Accuracy of 18O isotope ratio measurements on the ...

Research Article Received: 7 August 2015

Revised: 2 September 2015

Accepted: 7 September 2015

Published online in Wiley Online Library

Rapid Commun. Mass Spectrom. 2015, 29, 2252–2256 (wileyonlinelibrary.com) DOI: 10.1002/rcm.7390

Accuracy of δ18O isotope ratio measurements on the same sample by continuous-flow isotope-ratio mass spectrometry William W. Wong* and Lucinda L. Clarke Department of Pediatrics, USDA/ARS Children’s Nutrition Research Center, Baylor College of Medicine, Houston, Texas, USA RATIONALE: The doubly labeled water method is considered the reference method to measure energy expenditure.

Conventional mass spectrometry requires a separate aliquot of the same sample to be prepared and analyzed separately. With continuous-flow isotope-ratio mass spectrometry, the same sample could be analyzed sequentially for both 2H and 18 O content and thus minimize sample requirement, reduce analytical cost, and avoid memory effect. METHODS: The 2H contents of 197 urine samples collected from 22 doubly labeled water studies were determined using a Thermo Delta V Advantage continuous-flow isotope-ratio mass spectrometer. The 18O content of these samples was measured either using a separate aliquot of the same sample using a VG Isogas gas-isotope-ratio mass spectrometer or using the same sample following the 2H measurements on a Thermo Delta V continuous-flow isotope-ratio instrument. RESULTS: The δ18O values using the same aliquot of samples were accurate to 0.18 ± 2.61 ‰ (mean difference ± standard deviation (SD); 95% CI, –0.18 to 0.55 ‰; P = 0.33) compared with the values based on the standard conventional method. Bland and Altman pair-wise comparison also yielded a bias of 0.18 ‰ with a 95% limit of agreement between –4.94 and 5.30 ‰. CONCLUSIONS: The study demonstrated that continuous-flow isotope-ratio mass spectrometry is capable of producing accurate 18O measurements on the same sample after 2H measurements. The method greatly reduces the analytical cost and sample size requirement and could easily be adopted by any laboratories equipped with a continuous-flow isotope-ratio mass spectrometer. Copyright © 2015 John Wiley & Sons, Ltd.

Energy is required for muscular activity, growth, reproduction and synthesis of metabolites such as proteins, fatty acids, nucleic acids and steroids, which are essential to maintain basal metabolic functions as well as optimal growth and development. The doubly labeled water (DLW) method yields an average energy expenditure (EE) for a period of 5–14 days depending on the age of the study population. The procedure is noninvasive, nonrestrictive and reflective of the actual EE under free-living conditions.[1–8] Currently, the DLW method is considered to be the reference method for the estimation of EE or caloric requirements in free-living subjects.[9–15] With the DLW method, accurate measurements of the stable hydrogen and oxygen isotope ratios in physiological fluids such as urine, saliva or blood are required. Gasisotope-ratio mass spectrometry is considered to be the best instrumental method for the accurate and precise measurements of these isotopes in physiological samples.[16,17] For 2H measurements, the samples can be reduced to H2 either manually offline using the zinc reduction method[17,18] or online using the chromium reduction method.[19] For 18O measurements, the H2O-CO2 equilibration method is the preferred method.[20] However, two separate aliquots of the same sample must be processed and analyzed separately.

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* Correspondence to: W. W. Wong, USDA/ARS Children’s Nutrition Research Center, 1100 Bates Street, Houston, TX 77030, USA. E-mail: [email protected]

Rapid Commun. Mass Spectrom. 2015, 29, 2252–2256

Recently, we validated a platinum-catalyzed H2O-H2 equilibration method using a continuous-flow isotope-ratio mass spectrometry (CF-IRMS) system.[21] This method requires no pre-treatment of the physiological sample and offers an opportunity to measure the 18O content of the same sample following the 2H measurement. With the continuousflow method, the sample tube is flushed with the carrier gas, filled with the gas of interest (H2 or CO2), and then analyzed for its 2H or 18O content. However, it has not been documented whether significant isotope fractionation taking place during the sample preparation and mass spectrometric analysis for 2H measurements might affect the accuracy of the subsequent 18O measurements. In the current study, we sought to determine whether the 18 O content of 197 urine samples collected from 16 adult and 6 toddler DLW studies can be measured accurately following the 2H measurements on the same sample using CF-IRMS.

EXPERIMENTAL Mass spectrometer systems Continuous-flow isotope-ratio mass spectrometer system A Thermo Delta V Advantage continuous-flow isotope-ratio mass spectrometer system equipped with a GasBench II online gas preparation and introduction system (Thermo Electron North America, Palm Beach, FL, USA) was used to assess the accuracy and reproducibility of the continuous-

Copyright © 2015 John Wiley & Sons, Ltd.

δ18O isotope ratio measurements by CF-IRMS flow method for stable oxygen isotope ratio measurements following 2H measurements on the same sample. This mass spectrometer is equipped with a current-controlled rather than a field-regulated magnet. For 2H measurements, the mass spectrometer was set to H mode with high voltage at 3 kV and the magnet at 1579 steps, representing a voltage of 0.06516 V and an output current of 650 mA. The reference H2 (99.995%, Air Liquide Healthcare, Houston, TX, USA) pressure was set at 449 kPa and the helium carrier gas (99.999%, Air Liquide Healthcare) pressure was set at 414 kPa. For 18O measurements, the mass spectrometer was set to CO2 mode with high voltage at 3 kV and the magnet at 11,616 steps, representing a voltage of 0.31394 V and an output current of 3.14945 A. The reference CO2 (99.995%, Air Liquide Healthcare) pressure was set at 449 kPa and the helium carrier gas (99.999%, Air Liquide Healthcare) pressure was set at 414 kPa. Conventional gas-isotope-ratio mass spectrometer system A VG Isogas gas-isotope-ratio mass spectrometer equipped with an ISOPREP-18 H2O-CO2 equilibration system (VG Isogas Ltd, Middlewich, UK) was used to establish the 18O content of the urine samples collected in the DLW studies.[17] The mass spectrometer was set to CO2 mode with accelerating voltage at 2440 V, ion repeller at –9.84 V, electron energy at 69.95 eV, and trap current at 600 μA. The ISOPREP-18 H2O-CO2 equilibration system consists of 48 1-mL equilibration vessels, each of which is attached to a two-way solenoid valve through a 9.5-mm Ultra-torr fitting. The solenoid valve is connected to one of two common banks through a 0.015-mm i.d. capillary. The banks and the vessels are enclosed in a transparent plastic cabinet with a fan to circulate the air, and both banks can be made to oscillate under shaker control. Each bank can be either evacuated or connected to the inlet system of the mass spectrometer or to a CO2 reservoir through a three-way solenoid valve. Sample preparation procedures Continuous-flow isotope-ratio mass spectrometry


Conventional gas-isotope-ratio mass spectrometry A 100-μL aliquot of each urine sample was introduced into the 1-mL equilibration vessel with an Eppendorf variable-volume micropipette (Brinkmann Instruments, Inc., Westbury, NY, USA). The vessels were attached to the two-way solenoid valves, evacuated through the capillaries for 30 s, and then filled with CO2 (99.99%, Air Liquide Healthcare) to 30 kPa pressure. The sample-CO2 mixtures were allowed to equilibrate for 10 h at 25°C with constant shaking. At the end of the equilibration, the CO2 in each vessel was allowed to expand sequentially through the capillary into the cold trap for 30 s and finally into the inlet system of the VG Isogas mass spectrometer for 18O measurements. A desktop computer controlled the entire process of evacuation, CO2 injections, equilibration, CO2 extraction, and data collection automatically. Isotope-ratio measurements All stable isotope ratio measurements are expressed in delta (δ) per mil unit (‰) vs the international reference materials, Standard Mean Ocean Water (SMOW) and Standard Light Antarctic Precipitation (SLAP), as follows:[22,23] δ2 HSMOW=SLAP or δ18 OSMOW=SLAP ð‰Þ 18 0 2 1 H or 16 O 1H O sample sample B C 18 O 1A ¼ @2 H 1 H reference or 16 O reference where (2H/1H)sample and (2H/1H)reference represent the 2H/1H ratio of the sample and the reference H2, respectively, and (18O/16O)sample and (18O/16O)reference represent the 18O/16O ratio of the sample and the reference CO2, respectively. A change of 1 δ2HSMOW/SLAP unit is equivalent to a change of 0.2 ppm in 2H content and a change of 1 δ18OSMOW/SLAP unit is equivalent to a change of 2 ppm in 18O content. Institutional Review Boards The Institutional Review Boards for Human Studies at Baylor College of Medicine and Boston Children’s Hospital approved the human protocols used to generate the urine samples. Statistical analysis Paired samples t-test was used to compare the δ18O results obtained from the CF-IRMS method using the same sample with those generated from the conventional gas-isotope-ratio mass spectrometer method. The Bland and Altman pairwise comparison analysis[24] was used to evaluate the accuracy of the CF-IRMS for the measurements of δ18O values on urine samples against those generated using the reference gasisotope-ratio mass spectrometry method. The Bland and Altman pairwise comparison analysis also was used to compare the total energy expenditure (TEE) values calculated using the δ18O values measured using the continuous-flow


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Approx. 5 mg of activated charcoal (Fisher Scientific, Sugar Land, TX, USA) and ~200 mg of copper powder (Fisher Scientific) were introduced into an uncapped 12-mL Exetainer (Labco International Inc., Ceredigion, UK), followed by a platinum catalytic rod (Thermo Scientific, Madison, WI, USA). The activated charcoal and copper powder were added in order to remove any potential contaminants in the samples that might poison the catalyst. After 0.2 mL of urine had been put into the Exetainer, it was recapped, placed into the GasBench II and flushed with 2% H2 in helium at 483 kPa pressure (99.999% H2 and 99.996% helium, Air Liquide Healthcare) for 7 min. The sample was allowed to equilibrate with the H2 at room temperature for a minimum of 4 h. At the end of the equilibration, an aliquot of the H2 in the Exetainer was injected into the Thermo Delta V Advantage mass spectrometer for stable hydrogen isotope ratio measurement against the reference H2. After the hydrogen measurement, the punctured cap was replaced with a new cap and the Exetainer containing the urine was placed into the GasBench II and flushed with 0.3% CO2 in helium at 483 kPa pressure (99.999% CO2 and 99.996% helium, Air Liquide Healthcare)

for 7 min. The sample was allowed to equilibrate with the CO2 at room temperature for a minimum of 20 h. At the end of the equilibration, an aliquot of the CO2 in the Exetainer was injected into the mass spectrometer for 18O measurement.

W. W. Wong and L. L. Clarke method with the TEE values calculated using the δ18O values measured using the reference gas-isotope-ratio mass spectrometry method. An α value of 0.05 was used in all statistical analyses. All statistical analyses were performed using SPSS version 23 (IBM Corp., Armonk, NY, USA).

RESULTS As shown in Table 1, the δ18O values of the 197 urine samples collected from 16 adult and 6 toddler DLW studies ranged between –5.51 ‰ and 193.36 ‰ based on reference gasisotope-ratio mass spectrometry. Based on paired samples t-test, the δ18O values of the urine samples measured by CF-IRMS were not different (P = 0.33) from those measured by conventional gas-isotope-ratio mass spectrometry, with a mean difference of 0.18 ± 0.19 ‰ (mean ± SE) and a 95% confidence limit between –0.18 ‰ and 0.55 ‰. The Bland and Altman pairwise comparison analysis between the δ18O values measured by CF-IRMS and by conventional gas-isotope-ratio mass spectrometry is summarized in Fig. 1. On average, the δ18O values measured by CF-IRMS were higher than those measured by conventional gasisotope-ratio mass spectrometry by 0.18 ‰ (bias). The Bland and Altman pairwise analysis also yielded a 95% limit of agreement between –4.94 ‰ and 5.3 ‰. As shown in Fig. 1, only the differences of six urine samples were outside the 95% limits of agreement. Essentially 97% of the δ18O values measured by the two mass spectrometry systems fell within the 95% limits of agreement. Furthermore, linear regression analysis indicated that the differences between the δ18O values were not a function of the average δ18O values (P = 0.96). To further highlight the significance of our finding, the TEE values calculated using the δ18O values measured by the continuous-flow method were compared with those calculated using the δ18O values measured by conventional gas-isotope-ratio mass spectrometry using the Bland and Altman pairwise comparison analysis. As shown in Fig. 2, the Bland and Altman pairwise comparison yielded a bias of 31 kcal/day with a 95% limit of agreement between –97 kcal/day and 159 kcal/day. With one exception, all the TEE values calculated using the continuous-flow method were within –3.5% to 4.8% of the reference values, based on the δ18O values using conventional gas-isotope-ratio mass spectrometry. The one exception was observed among the toddlers but the TEE value by the continuous-flow method was still within 9.7% of the reference value.

Figure 1. Bland and Altman pair-wise comparison of the δ18O values of the 197 urine samples collected from 16 adults and 6 toddlers doubly labeled water studies measured by continuous-flow isotope-ratio mass spectrometry and by reference gas-isotope-ratio mass spectrometry. The solid line represents zero difference between the two measurements. The dashed line represents the bias between the two measurements. The two dotted lines represent the 95% limits of agreement between the two measurements. The plus symbols in the figure represent the individual differences between the two measurements. The P value is generated from a linear regression analysis on the differences in δ18O values and the average δ18O values between the two measurements.

DISCUSSION Our results showed that the δ18O values of urine samples measured by the CF-IRMS system following the δ2H measurements on the same sample are as accurate as those measured by conventional gas-isotope-ratio mass spectrometry. Since the same sample can be used for both 2 H and 18O measurements, the method will minimize sample size requirement. Minimizing sample size requirement is particularly important when performing DLW studies in a pediatric population and in small animals or when a blood sample is required. Although not described here, the method also works for saliva samples. The ability to measure both 2H and 18O content on the same sample using CF-IRMS also can reduce analytical cost and improve sample throughput. Although the offline zinc reduction method[18] and the online chromium reduction method[19] for 2H measurements require very small samples

Table 1. Comparison of the normalized δ18O values measured by the Thermo Delta V continuous-flow isotope-ratio mass spectrometer system (Delta V) and by the VG Isogas gas-isotope-ratio mass spectrometer system (VG) using paired samples t-test

Instruments Delta V VG

n

δ18O values (‰)a

Minimum δ18O values (‰)

Maximum δ18O values (‰)

Mean difference (‰)b

Lower limit (‰)c

Upper limit (‰)c

P

197 197

63.07 ± 3.56 62.89 ± 3.56

-8.14 -5.51

185.42 193.36

0.18 ± 0.19

-0.18

0.55

0.33

a

Values are mean ± SE. Mean difference = δ18O values by Delta V – c 95% confident intervals. b

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18

O values by VG, mean ± SE.



δ18O isotope ratio measurements by CF-IRMS followed by the H2O-CO2 equilibration method to measure the 18O content on the same sample will definitely reduce analytical cost, avoid memory effect, and improve sample throughput. For example, we estimated that one could reduce the cost of supplies by $2 per sample, which could add up quickly when large numbers of samples are being processed in the mass spectrometry laboratory. The major cost saving comes from technician time. It takes approximately 2 h to set up the ISOPREP-18 H2O-CO2 equilibration system to analyze 48 samples. However, with the continuous-flow method, it takes ~10 min to replace the caps for 48 samples.

CONCLUSIONS Figure 2. Bland and Altman pair-wise comparison of the total energy expenditure (TEE) values calculated using the δ18O values measured by the continuous-flow method and by the reference gas-isotope-ratio mass spectrometry. The sold line represents zero difference between the two measurements. The dashed line represents the bias between the two measurements. The two dotted lines represent the 95% limits of agreement between the two measurements. The open hexagon symbols in the figure represent the individual differences between the two measurements.


Acknowledgements Supported by USDA/ARS (Grant #6250-51000-053) and by a grant from Nutrition Science Initiative (NuSI). The contents of this publication do not necessarily reflect the views or policies of the USDA and NuSI, nor does mention of trade names, commercial products, or organizations imply endorsement.

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(1–10 μL), with the zinc reduction method, each sample must be prepared separately using a high-vacuum sample preparation system, custom-made reaction vessels, hightemperature vacuum grease, a high-temperature heating block, and a special zinc reagent that is available only from a single source. Although the zinc reduction method requires a very small amount of sample and avoids a memory effect, the offline sample preparation procedure is labor-intensive and requires the use of expensive equipment and reagents. The online chromium reduction method is currently the method of choice in many laboratories because it uses very tiny amounts of sample and the procedure of sample injection is automated. However, the sample must be pretreated offline with activated charcoal and filtered through a 0.2-μm filter. The pretreatment often requires several mL of the physiological fluid. The chromium reduction materials often are good for approximately 200 injections and have to be checked periodically with laboratory reference materials to ensure their reduction integrity. Since all samples are injected through the same chromium reduction tube, memory effect is an issue. Although the memory effect can be reduced by analyzing the samples with lower 2H content prior to samples with enriched levels of 2H, multiple injections of the same sample are required particularly between samples with different 2H content. The chromium reduction tubes are costly when purchased directly from the mass spectrometer manufacturer. Therefore, most laboratories using the online chromium reduction method purchase the reduction tubes and the reduction materials directly from the manufacturer and store the reduction tubes in their laboratories. In order to change the reduction tube, the heating block must be turned off and the inlet must be isolated from the mass spectrometer system. Therefore, the entire process of using the online chromium reduction method is also labor-intensive and costly. Therefore, the ability to use the platinum-catalyzed H2-water equilibration method to measure the 2H content

Our manuscript represents the first evaluation of continuousflow isotope-ratio mass spectrometry for accurate 18O measurements on the same sample following the 2H measurement using the platinum-catalyzed H2-water equilibration method. The method is simple, accurate, less labor-intensive and without memory effect. Therefore, any laboratories equipped with a continuous-flow isotope-ratio mass spectrometer system can easily adopt the method to support their nutrition studies.

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