Development of the stable isotope tracer approach for studies of copper turnover in the rat and mouse

The stable isotope tracer approach was explored for long-term investigations of copper turnover in the adult rat and mouse, with inductively coupled plasma mass spectrometry for isotope measurements. The isotopic measurement method permitted precision and accuracy of <1.0%, with an overall sample blank of <0.05 microg copper. Rats were fed a copper-deficient diet and deionized water with (+Cu) or without (-Cu) copper (20 microg/ml). Both groups underwent a single-day replacement of drinking water with 20 microg/ml of (65)Cu. Compared with the baseline isotope ratio ((65)Cu/(63)Cu) of 0.462 +/- 0.002, blood plasma ratios for the +Cu group on days 2, 7, and 14 postdosing were 0.702 +/- 0.021, 0.557 +/- 0.004, and 0.474 +/- 0.001, respectively. The corresponding data for liver were 1.652 +/- 0.018, 0.560 +/- 0.005, and 0.482 +/- 0.001, respectively. For the -Cu group, respective plasma ratios were 1.580 +/- 0.04. 0.917 +/- 0.02, and 0.664 +/- 0.01 for days 2, 7, and 14 postdosing, and the ratios for liver were 0.987 +/- 0.02, 0.876 +/- 0.04, and 0.739 +/- 0.03. Mice previously made copper deficient to varying degrees were given a single-day replacement with the label. When the 24-hour postdosing isotope ratios in the livers of these mice were correlated with the activity of plasma ceruloplasmin, a negative correlation (r = -0.85) was observed. Isotope enrichment in both rats and mice was greater in the copper-deficient animals compared with the controls.     

Tritium isotope effects in the reaction catalyzed by 4-hydroxyphenylpyruvate dioxygenase from pseudomonas sp. strain P.J. 874

Tritium isotope effects in the reaction catalyzed by 4-hydroxyphenylpyruvate dioxygenase (4-hydroxyphenyl-pyruvate:oxygen oxidoreductase (hydroxylating, decarboxylating), EC 1.13.11.27) from Pseudomonas sp. strain P.J. 874 were studied with 14C- and different 3H-labelled 4-hydroxyphenylpyruvate. Tritium of ring-2,6-3H2-labelled substrate was released into water in 1:2 stoichiometry to 14CO2 formation. The tritium release from ring-3,5-3H2- and side chain-3-3H1-labelled 4-hydroxyphenylpyruvate was low as compared with 14CO2 formation. The apparent tritium isotope effects were below two, as judged by comparison of 3H/14C ratios of 4-hydroxyphenylpyruvate and homogentisate. The ratios showed no dependence on oxygen concentrations between 1 and 21% in the gas phase. Thus, a tritium assay can be used to determine the activity of 4-hydroxyphenylpyruvate dioxygenase. Apparently, none of the substrate hydrogens is involved in any rate-limiting step up to the first irreversible step. enol-4-Hydroxyphenylpyruvate was excluded as the active substrate tautomer.     

Modelling non‐steady‐state isotope enrichment of leaf water in a gas‐exchange cuvette environment

The combined use of a gas‐exchange system and laser‐based isotope measurement is a tool of growing interest in plant ecophysiological studies, owing to its relevance for assessing isotopic variability in leaf water and/or transpiration under non‐steady‐state (NSS) conditions. However, the current Farquhar & Cernusak (F&C) NSS leaf water model, originally developed for open‐field scenarios, is unsuited for use in a gas‐exchange cuvette environment where isotope composition of water vapour (δ_v) is intrinsically linked to that of transpiration (δ_E). Here, we modified the F&C model to make it directly compatible with the δ_v–δ_E dynamic characteristic of a typical cuvette setting. The resultant new model suggests a role of ‘net‐flux’ (rather than ‘gross‐flux’ as suggested by the original F&C model)‐based leaf water turnover rate in controlling the time constant (τ) for the approach to steady sate. The validity of the new model was subsequently confirmed in a cuvette experiment involving cotton leaves, for which we demonstrated close agreement between τ values predicted from the model and those measured from NSS variations in isotope enrichment of transpiration. Hence, we recommend that our new model be incorporated into future isotope studies involving a cuvette condition where the transpiration flux directly influences δ_v. There is an increasing popularity among plant ecophysiologists to use a gas‐exchange system coupled to laser‐based isotope measurement for investigating non‐steady state (NSS) isotopic variability in leaf water (and/or transpiration); however, the current Farquhar & Cernusak (F&C) NSS leaf water model is unsuited for use in a gas‐exchange cuvette environment due to its implicit assumption of isotope composition of water vapor (δ_v) being constant and independent of that of transpiration (δ_E). In the present study, we modified the F&C model to make it compatible with the dynamic relationship between δ_v and δ_E as is typically associated with a cuvette setting. Using an experiment conducted on cotton leaves, we show that the modified NSS model performed well in predicting the time constant for the exponential approach of leaf water toward steady state under cuvette conditions. Such a result demonstrates the applicability of this new model to gas‐exchange cuvette conditions where the transpiration flux directly influences δ_v, and therefore suggests the need to incorporate this model into future isotope studies that employ a laser‐cuvette coupled system.     

Biosynthesis of the beta-diketones of barley spike epicuticular wax.

[1-¹⁴C]acetate and [2-¹⁴C]acetate were incorporated into the β-diketones of barley spike epicuticular wax via the peduncle. Utilizing column chromatography with dry copper acetate, the β-diketones were isolated and the labeling pattern in the hentriacontan-14, 16-dione determined after its degradation. A modified iodoform procedure was used to give myristic and palmitic acids. Radio-gas chromatography was then performed on the products of chemical α-oxidation of the separated fatty acids. This procedure, in effect, gave the specific activity of every carbon atom of hentriacontan-14,16-dione except carbon-1 to carbon-5 (from myristic acid) and carbon-27 to carbon-31 (from palmitic acid) for each labeled substrate. The specific activity of carbon-15 was determined by an indirect method. On the basis of these data it is suggested that the hentriacontan-14,16-dione is synthesized from the carbon-31 end of the molecule by elongation as follows. C₂ units are added, perhaps to a mixture of short chain precursors, to give a chain with 12 carbon atoms. This chain is then elongated to one with 16 carbon atoms so that the four added carbon atoms are uniformly labeled. Following this, the chain with 16 carbon atoms is elongated with C₂ units to give the complete molecule. Possibly some change in mechanism occurs in this last elongation process when the chain is 22 carbon atoms long. Barley spike wax β-diketones contain about 2% nonacosan-13, 15-dione which seems to be synthesized in an analogous manner.     

A method for in situ monitoring of the isotope composition of tree xylem water using laser spectroscopy

Field studies analyzing the stable isotope composition of xylem water are providing important information on ecosystem water relations. However, the capacity of stable isotopes to characterize the functioning of plants in their environment has not been fully explored because of methodological constraints on the extent and resolution at which samples could be collected and analysed. Here, we introduce an in situ method offering the potential to continuously monitor the stable isotope composition of tree xylem water via its vapour phase using a commercial laser‐based isotope analyser and compact microporous probes installed into the xylem. Our technique enables efficient high‐frequency measurement with intervals of only a few minutes per sample while eliminating the need for costly and cumbersome destructive collection of plant material and laboratory‐based processing. We present field observations of xylem water hydrogen and oxygen isotope compositions obtained over several days including a labelled irrigation event and compare them against results from concurrent destructive sampling with cryogenic distillation and mass spectrometric analysis. The data demonstrate that temporal changes as well as spatial patterns of integration in xylem water isotope composition can be resolved through direct measurement. The new technique can therefore present a valuable tool to study the hydraulic architecture and water utilization of trees.     

Quantitative autoradiography of 3H-thymidine-labelled nuclei: Reduction of grain count caused by washing in water

Summary Ehrlich ascites tumour cells and epithelial cells of intestinal crypts were labelled in vitro and in vivo with the pulse of either tritium or carbon-14-thymidine. Fixed cells were washed in either distilled or tap running water for 1/6–24 hr and autoradiographed. Quantitation of autoradiographs showed that the washing of cells labelled with tritiated thymidine caused remarkable diminution of grain count. The cells labelled with carbon-14-thymidine were not affected by washing. The strict control of washing in water of tritium-labelled specimens is recommended.     

稳定氢氧同位素定量植物水分来源的不确定性解析

稳定氢氧同位素技术被广泛运用于生态系统、特别是干旱区生态系统中植物水分来源的研究,其理论假设为"水分被植物根系吸收并向木质部运输过程中不发生氢氧同位素分馏"。生态系统中不同水源的氢氧同位素组成普遍存在显著差异,为从水源混合体中区分出各水源的贡献率提供了前提条件。但在实际应用过程中,诸多因素导致稳定氢氧同位素技术定量植物水分来源的结果具有不确定性。综合已有研究并加以分析,举证说明植物吸收水分相对于水源同位素变化的滞后性、水源同位素的季节性变化、蒸发作用和水源之间的混合作用对水源同位素的影响等导致植物水分来源定量结果不确定性的几个因素,以期为今后稳定氢氧同位素技术在植物水分来源领域的应用提供参考。     

Formation of lipoxin B by the pure reticulocyte lipoxygenase via sequential oxygenation of the substrate

The pure reticulocyte lipoxygenase converts 15LS-hydroxy-5,8,11,13(Z,Z,Z,E)-icosatetraenoic acid (15LS-HETE) methyl ester to a complex mixture of products containing 5DS,14LR,15LS-trihydro(pero)xy-6E,++ +8Z,10E,12E-icosatetraenoate methyl ester (lipoxin B methyl ester), 5DS,15LS-DiH(P)ETE methyl ester and four 8,15LS-DiH(P)ETE methyl ester isomers [DiH(P)ETE = dihydro(pero)xy-icosatetraenoic acid]. After a short incubation period (15 min) 5DS,15LS-DiH(P)ETE methyl ester was found to be the main product, whereas after a 3-h incubation lipoxin B methyl ester was the predominant product. The reaction shows a remarkable stereoselectivity since only small amounts of other trihydroxy tetraenes are formed. Anaerobiosis, heat inactivation of the enzyme, or incubation in the presence of lipoxygenase inhibitors (icosatetraynoic acid, nordihydroguaiaretic acid) completely abolished the reaction. The complete steric structure of the major tetraene product (lipoxin B methyl ester) was established by ultraviolet spectroscopy, HPLC on four different types of columns, gas chromatography/mass spectrometry, gas/liquid chromatography of the ozonolysis fragments of the menthoxycarbonyl derivatives, and by 400-MHz 1H-NMR. Atmospheric oxygen was incorporated at carbon-5 and carbon-14 into the major product. 5DS,15LS-DiH(P)ETE methyl ester was shown to be an intermediate in the synthesis. Lipoxin B was also formed during the oxygenation of arachidonic acid, 15LS-HETE and 5DS,15LS-DiHETE. The results presented here indicate that lipoxin B can be formed by pure lipoxygenases via a sequential oxygenation of arachidonic acid or its hydro(pero)xy derivatives.     

Isotope ratios in photosynthetic oxygen

Axenic suspensions of the fresh water green alga Ankistrodesmus braunii were illuminated under aerobic conditions. The released gas mixture was introduced into the ion source of an isotope mass spectrometer, which recorded the 18O/16O ratio. The 18O content of the photosynthetic oxygen (approximately 0.199%) exceeded that of the cell water (approximately 0.197%) significantly.     

The Effects of Gibberellin on the Metabolism of Ethanol-soluble Constituents in the Cotyledons of Hazel Seeds (Corylus avellana, L.)

Gibberellin pretreatment of hazel seeds induced significantincreases in the incorporation of carbon-14 from [2-¹⁴C] acetateinto both glutamate and sucrose, while the amounts of radioactivityincorporated into citrate, malate and succinate were significantlyhigher after pretreatment with sterile water. A ratio basedon the relative amounts of carbon-14 incorporated into glutamateand into the TCA cycle acids indicates that gibberellin treatmentcauses a diversion of metabolites from the essentially respiratoryTCA cycle into synthetic sequences such as glutamate formation.Gibberellin treatment also appears to stimulate the activityof a reversed glycolytic sequence, resulting in the productionof sucrose.     

利用稳定氢氧同位素定量区分白刺水分来源的方法比较

水是影响植物分布的重要生态因子之一,对植物水源的研究有助于在全球变化背景下了解植物的时空分布格局.根据同位素质量守恒,利用稳定氢氧同位素可以确定植物水分来源,相关的方法也不断改进.利用三源线性混合模型、多源线性混合模型、吸水深度模型以及动态模型分别对格尔木白刺(Nitraria Tangutorum)的水分来源进行了对比研究,发现格尔木白刺主要吸收利用50-100 cm处的土壤水及地下水.在研究方法上,各模型都有自己的应用范围和局限:三源线性混合模型一般只能在植物吸收的水分来源不超过3个的情况下运行;多源线性混合模型弥补了三源线性混合模型的不足,可以同时比较多种来源水各自对白刺的贡献率及贡献范围;吸水深度模型弥补了混合模型中不能计算白刺对土壤水的平均吸水深度的缺陷;动态模型则会为未来降水格局变化对植物的时空分布的影响研究起很大作用.针对不同的适用范围,模型的选择及综合应用会更广泛.但是,该技术还存在一些不足,需要结合测定土水势,富氘水的示踪等方法来弥补.     

Phenotyping hepatocellular metabolism using uniformly labeled carbon-13 molecular probes and LC-HRMS stable isotope tracing

Metabolite stable isotope tracing is a powerful bioanalytical strategy that has the potential to unravel phenotypic markers of early pharmaceutical efficacy by monitoring enzymatic incorporation of carbon-13 atoms into targeted pathways over time. The practice of probing biological systems with carbon-13 labeled molecules using broad MS-based screens has been utilized for many years in academic laboratories but has had limited application in the pharmaceutical R&D environment. The goal of this work was to establish a LCMS analytical workflow that was capable of monitoring carbon-13 isotope changes in glycolysis, the TCA and urea cycles, and non-essential amino acid metabolism. This work applies a standardized protein precipitation with 80% cold methanol and two distinct reverse-phase ion-pair liquid chromatography methods coupled to either a positive- or negative-ion mode high-resolution accurate mass spectrometry screening method. The data herein combines thousands of single-point peak integrations into a novel metabolite network map as a visualization aid to probe and monitor stable isotope incorporation in murine hepatocytes using uniformly labeled ¹³C₆ glucose, ¹³C₃ lactate, and ¹³C₅ glutamine. This work also demonstrates that nitrogen metabolism may have a large influence on the TCA cycle and gluconeogenic carbon fluxes in hepatocyte cell culture.     

Mechanistic Positron Emission Tomography Studies: Demonstration of a Deuterium Isotope Effect in the Monoamine Oxidase-Catalyzed Binding of [11C]l-Deprenyl in Living Baboon Brain

The application of positron emission tomography (PET) to the study of biochemical transformations in the living human and animal body requires the development of highly selective radiotracers whose concentrations in tissue provide a record of a discrete metabolic process. L-N-[11C-methyl]Deprenyl ([11C]L-deprenyl), a suicide inactivator of monoamine oxidase (MAO) type B, has been developed as a radiotracer for mapping MAO B in the living human and animal brain. In this investigation, [11C]L-deprenyl (1) and [11C]L-deprenyl-alpha, alpha-2H2 (2) have been compared in three different baboons by PET measurement of carbon-11 uptake and retention in the brain and the measurement of the amount of unchanged tracer in the arterial plasma over a 90-min time interval. For one baboon, N-[11C-methyl-2H3]L-deprenyl (3) was also studied. Kinetic parameters calculated using a three-compartment model revealed a deuterium isotope effect of 3.8 +/- 1.1. Comparison of the two tracers (1 and 2) in mouse brain demonstrated that deuterium substitution significantly reduced the amount of radioactivity bound to protein. HPLC and GLC analysis of the soluble radioactivity in mouse brain after injection of [11C]L-deprenyl showed the presence of [11C]methamphetamine as a major product along with unidentified labeled products. Sodium dodecyl sulfate-polyacrylamide electrophoresis with carbon-14-labeled L-deprenyl showed that a protein of molecular weight 58,000 was labeled. These results establish that MAO-catalyzed cleavage of the alpha carbon-hydrogen bond on the propargyl group is the rate limiting (or a major rate contributing) step in the retention of carbon-11 in brain and that the in vivo detection of labeled products in brain after the injection of [11C]L-deprenyl provides a record of MAO activity.     

通量梯度法在温室气体及同位素通量观测研究中的应用与展望

通量梯度法与涡度相关法均是微气象学的物质和能量通量观测方法, 在没有高频气体分析仪或下垫面风浪区较小的情况下, 通量梯度法可以有效观测生态系统(或土壤)与大气之间的温室气体及其同位素通量, 同时也可以作为涡度相关法的配套观测和有益补充。该文回顾了通量梯度法的基本原理、概念和假设, 重点综述了温室气体浓度梯度以及相关湍流扩散系数的观测与计算的方法和理论, 概述了通量梯度法在森林、农田、草地、湿地和水体等生态系统观测温室气体通量的应用进展, 特别是在稳定同位素通量观测中的应用, 最后从影响温室气体和同位素的浓度梯度以及湍流扩散系数测定与计算等方面概述了应用注意事项及建议。     

Oxygen-18 Content of Atmospheric Oxygen Does Not Affect the Oxygen Isotope Relationship between Environmental Water and Cellulose in a Submerged Aquatic Plant, Egeria densa Planch

We determined that the oxygen isotopic composition of cellulose synthesized by a submerged plant, Egeria densa Planch., is related to the isotopic composition of environmental water by a linear function, δ¹⁸O cellulose = 0.48 δ¹⁸O water + 24.1%‰. The observation of a slope of less than 1 indicates that a portion of cellulose oxygen is derived from an isotopically constant source other than water. We tested whether this source might be molecular oxygen by growing plants in the presence of high concentrations of ¹⁸O in the form of O₂ bubbled into the bottom of an aquarium. Cellulose synthesized during this experiment did not have significantly different oxygen isotope ratios than that synthesized by control plants exposed to O₂ of normal ¹⁸O abundance. We propose that oxygen in organic matter recycled from senescent portions of the plant is incorporated into cellulose. Our findings indicate that paleoclimatic models linking the oxygen isotope composition of environmental water to cellulose from fossil plants will have to be modified to account for contributions of oxygen from this or other sources besides water.     

Brief Report: Differential oxidations of estradiol-17β by the chimpanzee in vivo

The metabolism of estradiol-17β is primarily an oxidative process at either carbon-2 or carbon-16 in the human. The objective of this study was to determine the relative importance of these two oxygenation pathways in the chimpanzee. The rate of oxidation of estradiol-17β at each position was determined by measuring the release of tritium into body water from carbon-2 or carbon-16. [2-³H]-Estradiol-17β or [16-³H]-estradiol-17β was injected intravenously into three adult male chimpanzees, and blood samples were obtained at several time intervals between 1 and 48 hr. The blood was lyophilized, and the release of tritium from the specifically labeled estrogens into the body fluid pool was determined. The release of tritium from the 16α-position was very low and did not exceed 3% in any animal. The release of tritium from the carbon-2 was much faster, amounting to 29%, 34%, and 35%, respectively, by 24 hr. The ratio of tritium released from carbon-2:carbon-16 was 5.0, 13.2 and 16.9, respectively, at 24 hr after injection of the specifically labeled estradiol-17β. These results demonstrate clearly that the major pathway for oxidative metabolism of estradiol-17β in the chimpanzee is via oxygenation at carbon-2, with the formation of catechol estrogens, as in the human.     

Tracing carbon and oxygen isotope signals from newly assimilated sugars in the leaves to the tree-ring archive

The analysis of δ ¹³C and δ ¹⁸O in tree-ring archives offers retrospective insights into environmental conditions and ecophysiological processes. While photosynthetic carbon isotope discrimination and evaporative oxygen isotope enrichment are well understood, we lack information on how the isotope signal is altered by downstream metabolic processes.
In Pinus sylvestris , we traced the isotopic signals from their origin in the leaf water ( δ ¹⁸O) or the newly assimilated carbon ( δ ¹³C), via phloem sugars to the tree-ring, over a time-scale that ranges from hours to a growing season.
Seasonally, variable ¹³C enrichment of sugars related to phloem loading and transport did lead to uncoupling between δ ¹³C in the tree-ring, and the c _i/ c _a ratio at the leaf level. In contrast, the oxygen isotope signal was transferred from the leaf water to the tree-ring with an expected enrichment of 27‰, with time-lags of approximately 2 weeks and with a 40% exchange between organic oxygen and xylem water oxygen during cellulose synthesis.
This integrated overview of the fate of carbon and oxygen isotope signals within the model tree species P. sylvestris provides a novel physiological basis for the interpretation of δ ¹³C and δ ¹⁸O in tree-ring ecology.     

The origin of the sulfur atom in thiamine.

The mode of biosynthesis of the thiazole moiety of thiamine, 4-methyl-5beta-hydroxyethyl thiazole (MHET) was studied using Salmonella typhimurium as test organism. It was shown by isotope incorporation experiments, that the sulfur atom, but not carbon-3, of cysteine is incorporated into MHET, indicating a separation of the sulfur atom of cysteine from the carbon chain during incorporation. Isotope competition experiments revealed that the incorporation of [35S]cysteine is not significantly diluted by the presence of methionine, homocysteine, and glutathione. No incorporation of label from [14C]glutamate and [14C]formate was observed, leaving the origin of the five-carbon unit still in doubt.     

Design and synthesis of visible isotope-coded affinity tags for the absolute quantification of specific proteins in complex mixtures

Identification of proteins in complex mixtures by mass spectrometry is most useful when quantitative data is also obtained. We recently introduced isotope-coded affinity tags (ICAT reagents) for the relative quantification of proteins present in two or more biological samples. In this report, we describe a new generation of ICAT reagents that contain the following additional features: (1) a visible tag that allows the electrophoretic position of tagged peptides during separation to be easily monitored; (2) a photocleavable linker that allows most of the tag to be removed prior to mass spectrometric analysis; (3) an isotope tag that contains carbon-13 and nitrogen-15 atoms instead of deuterium to ensure precise comigration of light and heavy tagged peptides by reverse-phase HPLC. These reagents contain an iodoacetyl group that selectively reacts with peptide cysteine residues. Peptide modification chemistry is also reported that allows tagging of peptides that are devoid of cysteine. The synthesis of these visible isotope-coded affinity tags (VICAT reagents), and their reaction with peptides are described in this report. VICAT reagents containing a carbon-14 visible probe or an NBD fluorophore are described. These reagents are most useful for the determination of the absolute quantity of specific target proteins in complex protein mixtures such as serum or cell lysates.     

Variations in the natural abundance of oxygen-18 and deuterium in plant carbohydrates

Variations in the natural abundance of ¹⁸O and ²H in plant cellulose are influenced by the isotopic composition of the water directly involved in metabolism—the metabolic water fraction. The isotopic distinction between the metabolic source water and total tissue water must reflect the formation of isotopic gradients within the tissue that are influenced by the rate of water turnover, by properties of the water conducting system and by environmental conditions. It seems that the ¹⁸O abundance in the metabolic water is conserved in cellulose with a relatively constant isotope effect. The relationship of the ²H abundance between metabolic water and cellulose is more complex. Hydrogen incorporated into photosynthetic products during primary reduction steps is highly depleted in ²H. However, a large proportion of these hydrogens are subsequently replaced by exchange with water, leading to ²H enrichment during heterotrophic metabolism. Deciphering the oxygen isotope ratio of cellulose could help in providing insights into the carbon and oxygen fluxes exchanged between plants and the atmosphere. This is because the ¹⁸O abundance in cellulose records the ¹⁸O abundance in the metabolic water, which in turn, controls the oxygen isotopic signatures of the CO₂ and O₂ released by plants into the atmosphere. The hydrogen isotope effects associated with carbohydrate metabolism provide insights into the autotrophic state of a plant tissue. This is because the hydrogen isotope ratio of carbohydrates must reflect the net effects of the two opposing isotope effects associated with photosynthesis and heterotrophic metabolism.