for δ are parts per thousand or per million (‰).) for samples larger than 400 pmol and better than 0.5‰ for samples larger than 25 pmol (0.1‰ ¹⁵N is equivalent to 0.00004 atom % ¹⁵N). Results duplicated those of conventional, batchwise analyses to within 0.05‰. For combustion of organic compounds yielding CO₂/N₂ ratios between 14 and 28, in particular for N-acetyl n-propyl derivatives of amino acids, δ values were within 0.25‰ of results obtained using conventional techniques and standard deviations were better than 0.35‰. Pooled data for measurements of all amino acids produced an accuracy and precision of 0.04 and 0.23‰, respectively, when 2 mnol of each amino acid was injected on column and 20% of the stream of combustion products was delivered to the mass spectrometer.     

Supplemental Feeding of Laying Hens with Wood Vinegar to Decrease the Ratio of n-6 to n-3 Fatty Acids in Eggs

A balanced ratio of fatty acids n-6 to n-3 in chicken eggs is important for health and to help prevent and manage obesity and other diseases. Traditionally, fish oil or flax seed has been utilized as feed additives to decrease the ratio of n-6 to n-3(n-6:n-3) fatty acids in eggs. The hull of spina date seed(HSDS) is a common agricultural waste product in China, from which wood vinegar(HSDSWV) may be derived. This study evaluated HSDSWV as a supplement in hen feeds to improve the quality of eggs and decrease the ratio of fatty acids n-6:n-3. HSDSWV was obtained via carbonization, and refined. Six concentrations(nil to 0.5%) of HSDSWV were prepared and fed to 6 hen groups, respectively, for 50 d. The fatty acids of the hen's egg yolks were analyzed by gas chromatography/electron ionization-mass spectrometry(GC/EI-MS) in the selected ion monitoring(SIM) mode. The 0.2% HSDSWV resulted in the best egg yolk quality, with a lower percentage of linoleic acid(C18:2n6) and higher percentages of cis-5,8,11,14,17-eicosapentaenoic acid(C20:5n3) and cis-4,7,10,13,16,19-docosahexaenoic acid(C22:6n3), and thus a lower n-6:n-3 ratio compared with the other HSDSWV concentrations. In addition, the eggs contained higher levels of yolk fat and egg yolk than the controls did. In conclusion, to modify the fatty acid composition of hens' eggs and obtain a balanced ratio of n-6:n-3, 0.2% HSDSWV may be considered suitable as a dietary supplement in hens' feed.     

气相色谱-燃烧-同位素比值质谱法分析氨基酸氮稳定同位素并初步评估水生生物体营养级

氨基酸稳定氮同位素(δ15 N)分析能准确有效地评估生物体的营养级以及氮在食物链中的流动.本研究优化了氨基酸氮同位素的分析方法:样品在酸性条件下水解后,释放出的蛋白质氨基酸经阳离子交换树脂纯化后,衍生为对应的N-新戊酞基,O-异丙醇(N-pivaloyl-isopropyl,NPP)酯,利用气相色谱-燃烧-同位素比值质谱仪(Gas chromatography-combustion-isotope ratio mass spectrometry,GC-C-IRMS)测定其δ15 N.经非极性气相色谱柱DB-5ms分离后,13种氨基酸NPP酯衍生物均可得到良好的基线分离.在样品量不低于20 ng N条件下,GC-C-IRMS方法的精密度优于1‰,测得的δ15 N值与EA-IRMS法测得的δ15 N值没有明显差异.阳离子树脂纯化前后各氨基酸δ15 N值差异低于1‰,表明没有产生明显的同位素分馏.采用本方法成功地估算了阿哈湖生态系统中常见水生生物的营养级,可作为研究氨基酸代谢以及生态系统特征的新方法.     

Differences in the stable isotope signatures of seabird egg membrane and albumen – implications for non‐invasive studies

In many bird species, egg membranes can be obtained non‐invasively after the chicks have hatched, and stable isotope analysis of egg membranes can be used to study the diet and foraging distribution of these birds during egg formation. It has been suggested that the enrichment factors of albumen and egg membranes differ for ¹³C, but are similar for ¹⁵N. In this study, we compared carbon and nitrogen stable isotopes of the membranes and albumen of individual eggs of three wild seabird species, the Southern Rockhopper penguin Eudyptes chrysocome, the Imperial shag Phalacrocorax atriceps albiventer, and the Thin‐billed prion Pachyptila belcheri. We also included chicken eggs for comparison. Egg membranes were generally enriched in ¹³C, compared with albumen. The difference varied between species, with 2.1‰ in Rockhopper penguins, 1.6‰ in Imperial shags, but only 0.5‰ in Thin‐billed prions and 0.4‰ in chicken eggs. Egg membranes were slightly enriched in ¹⁵N in Imperial shags (0.9‰) and chickens (0.5‰), compared with albumen, while there was no difference for Thin‐billed prions and Rockhopper penguins. The isotopic values of carbon and nitrogen were correlated between albumen and egg membranes of individual eggs, suggesting that egg membranes can be used reliably to investigate trophic differences between individuals, seasons or colonies. Species‐specific mathematical corrections could be used to compare results across studies that use different egg components. Copyright © 2009 John Wiley & Sons, Ltd.     

Fractionation and metabolic turnover of carbon and nitrogen stable isotopes in black fly larvae

Diet-tissue fractionation factors and metabolic turnover rates of delta15N and delta13C were assessed in laboratory-reared black fly (Simulium vittatum IS-7) larvae fed isotopically distinct diets. Five treatments consisted of using food with different delta15N signatures throughout the experiments (19-26 days), a sixth shifted from a low to high delta15N signature diet (uptake) on day 14, and the last shifted from a high to low delta15N signature diet (elimination) on day 14. In the larvae, diet-tissue fractionation factors for delta13C, which were in steady state with food, ranged from -0.61 to 2.0, with a median of 1.87. The delta15N diet-tissue fractionation factors were mostly negative, ranging from +2.85 to -24.96 per thousand, with a single positive value from the elimination treatment in which larval delta15N did not achieve steady state with the food. Diet-tissue fractionation factors also had a significant negative relationship (r2 = 0.98) with delta15N values in the food suggesting that nitrogen diet-tissue fractionation factors are 15N concentration-dependent. The delta15N of shed head capsules and feces were enriched in 15N and could be mechanisms for elimination of 15N by the larvae. For delta15N, metabolic turnover values based on the Hesslein model were highly consistent (0.40 to 0.43 delta15N*day(-1)) between uptake and elimination phases and across experiments and were an order of magnitude greater than growth rates. The rapid turnover of nitrogen in black fly larvae, which was orders of magnitude greater than measured in vertebrates, makes them an excellent indicator of short-term changes in nitrogen inputs to aquatic systems.     

Metabolic turnover rates of carbon and nitrogen stable isotopes in captive juvenile snakes

Metabolic turnover rates (m) of δ¹⁵N and δ¹³C were assessed in different tissues of newly hatched captive‐raised corn snakes (Elaphe guttata guttata) fed maintenance diets consisting of earthworms (Eisenia foetida) that varied substantially in δ¹⁵N (by 644‰) and δ¹³C (by 5.0‰). Three treatments were used during this 144 day experiment that consisted of the same diet throughout (control), shifting from a depleted to an enriched stable isotope signature diet (uptake), and shifting from an enriched to depleted stable isotope signature diet (elimination). Values of δ¹³C in the liver, blood, and muscle of the control snakes reached equilibrium with and were, respectively, 1.73, 2.25 and 2.29 greater than in their diet, this increase is called an isotopic discrimination factor (Δδ¹³C = δ¹³C_snake ? δ¹³C_food). Values of δ¹⁵N in snake tissues did not achieve equilibrium with the diets in any of the exposures and thus Δ¹⁵N could not be estimated. Values of metabolic turnover rates (m) for δ¹³C and δ¹⁵N were greater in liver than in muscle and blood, which were similar, and relative results remained the same if the fraction of ¹⁵N and ¹³C were modeled. Although caution is warranted because equilibrium values of stable isotopes in the snakes were not achieved, values of m were greater for δ¹³C than δ¹⁵N, resulting in shorter times to dietary equilibrium for δ¹³C upon a diet shift, and for both stable isotopes in all tissues, greater during an elimination than in an uptake shift in diet stable isotope signature. Multiple explanations for the observed differences between uptake and elimination shifts raise new questions about the relationship between animal and diet stable isotope concentrations. Based on this study, interpretation of feeding ecology using stable isotopes is highly dependent on the kind of stable isotope, tissue, direction of diet switch (uptake versus elimination), and the growth rate of the animal. Copyright © 2009 John Wiley & Sons, Ltd.     

Carbon isotope analysis of bulk keratin and single amino acids from British and North American hair

The reconstruction of ancient diets using isotopic measurements of bone collagen, and other tissues, which survive in archaeological contexts, relies on known isotopic relationships between diet and body tissues. Examination of these relationships often requires the study of modern human and animal subjects. While hair keratin can act as a useful proxy for bone collagen in isotopic studies on living humans, where it is inappropriate to sample tissues such as collagen, it can, in addition, act as a chronological indicator of dietary change. This study investigates hair keratin delta13C values from current residents of the UK and the USA. Residents in the USA showed a clear bulk hair delta13C enrichment of approximately 3 per thousand over UK individuals, attributed to an elevated C4 dietary input from maize fed to livestock in North America. The keratin delta13C of subjects who moved between the UK and USA showed a pronounced change after relocation, taking approximately 4 months to reach isotopic equilibrium. To investigate these differences further, we measured delta13C values of dispensable and indispensable amino acids as a group, and selected individual amino acids. As a group, enrichment of dispensable amino acids compared with indispensable amino acids occurred in samples from both continents, averaging 7.2 per thousand in the UK and 7.9 per thousand in the USA. Dispensable and indispensable amino acids, as well as all individual amino acids measured, were enriched in samples from the USA compared with those from the UK.