Assessment of the amount of body water in the Red Knot (Calidris canutus): an evaluation of the principle of isotope dilution with 2H, (17)O, and (18)O as measured with laser spectrometry and isotope ratio mass spectrometry

We have used the isotope dilution technique to study changes in the body composition of a migratory shorebird species (Red Knot, Calidris canutus) through an assessment of the amount of body water in it. Birds were quantitatively injected with a dose of water with elevated concentrations of 2H, (17)O, and (18)O. Thereafter, blood samples were taken and distilled. The resulting water samples were analysed using an isotope ratio mass spectrometry (for 2H and (18)O only) and a stable isotope ratio infrared laser spectrometry (2H, (17)O, and (18)O) to yield estimates of the amount of body water in the birds, which in turn could be correlated to the amount of body fat. Here, we validate laser spectrometry against mass spectrometry and show that all three isotopes may be used for body water determinations. This opens the way to the extension of the doubly labelled water method, used for the determination of energy expenditure, to a triply labelled water method, incorporating an evaporative water loss correction on a subject-by-subject basis or, alternatively, the reduction of the analytical errors by statistically combining the (17)O and (18)O measurements.     

Analytics/Methods

An overview is presented of biomedical applications of stable isotopes in general, but mainly focused on the activities of the Center for Liver, Digestive and Metabolic Diseases of the University Medical Center Groningen. The aims of metabolic studies in the areas of glucose, fat, cholesterol and protein metabolism are briefly explained, as well as the principle of breath testing and the techniques to study body composition and energy expenditure. Much attention is paid to the analytical considerations based upon metabolite concentrations, sample size restrictions, the availability of stable isotope labelled substrates and dose requirements in relation to compound-specific isotope analysis. The instrumental advantages and limitations of the generally used techniques gas chromatography/reaction/isotope ratio mass spectrometry and gas chromatography/mass spectrometry are described as well as the novelties of the recently commercialised liquid chromatography/combustion/isotope ratio mass spectrometry. The present use and future perspective of infrared (IR) spectrometry for clinical and biomedical stable isotope applications are reviewed. In this respect, the analytical demands on IR spectrometry are discussed to enable replacement of isotope ratio mass spectrometry by IR spectrometry, in particular, for the purpose of compound-specific isotope ratio analysis in biological matrices.     

Analytical techniques in biomedical stable isotope applications: (isotope ratio) mass spectrometry or infrared spectrometry?

An overview is presented of biomedical applications of stable isotopes in general, but mainly focused on the activities of the Center for Liver, Digestive and Metabolic Diseases of the University Medical Center Groningen. The aims of metabolic studies in the areas of glucose, fat, cholesterol and protein metabolism are briefly explained, as well as the principle of breath testing and the techniques to study body composition and energy expenditure. Much attention is paid to the analytical considerations based upon metabolite concentrations, sample size restrictions, the availability of stable isotope labelled substrates and dose requirements in relation to compound-specific isotope analysis. The instrumental advantages and limitations of the generally used techniques gas chromatography/reaction/isotope ratio mass spectrometry and gas chromatography/mass spectrometry are described as well as the novelties of the recently commercialised liquid chromatography/combustion/isotope ratio mass spectrometry. The present use and future perspective of infrared (IR) spectrometry for clinical and biomedical stable isotope applications are reviewed. In this respect, the analytical demands on IR spectrometry are discussed to enable replacement of isotope ratio mass spectrometry by IR spectrometry, in particular, for the purpose of compound-specific isotope ratio analysis in biological matrices.     

Breath water-based doubly labelled water method for the noninvasive determination of CO2 production and energy expenditure in mice

ABSTRACT

We explored a novel doubly labelled water (DLW) method based on breath water (BW-DLW) in mice to determine whole body CO₂ production and energy expenditure noninvasively. The BW-DLW method was compared to the DLW based on blood plasma. Mice (n?=?11, 43.5?±?4.6?g body mass (BM)) were administered orally a single bolus of doubly labelled water (1.2?g H₂¹⁸O kg BM^?1 and 0.4?g ²H₂O kg BM^?1, 99 atom% (AP) ¹⁸O or ²H). To sample breath water, the mice were placed into a respiration vessel. The exhaled water vapour was condensed in a cold-trap. The isotope enrichments of breath water were compared with plasma samples. The ²H/¹H and ¹⁸O/¹⁶O isotope ratios were measured by means of isotope ratio mass spectrometry. The CO₂ production (RCO₂) was calculated from the ²H and ¹⁸O enrichments in breath water and plasma over 5 days. The isotope enrichments of breath water vs. plasma were correlated (R²?=?0.89 for ²H and 0.95 for ¹⁸O) linearly. The RCO₂ determined based on breath water and plasma was not different (113.2?±?12.7 vs. 111.4?±?11.0?mmol?d^–1), respectively. In conclusion, the novel BW-DLW method is appropriate to obtain reliable estimates of RCO₂ avoiding blood sampling.     

Use of dual isotope tracers in biomedical research

Biomedical stable isotope studies involve administration of tracer and measurement of isotope enrichment in blood, urine, feces or breath. The aim of the studies is to gather quantitative information about a specific metabolic function. However, the measured isotope enrichment may be affected by other metabolic events than only this function. In this case, a correction is necessary. The best approach is to add a second tracer simultaneously which is known to be metabolised by all interfering metabolic events but not by the function of interest. This dual isotope approach also enables simultaneous measurement of two interrelated functions. A summary of selected applications involving dual isotope tracer studies is presented. The applications deal with energy expenditure (doubly labelled water technique), cholesterol absorption, starch and lactose digestion, fat digestion, bile acid metabolism and the combination of stable and radioactive carbon isotopes in breath testing.     

Detection of hydrogen isotope atoms by mass spectrometry combined with resonant ionization

We examined the application of mass spectrometric methods using resonant ionization by a tunable laser and proposed its use for analyzing hydrogen isotopes. We conducted resonance ionization mass spectrometry (RIMS) to detect gas-phase hydrogen isotope atoms. The ionization efficiency was increased by more than 1000 times that obtained with conventional methods using nonresonant ionization. Resonant laser ablation mass spectrometry (RLAMS) was applied for deuterium detection in solid samples. A graphite substrate implanted with deuterium was used for ordinary laser ablation mass spectrometry (LAMS) and RLAMS. The deuterium signal was observed very clearly by RLAMS, in contrast to LAMS. Mass spectrometry combined with resonance ionization was very useful for hydrogen isotope detection, because components with equal mass numbers were resolved and the method demonstrated higher ionization efficiency. Received: 4 November 1998 / Revised version: 12 January 1999 / Published online: 7 April 1999     

Lead and strontium isotopes as indicators for mixing processes of waters in the former mine 'Himmelfahrt Fundgrube', Freiberg (Germany)

To pinpoint the origin and mixing processes of mine waters, different mine water types from the polymetallic sulphide ore deposit 'Himmelfahrt Fundgrube' (Freiberg, Germany) were analysed by thermal ionisation mass spectrometry using lead and strontium isotope ratios. Results show that the lead isotope composition of different mine waters results from a mixture of at least two sources: released lead from oxidised sulphide ores (mainly galena) and anthropogenic lead from groundwater. Furthermore, there are indications for an additional lead source. Strontium isotopes in mine waters identify at least three different sources: released strontium from weathered host rock (Grey Gneisses), released strontium from weathered gangue carbonates, and probably strontium from anthropogenic inputs. Contrary to former oxygen and sulphur isotope studies, strontium isotope compositions as well as hydrochemical parameters show the important role of gangue carbonates as an element source in mine waters.     

Evaluation of the performance of high temperature conversion reactors for compound-specific oxygen stable isotope analysis

In this study conversion conditions for oxygen gas chromatography high temperature conversion (HTC) isotope ratio mass spectrometry (IRMS) are characterised using qualitative mass spectrometry (IonTrap). It is shown that physical and chemical properties of a given reactor design impact HTC and thus the ability to accurately measure oxygen isotope ratios. Commercially available and custom-built tube-in-tube reactors were used to elucidate (i) by-product formation (carbon dioxide, water, small organic molecules), (ii) 2nd sources of oxygen (leakage, metal oxides, ceramic material), and (iii) required reactor conditions (conditioning, reduction, stability). The suitability of the available HTC approach for compound-specific isotope analysis of oxygen in volatile organic molecules like methyl tert-butyl ether is assessed. Main problems impeding accurate analysis are non-quantitative HTC and significant carbon dioxide by-product formation. An evaluation strategy combining mass spectrometric analysis of HTC products and IRMS ¹⁸O/¹⁶O monitoring for future method development is proposed.     

金属稳定同位素标记生物分析进展

细胞和组织的很多特定功能都由其在不同的生理条件下的生物分子含量决定,极少数分子的改变就有可能影响细胞生物功能并触发疾病生理过程,因此高灵敏的生物分子检测技术在疾病机理研究和疾病早期诊断方面具有重要作用。金属稳定同位素和放射同位素化学性质相近,借鉴放射同位素标记的成功经验,通过金属稳定同位素标记多组分生物分子,可以用原子质谱高灵敏地检测多组分生物分子。作为灵敏准确的金属元素检测工具,电感耦合等离子体质谱检出限低、基体效应低、线性范围广、同位素谱线分辨率高,因此适用于金属元素标记生物分子检测。金属稳定同位素标记已经被广泛应用到蛋白质、核酸、酶活性、生物小分子、甚至单个细胞的检测中,取得了一些可喜的进展,并展现了广阔未来应用前景。金属稳定同位素标记生物分析方法有三个特性：高灵敏度-大多数金属的稳定同位素有较高的标记灵敏度,并且可以通过纳米材料标记等方法实现信号放大;多组分同时分析-质谱仪同位素谱线高分辨率提供了多组分分析能力;高准确度-同位素稀释法提供了可溯源到SI国际单位制的高准确度检测结果。为了更好的推动相关研究,简要介绍金属稳定同位素标记生物分析的进展,主要内容包括以下几个部分：金属稳定同位素检测工具-无机质谱、金属稳定同位素标记高灵敏度分析、金属稳定同位素标记多组分同时分析、金属稳定同位素标记高准确度分析、金属稳定同位素标记单细胞分析的进展。     

Lead and strontium isotopes as indicators for mixing processes of waters in the former mine ‘Himmelfahrt Fundgrube’, Freiberg (Germany)†

To pinpoint the origin and mixing processes of mine waters, different mine water types from the polymetallic sulphide ore deposit ‘Himmelfahrt Fundgrube’ (Freiberg, Germany) were analysed by thermal ionisation mass spectrometry using lead and strontium isotope ratios.

Results show that the lead isotope composition of different mine waters results from a mixture of at least two sources: released lead from oxidised sulphide ores (mainly galena) and anthropogenic lead from groundwater. Furthermore, there are indications for an additional lead source. Strontium isotopes in mine waters identify at least three different sources: released strontium from weathered host rock (Grey Gneisses), released strontium from weathered gangue carbonates, and probably strontium from anthropogenic inputs. Contrary to former oxygen and sulphur isotope studies, strontium isotope compositions as well as hydrochemical parameters show the important role of gangue carbonates as an element source in mine waters.     

Box-modeling of bone and tooth phosphate oxygen isotope compositions as a function of environmental and physiological parameters

A time-dependent box model is developed to calculate oxygen isotope compositions of bone phosphate as a function of environmental and physiological parameters. Input and output oxygen fluxes related to body water and bone reservoirs are scaled to the body mass. The oxygen fluxes are evaluated by stoichiometric scaling to the calcium accretion and resorption rates, assuming a pure hydroxylapatite composition for the bone and tooth mineral. The model shows how the diet composition, body mass, ambient relative humidity and temperature may control the oxygen isotope composition of bone phosphate. The model also computes how bones and teeth record short-term variations in relative humidity, air temperature and delta18O of drinking water, depending on body mass. The documented diversity of oxygen isotope fractionation equations for vertebrates is accounted for by our model when for each specimen the physiological and diet parameters are adjusted in the living range of environmental conditions.     

Determination of cholesterol absorption in humans: from radiolabel to stable isotope studies

Hypercholesterolemia is a major health risk. Dietary cholesterol absorption is one important factor affecting levels of plasma and tissue cholesterol. Considerable effort has thus been devoted to develop reliable in vivo clinical methodologies to determine dietary cholesterol absorption in humans. The present paper summarises radiolabelled experiments and major advances in stable isotope technologies to determine cholesterol absorption. Initially, direct methods employing gastro-intestinal intubation were developed. Later, indirect methods using oral-faecal cholesterol balance permitted calculation of cholesterol mass absorption. Once the use of radiolabelled [3H, 14C]cholesterol balance was developed in healthy humans, it was finally possible to distinguish exogenous and endogenous cholesterol. Non-invasive and safer stable isotope (2H, 13C, 18O) labelled cholesterol tracers then replaced radioisotopes for use in infants and adults. Stable isotopes and radioisotopes showed identical cholesterol kinetics. The most promising contemporary stable isotope assessment of cholesterol absorption is a dual stable isotope dual tracer approach based on simultaneous administration of oral and intravenous differentially labelled cholesterol tracers, followed by plasma sampling for 3-4 d. Online GC/Combustion/IRMS and GC/Pyrolysis/IRMS allow minimal amounts of dual stable isotope cholesterol tracers to be detected. Using the dual stable isotope dual tracer approach, the percent cholesterol absorption in adult volunteers has been determined to be 50-70%.     

First results from the CRIS experiment

The ability to study rare isotopes with techniques such as mass spectrometry and laser spectroscopy is often prevented by low production rates and large isobaric contamination. This has necessitated the development of novel beam cleaning techniques that can efficiently isolate the isotope of interest. The Collinear Resonance Ionization Spectroscopy (CRIS) experiment at ISOLDE, achieves this by resonantly ionizing a bunched atom beam in a region of ultra high vacuum. This method is motivated by the need to measure the hyperfine structure and isotope shift at the extremes of isospin where typical production rates drop to 1 atom/s. The technique also offers the ability to purify an ion beam and even select long-lived isomeric states (> 1 ms) from the ground state, which can be subsequently studied by decay spectroscopy or mass spectrometry experiments. This paper will report on the successful commissioning of the CRIS beam line and the recent laser spectroscopy results and laser assisted nuclear decay spectroscopy on the neutron deficient francium isotopes.     

Accuracy and precision of a laser-spectroscopy approach to the analysis of δ²H and δ¹⁸O in human urine

The isotope ratio analysis of body water often involves large sample numbers and lengthy sample processing. Here we demonstrate the ability of isotope ratio infrared spectroscopy (IRIS) to rapidly and accurately analyse the isotope ratios of water in urine. We analysed water extracted from human urine using traditional isotope ratio mass spectrometry (IRMS) and compared those values with IRIS-analysed extracted water and un-extracted urine. Regression analyses for δ2H and δ1?O values between (1) extracted water analysed via IRMS and IRIS and (2) urine and extracted water analysed via IRIS were significant (R2=0.99). These results indicate that cryogenic distillation of urine was not required for an accurate estimate of the isotopic composition of urine when using IRIS.     

Box-modeling of bone and tooth phosphate oxygen isotope compositions as a function of environmental and physiological parameters*

A time-dependent box model is developed to calculate oxygen isotope compositions of bone phosphate as a function of environmental and physiological parameters. Input and output oxygen fluxes related to body water and bone reservoirs are scaled to the body mass. The oxygen fluxes are evaluated by stoichiometric scaling to the calcium accretion and resorption rates, assuming a pure hydroxylapatite composition for the bone and tooth mineral. The model shows how the diet composition, body mass, ambient relative humidity and temperature may control the oxygen isotope composition of bone phosphate. The model also computes how bones and teeth record short-term variations in relative humidity, air temperature and δ¹⁸O of drinking water, depending on body mass. The documented diversity of oxygen isotope fractionation equations for vertebrates is accounted for by our model when for each specimen the physiological and diet parameters are adjusted in the living range of environmental conditions.     

Linear responses in time-dependent Hartree-Fock-Bogoliubov method with Gogny interaction

A numerical method to integrate the time-dependent Hartree-Fock-Bogoliubov (TDHFB) equations with Gogny interaction is proposed. The feasibility of the TDHFB code is illustrated by the conservation of the energy, particle numbers, and center of mass in the small amplitude vibrations of ²⁰O . The TDHFB code is applied to the isoscalar quadrupole and/or isovector dipole vibrations in the linear (small amplitude) region in oxygen isotopes, ^{18, 20, 22, 24}O , titanium isotopes, ^{44, 50, 52, 54}Ti , neon isotope, ²⁶Ne , and magnesium isotopes, ^{24, 34}Mg . The isoscalar quadrupole and isovector dipole strength functions are calculated from the expectation values of the isoscalar quadrupole and isovector dipole moments.     

The reversibility of dissimilatory sulphate reduction and the cell-internal multi-step reduction of sulphite to sulphide: insights from the oxygen isotope composition of sulphate

Dissimilatory sulphate reduction (DSR) leads to an overprint of the oxygen isotope composition of sulphate by the oxygen isotope composition of water. This overprint is assumed to occur via cell-internally formed sulphuroxy intermediates in the sulphate reduction pathway. Unlike sulphate, the sulphuroxy intermediates can readily exchange oxygen isotopes with water. Subsequent to the oxygen isotope exchange, these intermediates, e.g. sulphite, are re-oxidised by reversible enzymatic reactions to sulphate, thereby incorporating the oxygen used for the re-oxidation of the sulphur intermediates. Consequently, the rate and expression of DSR-mediated oxygen isotope exchange between sulphate and water depend not only on the oxygen isotope exchange between sulphuroxy intermediates and water, but also on cell-internal forward and backward reactions. The latter are the very same processes that control the extent of sulphur isotope fractionation expressed by DSR. Recently, the measurement of multiple sulphur isotope fractionation has successfully been applied to obtain information on the reversibility of individual enzymatically catalysed steps in DSR. Similarly, the oxygen isotope signature of sulphate has the potential to reveal complementary information on the reversibility of DSR. The aim of this work is to assess this potential. We derived a mathematical model that links sulphur and oxygen isotope effects by DSR, assuming that oxygen isotope effects observed in the oxygen isotopic composition of ambient sulphate are controlled by the oxygen isotope exchange between sulphite and water and the successive cell-internal oxidation of sulphite back to sulphate. Our model predicts rapid DSR-mediated oxygen isotope exchange for cases where the sulphur isotope fractionation is large and slow exchange for cases where the sulphur isotope fractionation is small. Our model also demonstrates that different DSR-mediated oxygen isotope equilibrium values are observed, depending on the importance of oxygen isotope exchange between sulphite and water relative to the re-oxidation of sulphite. Comparison of model results to experimental data further leads to the conclusion that sulphur isotope fractionation in the reduction of sulphite to sulphide is not a single-step process.     

声波作用下液滴碰并沉降及其氢氧同位素特征

液滴沉降过程中会与环境水汽发生物质交换.论文通过小尺度室内实验,研究了低频声波作用下液滴碰并沉降及其与气室环境水汽氢氧同位素交换特征.研究结果证实液滴沉降过程中存在同位素交换,该过程受声波显著影响.声波作用后液滴粒径增大,液滴沉降后汇集水中重同位素出现富集.液滴与环境水汽氢氧同位素交换强度与液滴初始粒径呈正相关,声波对...     

Accuracy and precision of a laser-spectroscopy approach to the analysis of δ2H and δ18O in human urine

The isotope ratio analysis of body water often involves large sample numbers and lengthy sample processing. Here we demonstrate the ability of isotope ratio infrared spectroscopy (IRIS) to rapidly and accurately analyse the isotope ratios of water in urine. We analysed water extracted from human urine using traditional isotope ratio mass spectrometry (IRMS) and compared those values with IRIS-analysed extracted water and un-extracted urine. Regression analyses for δ²H and δ¹⁸O values between (1) extracted water analysed via IRMS and IRIS and (2) urine and extracted water analysed via IRIS were significant (R ²=0.99). These results indicate that cryogenic distillation of urine was not required for an accurate estimate of the isotopic composition of urine when using IRIS.     

Real-time field measurements of stable isotopes in water and CO2 by Fourier transform infrared spectrometry

Continuous records of isotope behaviour in the environment are invaluable to understanding mass and energy fluxes. Although techniques such as isotope ratio mass spectrometry provide high precision data, they are not well suited to the analysis of a large number of samples and are currently restricted to use in the laboratory. Fourier transform infrared spectrometers are relatively cheap and sufficiently portable and robust to be taken into the field to collect continuous records of gas-phase isotope behaviour. Several examples of the application of this technique will be presented. One data set provides half-hourly determinations of vertical profiles of D/H in water vapour above agricultural fields over a 3-week period; the same infrared spectra can also be used to determine 13C/12C in CO2. The technique has also been applied to the study of CO2 in ambient air and in a limestone cave system. Some of the features and complications associated with the method will also be considered.