Protein turnover models for LC-MS data of heavy water metabolic labeling.

Protein turnover is vital for cellular functioning and is often associated with the pathophysiology of a variety of diseases. Metabolic labeling with heavy water followed by liquid chromatography coupled to mass spectrometry is a powerful tool to study in vivo protein turnover in high throughput and large scale. Heavy water is a cost-effective and easy to use labeling agent. It labels all nonessential amino acids. Due to its toxicity in high concentrations (20% or higher), small enrichments (8% or smaller) of heavy water are used with most organisms. The low concentration results in incomplete labeling of peptides/proteins. Therefore, the data processing is more challenging and requires accurate quantification of labeled and unlabeled forms of a peptide from overlapping mass isotopomer distributions. The work describes the bioinformatics aspects of the analysis of heavy water labeled mass spectral data, available software tools and current challenges and opportunities.

A collagen extraction and deuterium oxide stable isotope tracer method for the quantification of bone collagen synthesis rates in vivo.

The development of safe and practical strategies to prevent weakening of bone tissue is vital, yet attempts to achieve this have been hindered by a lack of understanding of the short-term (days-weeks) physiology of bone collagen turnover. To address this, we have developed a method to quantify bone collagen synthesis in vivo, using deuterium oxide (D2 O) tracer incorporation techniques combined with gas chromatography pyrolysis isotope-ratio mass spectrometry (GC-pyrolysis-IRMS). Forty-six male and female rats from a selectively bred model ingested D2 O for 3 weeks. Femur diaphyses (FEM), tibia proximal (T-PRO), and distal (T-DIS) epiphyses-metaphyses and tibia mid-shaft diaphyses (T-MID) were obtained from all rats after necropsy. After demineralisation, collagen proteins were isolated and hydrolysed and collagen fractional synthetic rates (FSRs) determined by incorporation of deuterium into protein-bound alanine via GC-pyrolysis-IRMS. The collagen FSR for the FEM (0.131 ± 0.078%/day; 95% CI [0.106-0.156]) was greater than the FSR at T-MID (0.055 ± 0.049%/day; 95% CI [0.040-0.070]; p < 0.001). The T-PRO site had the highest FSR (0.203 ± 0.123%/day; 95% CI [0.166-0.241]) and T-DIS the lowest (0.027 ± 0.015%/day; 95% CI [0.022-0.031]). The three tibial sites exhibited different FSRs (p < 0.001). Herein, we have developed a sensitive method to quantify in vivo bone collagen synthesis and identified site-specific rates of synthesis, which could be applicable to studies of human bone collagen turnover.

Sweet taste of heavy water.

Hydrogen to deuterium isotopic substitution has only a minor effect on physical and chemical properties of water and, as such, is not supposed to influence its neutral taste. Here we conclusively demonstrate that humans are, nevertheless, able to distinguish D2O from H2O by taste. Indeed, highly purified heavy water has a distinctly sweeter taste than same-purity normal water and can add to perceived sweetness of sweeteners. In contrast, mice do not prefer D2O over H2O, indicating that they are not likely to perceive heavy water as sweet. HEK 293T cells transfected with the TAS1R2/TAS1R3 heterodimer and chimeric G-proteins are activated by D2O but not by H2O. Lactisole, which is a known sweetness inhibitor acting via the TAS1R3 monomer of the TAS1R2/TAS1R3, suppresses the sweetness of D2O in human sensory tests, as well as the calcium release elicited by D2O in sweet taste receptor-expressing cells. The present multifaceted experimental study, complemented by homology modelling and molecular dynamics simulations, resolves a long-standing controversy about the taste of heavy water, shows that its sweet taste is mediated by the human TAS1R2/TAS1R3 taste receptor, and opens way to future studies of the detailed mechanism of action.

Quantifying Moisture Penetration in Encapsulated Devices by Heavy Water Mass Spectrometry: A Standard Moisture Leak Using Poly(ether-ether-ketone).

Moisture penetration into active biomedical implants such as the bionic ear and eye is a major problem in healthcare since surgery is required to replace devices affected by corrosion. Existing methods for measuring moisture leak rates such as the commercially available dynamic relative humidity method are not sufficiently sensitive to guarantee security against moisture penetration. Helium leak detection is highly sensitive but is challenged by the unknown relation to the moisture leak rate because of mixed flow modes involving liquid water. A standard moisture leak traceable to fundamental units is not currently available, preventing direct comparison of moisture and helium leak rates in the same device. Here, we demonstrate a practical calibrated moisture leak based on the stable polymer poly(ether-ether-ketone), for calibrating heavy water mass spectrometry. Using biomedical test structures from manufactured encapsulations, we show that in the majority of cases, calibrated measurements of molar moisture leak rates exceed the helium leak rate, especially for very small and large leaks. Comparison with theory shows that LaPlace pressure is the driving force for the enhanced moisture flows. We recommend that the compliance limit for helium testing in biomedical devices be reduced by one order of magnitude.

Comparing saliva and urine samples for measuring breast milk intake with the 2H oxide dose-to-mother technique among children 2-4 months old.

Saliva and urine are the two main body fluids sampled when breast milk intake is measured with the 2H oxide dose-to-mother technique. However, these two body fluids may generate different estimates of breast milk intake due to differences in isotope enrichment. Therefore, we aimed to assess how the estimated amount of breast milk intake differs when based on saliva and urine samples and to explore whether the total energy expenditure of the mothers is related to breast milk output. We used a convenience sample of thirteen pairs of mothers and babies aged 2-4 months, who were exclusively breastfed and apparently healthy. To assess breast milk intake, we administered doubly labelled water to the mothers and collected saliva samples from them, while simultaneously collecting both saliva and urine from their babies over a 14-d period. Isotope ratio MS was used to analyse the samples for 2H and 18O enrichments. Mean breast milk intake based on saliva samples was significantly higher than that based on urine samples (854·5 v. 812·8 g/d, P = 0·029). This can be attributed to slightly higher isotope enrichments in saliva and to a poorer model fit for urine samples as indicated by a higher square root of the mean square error (14·6 v. 10·4 mg/kg, P = 0·001). Maternal energy expenditure was not correlated with breast milk output. Our study suggests that saliva sampling generates slightly higher estimates of breast milk intake and is more precise as compared with urine and that maternal energy expenditure does not influence breast milk output.

Assessing Breastfeeding Practices Objectively Using Stable Isotope Techniques.

An objective method of assessing breastfeeding practices is required to evaluate progress toward the World Health Organization Global Target 2025: to increase exclusive breastfeeding (EBF) rates in the first 6 months to at least 50% by 2025. Currently, assessment of EBF at the population level is based on mother or caregiver reporting, which risks recall and social desirability bias. A more objective method is the deuterium oxide dose to mother (DTM) technique, in which lactating mothers are given a small amount of deuterium-labeled water. The infant receives deuterium during breastfeeding, and a compartmental model is used to determine the amount of human milk consumed by the infant, and the exclusivity of breastfeeding practices. If the amount of human milk consumed by an infant is determined using the DTM technique and the concentration of nutritional components or potentially toxic contaminants is measured, then the infant's intake of essential nutrients or environmental contaminants can be ascertained.

Training-induced changes in daily energy expenditure: Methodological evaluation using wrist-worn accelerometer, heart rate monitor, and doubly labeled water technique.

INTRODUCTION: Wrist-mounted motion sensors can quantify the volume and intensity of physical activities, but little is known about their long-term validity. Our aim was to validate a wrist motion sensor in estimating daily energy expenditure, including any change induced by long-term participation in endurance and strength training. Supplemental heart rate monitoring during weekly exercise was also investigated. METHODS: A 13-day doubly labeled water (DLW) measurement of total energy expenditure (TEE) was performed twice in healthy male subjects: during two last weeks of a 12-week Control period (n = 15) and during two last weeks of a 12-week combined strength and aerobic Training period (n = 13). Resting energy expenditure was estimated using two equations: one with body weight and age, and another one with fat-free mass. TEE and activity induced energy expenditure (AEE) were determined from motion sensor alone, and from motions sensor combined with heart rate monitor, the latter being worn during exercise only. RESULTS: When body weight and age were used in the calculation of resting energy expenditure, the motion sensor data alone explained 78% and 62% of the variation in TEE assessed by DLW at the end of Control and Training periods, respectively, with a bias of +1.75 (p <.001) and +1.19 MJ/day (p = .002). When exercise heart rate data was added to the model, the combined wearable device approach explained 85% and 70% of the variation in TEE assessed by DLW with a bias of +1.89 and +1.75 MJ/day (p <.001 for both). While significant increases in TEE and AEE were detected by all methods as a result of participation in regular training, motion sensor approach underestimated the change measured by DLW: +1.13±0.66 by DLW, +0.59±0.69 (p = .004) by motion sensor, and +0.98±0.70 MJ/day by combination of motion sensor and heart rate. Use of fat-free mass in the estimation of resting energy expenditure removed the biases between the wearable device estimations and the golden standard reference method of TEE and demonstrated a training-induced increase in resting energy expenditure by +0.18±0.13 MJ/day (p <.001). CONCLUSIONS: Wrist motion sensor combined with a heart rate monitor during exercise sessions, showed high agreement with the golden standard measurement of daily TEE and its change induced by participation in a long-term training protocol. The positive findings concerning the validity, especially the ability to follow-up the change associated with a lifestyle modification, can be considered significant because they partially determine the feasibility of wearable devices as quantifiers of health-related behavior.

Deuterium Oxide Labeling for Global Omics Relative Quantification: Application to Lipidomics.

A novel quantitative mass spectrometric method based on partial metabolic deuterium oxide (D2O) labeling, named "Deuterium Oxide Labeling for Global Omics Relative Quantification (DOLGOReQ)", was developed for relative quantification of lipids on a global scale. To assess the precision and robustness of DOLGOReQ, labeled and unlabeled lipids from HeLa cells were mixed in various ratios based on their cell numbers. Using in-house software developed for automated high-throughput data analysis of DOLGOReQ, the number of detectable mass isotopomers and the degree of deuterium labeling were exploited to filter out low quality quantification results. Quantification of an equimolar mixture of HeLa cell lipids exhibited high reproducibility and accuracy across multiple biological and technical replicates. Two orders of magnitude of effective dynamic range for reasonable relative quantification could be established with HeLa cells mixed from 10:1 to 1:10 ratios between labeled and unlabeled samples. The quantification precision of DOLGOReQ was also illustrated with lipids commonly detected in both positive and negative ion modes. Finally, quantification performance of DOLGOReQ was demonstrated in a biological sample by measuring the relative change in the lipidome of HeLa cells under normal and hypoxia conditions.

Total Energy Expenditure, Physical Activity Level, and Water Turnover of Collegiate Dinghy Sailors in a Training Camp.

Prior studies have examined offshore sailing and energy strategies using accurate total energy expenditure (TEE) measurement in free-living conditions. However, no research has studied energy and water requirement during dinghy class sailing such as an Olympic event with concentrated training. This study aimed to investigate the TEE, physical activity level (PAL), and water turnover (rH2O) of collegiate dinghy sailors in a training camp using the doubly labeled water (DLW) method. Eleven dinghy sailing collegiate athletes (9 males and 2 females) participated. The DLW method was used to determine the participants' TEE and PAL over 8 days (6 training, 2 non-training days). Participants trained approximately 7 h/day on water. Body fat was measured using a stable-isotope dilution method. The rH2O was estimated using deuterium turnover. The mean TEE, PAL, and rH2O were 17.30 ± 4.22 MJ/day (4133 ± 1009 kcal/day), 2.8 ± 0.3 (range, 2.1 to 4.1), and 3.3±0.7 (range, 2.6 to 4.5) L/day, respectively. To our knowledge, this was the first study to use the DLW method to determine TEE, PAL, and rH2O as references for competitive dinghy sailors in a spring training camp. Our results may serve as a reference to assist competitive dinghy sailors in determining their required nutritional support.

Energy Expenditure during Extreme Endurance Exercise: The Giro d'Italia.

PURPOSE: Little data are available on doubly labeled water (DLW) assessed total daily energy expenditure (TDEE) during extreme endurance exercise. Doubly labeled water is considered the gold standard to measure TDEE, but different calculations are being used, which may have a large impact on the results. The aim of the current study was to measure TDEE during the Giro d'Italia and apply two different calculation methods. METHODS: Seven male cyclists (age, 28 ± 5 yr; body mass index, 22.1 ± 2.1 kg·m) completed the 24-d professional cycling race "Giro d'Italia" in which a total distance of 3445 km was covered, including 10 mountain stages. Total daily energy expenditure was measured over the entire duration of the race, with the ingestion of DLW at three different time points. To calculate TDEE and body composition, the isotope dilution space was calculated using two different techniques, the "plateau" and "intercept" technique. RESULTS: The %fat mass at baseline was 7.8% and 16.8% with the plateau and intercept technique respectively and did not significantly change over the course of the race. Total daily energy expenditure was on average 32.3 ± 3.4 MJ·d using the plateau technique versus 28.9 ± 3.2 using the intercept technique, resulting in an average physical activity level (PAL) of 4.37 ± 0.43 versus 3.91 ± 0.39, respectively. The dilution space ratio was on average 1.030 with the plateau and 1.060 with the intercept technique. CONCLUSIONS: Given that the observed dilution space ratio with the plateau technique is similar as the expected ratio from literature and the % fat mass of 7.8% is more realistic for the athletes being studied, we propose the application of the plateau rather than the intercept method, when using DLW during extreme endurance exercise.

Active Image-Assisted Food Records in Comparison to Regular Food Records: A Validation Study against Doubly Labeled Water in 12-Month-Old Infants.

Overreporting of dietary intake in infants is a problem when using food records (FR), distorting possible relationships between diet and health outcomes. Image-assisted dietary assessment may improve the accuracy, but to date, evaluation in the pediatric setting is limited. The aim of the study was to compare macronutrient and energy intake by using an active image-assisted five-day FR against a regular five-day FR, and to validate image-assistance with total energy expenditure (TEE), was measured using doubly labeled water. Participants in this validation study were 22 healthy infants randomly selected from the control group of a larger, randomized intervention trial. The parents reported the infants' dietary intake, and supplied images of main course meals taken from standardized flat-surfaced plates before and after eating episodes. Energy and nutrient intakes were calculated separately using regular FR and image-assisted FRs. The mean (± standard deviations) energy intake (EI) was 3902 ± 476 kJ/day from the regular FR, and 3905 ± 476 kJ/day from the FR using active image-assistance. The mean EI from main-course meals when image-assistance was used did not differ (1.7 ± 55 kJ, p = 0.89) compared to regular FRs nor did the intake of macronutrients. Compared to TEE, image-assisted FR overestimated EI by 10%. Without validation, commercially available software to aid in the volume estimations, food item identification, and automation of the image processing, image-assisted methods remain a more costly and burdensome alternative to regular FRs in infants. The image-assisted method did, however, identify leftovers better than did regular FR, where such information is usually not readily available.

High-Throughput Measurement of Lipid Turnover Rates Using Partial Metabolic Heavy Water Labeling.

Novel analytical platforms for high-throughput determination of lipid turnover in vivo have been developed based on partial metabolic 2H2O labeling. The performance on lipid kinetics measurement of our methods was validated in three different liquid chromatography-mass spectrometry (LC-MS) setups: MS-only, untargeted MS/MS, and targeted MS/MS. The MS-only scheme consisted of multiple LC-MS runs for quantification of lipid mass isotopomers and an extra LC-MS/MS run for lipid identification. The untargeted MS/MS format utilized multiple data-dependent LC-MS/MS runs for both quantification of lipid mass isotopomers and lipid identification. An in-house software was also developed to streamline the data processing from peak area quantification of mass isotopomers to exponential curve fitting for extracting the turnover rate constant. With HeLa cells cultured in 5% 2H2O media for 48 h, we could deduce the species-level turnover rates of 108 and 94 lipids in the MS-only and untargeted MS/MS schemes, respectively, which covers 13 different subclasses and spans 3 orders of magnitude. Furthermore, the targeted MS/MS setup, which performs scheduled LC-MS/MS experiments for some targeted lipids, enabled differential measurement between the turnover rates of the head and tail groups of lipid. The reproducibility of our lipid kinetics measurement was also demonstrated with lipids that commonly detected in both positive and negative ion modes or in two different adduct forms.

Development and validation of bioelectrical impedance analysis equations for prediction total body water and fat-free mass using D2O technique in Moroccan children aged between 8 and 11 years old.

BACKGROUND/OBJECTIVES: Estimating body composition using bioelectric impedance analysis (BIA) requires specific prediction equations. The purpose of our study was to examine the validity of published BIA equations for assessing total body water (TBW) and fat-free mass (FFM) using deuterium oxide dilution (D2O) as a reference method and to develop new FFM and TBW BIA equations if needed for Moroccan pre-pubertal children. SUBJECTS/METHODS: Data were collected from 247 schoolchildren aged 8-11 years old. Children were sorted by gender and age group and assigned in development and validation groups. D2O dilution was used as reference method for estimating TBW and FFM. Bland and Altman test, effect size, pure error, and proportional bias were used to assess the reliability of previous published equations. Cross-validation was performed by Bland and Altman test and BIA new equations were developed by linear regression. RESULTS: Previously published equations were tested and showed significant bias values indicating that if used they would provide biased values of TBW and FFM. The new prediction equations developed were: [Formula: see text] (l) = 0.269 + 0.292 Ht2/R (cm2/Ω) + 0.221 weight (kg) + 0.824 sex (boys = 1, girls = 0) + 0.291 age (years) (R2 = 0.91, RMSE = 1.54 kg); [Formula: see text] (kg) = -0.450 + 0.380 Ht2/R (cm2/Ω) + 0.291 weight (kg) + 1.294 sex (boys = 1, girls = 0) + 0.446 age (years) (R2 = 0.91, RMSE = 1.97 kg). These equations provide better values of proportional bias, agreement, and pure error than the other tested equations. CONCLUSIONS: The new BIA prediction equations seem to be the most accurate for Moroccan pre-pubertal children.

Predictive validity of body fat percentage by bioimpedance compared with deuterium oxide dilution in Costa Rican schoolchildren.

OBJECTIVE: The purpose of this study was to verify the validity of body fat percentage (BF%) by bioelectrical impedance analysis (BIA) in a sample of Costa Rican schoolchildren. METHODS: The sample consisted of 52 male (mean age 8.1 ± 0.9 years) and 49 female (mean age 7.5 ± 1.0 years) schoolchildren. Deuterium oxide dilution was the reference method used as the "gold standard" to determine the fat mass (FM) expressed as BF%. Linear regression analyses and paired sample t-tests were used to test the association and mean differences between BIA and deuterium oxide dilution BF%. Concordance between BIA and deuterium oxide dilution BF% was determined by Lin's concordance correlation coefficient. Measurement of agreement between the two methods was analyzed using the Bland-Altman procedure. RESULTS: Significant associations between BIA and deuterium oxide dilution BF% were found in males (R = 0.97, R2 = 0.95, P < .0001) and in females (R = 0.98, R2 = .96, P < .0001). The measurements of BF% (mean ± SD) were not significantly different between males (BIA 31.1 ± 7.6% vs. deuterium oxide dilution 31.0 ± 7.4%) and females (BIA 25.9 ± 7.9% vs. deuterium oxide dilution 26.3 ± 7.8%) by t-test. Lin's concordance correlation coefficient identified an almost perfect strength of agreement between males and females. Bland-Altman plots showed that the Bodystat measured similar BF% compared to the deuterium oxide dilution in both males and females. CONCLUSIONS: BIA Bodystat presented high agreement with BF% measured by deuterium oxide dilution; therefore BIA Bodystat is recommended for estimating BF% in Costa Rican schoolchildren.

Determination of (2)H-enrichment of rat brain interstitial fluid and rat plasma by headspace-gas-chromatography - quadrupole-mass-spectrometry.

(2)H2O as nonradioactive, stable marker substance is commonly used in preclinical and clinical studies and the precise determination of (2)H2O concentration in biological samples is crucial. However, aside from isotope ratio mass spectrometry (IRMS), only a very limited number of methods to accurately measure the (2)H2O concentration in biological samples are routinely established until now. In this study, we present a straightforward method to accurately measure (2)H-enrichment of rat brain interstitial fluid (ISF) and rat plasma to determine the relative recovery of a cerebral open flow microperfusion (cOFM) probe, using headspace-gas-chromatography - quadrupole-mass-spectrometry. This method is based on basic-catalyzed hydrogen/deuterium exchange in acetone and detects the (2)H-labelled acetone directly by the headspace GC-MS. Small sample volumes and limited number of preparation steps make this method highly competitive. It has been fully validated. (2)H enriched to 8800 ppm in plasma showed an accuracy of 98.9% and %Relative Standard Deviation (RSD) of 3.1 with n = 18 over three days and with two operators. Similar performance was obtained for cerebral ISF enriched to 1100 ppm (accuracy: 96.5%, %RSD: 3.1). With this highly reproducible method we demonstrated the successful employment of (2)H2O as performance marker for a cOFM probe.

Stratum corneum lipid matrix: Location of acyl ceramide and cholesterol in the unit cell of the long periodicity phase.

The extracellular lipid matrix in the skin's outermost layer, the stratum corneum, is crucial for the skin barrier. The matrix is composed of ceramides (CERs), cholesterol (CHOL) and free fatty acids (FFAs) and involves two lamellar phases: the short periodicity phase (SPP) and the long periodicity phase (LPP). To understand the skin barrier thoroughly, information about the molecular arrangement in the unit cell of these lamellar phases is paramount. Previously we examined the molecular arrangement in the unit cell of the SPP. Furthermore X-ray and neutron diffraction revealed a trilayer arrangement of lipids within the unit cell of the LPP [D. Groen et al., Biophysical Journal, 97, 2242-2249, 2009]. In the present study, we used neutron diffraction to obtain more details about the location of lipid (sub)classes in the unit cell of the LPP. The diffraction pattern revealed at least 8 diffraction orders of the LPP with a repeating unit of 129.6±0.5Å. To determine the location of lipid sub(classes) in the unit cell, samples were examined with either only protiated lipids or selectively deuterated lipids. The diffraction data obtained by means of D2O/H2O contrast variation together with a gradual replacement of one particular CER, the acyl CER, by its partly deuterated counterpart, were used to construct the scattering length density profiles. The acyl chain of the acyl CER subclass is located at a position of ~21.4±0.2Å from the unit cell centre of the LPP. The position and orientation of CHOL in the LPP unit cell were determined using tail and head-group deuterated forms of the sterol. CHOL is located with its head-group positioned ~26±0.2Å from the unit cell centre. This allows the formation of a hydrogen bond with the ester group of the acyl CER located in close proximity. Based on the positions of the deuterated moieties of the acyl CER, CHOL and the previously determined location of two other lipid subclasses [E.H. Mojumdar et al., Biophysical Journal, 108, 2670-2679, 2015], a molecular model is proposed for the unit cell of the LPP. In this model CHOL is located in the two outer layers of the LPP, while CER EOS is linking the two outer layers with the central lipid layers. Finally the two other lipid subclasses are predominantly located in the central layer of the LPP.

Using doubly-labelled water to measure free-living energy expenditure: Some old things to remember and some new things to consider.

The doubly-labelled water (DLW) method provides the ability to measure the energy expenditure of free-living animals based only on the injection of two isotopes in water (one of oxygen and one of hydrogen) and traditionally the collection of 2 blood samples. We review here the fundamental basis of how the method works, and highlight how the choice of the appropriate calculation equation can have a large impact on the resultant estimates, particularly in species where the difference between the isotope elimination constants is small. This knowledge is not new, but is worth reiterating given the potential for error by making the wrong choice. In particular, it is important to remember that for mammals weighing less than 5kg, and birds weighing less than 2kg, the single pool models perform best in validation studies, while in mammals above 15kg the two-pool models perform best. Above 2kg in birds and between 5 and 15kg in mammals, however, the model superiority is uncertain. Even where the choice based on body mass would appear clear, the decision may need to be tempered by species specific information regarding potential additional sources for hydrogen turnover, such as de novo lipogenesis or methanogenesis. Recent advances in the technique have included attempts to make the method less invasive by using innovative methods for dosing and sample collection. In addition, the advent of laser spectroscopy, as a replacement technology for mass spectrometry, may open up many new opportunities in the field. These potentially include direct sampling of breath in the field and tracking background isotope drift using 17oxygen levels.

Total energy expenditure assessed by salivary doubly labelled water analysis and its relevance for short-term energy balance in humans.

RATIONALE: The doubly labelled water (DLW) method is a stable isotopic technique for measuring total energy expenditure (TEE). Saliva is the easiest sampling fluid for assessing isotopic enrichments, but blood is considered superior because of its rapid exchange with body water. Therefore, we compared a large range of isotopic enrichments in saliva and blood, and related TEE in subjects with their ad libitum total energy intake (TEI). The relevance of these parameters to body weight and fat change over an 8-day interval was also assessed. METHODS: Thirty subjects underwent DLW analysis over either 8 or 14 days, during which time initial and final blood and saliva enrichments were compared. TEI was assessed by dieticians over the 8-day period only. Isotope ratio mass spectrometry was used for the measurement of δ(2)H and δ(18)O values. RESULTS: No discrepancies were observed between sampling fluids over a wide range of enrichments. During the 8-day period, average TEI exceeded TEE by ~5% or less. Using saliva as sampling fluid, TEI and TEI-TEE, but not TEE, were positively correlated to body weight change. TEI-TEE and physical activity EE (AEE), but not TEI, correlated, respectively, positively and negatively to changes in fat mass. CONCLUSIONS: The DLW method in humans can be reliably applied using saliva as sampling fluid. TEI-TEE as well as AEE contributes significantly to changes in fat mass over an 8-day period.

Reconciling Estimates of Cell Proliferation from Stable Isotope Labeling Experiments.

Stable isotope labeling is the state of the art technique for in vivo quantification of lymphocyte kinetics in humans. It has been central to a number of seminal studies, particularly in the context of HIV-1 and leukemia. However, there is a significant discrepancy between lymphocyte proliferation rates estimated in different studies. Notably, deuterated (2)H2-glucose (D2-glucose) labeling studies consistently yield higher estimates of proliferation than deuterated water (D2O) labeling studies. This hampers our understanding of immune function and undermines our confidence in this important technique. Whether these differences are caused by fundamental biochemical differences between the two compounds and/or by methodological differences in the studies is unknown. D2-glucose and D2O labeling experiments have never been performed by the same group under the same experimental conditions; consequently a direct comparison of these two techniques has not been possible. We sought to address this problem. We performed both in vitro and murine in vivo labeling experiments using identical protocols with both D2-glucose and D2O. This showed that intrinsic differences between the two compounds do not cause differences in the proliferation rate estimates, but that estimates made using D2-glucose in vivo were susceptible to difficulties in normalization due to highly variable blood glucose enrichment. Analysis of three published human studies made using D2-glucose and D2O confirmed this problem, particularly in the case of short term D2-glucose labeling. Correcting for these inaccuracies in normalization decreased proliferation rate estimates made using D2-glucose and slightly increased estimates made using D2O; thus bringing the estimates from the two methods significantly closer and highlighting the importance of reliable normalization when using this technique.

Double and zero quantum filtered (2)H NMR analysis of D2O in intervertebral disc tissue.

The analysis of double and zero quantum filtered (2)H NMR spectra obtained from D2O perfused in the nucleus pulposus of human intervertebral disc tissue samples is reported. Fitting the spectra with a three-site model allows for residual quadrupolar couplings and T2 relaxation times to be measured. The analysis reveals changes in both the couplings and relaxation times as the tissue begins to show signs of degradation. The full analysis demonstrates that information about tissue hydration, water collagen interactions, and sample heterogeneity can be obtained and used to better understand the biochemical differences between healthy and degraded tissue.