Search

The doubly labeled water (DLW) method is considered the reference method for the measurement of energy expenditure under free-living conditions. However, the reproducibility of the DLW method in longitudinal studies is not well documented. This study was designed to evaluate the longitudinal reproducibility of the DLW method using 2 protocols developed and implemented in a multicenter clinical trial—the Comprehensive Assessment of Long-term Effects of Reducing Intake of Energy (CALERIE). To document the longitudinal reproducibility of the DLW method, 2 protocols, 1 based on repeated analysis of dose dilutions over the course of the clinical trial (dose-dilution protocol) and 1 based on repeated but blinded analysis of randomly selected DLW studies (test-retest protocol), were carried out. The dose-dilution protocol showed that the theoretical fractional turnover rates for 2 H and 18 O and the difference between the 2 fractional turnover rates were reproducible to within 1% and 5%, respectively, over 4.5 y. The Bland-Altman pair-wise comparisons of the results generated from 50 test-retest DLW studies showed that the fractional turnover rates and isotope dilution spaces for 2 H and 18 O, and total energy expenditure, were highly reproducible over 2.4 y. Our results show that the DLW method is reproducible in longitudinal studies and confirm the validity of this method to measure energy expenditure, define energy intake prescriptions, and monitor adherence and body composition changes over the period of 2.5–4.4 y. The 2 protocols can be adopted by other laboratories to document the longitudinal reproducibility of their measurements to ensure the long-term outcomes of interest are meaningful biologically. This trial was registered at clinicaltrials.gov as NCT00427193. J. Nutr. 144: 777–783, 2014.

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 (2)H and (18)O content and thus minimize sample requirement, reduce analytical cost, and avoid memory effect.The (2)H 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 (18)O 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 (2)H measurements on a Thermo Delta V continuous-flow isotope-ratio instrument.The δ(18)O 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‰.The study demonstrated that continuous-flow isotope-ratio mass spectrometry is capable of producing accurate (18)O measurements on the same sample after (2)H 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.

We evaluated the precision, accuracy and linearity of three commercial gas chromatography/combustion isotope ratio mass spectrometry (GC/C-IRMS) instruments in regard to stable carbon isotope ratio measurements of fatty acids with 13C enrichment levels ranging from natural abundance to 924‰, in comparison to measurements by conventional gas isotope ratio mass spectrometry (GIRMS). Memory effect was also evaluated with a fatty acid mixture containing methyl myristate and methyl stearate at natural abundance levels of 13C but methyl palmitate at 13C enrichment levels of 460 or 924‰. At natural abundance, the δ13C values of fatty acids measured by the GC/C-IRMS instruments were reproducible to within 0.34‰ (range: 0.11‰ to 0.32‰) and accurate to −0.38±0.40‰ (mean difference ± standard deviation; range: −0.03±0.25‰ to −0.84±0.39‰). At enrichment levels of 13C (23 to 924‰), the δ13C values were reproducible to within 1.48‰ (range 0.20‰ to 1.00‰) and accurate to −1.11±3.16‰ (range: −1.02±3.23‰ to +4.56±13.42‰). Stable carbon isotope ratio measurements by all three GC/C-IRMS instruments were found to be linear from −30 to 924‰ (r2 > 0.9999; p ⩽ 0.004) in comparison to those measured by GIRMS. Significant memory effect was found in only one system. Based on our data, the GC/C-IRMS instruments can provide linear, precise and accurate stable carbon isotope ratio measurements and require only pico-and nanogram quantities of samples.

Stable hydrogen isotope methodology is used in nutrition studies to measure growth, breast milk intake, and energy requirement. Isotope ratio MS is the best instrumentation to measure the stable hydrogen isotope ratios in physiological fluids. Conventional methods to convert physiological fluids to hydrogen gas (H(2)) for mass spectrometric analysis are labor intensive, require special reagent, and involve memory effect and potential isotope fractionation. The objective of this study was to determine the accuracy and precision of a platinum catalyzed H(2)-water equilibration method for stable hydrogen isotope ratio measurements. Time to reach isotopic equilibrium, day-to-day and week-to-week reproducibility, accuracy, and precision of stable hydrogen isotope ratio measurements by the H(2)-water equilibration method were assessed using a Thermo DELTA V Advantage continuous-flow isotope ratio mass spectrometer. It took 3 h to reach isotopic equilibrium. The day-to-day and week-to-week measurements on water and urine samples with natural abundance and enriched levels of deuterium were highly reproducible. The method was accurate to within 2.8 (o)/oo and reproducible to within 4.0 (o)/oo based on analysis of international references. All the outcome variables, whether in urine samples collected in 10 doubly labeled water studies or plasma samples collected in 26 body water studies, did not differ from those obtained using the reference zinc reduction method. The method produced highly accurate estimation on ad libitum energy intakes, body composition, and water turnover rates. The method greatly reduces the analytical cost and could easily be adopted by laboratories equipped with a continuous-flow isotope ratio mass spectrometer.

We report on an improved HPLC method to purify erythrocyte cholesterol for use in measuring in vivo cholesterol synthesis from 2H2O. The new procedure uses a smaller compression cartridge column at a flow rate of 3 mL/min. The new method reduces by almost tenfold the amount of solvent required to maintain the analytical purity of the isolated cholesterol.

To test the validity of cotton balls as a urine collection medium for hydrogen and oxygen isotope ratio measurements, we moistened cotton balls with 1-, 5-, and 10-mL aliquots of urine that contained either natural abundances or one of two enriched levels of 2H and 18O. The liquids were then expressed from the cotton balls, either at once, or after 30 or 60 min of atmospheric exposure, and analyzed for their 2H/1H and 18O/16O isotope ratios. The 2H abundances of the fluids expressed from the cotton balls were significantly lower than the original values. This dilution of 2H, however, diminished in the samples that had a greater volume of urine. We observed no effects of volume or time on 18O. Our results indicate that at low urine volumes, ambient moisture dilutes 2H in the cotton balls, but isotope exchanges and evaporation have little or no effect on the hydrogen and oxygen isotopic abundances of the urine samples. Total body water and energy expenditure values calculated from the 2H and 18O enrichments of 5- and 10-mL urine samples were within 1% of the theoretical values. Therefore, cotton balls are suitable for collection of infant urine samples for hydrogen and oxygen isotope ratio measurements, if the volume of urine that can be expressed from a cotton ball is ⩾ 5 mL.

Two commercial elemental-analyzer gas-isotope-ratio mass spectrometer systems (Finnigan Heraeus-Delta E and VG Carlo Erba-PRISM) were evaluated for their ability to analyze 13 C/ 12 C isotope ratios (δ PDB 13 C, -30.71-1433.67‰) and carbon content (36-100%) in a variety of organic samples (isotope standards, chemical standards, agricultural products, and physiological fluids)

A rapid preparative scale purification of erythrocyte free cholesterol has been developed for measurements of in vivo cholesterol synthesis from 2H2O. The quantity and purity of cholesterol obtained is suitable for combustion, zinc reduction of the water formed, and determination of deuterium isotopic content by gas isotope ratio mass spectrometry. The ability to detect and to quantitate a range of cholesterol synthesis rates is illustrated by measurements on young pigs receiving diets without and with added dietary cholesterol.

To assess the possibility of ethnic differences in mineral metabolism in prepubertal children, we compared measures of calcium metabolism in 7- and 8-y-old Mexican-American (MA) and non-Hispanic Caucasian (CAU) girls (n = 38) living in southeastern Texas. We found similar fractional calcium absorption, urinary calcium excretion, calcium kinetic values and total-body bone mineral content in the MA and CAU girls. In contrast, parathyroid hormone (PTH) concentrations were greater in MA girls (4.01 +/- 0.47 vs. 1. 96 +/- 0.50 pmol/L, P = 0.005) than in CAU girls. Serum 25-hydroxyvitamin D concentrations were lower in MA girls (68.9 +/- 7.7 vs. 109.4 +/- 8.4 nmol/L, P = 0.001) than in CAU girls, but 1, 25-dihydroxyvitamin D concentrations did not differ between groups. Seasonal variability was seen for 25-hydroxyvitamin D concentrations in girls of both ethnic groups, but values in all of the girls were >30 nmol/L (12 ng/mL). We conclude the following: 1) greater PTH levels in MA girls than CAU girls are present without evidence of vitamin D deficiency; and 2) differences in 25-hydroxyvitamin D and PTH concentrations between MA and CAU girls do not have a large effect on calcium absorption, excretion or bone calcium kinetics. These data do not provide evidence for adjusting dietary recommendations for mineral or vitamin D intake by MA girls.

We describe a compact ultrafiltration system for the removal of pyrogens and bacteria from water labeled with the stable isotopes of deuterium and oxygen-18. The ultrafiltration system is constructed from readily available commercial components and can achieve complete removal of pyrogens and bacteria from 1L contaminated water within 30 min. By use of our procedure, loss of the isotopically labeled water by retention in the filtration system was minimal. The purified water is suitable for both oral and intravenous administration to healthy human subjects participating in nutrition studies.

Extracellular body water can be determined from plasma bromide dilution. Plasma Br is separated from other anions by ion chromatography and is detected at an ultraviolet wavelength of 210 nm. Plasma proteins are removed by ultrafiltration, and interference by plasma chloride is minimized by dilution and the use of 5 mmol NaCl/L as the eluant. Human plasma samples were spiked with known quantities of Br (between 37.54 and 125.14 mumol/L) and were measured by ion chromatography. The results were reproducible to within 0.72 mumol/L (SD) and differed from the gravimetric values by -1.88 +/- 4.27 mumol/L (mean +/- SD). The difference, however, was not significantly different from 0 (p = 0.19). Extracellular water volumes of 10 newborn minipigs measured by Br dilution by using the chromatographic technique (400 +/- 63 mL/kg) were comparable with literature values reported for premature infants.