稳定同位素红外光谱技术测定CO_2同位素校正方法的研究进展

稳定同位素红外光谱(IRIS)技术克服了传统的大气CO_2气瓶采样-同位素质谱(IRMS)技术时间分辨率低且耗时费力的缺点,可以实现高时间分辨率和高精度的大气CO_2碳同位素组成(δ~(13)C)和氧同位素组成(δ~(18)O)的原位连续测定。基于IRIS技术测量CO_2δ~(13)C和δ~(18)O的误差来源主要包括δ~(13)C和δ~(18)O测量值对CO_2浓度变化的非线性响应(浓度依赖性)以及对环境条件变化的敏感性导致的漂移(时间漂移)。如何有效地校正浓度依赖性和时间漂移导致的误差是IRIS仪器应用的前提。该综述阐述了δ~(13)C和δ~(18)O测量值的浓度依赖性产生的理论基础,回顾了浓度依赖性的理论校正和经验方程校正方法和应用;回顾了时间漂移的校正原理、方法和应用;概述了数据溯源至国际标准的原理、方法与应用现状。结合实际情况推荐利用3个或3个以上已知CO_2浓度和δ~(13)C、δ~(18)O真值的CO_2标准气体涵盖待测气体CO_2浓度的浓度依赖性校正,设置适当的校正频率校正时间漂移并进行数据溯源。指出应该加强不同仪器和校正方法的比对研究;采用IRIS技术测定CH_4、N_2O和H_2O同位素组成也可以采取类似的校正方法。     

中国农业科学院稳定同位素实验室

中国农业科学院稳定同位素实验室成立于2002年,经过近10年的努力,在同位素的研究与测试领域取得一些重要进展,实验室与欧洲7个稳定同位素实验室展开积极的合作,参加了国际比对,实验结果得到了国际同行的认可和高度评价。实验室利用国际原子能机构的标准物质对相关结果进行标定,采用国际标准测定方法进行C、H、O、N、S稳定同位素测定,保证了结果的准确性。     

中国农业科学院稳定同位素实验室

<正>中国农业科学院稳定同位素实验室成立于2002年,经过近10年的努力,在同位素的研究与测试领域取得一些重要进展,实验室与欧洲7个稳定同位素实验室展开积极的合作,参加了国际比对,实验结果得到了国际同行的认可和高度评价。实验室利用国际原子能机构的标准物质对相关结果进行标定,采用国际标准测定方法进行C、H、O、N、S稳定同位素测定,保证了结果的准确性。     

中国农业科学院稳定同位素实验室

<正>中国农业科学院稳定同位素实验室成立于2002年,经过近10年的努力,在同位素的研究与测试领域取得一些重要进展,实验室与欧洲7个稳定同位素实验室展开积极的合作,参加了国际比对,实验结果得到了国际同行的认可和高度评价。实验室利用国际原子能机构的标准物质对相关结果进行标定,采用国际标准测定方法进行C、H、O、N、S稳定同位素测定,保证了结果的准确性。2010年8月本实验室新购置的最新一代Isoprime100稳定同位素质谱安装完毕,     

碳氮稳定同位素技术在青藏高原高寒草甸生态系统研究中的应用:进展与展望

碳氮稳定同位素技术在草地生态系统研究中的应用日渐广泛,本文针对其在青藏高原高寒草甸生态系统中的研究与应用进行了总结。首先,探讨了环境因子(海拔、水肥、草地退化、温度)对青藏高原高寒草甸碳氮同位素组成(δ~(13)C、δ~(15)N)的影响:高寒草甸植物δ~(13)C值与海拔呈正相关,与大气压强、草地退化和温度均呈负相关,与降水的关系尚有争议;土壤δ~(13)C值与海拔和草地退化呈正相关;植被的δ~(15)N值与水肥呈正相关,土壤的δ~(15)N值与草地退化呈负相关。其次,综述了近年来该技术在高寒草甸植物光合型鉴定、植物水分利用、食物链营养关系、碳氮循环等方面的研究进展。最后,对碳氮稳定同位素技术在研究高寒草甸土壤有机碳与土壤呼吸、重现植被类型更替和气候演化历史、土壤N_2O溯源、探究高寒草甸退化的原因、藏药与动物食品产地溯源等方面的应用前景进行了展望,以期进一步发挥其在青藏高原高寒草甸研究中的潜力。     

早期鲜卑人和动物骨骼的稳定同位素分析

本文对呼伦贝尔地区扎赉诺尔、团结和东乌珠尔早期鲜卑墓地出土的人和动物骨骼进行了稳定同位素分析,以期揭示早期鲜卑的食物结构和生计方式。C、N稳定同位素分析结果表明,早期鲜卑人的食物来源兼具C_3类和C_4类,其中C_4类食物很可能来自其从事的少量粟作农业。人具有较高的δ~(15)N值,且与动物的δ~(15)N值差异较大,表明人的营养级较高,肉食资源在食物结构中占据较大比重,这可能来自于早期鲜卑人所从事的游牧及狩猎经济的贡献。C、N、S稳定同位素分析结果表明,动物和人的食物来源来自于当地的陆生系统,而个体马(DW M3(1))具有较高的δ~(13)C和δ~(15)N值以及明显不同于其他样品的异常高的δ~(34)S值,说明此个体可能属于来自于其他地质环境的"外来者"。     

双向标记培养植物测定大气二氧化碳稳定碳同位素组成

基于植物能够利用体内的碳酸酐酶来催化碳酸氢根离子生成二氧化碳和水作为底物进行光合作用的特性,采用两种δ13CPDB值差值大于10‰的碳酸氢钠分别作为外源碳酸氢根离子的碳同位素标记物,通过室内双向水培诸葛菜和芥菜型油菜实验,分别向水培处理液里添加已知δ13CPDB值的碳酸氢钠并培养24 h,利用同位素比值质谱(IRMS)技术,测定并计算了两个时间、两种环境下的大气二氧化碳稳定碳同位素日平均组成。结果表明:在环境1(不同浓度的Na HCO3处理液)下所得到的δCa值与添加到处理液中的碳酸氢根离子的浓度有关;在环境2(不同浓度的PEG处理液)下所得到的δCa值与添加到处理液中的PEG的浓度无关;两种环境下所测得的大气二氧化碳稳定碳同位素日平均组成δCa值与实验中培养的植物种类无关,而与添加到培养液中碳酸氢根离子的浓度及植物的生长速率有关。数据重现性好,结果准确可靠,可以高精度的测定不同待测环境下大气二氧化碳稳定碳同位素比值,其可为以后监测不同时间、不同地点的大气二氧化碳碳同位素组成及来源提供非常有效的方法和信息。     

前处理与测试条件差异对化石牙釉质羟磷灰石稳定同位素数据的影响：以步氏巨猿动物群为例（英文）

牙釉质羟磷灰石的稳定同位素分析被广泛应用于古生物学研究之中,以重建古生态和古环境信息。在对不同研究中的同位素结果进行对比分析时,往往会忽略不同实验室、不同前处理方法可能引发的数据误差。为了探讨这些因素对牙釉质羟磷灰石同位素值的影响,重新测量了湖北省龙骨洞步氏巨猿动物群动物牙釉质样本的碳氧稳定同位素值,该批样本曾使用不同的前处理和实验方法进行过测试(Zhao et al., 2011; Nelson, 2014)。研究结果显示,重测的数据与Zhao et al.(2011)、Nelson (2014)发表的数据结果均存在一定差异。前处理方法与实验室测试差异都会造成牙釉质碳、氧稳定同位素结果的偏差。相较氧同位素而言,碳同位素值会更容易被前处理过程中反应试剂、反应时间等的不同所影响。但上述因素所导致的数据差异较小,不会对后续的分析产生实质性影响。本研究为直接对比不同来源牙釉质同位素值的可行性提供了初步的理论支持。建议为减少由于样品前处理和实验测试方案引发的数据误差,获得更加精确的研究结果,应尽可能采用同样的前处理与测试方案,多进行实验室间数据校正对比分析。     

箱式通量观测技术和方法的理论假设及其应用进展

碳(CO_2、CH_4)、氮(N_2O)和水汽(H_2O)等温室气体的交换通量是生态系统物质循环的核心,是地圈-生物圈-大气圈相互作用的纽带。稳定同位素光谱和质谱技术和方法的进步使碳稳定同位素比值(δ~(13)C)和氧稳定同位素比值(δ~(18)O)(CO_2)、δ~(13)C(CH_4)、氮稳定同位素比值(δ~(15)N)和δ~(18)O (N_2O)、氢稳定同位素比值(δD)和δ~(18)O (H_2O)的观测成为可能,与箱式通量观测技术和方法结合可以实现土壤、植物乃至生态系统尺度温室气体及其同位素通量观测研究。该综述以CO_2及其δ~(13)C通量的箱式观测技术和方法为例,概述了箱式通量观测系统的基本原理及分类,阐述了系统设计的理论要求和假设,综述了从野外到室内土壤、植物叶-茎-根以及生态系统尺度箱式通量观测研究的应用进展及问题,展望了气体分析精度和准确度、观测数据精度和准确度以及观测数据的代表性评价在箱式通量观测研究中的重要性。     

利用原位连续测定水汽δ~(18)O值和Keeling Plot方法 区分麦田蒸散组分

利用稳定同位素技术和Keeling Plot方法可以有效分割地表蒸散量,进而加深对陆地生态系统水循环的理解.该研究通过原位连续测定麦田的水汽同位素数据,评价Keeling Plot方法在分割地表蒸散中的应用,并揭示华北冬小麦(Triticum aes-tivum)蒸腾在总蒸散中的比例.实验于2008年3-5月在中国科学院栾城农业生态站进行,利用国际上先进的H_2~(18)O、HD~(16)O激光痕量气体分析仪(TDLAS)为基础构建的大气水汽~(18)O/~(16)O和D/H同位素比原位连续观测系统,同时利用涡度相关技术、真空抽提技术、同位素质谱仪技术,获取了必要的数据.研究分析了一天中不同时间段的连续的大气水汽δ~(18)O与水汽浓度倒数拟合Keeling Plot曲线的差异和可能的原因.结果显示,中午时段的拟合结果较好,这也暗示中午时段蒸腾速率高时最可能满足植物蒸腾的同位素稳定态假设.进一步的分析发现植物蒸腾的同位素稳定态并不总是成立,尤其是水分胁迫下进入成熟期的小麦,其蒸腾水汽同位素一般处于非稳定态.利用同位素分割结果显示,生长盛期麦田94%-99%的蒸散来源于植物蒸腾.     

Blood-sucking bugs as a gentle method for blood-collection in water budget studies using doubly labelled water

During doubly-labelled water (DLW) experiments, blood collection by venous puncture may traumatize animals and consequently affect the animals' behaviour and energy budget. Recent studies have shown that blood-sucking bugs (Triatominae; Heteroptera) can be used instead of conventional needles to obtain blood from animals. In this paper, we validate the bug method in captive nectar-feeding bats, Glossophaga soricina, for water budget analysis by comparing the daily water flux estimated with the DLW method with values measured by an energy balance method. As the mean daily water flux of the DLW method was not significantly deviating from the expected value, blood-sucking bugs may substitute more invasive methods of blood collection in DLW experiments. Based on the DLW estimates, daily energy and water intake rates were calculated and compared to values measured with the energy balance method. The DLW method and the energy balance method yielded on average similar results regarding the daily energy intake (DLW method: 48.8+/-14.2 kJ d(-1) versus energy balance method: 48.1+/-9.9 kJ d(-1)) and daily water intake (DLW method: 13.7+/-2.4 mL d(-1) versus energy balance method: 14.7+/-3.0 mL d(-1)). Based on the calculated water and sugar intake per day, we estimated the sugar concentration of ingested nectar to equal on average 16.2+/-2.4% (mass/mass), which fell close to the measured sugar concentration of 17% (mass/mass) bats fed on during the experiment. We conclude that it is possible to extrapolate mean daily energy and water intake for animal groups, populations and species based on DLW estimates, but due to the large variance of results (low accuracy), it seems inadequate to calculate values for single individuals.     

Correction Approaches for Doubly Labeled Water in Situations of Changing Background Water Abundance

Doubly labeled water (DLW) is an accurate, portable method for measuring free-living energy expenditure. However, under certain conditions shifts in baseline abundance of deuterium and oxygen-18 tracers used in the method may produce errors in derivation of both turnover (k) rates and calculated energy expenditure. Present objectives were to examine during what experimental situations baseline errors arise and to address means of correcting for such baseline shifts so that consequent errors in energy expenditure calculations are minimized. Under conditions where shifts in baseline abundance for deuterium and oxygen-18 parallel abundances corresponding to the natural meteoric water ratio, self-compensating changes in k values for both deuterium and oxygen will result in minimal error to the DLW energy expenditure calculations, provided that the dose ratio of isotopes also mimics the meteoric water line. However, in situations where relative shifts in abundance of each isotope across the measurement period are not in parallel relative to the natural meteoric water line, then the potential for larger DLW errors exists. Optimally, subjects should equilibrate with the new water source. Failing this, correction for shifting baseline can be accomplished by measuring isotopic abundance changes in a control group of subjects not given the DLW dose, but performing similar tasks and consuming the same diet as the group given DLW. Alternatively, theoretically based correction values can be calculated given knowledge of the abundances of the final drinking water and the interval time that subjects consumed the new fluid.     

Comparison of different approaches for the calculation of energy expenditure using doubly labeled water in a small mammal

The doubly labeled water (DLW) method is an isotope-based technique for the estimation of the CO(2) production, and hence energy expenditure, of free-living animals and humans. Several methods are available for the calculation of CO(2) production from the isotope fluxes, depending on different assumptions about the behavior of isotopes during the elimination process. We used the DLW method to estimate the daily energy expenditures (DEE) of 55 field voles (Microtus agrestis) held in a captive facility at 8 degrees C. We calculated DEE using both plateau and intercept approaches for estimating the sizes of the isotope dilution spaces, three different assumptions about fractionation processes, and two ways of treating the different dilution spaces of the oxygen and hydrogen isotopes. We compared the resultant DEE estimates with metabolizable energy intake (MEI) measured during a 3-d feeding trial immediately before the DLW measurements, during which the animals were in energy balance. By making different assumptions about the apparent energy absorption efficiency, we generated a range of direct estimates of MEI. When we compared DEE and MEI, we found that the two-pool model formulations consistently underestimated energy demands by up to 29.8%, depending on the assumptions made in the reference calculation. However, while our data suggest that some correction for fractionation is necessary, with the present data we were unable to separate the two most common treatments of fractionation. These data strongly support the previous suggestion that for small mammals single-pool models provide more accurate estimates of energy demands than two-pool formulation of the DLW method.     

Validation of a non-invasive blood-sampling technique for doubly-labelled water experiments

Two techniques for bleeding small mammals have been used in doubly-labeled water (DLW) studies, including vena puncture and the use of starved nymphal stages of hematophagous reduviid bugs (Reduviidae, Hemiptera). In this study, we tested the validity of using reduviid bugs in doubly-labeled water experiments. We found that the isotope enrichment in initial blood samples collected with bugs was significantly lower compared to isotope enrichment in blood samples obtained using vena puncture. We therefore used the desiccation method for estimating total body water (TBW) in DLW experiments because TBW calculated using the isotope dilution method was overestimated when blood samples were collected using reduviid bugs. In our validation experiment with nectar-feeding bats (Glossophaga soricina), we compared estimates of daily energy expenditure (DEE) using DLW with those derived from the energy balance method. We considered Speakman's equation (controlling for 25% fractionated water loss) as the most appropriate for our study animal and calculated DEE accordingly. On average, DEE estimated with DLW was not significantly different from the mean value obtained with the energy balance method (mean deviation 1.2%). We conclude that although bug hemolymph or intestinal liquids most likely contaminate the samples, estimates of DEE are still valid because the DLW method does not depend on absolute isotope enrichments but on the rate of isotope decrease over time. However, dilution of blood with intestinal liquids or hemolymph from a bug may lead to larger variation in DEE estimates. We also tested how the relative error of DLW estimates changed with varying assumptions about fractionation. We used three additional equations for calculating DEE in DLW experiments. The basic equation for DLW experiments published by Lifson and McClintock (LM-6) assumes no fractionation, resulted in an overestimate of DEE by 10%. Nagy's equation (N-2) controls for changes in body mass but not for fractionation. Using Nagy's equation, DEE was overestimated by 8%. Under the assumption that 50% of total water flux fractionates, the alternative equation by Lifson and McClintock (LM-35) DEE was underestimated by 5%. The best fit between estimates of DEE based on DLW and energy balance measurements was derived by assuming that 32% of total water flux (TWF) is fractionated. We conclude that the outcome of DLW experiments is sensitive to assumptions regarding evaporative water loss, and thus recommend Speakman's equation 7.17 for use with bats.     

Measurement of energy expenditure in humans by doubly labeled water method

The utility of the doubly labeled water method for the determination of energy expenditure and water output was investigated in humans. Approximately 10 g of 18O and 0.5 g of 2H as water was orally administered to four healthy adults. Total body water was determined from the isotope dilution, and the ensuing 18O and 2H disappearance rates from body water were determined for 13 days by mass spectrometric isotope ratio analysis of the urinary water. During this period, subjects were maintained on a measured diet to determine energy and water intake. The energy expenditure from the doubly labeled water method differed from dietary intake plus change in body composition by an average of 2%, with a coefficient of variation of 6%. The water outputs determined by the two methods differed by 1%, with a coefficient of variation of 7%. The doubly labeled water method is noninvasive, and the subjects could maintain their daily activities without restriction.     

Validation of the doubly labeled water technique for measuring energy metabolism in starlings and sparrows

I have tested the idea that doubly labeled water (DLW) can accurately predict CO2 production in savannah sparrows, song sparrows, white-throated sparrows, starlings, and a single house sparrow by comparing DLW estimates with those obtained simultaneously by capturing expired CO2 in Ascarite. In addition I used the energy balance method to see if metabolic rates generated from DLW measurements accurately reflected the actual metabolic rates of these birds. I found close agreement in DLW and the gravimetric and energy balance methods, with DLW underestimating CO2 production on average by -3.5% in sparrows, and -7.1% in starlings. Similarly, the energy balance method indicated a -3.1% underestimate by DLW for sparrows and a -5.1% for starlings. The DLW method can yield reasonable estimates of CO2 production in a variety of passerine birds.     

Validation of the doubly labeled water method in growing precocial birds: the importance of assumptions concerning evaporative water loss

The doubly labeled water (DLW) method was validated against respiration gas analysis in growing precocial chicks of the black-tailed godwit (Limosa limosa) and the northern lapwing (Vanellus vanellus). To calculate the rate of CO2 production from DLW measurements, Lifson and McClintock's equations (6) and (35) were employed, as well as Speakman's equation (7.17) (all single-pool models). The average errors obtained with the first two equations (+7.2% and -11.6%, respectively) differed significantly from zero but not the error obtained with Speakman's equation (average: -2.9%). The latter error could be reduced by taking a fractional evaporative water loss of 0.13, instead of the value of 0. 25 recommended by Speakman. Application of different two-pool models resulted in relative errors of the DLW method of -15.9% or more. After employing the single-pool model with a fractional evaporative water loss value of 0.13, it was found that there was no relationship between the relative growth rate of the chick and the relative error of the DLW method. Recalculation of previously published results on Arctic tern (Sterna paradisaea) chicks revealed that the fit of the validation experiment could be considerably improved by employing a single-pool model and assuming a fractional evaporative water loss of 0.20 instead of the value of 0.50 taken originally. After employing the value of 0.20, it was found that there was no relationship between the relative growth rate of the chick and the relative error of the DLW method. This suggests that isotope incorporation into new body substances does not cause a detectable error. Thus, the DLW method seems to be applicable in young birds growing as fast as 20% d-1, after making adjustments for the fractional evaporative water loss. We recommend Speakman's equation (7.17) for general use in growing birds when evaporation is unknown.     

Doubly labeled water measurement of human energy expenditure during strenuous exercise

The energy expenditures (EE) of 23 adult male Marines were measured during a strenuous 11-day cold-weather field exercise at 2,200- to 2,550-m elevation by both doubly labeled water (2H2 18O, DLW) and intake balance methods. The DLW EE calculations were corrected for changes in baseline isotopic abundances in a control group that did not receive 2H2 18O. Intake balance EE was estimated from the change in body energy stores and food intake. Body energy-store changes were calculated from anthropometric [-1,574 +/- 144 (SE) kcal/day] and isotope dilution (-1,872 +/- 293 kcal/day) measurements made before and after the field exercise. The subjects kept daily logbook records of ration consumption (3,132 +/- 165 kcal/day). Mean DLW EE (4,919 +/- 190 kcal/day) did not differ significantly from intake balance EE estimated from food intake and either anthropometric (4,705 +/- 181 kcal/day) or isotope dilution (5,004 +/- 240 kcal/day) estimates of the change in body energy stores. The DLW method can be used with at least the same degree of confidence as the intake balance method to measure the EE of active free-living humans.     

Validation of the DLW method in Japanese quail at different water fluxes using laser and IRMS.

In Japanese quail (Coturnix c. japonica; n = 9), the doubly labeled water (DLW) method ((2)H, (18)O) for estimation of CO(2) production (l/day) was validated. To evaluate its sensitivity to water efflux levels (r(H(2))O(e); g/day) and to assumptions of fractional evaporative water loss (x; dimensionless), animals were repeatedly fed a dry pellet diet (average r(H(2))O(e) of 34.8 g/day) or a wet mash diet (95.8 g/day). We simultaneously compared the novel infrared laser spectrometry (LS) with isotope ratio mass spectrometry. At low r(H(2))O(e), calculated CO(2) production rate exhibited little sensitivity to assumptions concerning x, with the best fit being found at 0.51, and only little error was made employing an x value of 0.25. In contrast, at high r(H(2))O(e), sensitivities were much higher with the best fit at x = 0.32. Conclusions derived from isotope ratio mass spectrometry and LS were similar, proving the usefulness of LS. Within a threefold range of r(H(2))O(e), little error in the DLW method is made when assuming one single x value of 0.25 (recommended by Speakman JR, Doubly Labelled Water. Theory and Practice. London: Chapman & Hall, 1997), indicating its robustness in comparative studies.     

Multiple Laboratory Comparison of the Doubly Labeled Water Technique*

A double-blind study was conducted to determine between-laboratory variability in the doubly labeled water method for measurement of total energy expenditure in humans, and to compare the accuracy and precision of three widely-used procedures for calculating rates of carbon dioxide production from the original isotope data. Eighteen laboratories from five countries participated in the study. All laboratories were provided with five water standards containing varying amounts of ²H and ¹⁸O, and in addition 11 laboratories were provided with urine and dose specimens from one (six laboratories) or two (five laboratories) healthy elderly subjects of normal height and weight undergoing a calorimetric validation of the doubly labeled water method. The data from the five water standards were analyzed to predict between-laboratory variability in the doubly labeled water technique in all laboratories. In addition, data from the subjects were analyzed using the “slope-intercept”, “2-point” and “modified” methods of calculation. The results confirm that the doubly labeled water method can be an accurate technique for the measurement of energy expenditure in adult human subjects in some laboratories. However, there was substantial between-laboratory variability in the results and some laboratories returned physiologically impossible results. There was no significant effect of calculation procedure on the accuracy of the technique in this limited comparison, although the slope-intercept procedure appeared to be more susceptible to analytical error than the other procedures. The isotope standards analyzed by participants in this study will be made available to other investigators on request.