A hypothesis-testing framework for studies investigating ontogenetic niche shifts using stable isotope ratios

Ontogenetic niche shifts occur across diverse taxonomic groups, and can have critical implications for population dynamics, community structure, and ecosystem function. In this study, we provide a hypothesis-testing framework combining univariate and multivariate analyses to examine ontogenetic niche shifts using stable isotope ratios. This framework is based on three distinct ontogenetic niche shift scenarios, i.e., (1) no niche shift, (2) niche expansion/reduction, and (3) discrete niche shift between size classes. We developed criteria for identifying each scenario, as based on three important resource use characteristics, i.e., niche width, niche position, and niche overlap. We provide an empirical example for each ontogenetic niche shift scenario, illustrating differences in resource use characteristics among different organisms. The present framework provides a foundation for future studies on ontogenetic niche shifts, and also can be applied to examine resource variability among other population sub-groupings (e.g., by sex or phenotype).     

An HPLC/push-pull perfusion technique for investigating peptide metabolism

We have combined the push-pull perfusion of radiolabelled peptide, with reverse phase--HPLC procedures as a valuable method for monitoring peptide metabolism in the CNS. This model not only permits studies on metabolism of neuropeptides at specific sites in the CNS, but more importantly, allows (unlike in vitro methods) the study to be made under the appropriate physiological conditions. We have demonstrated unambiguously for the first time, the metabolism of CCK-8 in situ.     

A compartmental model of magnesium metabolism in healthy men based on two stable isotope tracers

The aim of this study was to build a compartmental model of magnesium (Mg) kinetics by using data collected from six healthy adult men after oral administration of 26Mg and intravenous administration of 25Mg. Blood, urine, and feces were collected for 12 days after administration of the isotopes. Isotopic ratios were determined by inductively coupled plasma-mass spectrometry. Data were analyzed for each subject using SAAMII. We began with a compartmental model previously proposed (Avioli LV and Berman M. J Appl Physiol 21: 1688-1694, 1966) and developed an alternative approach to resolve the discrepancy between model-predicted curves and experimental data. This analysis enables the exploration of 25% of total body Mg that exchanges rapidly from plasma compartment with two extraplasma pools. One of the extraplasma compartments contains 80% of the exchangeable Mg with a transport rate of 48 +/- 13 mg/h. The second exchanges 179 +/- 88 mg of Mg/h. The model permitted estimation of kinetic parameters as well as fractional Mg absorption and fecal endogenous excretion.     

定量稳定性同位素探针技术及其在微生物生态学研究中的应用

定量稳定性同位素探针技术(qSIP)是将生态系统中微生物分类性状与代谢功能联系起来的有效工具,能够定量测定特定环境中单个微生物类群暴露于同位素示踪剂后微生物代谢活动或生长速率.qSIP技术采用定量PCR与高通量测序技术并结合稳定同位素探针技术(SIP),通过向环境样品添加标记底物进行培养,提取微生物生物标记物,利用超高...     

Kinetic study on p-Tyramine metabolism in humans using stable isotope-labeled tracers

A sensitive method for the determination of p-tyramine was developed using gas chromatographychemical ionization mass spectrometry. This method was combined with a stable isotope tracer technique to study p-tyramine metabolism in humans. [²H]₅-Phenylalanine was administered orally to men (5 mg/kg) as a tracer and the amounts of [²H]₄-p-tyramine excreted into urine were determined at each hour. Excretion of [²H]₄-p-tyramine was maximal, between 1 and 2 hours following administration, at about 15 ng/kg·h in healthy adult men. Possible application in the study of metabolic disorders in human was demonstrated.     

A stable isotope assay for phenprocoumon and its metabolites

A gas chromatographic/mass spectrometric assay for quantifying phenprocoumon and its 4'-, 6-, 7- and 8-hydroxy metabolites in microsomal preparations is described. This assay which uses deuterium-labeled analogs of the phenprocoumon metabolites as internal standards has a lower limit of quantitation of 20 ng ml-1. Diazomethane is used to derivatize both metabolites and parent compound yielding along with the expected 4-methoxy derivative a minor amount of the 2-methoxychromone. Resolution of the methylated metabolites is accomplished by capillary gas chromatography.     

稳定同位素技术在植物水分利用研究中的应用

近20a稳定同位素技术在植物生态学研究中的应用得到了长足发展,使得对植物与水分关系也有了更深一步的了解。介绍稳定同位素性碳、氢、氧同位素在研究植物水分关系中的应用及进展,以期能为国内植物水分利用研究提供参考。由于植物根系从土壤中吸收水分时并不发生同位素分馏,对木质部水分同位素分析有助于对植物利用水分来源,生态系统中植物对水分的竞争和利用策略的研究,更好地了解生态系统结构与功能。稳定碳同位素作为植物水分利用效率的一个间接指标,在不同水分梯度环境中,及植物不同代谢产物与水分关系中有着广泛的应用。同位素在土壤-植被-大气连续体水分中的应用,有助于了解生态系统的水分平衡。随着稳定同位素方法的使用,植物与水分关系的研究将取得更大的进展。     

Measurement of parameters of cholic acid kinetics in plasma using a microscale stable isotope dilution technique: application to rodents and humans.

A stable isotope dilution method is described that allows measurement of cholic acid (CA) kinetics, that is, pool size, fractional turnover rate (FTR), and synthesis rate in mice, rats, and humans. Decay of administered [2,2,4,4-2H4]CA enrichment was measured in time in 50-microl plasma samples by gas-liquid chromatography/electron capture negative chemical ionization-mass spectrometry, applying the pentafluorobenzyl-trimethylsilyl derivative. The kinetic data expressed species-dependent differences. The CA pool sizes were 16.8 +/- 2.1, 10.6 +/- 1.2, and 2.4 +/- 0.7 micromol/100 g body weight for mice, rats, and humans, respectively. The FTR values were 0.44 +/- 0.03, 0.88 +/- 0.10, and 0.46 +/- 0.14 per day for mice, rats, and humans. The corresponding synthesis rates were 7.3 +/- 1.6, 9.3 +/- 0.1, and 1.0 +/- 0.2 micromol/100 g body weight per day. The human data agreed well with literature data obtained by conventional isotope dilution techniques. For rats and mice these are the first reported isotope dilution data. The method was validated by confirmation of isotopic equilibrium between biliary CA and plasma CA in the rat. Its applicability was demonstrated by the observation of increased CA FTR and CA synthesis rate in rats fed cholestyramine, which is known to increase fecal bile acid excretion. The presented stable isotope dilution method enables the determination of CA kinetic parameters in small plasma samples. The method can be applied in unanesthetized rodents with an intact enterohepatic circulation and may also be valuable when studying the development of human neonatal bile acid kinetics.     

稳定同位素技术在土壤N2O溯源研究中的应用

氧化亚氮(N₂O)作为三大温室气体之一,追溯其来源已经成为研究热点,稳定同位素技术在土壤N₂O来源追踪方面已得到广泛应用.本文概述了产生N₂O的微生物、产生过程及其主要影响因素,综述了国内外关于利用N₂O同位素特征值δ¹⁵N、δ¹⁸O和SP值(N₂O分子内¹⁵N在不同位置上的位嗜值)分析其来源的研究进展,以期为今后相关研究提供参考.其中关于SP的研究在国内尚属初级阶段,因此本文重点介绍国外SP在其溯源上的研究情况,并对今后国内相关研究提出一点看法.     

The impact of metabolism on stable isotope dynamics: a theoretical framework

Stable isotope analysis is a powerful tool used for reconstructing individual life histories, identifying food-web structures and tracking flow of elemental matter through ecosystems. The mechanisms determining isotopic incorporation rates and discrimination factors are, however, poorly understood which hinders a reliable interpretation of field data when no experimental data are available. Here, we extend dynamic energy budget (DEB) theory with a limited set of new assumptions and rules in order to study the impact of metabolism on stable isotope dynamics in a mechanistic way. We calculate fluxes of stable isotopes within an organism by following fluxes of molecules involved in a limited number of macrochemical reactions: assimilation, growth but also structure turnover that is here explicitly treated. Two mechanisms are involved in the discrimination of isotopes: (i) selection of molecules occurs at the partitioning of assimilation, growth and turnover into anabolic and catabolic sub-fluxes and (ii) reshuffling of atoms occurs during transformations. Such a framework allows for isotopic routing which is known as a key, but poorly studied, mechanism. As DEB theory specifies the impact of environmental conditions and individual state on molecule fluxes, we discuss how scenario analysis within this framework could help reveal common mechanisms across taxa.     

A neutron activation technique for manganese measurements in humans

Manganese (Mn) is an essential element for humans, animals, and plants and is required for growth, development, and maintenance of health. Studies show that Mn metabolism is similar to that of iron, therefore, increased Mn levels in humans could interfere with the absorption of dietary iron leading to anemia. Also, excess exposure to Mn dust, leads to nervous system disorders similar to Parkinson's disease. Higher exposure to Mn is essentially related to industrial pollution. Thus, there is a benefit in developing a clean non-invasive technique for monitoring such increased levels of Mn in order to understand the risk of disease and development of appropriate treatments.To this end, the feasibility of Mn measurements with their minimum detection limits (MDL) has been reported earlier from the McMaster group. This work presents improvement to Mn assessment using an upgraded system and optimized times of irradiation and counting for induced gamma activity of Mn. The technique utilizes the high proton current Tandetron accelerator producing neutrons via the ⁷Li(p,n)⁷Be reaction at McMaster University and an array of nine NaI (Tl) detectors in a 4π geometry for delayed counting of gamma rays. The neutron irradiation of a set of phantoms was performed with protocols having different proton energy, current and time of irradiation. The improved MDLs estimated using the upgraded set up and constrained timings are reported as 0.67 μgMn/gCa for 2.3 MeV protons and 0.71 μgMn/gCa for 2.0 MeV protons. These are a factor of about 2.3 times better than previous measurements done at McMaster University using the in vivo set-up. Also, because of lower dose-equivalent and a relatively close MDL, the combination of: 2.0 MeV; 300 μA; 3 min protocol is recommended as compared to 2.3 MeV; 400 μA; 45 s protocol for further measurements of Mn in vivo.     

Development of stable isotope dilution assays for ochratoxin A in blood samples

Two new stable isotope dilution assays were developed for the quantification of ochratoxin A in human blood samples for exposure studies. The methods based on two different sample extraction and cleanup procedures including liquid–liquid extraction with following immunoaffinity chromatography (IA) as well as a dispersive solid-phase extraction (DSPE) method. For detection, LC–MS/MS was applied. For the first time, exact quantitation of the reference compound ochratoxin A was performed by quantitative NMR spectroscopy (qNMR). Additionally, a comparison of different blood-drawing procedures revealed no differences for heparin plasma and serum whereas citrate plasma gave significantly lower results for the mycotoxin. Limits of detection (LOD: 0.02 ng/g (IA) vs 0.03 ng/g (DSPE)), limits of quantification (LOQ: 0.07 ng/g (IA) vs 0.08 ng/g (DSPE)), relative recovery (?94%), precision, and linearity indicated excellent performance of the developed methods.     

Effect of exercise on protein metabolism in humans as explored with stable isotopes

Exercising for 3.75 h on a treadmill at 50% VO2 max in the fed state induced an increased excretion of 71 mg nitrogen/kg over the 18 h after exercise. However, measurements of the time course of changes in 13CO2 excretion from ingested [1-13C]leucine indicated that all of this increased nitrogen production occurs during the exercise period. Because of the reduced renal clearance and slow turnover of the urea pool, urea excretion lags behind urea production. Measurements of nitrogen flux from the plateau labeling of urinary ammonia achieved by repeated oral doses of 15N-labeled glycine indicated that the nitrogen loss resulted from an increase in protein degradation and a decrease in protein synthesis. Further studies with [1-13C]leucine indicated that a 2-h treadmill exercise induced an increase in the nitrogen loss from 5.4 to 16 mg . kg-1 . h-1 measured with a primed constant infusion of [1-13C]leucine. This resulted from a fall in whole-body protein synthesis. Glucose given at the rate of 0.88 g . kg-1 . h-1 depressed the rate of whole-body protein degradation and appeared to suppress the exercise-induced increase in nitrogen excretion. When leucine oxidation rates were measured at increasing work rates, a linear relationship between percentage of VO2 max and leucine oxidation was observed up to 89% VO2 max when 54% of the flux of leucine was oxidized. These changes may involve nonmuscle as well as muscle tissue. Thus the source of the increased nitrogen losses is probably liver. In muscle, protein degradation is actually decreased judged by methylhistidine excretion, whereas in liver, protein degradation may be increased. Also the fall in whole-body protein synthesis may reflect changes in nonmuscle tissues because in running rats protein synthesis in muscle is maintained. As far as leucine metabolism is concerned, because the increase in leucine oxidation occurs when leucine and its keto acid concentration falls, exercise must specifically activate the 2-oxoacid dehydrogenase.     

Analysis of mitochondrial metabolism in situ: Combining stable isotope labeling with selective permeabilization

To date, it is well-established that mitochondrial dysfunction does not only play a vital role in cancer but also in other pathological conditions such as neurodegenerative diseases and inflammation. An important tool for the analysis of cellular metabolism is the application of stable isotope labeled substrates, which allow for the tracing of atoms throughout metabolic networks. While such analyses yield very detailed information about intracellular fluxes, the determination of compartment specific fluxes is far more challenging. Most approaches for the deconvolution of compartmented metabolism use computational models whereas experimental methods are rare. Here, we developed an experimental setup based on selective permeabilization of the cytosolic membrane that allows for the administration of stable isotope labeled substrates directly to mitochondria. We demonstrate how this approach can be used to infer metabolic changes in mitochondria induced by either chemical or genetic perturbations and give an outlook on its potential applications.     

A new radiographic cadaver injection technique for investigating the lymphatic system

Studies of the gross anatomy of the lymphatic system are few and far between when compared with those of other vascular systems. Our knowledge of the anatomy of the lymphatic system is so limited that it seems vastly inadequate in explaining the clinical manifestations caused by its disorder. This study has developed an effective method to identify the lymphatics using hydrogen peroxide, to demonstrate the lymphatic vessels radiographically using a lead oxide suspension, and to dissect them out in adult human cadavers.     

Analytical error in stable isotope ecology

The increasing popularity of stable isotope analysis (SIA) as an ecological research tool and the ease of automated analysis have created a knowledge gap between ecologists using SIA and the operators of isotope ratio mass spectrometry (IRMS) equipment. This has led to deterioration in the understanding of IRMS methodology and its proper dissemination in the ecological literature. Of 330 ecological research papers surveyed, 63 (19%) failed to report any form of analytical error associated with IRMS. Of the 267 papers that reported analytical error, there was considerable variation both in the terminology and approach used to quantify and describe error. Internal laboratory standards were often used to determine the analytical error associated with IRMS, so chosen because they are homogenous and have isotopic signatures that do not vary over time. We argue that true ecological samples collected in the field are complex bulk mixtures and often fail to adhere to these two criteria. Hence the analytical error associated with samples is potentially greater than that of standards. A set of standard data run over time with a precision typically reported in the ecological literature (1 standard deviation: 1SD=0.26‰) was simulated to determine the likelihood of spurious treatment effects depending on timing of analysis. There was a 90% likelihood of detecting a significant difference in the stable nitrogen ratio of a single sample (homogenized bovine liver) run in two time periods when n>30. Minor protocol adjustments, including the submission of blind replicates by researchers, random assignment of sample repeats within a run by analytical labs, and reporting 1SD of a single sample analyzed both within and between runs, will only serve to strengthen the interpretation of true ecological processes by both researchers and reviewers.     

Effects of live high, train low hypoxic exposure on lactate metabolism in trained humans.

We determined the effect of 20 nights of live high, train low (LHTL) hypoxic exposure on lactate kinetics, monocarboxylate lactate transporter proteins (MCT1 and MCT4), and muscle in vitro buffering capacity (betam) in 29 well-trained cyclists and triathletes. Subjects were divided into one of three groups: 20 consecutive nights of hypoxic exposure (LHTLc), 20 nights of intermittent hypoxic exposure [four 5-night blocks of hypoxia, each interspersed with 2 nights of normoxia (LHTLi)], or control (Con). Rates of lactate appearance (Ra), disappearance (Rd), and oxidation (Rox) were determined from a primed, continuous infusion of l-[U-14C]lactic acid tracer during 90 min of steady-state exercise [60 min at 65% peak O2 uptake (VO(2 peak)) followed by 30 min at 85% VO(2 peak)]. A resting muscle biopsy was taken before and after 20 nights of LHTL for the determination of betam and MCT1 and MCT4 protein abundance. Ra during the first 60 min of exercise was not different between groups. During the last 25 min of exercise at 85% VO(2 peak), Ra was higher compared with exercise at 65% of VO(2 peak) and was decreased in LHTLc (P < 0.05) compared with the other groups. Rd followed a similar pattern to Ra. Although Rox was significantly increased during exercise at 85% compared with 65% of VO(2 peak), there were no differences between the three groups or across trials. There was no effect of hypoxic exposure on betam or MCT1 and MCT4 protein abundance. We conclude that 20 consecutive nights of hypoxia exposure decreased whole body Ra during intense exercise in well-trained athletes. However, muscle markers of lactate metabolism and pH regulation were unchanged by the LHTL intervention.     

TDFCAM: A method for estimating stable isotope trophic discrimination in wild populations

1. Stable isotope mixing models (SIMMs) are widely used for characterizing wild animal diets. Such models rely upon using accurate trophic discrimination factors (TDFs) to account for the digestion, incorporation, and assimilation of food. Existing methods to calculate TDFs rely on controlled feeding trials that are time-consuming, often impractical for the study taxon, and may not reflect natural variability of TDFs present in wild populations.
2. We present TDF_CAM as an alternative approach to estimating TDFs in wild populations, by using high-precision diet estimates from a secondary methodological source—in this case nest cameras—in lieu of controlled feeding trials, and provide a framework for how and when it should be applied.
3. In this study, we evaluate the TDF_CAM approach in three datasets gathered on wild raptor nestlings (gyrfalcons Falco rusticolus; peregrine falcons Falco perigrinus; common buzzards Buteo buteo) comprising contemporaneous δ¹³C & δ¹⁵N stable isotope data and high-quality nest camera dietary data. We formulate Bayesian SIMMs (BSIMMs) incorporating TDFs from TDF_CAM and analyze their agreement with nest camera data, comparing model performance with those based on other relevant TDFs. Additionally, we perform sensitivity analyses to characterize TDF_CAM variability, and identify ecological and physiological factors contributing to that variability in wild populations.
4. Across species and tissue types, BSIMMs incorporating a TDF_CAM outperformed any other TDF tested, producing reliable population-level estimates of diet composition. We demonstrate that applying this approach even with a relatively low sample size (n < 10 individuals) produced more accurate estimates of trophic discrimination than a controlled feeding study conducted on the same species. Between-individual variability in TDF_CAM estimates for ∆¹³C & ∆¹⁵ N increased with analytical imprecision in the source dietary data (nest cameras) but was also explained by natural variables in the study population (e.g., nestling nutritional/growth status and dietary composition).
5. TDF_CAM is an effective method of estimating trophic discrimination in wild animal populations. Here, we use nest cameras as source dietary data, but this approach is applicable to any high-accuracy method of measuring diet, so long as diet can be monitored over an interval contemporaneous with a tissue's isotopic turnover rate.