Abstract:
A method of measuring energy expenditure in a living subject comprises: (a) administering a specified dose of doubly-labeled water ( 2 H 2   18 O) to a living subject; (b) obtaining samples at three or more times of body water from the living subject; (c) measuring  2 H/ 1 H,  17 O/ 16 O and  18 O/ 16 O ratios in each of the obtained samples using optical spectroscopy; and (d) determining (1) a combined value of flux of body water and exhaled carbon dioxide from a change in measured  18 O/ 16 O over time, (2) a value of flux of body water alone from a change in measured  2 H/ 1 H over time, and (3) a reference value of isotopic background fluctuation from a change in measured  17 O/ 16 O over time. Using  17 O measurements to estimate background fluctuations of the  2 H and  18 O decreases the required isotope dosing of subjects or decreases uncertainty at current dosing levels.

Description:
CROSS-REFERENCE TO RELATED APPLICATION 
       [0001]    The present application claims priority under 35 U.S.C. 119(e) from U.S. provisional patent application 61/511,652, filed Jul. 26, 2011. 
     
    
     TECHNICAL FIELD 
       [0002]    The present invention relates to isotopic analysis of water samples and instruments therefor. The invention also relates to diagnostic methods for measuring total energy expenditure in living subjects using such isotopic analysis of water. 
       BACKGROUND ART 
       [0003]    The high prevalence of obesity in the US is a major public health concern, as overweight and obese individuals are at increased risk for many chronic diseases. Obesity stems from an imbalance between total caloric consumption and total energy expenditure (TEE), although the causes of this imbalance remain debated. Accurate measurements of TEE therefore play a pivotal role in understanding and ultimately reversing this epidemic. 
         [0004]    TEE can be measured using direct (measurement of heat production) or indirect (measurement of respiratory gas exchange) calorimetry, but neither of these approaches are practical for measuring TEE in free living subjects. The gold standard for measuring TEE in free-living individuals is the doubly labeled water (DLW) method, which is based on the principle that the oxygen in body water is in complete isotopic equilibrium with the oxygen in dissolved respiratory carbon dioxide due to the action of carbonic anhydrase. The consequence of this exchange is that an isotopic label of oxygen introduced into body water is eliminated by the combined flux of body water and the exhaled carbon dioxide. Lifson and colleagues reasoned that, since hydrogen is found only in water and not in carbon dioxide, the elimination of a hydrogen isotope would be affected solely by the flux of body water. Thus the difference in the rates of isotope elimination of simultaneously administered oxygen and hydrogen labels is a measure of CO 2  production. Review of the doubly labeled water technique, validation of its assumptions, and equations for calculating metabolic CO 2  production from the isotopic measurements may be found, e.g., in D. A. Schoeller, “Measurement of Energy Expenditure in Free-Living Humans by Using Doubly Labeled Water”, Journal of Nutrition 118, pages 1278-89 (1988); J. R. Speakman, S. Nair, and M. I. Goran, “Revised equations for calculating CO2 production from doubly labeled water in humans”, American Journal of Physiology 264, pages E912-7 (1993); J. R. Speakman, “The history and theory of the doubly labeled water technique”, American Journal of Clinical Nutrition 68(suppl), pages 932S-938S (1998); and others, including references cited in the aforementioned papers. 
         [0005]    Despite its usefulness, the DLW, method has some limitations. First, the test is expensive to perform due to the need for large quantities of H 2   18 O (approx. 0.25 gram per kilogram of a subject&#39;s fat-free mass) in addition to  2 H 2 O. This expense is predominantly due to the cost of the  18 O that is used to label subjects. High levels of  18 O are required to distinguish the dose from fluctuating background isotope levels after 14-28 days of elimination; it currently costs $300-$400 for the  18 O required to perform a DLW measurement on an adult subject (50-100 kg fat-free mass). Thus, widespread adoption of the DLW method has been limited by its high cost. 
         [0006]    High levels of  18 O tracer are needed to ensure that unknown fluctuations in the background isotope levels over time do not contribute excessively to measurement uncertainty. While the isotopic composition of atmospheric oxygen (O 2 ) is itself essentially constant within the time frame of TEE testing, living test subjects also require regular food and water intake for good health, both of which are background sources of hydrogen and oxygen intakes. The isotopic composition of both natural water and various water-bearing foodstuffs vary according to factors such as local evaporation and precipitation rates at their source. Accordingly, daily variations in dietary and beverage intake by the test subjects contribute to the uncertainty in background isotope levels. This uncertainty in the background levels increases the isotope dose that must be administered and contributes to the uncertainty in the DLW measurements as compared to the reference calorimetry measurements of TEE in validation studies. Individual measurements are only precise to ±5%, so the method is currently most suitable for studies of groups rather than individual variation. 
         [0007]    The proposed invention aims to address these two problems by significantly reducing the cost of the DLW method and improving the individual accuracy of the measurements. 
         [0008]    E. R. T. Kerstel, R. Van Trigt, N. Dam, J. Reuss, H. A. J. Meijer, “Laser spectrometry applied to the simultaneous determination of the δ 2 H, δ 17 O, and δ 18 O isotope abundances in water”, IAEA-TECDOC-1247,pp. 7-13 (2001) describes application of infrared laser spectrometry to the simultaneous determination of the relative  2 H/ 1 H,  17 O/ 16 O, and  18 O/ 16 O isotope abundances in natural water. The method uses a narrow line width color center laser directed into gas cells equipped with multiple-pass reflection optics (for≈20 m path length) to record the direct absorption spectrum of low-pressure gas-phase water samples in the 3 μm spectral region (ro-vibrational transitions around 3663 cm −1 ). The precision of the technique is shown to be 0.7% for δ 2 H and 0.5% for δ 17 O and δ 18 O, while the calibrated accuracy is about 3% and 1%, respectively. 
         [0009]    G. Lis, L. I. Wassenaar, and M. J. Hendry, “High-Precision Laser Spectroscopy D/H and 180/160 Measurements of Microliter Natural Water Samples”, Anal. Chem. 80(1), pp. 287-293 (Jan. 1, 2008) describes use of off-axis integrated cavity output spectroscopy (OA-ICOS) for isotopic analysis (δD and δ 18 O) of water samples. A liquid autosampler injects from 0.2 to 1.0 μL of H 2 O into a pre-evacuated optical cavity via heated (70° C.) injection port to facilitate complete evaporation and vapor transfer through a tube. The highly reflective mirrors of the optical cavity extend the average optical path length to ˜3000 m allowing the use of infrared diode lasers operated at room temperature. The laser wavelength is tuned over the absorption spectrum of the isotopologues of interest of the injected H 2 O sample. Random instrumental drift was corrected by systematically spacing standard injections within the autorun and conducting linear interpolations. Potential intersample memory effects and mixing of water samples were overcome by using five sequential injections of each sample, discarding the first two injection results and accepting the mean of the final three injection results. Measurement accuracies of ±0.8% for δD and ±0.1% for δ 18 O were achieved. 
       SUMMARY DISCLOSURE 
       [0010]    The innovation involves measuring the changes over the sampling time in the background isotope levels of  18 O and  2 H indirectly by measuring the undosed  17 O stable isotope of oxygen in the respective body water samples. Natural factors (e.g. fractionation) affect isotopes similarly leading to a correlation in the abundances of different isotope species, such as that found in the meteoric water line. Previous studies have shown in body water measurements that background fluctuations in  2 H and  18 O are correlated with an average R value of 0.79. Assuming a similar correlation with  17 O, using  17 O measurements to estimate the background fluctuations of the  2 H and  18 O will allow researchers to substantially decrease the required isotope, reducing the cost of the  18 O label to an estimated $60-$80, and thus addressing the first major problem with the DLW technique. Alternatively the dose could be maintained at its current level, and the  17 O measurement would provide an estimated five-fold decrease in the uncertainty of the method due to background fluctuation (addressing the second major problem with the method detailed above). 
         [0011]    Measurement of the  17 O isotope by conventional Isotope Ratio Mass Spectrometry (IRMS) with adequate precision to measure the background correlation is sufficiently expensive (e.g. $500-$1000 per sample) to obviate the potential price gains available by measuring the background fluctuations. However, recently developed optical spectroscopy instruments (e.g. using off-axis integrated cavity output spectroscopy, Off-Axis ICOS) can simultaneously and inexpensively (&lt;$50 per sample) measure  2 H,  18 O, and  17 O in liquid water samples. 
     
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
         [0012]      FIG. 1  shows a schematic plan view of an isotopic water analyzer for use with a doubly-labeled water experiment in accord with the present invention. 
           [0013]      FIG. 2A and 2B  shows graphs of absorption spectrum of water isotopomers near 1365 and 1390 nm, respectively, allowing precise measurements of the  17 O/ 16 O,  18 O/ 16 O, and  2 H/ 1 H isotope ratios. 
           [0014]      FIG. 3  shows a graph of repeated measurements of δ 17 O for a single water sample against internal standards. 
           [0015]      FIG. 4  shows a graph of enrichments measured by IRMS over time of  18 O and  17 O following a bolus dose administration in a human subject (Adolescent in Kenya—hence high background level of  18 O). There is no discernible elevation of the  17 O enrichment coincident with the rise in  18 O. 
       
    
    
     DETAILED DESCRIPTION 
       [0016]    We have demonstrated the technical feasibility of utilizing optical spectrometry for DLW measurements ( 2 H/ 1 H and  18 O/ 16 O) of TEE. The test results prove that these instruments are capable of analyzing samples for standard TEE measurements with accuracy comparable to (or exceeding that) obtained with a conventional IRMS. We have additionally demonstrated the use of optical spectrometry to precisely measure  17 O/ 16 O. Furthermore, preliminary IRMS studies have shown that enrichment of  18 O does not result in a concurrent enrichment of  17 O, opening up the possibility of using the  17 O/ 16 O isotope ratio to measure the isotopic background fluctuations during DLW experiments. 
         [0017]    The ratios  2 H/ 1 H,  17 O/ 16 O and  18 O/ 16 O are conventionally described relative to Vienna Standard Mean Ocean Water (VSMOW), available as NIST RM 8535a, which has  2 H/ 1 H of 155.76±0.05 ppm,  17 O/ 16 O of 379.9±0.8 ppm, and  18 O/ 16 O of 2005.20±0.45 ppm. The observed natural range of  2 H/ 1 H is from −836% to +180% (0.0000255 to 0.0001838 molar fractions of  2 H) for all forms of hydrogen (including from natural gas and atmospheric hydrogen) and from −495% to +129% for natural water originating as precipitation. The observed natural range of  18 O/ 16 O is from −62.8% to +109% (0.001875 to 0.002218 molar fractions of  18 O) for all forms of oxygen (including that found in marine carbonates, atmospheric nitrogen oxides, etc.) and from −62.8% to +31.3% for natural water originating as precipitation. In natural waters, δ 17 O variation relative to VSMOW has been found to be 0.5281±0.0015 times the variation in δ 18 O. [T. B. Coplen et al, Isotope-Abundance Variations of Selected Elements, Pure Appl. Chem., Vol. 74, No. 10, pp. 1987-2017, 2002] For purposes of the present invention, the measured values of δ 2 H, δ 17 O and δ 18 O are likewise described relative to the VSMOW standard, using any of a variety of reference samples of known isotopic composition to calibrate the measurements from the OA-ICOS instrumentation. Measured δ 17 O is used to adjust for the effect of isotopic background fluctuations upon the isotope elimination rate from the body water. 
         [0018]    With reference to  FIG. 1 , an isotopic water analyzer uses off-axis integrated cavity output spectroscopy (OA-ICOS) to obtain fully resolved absorption spectra containing all of water&#39;s major isotopologues (H 2 O, H 2 HO, H 2   17 O and H 2   18 O) simultaneously. 
         [0019]    Isotopic water analyzers are available that can measure liquid water samples (as described, e.g., in U.S. Pat. No. 6,839,140 to O&#39;Keefe et al.) as well as water vapor samples. Los Gatos Research, Inc. of Mountain View, Calif. (the assignee of the present invention) supplies isotopic water analyzers (e.g. model 911-0034) that performs water vapor measurements, as well as other analyzers (e.g. model IWA-45EP) that can perform both water vapor and liquid water measurements. 
         [0020]    OA-ICOS instruments and their operation are described in U.S. Pat. No. 6,795,190 to Paul et al., as well as in U.S. Pat. No. 7,468,797 to O&#39;Keefe et al. Off-axis light injection into the optical cavity provides spatial separation of reflections from the cavity mirrors that extend the effective optical path length to −3000 m, resulting in increased absorption and allowing the use of economical, room-temperature, infrared diode lasers. The laser wavelength from one or more laser sources is tuned over a selected absorption spectrum band of water capable of distinguishing between the various major isotopologues of water, such as in the vicinity of 1350 to 1400 nm, and the resulting absorption is measured to high resolution. 
         [0021]    Thus, as seen in  FIG. 1 , an isotopic water analyzer  10  includes a sample cell  12  having a set of access ports  14 ,  20  and  24 . The access port  14  is an inlet for dry air. Room air can enter from an intake  16 , flow through a drierite dryer  18  and then pass through the access port  14  into the sample cell  12 . The dry air flow serves to help remove any residual water from a previous sample by means of flushing and dilution. The access port  20  is a water sample inlet coupled to an autoloader  22  to receive injections of a water sample to be measured. A typical sample volume is approximately 1 μL of liquid water per injection. The third access port  24  is an outlet coupled through a diaphragm pump  26  to an exhaust  28 . 
         [0022]    The sample cell  12  has two highly reflective mirrors  30  and  32  at opposite ends, which define an optical cavity. In the depicted embodiment, two laser sources  34  and  36  direct laser beams through respective collimation lenses  38  and  40  and through one of the mirrors  32  into the optical cavity. In this embodiment, two lasers of slightly different wavelength bands are used to extend the available wavelengths to a full absorption spectrum of all of the major water isotopomers. The lasers can be tunable diode lasers or diode-pumped tunable fiber lasers, for example, and may have respective infrared wavelengths centered near 1365 nm and 1390 nm, respectively. Since the laser beams do not need to be resonantly coupled into the sample cell (i.e. precise beam alignment is not critical), the analyzer is inherently robust thermally and mechanically. The long effective path length of the light inserted into the cavity means that a far wider range of absorbance values (optical depth) can be recorded. 
         [0023]    Light exiting the optical cavity, such as through one of the mirrors  30  of the sample cell  12 , is collected by a lens  42  and detected by an optical detector  44  responsive to the laser wavelengths, such as an InGaAs detector. The analyzer may operate in a ringdown mode, wherein pulses of laser light are injected and the intensity decay time corresponds to the absorbance of the sample at that particular laser wavelength. The pulses may sweep through the absorption spectrum of the water sample and the data analyzed to determine peaks of absorbance for each of the isotopomers of water. 
         [0024]    A temperature probe  46  may be provided to measure sample cell temperature and thermal control may be used for ultra-stable measurement with essentially no drift. 
       Demonstration of Precise Optical Measurements of the  17 O/ 16 O Isotopic Ratio 
       [0025]    We have fabricated an instrument which measures  17 O,  16 O and the  17 O/ 16 O isotopic ratio using optical spectroscopy.  FIG. 2A  shows the optical absorption spectrum in a region in the near-infrared near 1365 nm. Possible wavelength regions for the  17 O measurements include those near 1365 nm (seen in the Figure), near 1390 nm and near 1400 nm, as well as numerous others (such as near 2730 nm for the Kerstel et al. paper described above) provided a suitable laser source is available. 
         [0026]    We have also fabricated instruments which measure  18 O,  16 O,  2 H,  1 H, and the  18 O/ 16 O and  2 H/ 1 H isotopic ratios using optical spectroscopy.  FIG. 2B  shows the optical absorption spectrum in a region in the near-infrared near 1390 nm. While there are dozens of other possible absorption regions, this particular one is well characterized for  18 O and  2 H measurements. 
         [0027]    A single instrument as in  FIG. 1  may combine capabilities to obtain both of the absorption spectra in  FIGS. 2A and 2B . Alternatively, the spectra in  FIGS. 2A and 2B  and corresponding measurements of their respective isotopic ratios can be obtained using two separate instruments. The relative quantities of each isotope in a sample can be determined directly from the relative absorption peaks for each major isotopomer of water. 
         [0028]    While this embodiment uses absorption of infrared light by the water in the 1350-1400 nm range, water&#39;s many other absorption bands could also be used, provided the various isotopomers can be readily distinguished. 
         [0029]      FIG. 3  demonstrates the preliminary measurement precision which we are able to achieve with this instrument, ±0.093% in δ 17 O at 150 samples a day averaging down, to ±0.026% at 10 samples per day. Further improvements in the precision are underway and are expected to improve the precision to better than ±0.010%. 
         [0000]    Demonstration that  18 O Enrichment Does Not Result in  17 O Enrichment 
         [0030]    The industrial process by which  18 O is enriched depends on physical fractionation which may inadvertently also enrich  17 O. Estimates of the levels of  17 O in dose solutions suggest that it is only enriched to about 1/300 th  the level of  18 O. Consequently, if subjects were dosed with  18 O to a level of 1% ppm above background, the enrichment in  17 O would be only 0.3 ppm. In a pilot study we have measured  17 O levels using IRMS in 19 subjects involved in a DLW protocol where urine samples were collected daily.  FIG. 4  shows that there is no detectable effect of the dose water on background  17 O levels. This observation is critical in utilizing  17 O to correct for naturally occurring background isotopic fluctuations in  18 O and  2 H. 
         [0031]    Hence, after measuring  17 O/ 16 O and  18 O/ 16 O, the measured change in  18 O/ 16 O can be proportionally adjusted by dividing its value by the corresponding change in  17 O/ 16 O. That is, measured changes in δ 17 O are entirely due to background enrichment levels and can serve as a proxy for otherwise unknown background fluctuations in δ 18 O. It is also assumed that measured changes in δ 17 O can also serve as a proxy for background fluctuations in δ 2 H, whereby the measured change in  2 H/ 1 H is likewise proportionally adjusted by division with the  17 O/ 16 O. corresponding change in 
       Testing Protocol 
       [0032]    The innovation involves no change in current DLW protocols other than the permitted change in isotope dose and subsequent analysis of  17 O in addition to  2 H and  18 O. Any DLW protocol can be used with the innovation. In general, the DLW technique involves introducing stable isotopes of both oxygen ( 18 O) and hydrogen ( 2 H) into a human or animal subject, often but not exclusively by oral administration of labeled water. The subsequent enrichment of the body water is then measured over time to ascertain the differential rates of elimination of the  18 O and  2 H. Body water can be measured from blood, plasma, urine, saliva, and/or exhaled breath. The difference in the elimination rates of the  18 O and  2 H can then be used to calculate the total energy expenditure (TEE) of the free-living subject. The innovation involves measuring the  17 O in the body water samples and using this measurement to improve the calculations of TEE. Specific examples of TEE testing protocols utilizing the innovation ( 17 O measurements) to either reduce the uncertainty in the calculations or to reduce the cost of the test are provided below. Also included below is a specific example of how to analyze the sample using the preferred instrument to measure the isotopes. 
       Example Testing Protocol for Reduced Uncertainty 
       [0033]    TEE is measured for a 14 day period using DLW. Upon subject arrival for the study, body weight is measured to ±0.1 kg and a baseline urine sample is obtained for determination of background enrichments of  2 H,  18 O and  17 O. Subjects are then given an oral dose of 0.23 g H 2   18 O and 0.12 g  2 H 2 O per kg of total body water (estimated as 73% of the fat-free mass). Urine samples are obtained 4 and 5 hours after the dosing. On the mornings of days 7 and 14, subjects are instructed to discard their first urine void and collect the second urine void of the day. The aliquots are then analyzed in triplicate on the Triple Isotope Water Analyzer. Background fluctuations of  2 H and  18 O are calculated using the measured change in  17 O. Turnover rates and the extrapolated dilution spaces at the time of dosing are calculated from the slope and intercept of the semi-log plot of urine isotope enrichment relative to the time after dosing. CO 2  production rate is calculated using a modification [J. Speakman, K. Nair, M. Goran, “Revised equations for calculating CO2 production from doubly labeled water in humans”, American Journal of Physiology, 264 (1993) E912-917] of the original equation of Schoeller et al. [D. Schoeller, E. Ravussin, Y. Schutz, K. Acheson, P. Baertschi, E. Jequier, “Energy expenditure by doubly labeled water: validation in humans and proposed calculation”, American Journal of Physiology, 250 (1986) R823-830]. O 2  consumption rate is calculated by dividing the CO 2  production rate assuming an RQ of 0.82. Total EE is then calculated using the equation of Weir [J. Weir, “New methods for calculating metabolic rate with special reference to protein metabolism”, Nutrition, 6 (1990) 213-221]. TEE (kcal/d) is determined as the average daily EE over the 14 days of measurement. Using the  17 O to calculate background fluctuations will provide an estimated five-fold decrease in the uncertainty of the TEE calculations due to background fluctuation. 
         [0000]    Example Testing Protocol with Reduced  18 O Dose 
         [0034]    TEE is measured for a 14 day period using DLW. Upon subject arrival for the study, body weight is measured to ±0.1 kg and a baseline urine sample is obtained for determination of background enrichments of  2 H,  18 O and  17 O. Subjects are then given an oral dose of 0.05 g H 2   18 O and 0.12 g  2 H 2 O per kg of total body water (estimated as 73% of the fat-free mass). Urine samples are obtained 4 and 5 hours after the dosing. On the mornings of days 7 and 14, subjects are instructed to discard their first urine void and collect the second urine void of the day. The aliquots are then analyzed in triplicate on the Triple Isotope Water Analyzer. Background fluctuations of  2 H and  18 O are calculated using the measured change in  17 O. Turnover rates and the extrapolated dilution spaces at the time of dosing are calculated from the slope and intercept of the semi-log plot of urine isotope enrichment relative to the time after dosing. CO 2  production rate is calculated using a modification [J. Speakman et al., op. cit.] of the original equation of Schoeller et al. [op. cit.]. O 2  consumption rate is calculated by dividing the CO 2  production rate assuming an RQ of 0.82. Total EE is then calculated using the equation of Weir [op. cit.]. TEE (kcal/d) is determined as the average daily EE over the 14 days of measurement. Using the  17 O to calculate background fluctuations in this case reduces the cost of the measurement approximately 5-fold while, we estimate, maintaining current levels of uncertainty in TEE calculations. 
       Sample Analysis Example 
       [0035]    The preferred instrument is an optical spectrometer capable of simultaneously measuring δ 2 H, δ 18 O, and δ 17 O. Samples collected from any doubly labeled water experiment can be analyzed using the instrument. The recommended protocol is as follows. Prepare the samples for analysis by the instrument. For urine samples, this involves centrifuging the samples and removing the supernatant for analysis, or samples can be carefully distilled. Preparation steps vary depending on the body water that is to be measured. Samples are pipetted into autosampler vials and loaded into an autosampler for automated analysis, or can be manually injected into the instrument. Calibration standards are measured throughout the analysis, interleaved between samples to continuously calibrate the instrument. The specific number of injections and frequency of calibration is dependent on the salinity and isotopic enrichment of the samples being analyzed. After analysis, the calibration standards are used to adjust the raw output of the instrument to give final measured values for δ 2 H, δ 18 O, and δ 17 O.