Determinants of Free-Living Energy Expenditure in Normal Weight and Obese Women Measured by Doubly Labeled Water

Total free-living energy expenditure (TEE) was measured in 9 normal weight controls and 5 obese women using the doubly labeled water (DLW) method. Resting energy expenditure (REE) and the thermic effect of food (TEF) were measured by indirect calorimetry and the energy cost of physical activity (PA) calculated by deduction, in order to quantify the components and identify determinants of free-living TEE. Although REE was quantitatively the major component of TEE in both groups, PA best explained the variability, contributing 76% to the variance in free-living TEE. The obese women had elevated values for TEE (12397+/-2565 vs. 8339+/-1787 kJ/d, mean+/-SD; p<0.00S), compared with the control women. PA (5071+/-2385 vs. 2552+/-1452; p<0.0S) and REE (6393+/-678 vs. 5084+/-259; p<0.000S) were also raised in the obese, whereas TEF was not significantly different between the groups, accounting for 7.6% of energy expenditure for the obese and 8% for the control subjects. Body weight was the single best determinant of mean daily free-living TEE across both groups. We conclude that PA and body weight are the main determinants of free-living TEE .     

Analytic Requirements for the Doubly Labeled Water Method

The doubly labeled water method is the first method that accurately measures total daily energy expenditure in free-living subjects over periods of days to weeks. Validations have indicated that the method can be performed with a coefficient of variation of between 3% and 5%. This precision, however, is dependent on the quality of the isotopic analyses. A recent interlaboratory comparison has indicated that there is a wide variation in the accuracy and precision with which deuterium and ¹⁸O enrichments are measured. This reduces the accuracy and precision with which a laboratory will perform the doubly labeled water method and in some cases may limit the application of this technique. Herein we review the analytical requirements for optimal use of the method and some of the potential sources of error in the stable isotope analysis.     

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.     

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.     

The Maastricht Protocol for the Measurement of Body Composition and Energy Expenditure with Labeled Water

An update of practical aspects of the use of labeled water for the measurement of total body water (TBW) and energy expenditure (EE) is presented as applied in Maastricht, The Netherlands. We use a 10-hour equilibration period. The isotopes for the measurement of TBW and EE are routinely administered, after collecting a background urine sample, as a last consumption before the night. Our data show an underestimate of TBW measured with isotope dilution after 4 hours (in the morning), a discrepancy which increases with the size of TBW. No such relation and no significant differences were found after 10-hour (overnight) equilibration. The ratio between the dilution space for deuterium and oxygen-18 is higher than the earlier figure of 1.03, especially in adult subjects with a high body fat content. For an observation period of EE over two weeks, samples from the second and the last voiding on the first, mid, and last day of the observation period are collected. Differences in EE calculated from morning and evening samples within the first and second week allow detection of sampling errors and if so, samples are excluded from the final calculation. Differences of EE between weeks 1 and 2 allow a check for the consistency of the subjects' physical activity level and usually fall within 10% of the average EE over the total observation interval.     

Comparison of Zinc Reduction with Platinum Reduction for Analysis of Deuterium‐Enriched Water Samples for the Doubly Labeled Water Technique

Objective: Isotope ratio mass spectrometry of hydrogen and oxygen is frequently used to determine total energy expenditure (TEE) using doubly labeled water. Conventionally, hydrogen isotope ratio is determined in hydrogen gas generated from water samples using zinc reduction. We compare this with a new automated platinum method to determine the ratios of hydrogen isotopes in deuterium‐enriched water samples. Research Methods and Procedures: The platinum method of sample preparation was compared with the zinc method in three ways: analytical variation in deuterium enrichment (within sample; n = 51), analytical variation in TEE estimates (within sample set; n = 10), and level of agreement of TEE estimates between both methods (n = 14). Results: For the zinc method, the standard deviation for multiple sets of triplicate ²H₂O sample analysis was ±4.36‰ and ±2.07‰ for platinum. The correlation between TEE estimates when sample sets were analyzed in duplicate was r = 0.89 for zinc and r = 0.83 for platinum. The intercept and slope of the regression line were significantly different from the line of identity for duplicate TEE estimates by zinc but were not different from the line of identity for platinum. After correction for the intra‐assay variation of each method, the correlation between zinc and platinum for TEE was 0.77, and the intercept, but not the slope, of the regression was significantly different from the line of identity. The mean difference between the zinc method and the platinum method was 56 kcal/day, and the 95% confidence interval was ?438 to 550 kcal/day. Discussion: These data suggest that the platinum method is at least as reliable as the zinc method as a sample preparation technique for isotope ratio mass spectrometry of deuterium‐enriched water samples. The platinum method is also less costly and less labor‐intensive than the zinc method.     

Physiological Daily Inhalation Rates for Free-Living Pregnant and Lactating Adolescents and Women Aged 11 to 55 Years,Using Data from Doubly Labeled Water Measurements for Use in Health Risk Assessment

The distribution of physiological daily inhalation rates for pregnant and lactating females aged 11 to 55 years was determined according to total daily energy expenditures, energy costs for growth, pregnancy and lactation (maternal milk-energy synthesis and breast-energy output) in free-living females. Such published data were obtained using a methodology based on the disappearance rates of predetermined doses of doubly labeled water (²H₂O and H₂ ¹⁸O) in urine from non-pregnant and non-lactating females (n = 357), as well as saliva from gravid and breastfeeding females (n = 91), monitored by gas-isotope-ratio mass spectrometry over an aggregate period of about 6,000 days. Monte Carlo simulations were necessary to integrate total daily energy requirements of non-pregnant and non-lactating females into energy costs and weight changes at the 9th, 22nd, and 36th week of pregnancy and at the 6th and 27th, postpartum week: 540,000 data were simulated. The present article confirms that physiological daily inhalation rates for under-, normal-, and overweight/obese pregnant and lactating females expressed in m³/day and m³/kg-day are higher than those for males. For instance, in normal-weight subjects, inhalation rates are higher by 18 to 41% throughout pregnancy and 23 to 39% during postpartum weeks: actual values were higher in females by 1.13 to 2.01 m³/day at the 9th week of pregnancy, 3.74 to 4.53 m³/day at the 22nd week and 4.41 to 5.20 m³/day at the 36th week, and by 4.43 to 5.30 m³/day at the 6th postpartum week and 4.22 to 5.11 m³/day at the 27th postpartum week. The highest 99th percentiles were found to be 0.622 m³/kg-day in pregnant females and 0.647 m³/kg-day in lactating females. By comparison, the highest 99th percentile value for individuals aged 2.6 months to 96 years was determined to be 0.725 m³/kg-day in Brochu et al. (2006a) Brochu, P, Ducré-Robitaille, J F and Brodeur, J. 2006a. Physiological daily inhalation rates for free-living individuals aged 1 month to 96 years, using data from doubly labeled water measurements: a proposal for air quality criteria, standard calculations and health risk assessment. Hum Ecol Risk Assess, 12: 675–701. [CSA][Taylor & Francis Online], [Web of Science ®] [Google Scholar]. Air quality criteria and standard calculations based on the latter value for non-carcinogenic toxic compounds should therefore be protective for virtually all pregnant and lactating females. The present article highlights evidence that the current default assumption regarding the total daily air intake used by the Integrated Risk Information System (IRIS) to derive human equivalent concentrations in reference dose calculations is also underestimated compared to some higher 75th and 90th percentiles of physiological daily inhalation rates in pregnant and lactating females.     

Physiological Daily Inhalation Rates for Free-Living Individuals Aged 1 Month to 96 Years,Using Data from Doubly Labeled Water Measurements: A Proposal for Air Quality Criteria,Standard Calculations and Health Risk Assessment

Reported disappearance rates of oral doses of doubly labeled water (²H₂O and H₂ ¹⁸O) in urine, monitored by gas-isotope-ratio mass spectrometry for an aggregate period of over 30,000 days and completed with indirect calorimetry and nutritional balance measurements, have been used to determine physiological daily inhalation rates for 2210 individuals aged 3 weeks to 96 years. Rates in m³/kg-day for healthy normal-weight individuals (n = 1252) were higher by 6 to 21% compared to their overweight/obese counterparts (n = 679). Rates for healthy normal-weight males and females drop by about 66 to 75% within the course of a lifetime. Infants and children between the age of 3 weeks to less than 7 years inhale 1.6 to 4.3 times more air (0.395 ± 0.048 to 0.739 ± 0.071 m³/kg-day, mean ± S.D., n = 581) than adults aged 23 to 96 years (0.172 ± 0.037 to 0.247 ± 0.039 m³/kg-day, n = 388). The 99th percentile rate of 0.725 m³/kg-day based on measurements for boys aged 2.6 to less than 6 months is recommended for air quality criteria and standard calculation for non-carcinogenic compounds pertaining to individuals of any age or gender (normality confirmed using the Shapiro-Wilk test, p ≥ 0.05). This rate is 2.5-fold more protective than the daily inhalation estimate of 0.286 m³/kg-day published by the Federal Register in 1980 (i.e., 20 m³/day for a 70-kg adult). It ensures that very few newborns aged 1 month and younger, less than 1% of infants aged 2.6 to less than 6 months and of course no older individuals up to 96 years of age inhale more toxic chemicals than associated safe doses which are not anticipated to result in any adverse effects in humans, when air concentration reaches the resulting air quality criteria and standard values. This rate is also protective for underweight, overweight, and obese individuals. Finally, as far as newborns are concerned, a rate of 0.956 m³/kg-day based on the 99th percentile estimates is recommended for short-term criteria and standard calculations for toxic chemicals that yield adverse effects over instantaneous to short-term duration.     

Pulse Dipolar ESR of Doubly Labeled Mini TAR DNA and Its Annealing to Mini TAR RNA

Pulse dipolar electron-spin resonance in the form of double electron electron resonance was applied to strategically placed, site-specifically attached pairs of nitroxide spin labels to monitor changes in the mini TAR DNA stem-loop structure brought on by the HIV-1 nucleocapsid protein NCp7. The biophysical structural evidence was at Ångstrom-level resolution under solution conditions not amenable to crystallography or NMR. In the absence of complementary TAR RNA, double labels located in both the upper and the lower stem of mini TAR DNA showed in the presence of NCp7 a broadened distance distribution between the points of attachment, and there was evidence for several conformers. Next, when equimolar amounts of mini TAR DNA and complementary mini TAR RNA were present, NCp7 enhanced the annealing of their stem-loop structures to form duplex DNA-RNA. When duplex TAR DNA-TAR RNA formed, double labels initially located 27.5 Å apart at the 3′- and 5′-termini of the 27-base mini TAR DNA relocated to opposite ends of a 27 bp RNA-DNA duplex with 76.5 Å between labels, a distance which was consistent with the distance between the two labels in a thermally annealed 27-bp TAR DNA-TAR RNA duplex. Different sets of double labels initially located 26–27 Å apart in the mini TAR DNA upper stem, appropriately altered their interlabel distance to ∼35 Å when a 27 bp TAR DNA-TAR RNA duplex formed, where the formation was caused either through NCp7-induced annealing or by thermal annealing. In summary, clear structural evidence was obtained for the fraying and destabilization brought on by NCp7 in its biochemical function as an annealing agent and for the detailed structural change from stem-loop to duplex RNA-DNA when complementary RNA was present.     

Using Doubly-Labeled Water to Measure Energy Expenditure in an Important Small Ectotherm Drosophila melanogaster

Energy expenditure is a key variable in the study of ageing,and the fruit fly Drosophila melanogaster is a model organism that has been used to make step changes in our understanding of the ageing process.Standard methods for measurement of energy expenditure involve placing individuals in metabolic chambers where their oxygen consumption and CO:production can be quantified.These measurements require separating individuals from any social context,and may only poorly reflect the environment in which the animals normally live.The doubly-labeled water(DLW) method is an isotope-based technique for measuring energy expenditure which overcomes these problems.However,technical challenges mean that the smallest animals this method has been previously applied to weighed 50-200 mg.We overcame these technical challenges to measure energy demands in Drosophila weighing 0.78 mg.Mass-specific energy expenditure varied between 43 and 65 raW-g.These estimates are considerably higher than estimates using indirect calorimetry of Drosophila in small metabolic chambers(around 18 mW-g).The methodology we have established extends downwards by three orders of magnitude the size of animals that can be measured using DLW.This approach may be of considerable value in future ageing research attempting to understand the genetic and genomic basis of ageing.     

Physiological Daily Inhalation Rates for Free-Living Individuals Aged 2.6 Months to 96 Years Based on Doubly Labeled Water Measurements: Comparison with Time-Activity-Ventilation and Metabolic Energy Conversion Estimates

In the first part of this article, a critical review of traditional approaches used to estimate daily inhalation rates as a function of age for health risk assessment purposes shows that such rates are not totally reliable due to various biases introduced by both quantitative and qualitative deficiencies regarding certain input parameters. In the second part, the magnitude of under- and overestimations of published inhalation rates derived from each approach is described by a comparison with new sets of physiological daily inhalation rates and distribution percentile values based on total daily energy expenditures (TDEEs) measured by the doubly labeled water (DLW) method. TDEEs are derived from the analysis of deuterium (²H) and heavy oxygen-18 (¹⁸O) in urine samples by gas-isotope-ratio mass spectrometry during an aggregate period of over 20,000 days for unrestrained free-living normal-weight individuals aged 2.6 months to 96 years (n = 1252). Regarding physiological values based on DLW measurements, opposite tendencies have been observed between two sets of estimates using time-activity patterns both biased by conservative input data assumptions during sleep and light activities: most estimates based on the time-activity-ventilation approach are overestimated, whereas most of those using metabolic equivalent approach are underestimated. Erroneous food intakes have clearly lead to underestimated rates when used in daily food-energy intake (EFD) and Parameter A approaches. With the latter approach, overestimated basal metabolic rates (BMRs) used in EFD/BMR ratios contribute to the underestimation of inhalation values. Few mean daily inhalation rates and Monte Carlo simulation percentiles based on traditional approaches (57 out of 253) are close to physiological values within a gap of ± 5% or less. Aggregate errors in all estimates (in m³/day and m³/kg-day) vary from -52 to +126%. The most accurate daily inhalation rates are those based on DLW measurements with an error of about ± 5%, as calculated in previous studies for free-living males and females aged 1 month to 96 years and pregnant and lactating adolescents and women aged 11 to 55 years during real-life situations in their normal surroundings.     

利用转座质粒plasposon构建荧光标记的脱色希瓦氏菌S12

采用分子生物学手段将具有转座功能的自杀性质粒pTnMod-okm与荧光蛋白基因eyfp构建重组质粒pTE-okm。pTE-okm通过结合转移进入脱色希瓦氏菌S12中,质粒上的转座子元件转座到S12的染色体上,而质粒本身的窄宿主复制位点使其在S12中不能得到有效的复制而"自杀"。荧光显微镜下筛选表达荧光蛋白的脱色希瓦氏菌克隆,通过对其提取质粒确定pTE-okm已经在脱色希瓦氏菌中自杀。筛选得到生长速度未发生延迟、脱色能力不受影响的荧光标记菌株S12-40。标记的脱色希瓦氏菌在无抗生素压力的情况下培养,传代20次(8h/次)后在荧光显微镜下依然查看到荧光蛋白的表达。该菌株的构建为研究其生态学行为奠定了基础。     

Bio-Imaging of Colorectal Cancer Models Using Near Infrared Labeled Epidermal Growth Factor

Novel strategies that target the epidermal growth factor receptor (EGFR) have led to the clinical development of monoclonal antibodies, which treat metastatic colorectal cancer (mCRC) but only subgroups of patients with increased wild type KRAS and EGFR gene copy, respond to these agents. Furthermore, resistance to EGFR blockade inevitably occurred, making future therapy difficult. Novel bio-imaging (BOI) methods may assist in quantization of EGFR in mCRC tissue thus complementing the immunohistochemistry methodology, in guiding the future treatment of these patients. The aim of the present study was to explore the usefulness of near infrared-labeled EGF (EGF-NIR) for bio-imaging of CRC using in vitro and in vivo orthotopic tumor CRC models and ex vivo human CRC tissues. We describe the preparation and characterization of EGF-NIR and investigate binding, using BOI of a panel of CRC cell culture models resembling heterogeneity of human CRC tissues. EGF-NIR was specifically and selectively bound by EGFR expressing CRC cells, the intensity of EGF-NIR signal to background ratio (SBR) reflected EGFR levels, dose-response and time course imaging experiments provided optimal conditions for quantization of EGFR levels by BOI. EGF-NIR imaging of mice with HT-29 orthotopic CRC tumor indicated that EGF-NIR is more slowly cleared from the tumor and the highest SBR between tumor and normal adjacent tissue was achieved two days post-injection. Furthermore, images of dissected tissues demonstrated accumulation of EGF-NIR in the tumor and liver. EGF-NIR specifically and strongly labeled EGFR positive human CRC tissues while adjacent CRC tissue and EGFR negative tissues expressed weak NIR signals. This study emphasizes the use of EGF-NIR for preclinical studies. Combined with other methods, EGF-NIR could provide an additional bio-imaging specific tool in the standardization of measurements of EGFR expression in CRC tissues.     

Unveiling Undercover Cropland Inside Forests Using Landscape Variables: A Supplement to Remote Sensing Image Classification

The worldwide demand for food has been increasing due to the rapidly growing global population, and agricultural lands have increased in extent to produce more food crops. The pattern of cropland varies among different regions depending on the traditional knowledge of farmers and availability of uncultivated land. Satellite images can be used to map cropland in open areas but have limitations for detecting undergrowth inside forests. Classification results are often biased and need to be supplemented with field observations. Undercover cropland inside forests in the Bale Mountains of Ethiopia was assessed using field observed percentage cover of land use/land cover classes, and topographic and location parameters. The most influential factors were identified using Boosted Regression Trees and used to map undercover cropland area. Elevation, slope, easterly aspect, distance to settlements, and distance to national park were found to be the most influential factors determining undercover cropland area. When there is very high demand for growing food crops, constrained under restricted rights for clearing forest, cultivation could take place within forests as an undercover. Further research on the impact of undercover cropland on ecosystem services and challenges in sustainable management is thus essential.     

Molecular Imaging of Oral Premalignant and Malignant Lesions Using Fluorescently Labeled Lectins

Aberrant glycosylation during carcinogenesis results in altered glycan expression on oral cancer cells. The objective of this study was to detect this atypical glycosylation via imaging of fluorophore-conjugated lectins. Paired normal and tumor tissue from seven patients with oral squamous cell carcinoma were investigated for sialic acid expression via the legume protein wheat germ agglutinin (WGA). Fluorophore (Alexa Fluor 350 and Alexa Fluor 647) conjugated WGA was topically applied to the tissue samples and imaged using a custom wide-field fluorescence imaging system. All seven patients had histologically confirmed disease with 6/7 exhibiting squamous cell carcinoma and 1/7 exhibiting dysplasia. Fluorescent data collected from all patients demonstrated that fluorophore conjugated WGA could distinguish between pathologically normal and diseased tissue with the average signal-to-noise ratio (SNR) among all patients being 5.88 (P = .00046). This SNR was statistically significantly higher than the SNR from differences in tissue autofluorescence (P = .0049). A lectin inhibitory experiment confirmed that lectin binding is molecularly specific to overexpressed tumor glycans and that fluorescence is not due to tissue optical properties or tissue diffusion differences. These results illustrate that changes in tumor glycan content of oral neoplasms can be detected with optical imaging using topically applied fluorescently labeled WGA. Lectin targeting of oral lesions using optical imaging may provide a new avenue for the early detection of oral cancers.     

Choline Uptake and Acetylcholine Synthesis in Synaptosomes: Investigations Using Two Different Labeled Variants of Choline

Abstract: Using sequential incubations in media of different K⁺ composition, we investigated the dynamics of choline (Ch) uptake and acetylcholine (ACh) synthesis in rat brain synaptosomal preparations, using two different deuterated variants of choline and a gas chromatographic-mass spectrometric (GC-MS) assay for ACh and Ch. Synaptosomes were preincubated for 10 min in a Krebs medium with or without high K⁺ and with 2 μM-[²H₉]Ch. At the end of the preincubation all variants of ACh and Ch were measured in samples of the pellet and medium. In the second incubation (4 min) samples of synaptosomes were resuspended in normal or high K⁺ solutions containing [²H₄]Ch (2 μM) and all variants of ACh and Ch were measured in the pellet and medium at the end of this period. This protocol allowed us to compare the effects of preincubation in normal or high K⁺ solution on the metabolism during a second low or high K⁺ incubation of a [²H₉]Ch pool accumulated during the preincubation period. Moreover, we were able to compare and contrast the effects of this protocol on [²H₉]Ch metabolism versus [²H₄]Ch metabolism. The most striking result we obtained was that [²H₉]Ch that had been retained by the synaptosomes after the preincubation was not acetylated during a subsequent incubation in normal or high K⁺ media. This result suggests that if an intraterminal pool of Ch is involved in ACh synthesis, the size of this pool is below the limits of detection of our assay. We have confirmed the observation that a prior depolarizing incubation results in an enhanced uptake of Ch during a second incubation in normal K⁺ Krebs. Moreover, Ch uptake is stimulated by prior incubation under depolarizing conditions relative to normal preincubation when the second incubation is in a high K⁺ solution. These results are discussed in terms of current models of the regulation of ACh synthesis in brain.