Measuring acetyl-CoA and acetylated histone turnover in vivo: Effect of a high fat diet.

Cellular availability of acetyl-CoA, a central intermediate of metabolism, regulates histone acetylation. The impact of a high-fat diet (HFD) on the turnover rates of acetyl-CoA and acetylated histones is unknown. We developed a method for simultaneous measurement of acetyl-CoA and acetylated histones kinetics using a single 2H2O tracer, and used it to examine effect of HFD-induced perturbations on hepatic histone acetylation in LDLR-/- mice, a mouse model of non-alcoholic fatty liver disease (NAFLD). Mice were given 2H2O in the drinking water and the kinetics of hepatic acetyl-CoA, histones, and acetylated histones were quantified based on their 2H-labeling. Consumption of a high fat Western-diet (WD) for twelve weeks led to decreased acetylation of hepatic histones (p< 0.05), as compared to a control diet. These changes were associated with 1.5-3-fold increased turnover rates of histones without any change in acetyl-CoA flux. Acetylation significantly reduced the stability of histones and the turnover rates of acetylated peptides were correlated with the number of acetyl groups in neighboring lysine sites. We conclude that 2H2O-method can be used to study metabolically controlled histone acetylation and acetylated histone turnover in vivo.

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.

Discovery of Half-life of Circulating Hepatitis B Surface Antigen in Patients With Chronic Hepatitis B Infection Using Heavy Water Labeling.

In a pilot study, heavy water labeling was used to determine hepatitis B surface antigen (HBsAg) turnover rates in chronic hepatitis B (CHB) patients. The mean (standard deviation) half-life of HBsAg in blood was 6.7 (5.5) days, which reflects recent production in the liver and supports strategies aimed at reducing HBsAg production in CHB patients.

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.

Renal MR angiography and perfusion in the pig using hyperpolarized water.

PURPOSE: To study hyperpolarized water as an angiography and perfusion tracer in a large animal model. METHODS: Protons dissolved in deuterium oxide (D2 O) were hyperpolarized in a SPINlab dissolution dynamic nuclear polarization (dDNP) polarizer and subsequently investigated in vivo in a pig model at 3 Tesla (T). Approximately 15 mL of hyperpolarized water was injected in the renal artery by hand over 4-5 s. RESULTS: A liquid state polarization of 5.3 ± 0.9% of 3.8 M protons in 15 mL of deuterium oxide was achieved with a T1 of 24 ± 1 s. This allowed injection through an arterial catheter into the renal artery and subsequently high-contrast imaging of the entire kidney parenchyma over several seconds. The dynamic images allow quantification of tissue perfusion, with a mean cortical perfusion of 504 ± 123 mL/100 mL/min. CONCLUSION: Hyperpolarized water MR imaging was successfully demonstrated as a renal angiography and perfusion method. Quantitative perfusion maps of the kidney were obtained in agreement with literature and control experiments with gadolinium contrast. Magn Reson Med 78:1131-1135, 2017. © 2016 International Society for Magnetic Resonance in Medicine.

Transition duration of ingested deuterium oxide to eccrine sweat during exercise in the heat.

The time necessary for the initial appearance of ingested water as sweat during exercise in the heat remains unknown. Based on the current literature, we estimated fluid transition through the body, from ingestion to appearance as sweat, to have a minimum time duration of approximately three minutes. The purpose of this study was to test this prediction and identify the time necessary for the initial enrichment of deuterium oxide (D2O) in sweat following ingestion during exercise in the heat. Eight participants performed moderate intensity (40% of maximal oxygen uptake) treadmill exercise in an environmental chamber (40°C, 40% rH) to induce active sweating. After fifteen minutes, while continuing to walk, participants consumed D2O (0.15mlkg-1) in a final volume of 50ml water. Scapular sweat samples were collected one minute prior to and ten minutes post-ingestion. Samples were analyzed for sweat D2O concentration using isotope ratio mass spectrometry and compared to baseline. Mean±SD ∆ sweat D2O concentration at minutes one and two post-ingestion were not significantly higher than baseline (0min). Minutes three (9±3ppm) through ten (23±11ppm) post-ingestion had ∆ sweat D2O concentrations significantly (P<0.05) higher than baseline. Such results suggest that ingested water rapidly transports across the mucosal membrane of the alimentary canal into the vasculature space, enters the extravascular fluid, and is actively secreted by the eccrine sweat glands onto the surface of the skin for potential evaporation in as little as three minutes during exercise in the heat.

Skeletal muscle hypertrophy adaptations predominate in the early stages of resistance exercise training, matching deuterium oxide-derived measures of muscle protein synthesis and mechanistic target of rapamycin complex 1 signaling.

Resistance exercise training (RET) is widely used to increase muscle mass in athletes and also aged/cachectic populations. However, the time course and metabolic and molecular control of hypertrophy remain poorly defined. Using newly developed deuterium oxide (D2O)-tracer techniques, we investigated the relationship between long-term muscle protein synthesis (MPS) and hypertrophic responses to RET. A total of 10 men (23 ± 1 yr) undertook 6 wk of unilateral (1-legged) RET [6 × 8 repetitions, 75% 1 repetition maximum (1-RM) 3/wk], rendering 1 leg untrained (UT) and the contralateral, trained (T). After baseline bilateral vastus lateralis (VL) muscle biopsies, subjects consumed 150 ml D2O (70 atom percentage; thereafter 50 ml/wk) with regular body water monitoring in saliva via high-temperature conversion elemental analyzer:isotope ratio mass spectrometer. Further bilateral VL muscle biopsies were taken at 3 and 6 wk to temporally quantify MPS via gas chromatography:pyrolysis:isotope ratio mass spectrometer. Expectedly, only the T leg exhibited marked increases in function [i.e., 1-RM/maximal voluntary contraction (60°)] and VL thickness (peaking at 3 wk). Critically, whereas MPS remained unchanged in the UT leg (e.g., ∼1.35 ± 0.08%/d), the T leg exhibited increased MPS at 0-3 wk (1.6 ± 0.01%/d), but not at 3-6 wk (1.29 ± 0.11%/d); this was reflected by dampened acute mechanistic target of rapamycin complex 1 signaling responses to RET, beyond 3 wk. Therefore, hypertrophic remodeling is most active during the early stages of RET, reflecting longer-term MPS. Moreover, D2O heralds promise for coupling MPS and muscle mass and providing insight into the control of hypertrophy and efficacy of anabolic interventions.

The Deuterium Oxide-to-the-Mother Method Documents Adequate Breast-Milk Intake among Sri Lankan Infants.

BACKGROUND: The WHO recommends that exclusive breastfeeding should last up to 6 mo. However, human milk intake of Sri Lankan infants has not been quantified scientifically. OBJECTIVES: The objectives of this study were to measure the human milk intake of Sri Lankan infants during the first 6 mo of age and to document the breastfeeding practices of their mothers. METHODS: Forty-eight healthy mother-infant dyads were randomly recruited for this cross-sectional study at well-baby clinics. Milk intake was measured using the deuterium oxide-to-the-mother technique over a period of 2 wk. Information on breastfeeding practice and living standards of the participants were gathered using an interviewer-administered questionnaire. RESULTS: Human milk intake was 672 ± 123 g ⋅ d(-1) (mean ± SD), 776 ± 212 g ⋅ d(-1), and 801 ± 51 g ⋅ d(-1) for infants <2 mo, 2 to <4 mo, and 4-6 mo of age, respectively. The milk intakes were not different among the age groups. Maternal body composition, age, or parity had no effects on milk intake of the infants. However, mother's education : P < 0.05, r = 0.35), infant's age (P < 0.05, r = 0.30), and body mass index (P < 0.05, r = 0.41 : positively correlated with the milk intake. Over 63% of mothers had commenced breastfeeding within 30 min of delivery. About 60% of the mothers were feeding the baby 6-10 times during the daytime and >81% intended to continue exclusive breastfeeding until 6 mo of age. CONCLUSION: This study, for the first time, documented the adequacy of breast-milk intake among Sri Lankan infants and the nutrition status of the mothers.

Determination of low levels of 2H-labeling using high-resolution mass spectrometry: application in studies of lipid flux and beyond.

RATIONALE: The ability to measure low levels of (2)H-labeling is important in studies of metabolic flux, e.g. one can estimate lipid synthesis by administering (2)H2O and then measuring the incorporation of (2)H into fatty acids. Unfortunately, the analyses are complicated by the presence of more abundant naturally occurring stable isotopes, e.g. (13)C. Conventional approaches rely on coupling gas chromatographic separation of lipids with either quadrupole-mass spectrometry (q-MS) and/or pyrolysis-isotope ratio mass spectrometry (IRMS). The former is limited by high background labeling (primarily from (13)C) whereas the latter is not suitable for routine high-throughput analyses. METHODS: We have contrasted the use of continuous flow-pyrolysis-IRMS against high-resolution mass spectrometry (i.e. Qq-FT-ICR MS) for measuring the (2)H-enrichment of fatty acids and peptides. RESULTS: In contrast to IRMS, which requires ~30 min per analysis, it is possible to measure the (2)H-enrichment of palmitate via direct infusion high-resolution mass spectrometry (HRMS) in ~3 min per sample. In addition, Qq-FT-ICR MS enabled measurements of the (2)H-enrichment of peptides (which is not possible using IRMS). CONCLUSIONS: High-resolution mass spectrometry can be used to measure low levels of (2)H-labeling so we expect that this approach will enhance studies of metabolic flux that rely on (2)H-labeled tracers, e.g. (2)H2O. However, since the high-resolution analyses require greater amounts of a given analyte one potential limitation centers on the overall sensitivity. Presumably, future advances can overcome this barrier.

Appearance of D2O in sweat after oral and oral-intravenous rehydration in men.

Intravenous (IV) rehydration is common in athletics, but its thermoregulatory benefits and ergogenicity have not been elucidated. Availability of orally ingested fluid is dependent on gastric emptying and intestinal absorption rate. Deuterium oxide (D2O) has been used to demonstrate that fluid ingested during exercise appears in sweat within 10 minutes. The purpose of this study was to determine the effect of concurrent IV rehydration on D2O appearance in sweat samples after per ora rehydration with D2O labeled fluid. We hypothesized that the combination method would not be superior to the oral method. Ten fit men (age 23 ± 4, VO2max 59.49 ± 4.09 L·min(-1)) underwent 20 hours of fluid restriction resulting in 1.95 ± 0.25% body weight loss before beginning treadmill exercise and cycling. Exercise was performed in an environmental chamber (35.6 ± 0.2° C, 35.0 ± 1.8% relative humidity) for 2 hours at 55% VO2max, and the participants exhibited a mean body weight deficit of 4.50 ± 0.04%. Thermoregulatory measures were recorded while subjects were rehydrated with oral (OR) or oral combined with intravenous (IVO) fluid traced with D2O. After 30 minutes of rehydration and 30 minutes of seated recovery, the subjects began treadmill exercise at 55-60% VO2max. Forehead sweat samples were collected 0, 5, 10, 20, and 75 minutes from the start of rehydration. The samples were analyzed for D2O via isotope ratio mass spectrometry. D2O did not appear in the sweat within 20 minutes of rehydration; however, it did appear during the subsequent exercise bout. There was no significant difference between rehydration modes. Plasma volume increases and decreased volume of orally ingested fluid did not significantly alter transit time from ingestion to appearance in excreted sweat. The IVO method does not appear to be superior to the traditional OR method of rehydration.

NUTRITIONAL STATUS IN CHILDREN WITH CANCER: COMPARISON OF DEUTERIUM OXIDE DILUTION WITH BIOELECTRIC IMPEDANCE ANALYSIS AND ANTHROPOMETRY.

OBJECTIVE: To explore changes in the nutritional status of pediatric cancer patients before and after chemotherapy and evaluate the correlation between deuterium oxide dilution, bioelectric impedance analysis, and anthropometry for assessment of body composition. METHODS: This study included 14 children (aged 5.6 to 13.6 years) and classified them as having hematologic or solid tumors. They had their body composition analyzed according to deuterium oxide, bioelectric impedance, and anthropometric measurements before the first chemotherapy cycle and after three and six months of therapy. RESULTS: The patients in the hematologic tumor group had an increase in weight, height, body mass index, waist, hip, and arm circumference, subscapular skinfold thickness, and fat mass with the isotope dilution technique during chemotherapy. In the solid tumor group, the children showed a reduction in fat-free mass when assessed by bioimpedance analysis. We found a positive correlation between the triceps skinfold thickness and fat mass determined by bioimpedance analysis and deuterium oxide. The arm muscle circumference correlated with the fat-free mass estimated by bioimpedance analysis and deuterium oxide. CONCLUSIONS: Patients with hematologic tumors had an increase in body weight, height, and fat mass, which was not identified in the solid tumor group. The positive correlation between anthropometry (triceps skinfold thickness and arm muscle circumference), deuterium oxide dilution, and bioelectric impedance analysis shows the applicability of anthropometry in clinical practice.

Effects of Breastfeeding on Maternal Body Composition in Moroccan Lactating Women during Twelve Months after Birth Using Stable Isotopic Dilution Technique.

BACKGROUND: Exclusive breastfeeding during the first six months of an infant's life is an important factor for their optimal growth and health. Breastfeeding also has maternal benefits and can assist with postpartum weight loss. As shown by previous studies, postpartum weight retention can contribute to obesity. OBJECTIVE: To quantify the human milk and evaluate the effect of breastfeeding on maternal weight loss during the 12 months postpartum. METHOD: This study included 70-mother-baby pairs. Infants' intake of human milk and water from other sources, as well as the body composition of the mothers, were measured at the 1st, 3rd, 6th, 9th and 12th month postpartum by using the deuterium oxide dose-to-mother technique. RESULTS: There was a significant change in the mothers' body composition between the first and twelfth months in exclusive breastfeeding women compared to not-exclusive ones. Similarly, the difference between the quantities of human milk intake was highly significant in exclusive breastfeeding women compared to women who were not exclusively breastfeeding. CONCLUSION: Our results showed that exclusive breastfeeding for twelve months has a significant effect on postpartum weight loss among Moroccan women and that it is an effective way to control overweight and obesity among lactating women.

Feasibility, Acceptability, and Preliminary Validity of Self-Report Dietary Assessment in Adults with Multiple Sclerosis: Comparison with Doubly Labeled Water Measured Total Energy Expenditure.

BACKGROUND: Diet is a modifiable behavior of interest in multiple sclerosis (MS); however, measures of diet in persons with MS have not been vetted for feasibility, acceptability, and validity. METHODS: This cross-sectional study examined the Automated Self-Administered 24-H (ASA24) Dietary Assessment Tool in 30 persons with MS and 15 healthy control (HC) participants. Participants were prompted to complete six ASA24 recalls and undergo a standard doubly labeled water (DLW) protocol. Acceptability of ASA24 was assessed using an online questionnaire. Total energy expenditure (TEE) from DLW was compared with ASA24-reported energy intake for assessing validity. RESULTS: All participants completed four or more ASA24 recalls, indicating feasibility of ASA24. Regarding acceptability, the hardest part of completing the ASA24 was remembering everything eaten the previous day. Pearson correlation coefficients between DLW TEE and ASA24 kcal/day were not significant among HC (r = 0.40; p = 0.14) or MS (r = 0.26; p = 0.16) participants. The absolute mean error between DLW TEE and ASA24 among HC participants was 694.96 ± 506.25 mean kcal/day and among MS participants was 585.37 ± 529.02 mean kcal/day; this represents a mean difference of 30 and 25%, respectively. CONCLUSION: This study established the feasibility and acceptability of ASA24 in persons with MS and provides a foundation regarding the need for further validation research examining appropriate outcomes for supporting dietary interventions.

Deuterium equilibrium time in saliva of newborn infants.

UNLABELLED: There is an increasing interest about the use of stable isotopes for body composition analysis in pediatrics. To ensure the success of total body water analysis by the deuterium dilution method, it is fundamental to determine the equilibrium time (plateau) of deuterium in the body fluid studied. OBJECTIVES: We report here the equilibration time of deuterium oxide in the saliva of newborns after oral intake of the isotope. METHODS: Twenty healthy term newborn infants, 10 males and 10 females, were analyzed. Saliva was collected from each newborn before the oral administration of a 100 mg/kg dose of deuterium oxide (baseline sample) and then at 1-hour intervals for 5 hours after administration. Deuterium enrichment of saliva was determined by isotope ratio mass spectrometry according to the recommendations of the International Atomic Energy Agency. RESULTS: The plateau time of deuterium in saliva occurred 3 hours after oral administration of the stable isotope. CONCLUSION: These data are essential for further studies on the body composition of newborn infants. To the best of our knowledge, this is the first study regarding the equilibration time of deuterium in the saliva of term newborns.

Change in body mass accurately and reliably predicts change in body water after endurance exercise.

This study tested the hypothesis that the change in body mass (DeltaBM) accurately reflects the change in total body water (DeltaTBW) after prolonged exercise. Subjects (4 men, 4 women; 22-36 year; 66 +/- 10 kg) completed 2 h of interval running (70% VO(2max)) in the heat (30 degrees C), followed by a run to exhaustion (85% VO(2max)), and then sat for a 1 h recovery period. During exercise and recovery, subjects drank fluid or no fluid to maintain their BM, increase BM by 2%, or decrease BM by 2 or 4% in separate trials. Pre- and post-experiment TBW were determined using the deuterium oxide (D(2)O) dilution technique and corrected for D(2)O lost in urine, sweat, breath vapor, and nonaqueous hydrogen exchange. The average difference between DeltaBM and DeltaTBW was 0.07 +/- 1.07 kg (paired t test, P = 0.29). The slope and intercept of the relation between DeltaBM and DeltaTBW were not significantly different from 1 and 0, respectively. The intraclass correlation coefficient between DeltaBM and DeltaTBW was 0.76, which is indicative of excellent reliability between methods. Measuring pre- to post-exercise DeltaBM is an accurate and reliable method to assess the DeltaTBW.

Measurement of Leukemic B-Cell Growth Kinetics in Patients with Chronic Lymphocytic Leukemia.

Cell proliferation plays a central role in the pathogenesis of every neoplastic disease as well as many other types of illness. Labeling of newly replicated DNA with deuterium (2H), a nonradioactive isotope of hydrogen, administered to the patients in drinking water (2H2O) is a safe and reliable method to measure the in vivo birth rates of cells. Here, we describe a protocol to measure chronic lymphocytic leukemia B-cell birth/proliferation and death rates over time using this approach.

Sources of hepatic glucose production by 2H2O ingestion and Bayesian analysis of 2H glucuronide enrichment.

The contribution of gluconeogenesis to hepatic glucose production (GP) was quantified after (2)H(2)O ingestion by Bayesian analysis of the position 2 and 5 (2)H-NMR signals (H2 and H5) of monoacetone glucose (MAG) derived from urinary acetaminophen glucuronide. Six controls and 10 kidney transplant (KTx) patients with cyclosporine A (CsA) immunosuppressant therapy were studied. Seven KTx patients were lean and euglycemic (BMI = 24.3 +/- 1.0 kg/m(2); fasting glucose = 4.7 +/- 0.1 mM) while three were obese and hyperglycemic (BMI = 30.5 +/- 0.7 kg/m(2); fasting glucose = 7.1 +/- 0.5 mM). For the 16 spectra analyzed, the mean coefficient of variation for the gluconeogenesis contribution was 10% +/- 5%. This uncertainty was associated with a mean signal-to-noise ratio (SNR) of 79:1 and 45:1 for the MAG H2 and H5 signals, respectively. For control subjects, gluconeogenesis contributed 54% +/- 7% of GP as determined by the mean and standard deviation (SD) of individual Bayesian analyses. For the lean/normoglycemic KTx subjects, the gluconeogenic contribution to GP was 62% +/- 7% (P = 0.06 vs. controls), while hyperglycemic/obese KTx patients had a gluconeogenic contribution of 68% +/- 3% (P < 0.005 vs. controls). These data suggest that in KTx patients, an increased gluconeogenic contribution to GP is strongly associated with obesity and hyperglycemia.

Determination of protein synthesis in vivo using labeling from deuterated water and analysis of MALDI-TOF spectrum.

This paper describes a method of determining protein synthesis and turnover using in vivo labeling of protein with deuterated water and analysis of matrix-assisted laser desorption time-of-flight mass spectrometer (MALDI-TOF) spectrum. Protein synthesis is calculated using mass isotopomer distribution analysis instead of precursor to product amino acid enrichment ratio. During protein synthesis, the incorporation of deuterium from water changes the mass isotopomer distribution (isotope envelop) according to the number of deuterium atoms (0, 1, 2, 3, etc.) incorporated, and the distribution of the protein with 0, 1, 2, 3,... atoms of deuterium follows a binomial distribution. A mathematical algorithm by which the distribution of deuterium isotopomers can be extracted from the observed MALDI-TOF spectrum is presented. Since deuterium isotopomers are unique to newly synthesized proteins, the quantitation of their distribution provides a method for the quantitation of newly synthesized proteins. The combined use of postsource decay sequence identification and mass isotopomer distribution analysis makes the use of in vivo labeling with deuterated water a precise method to determine specific protein synthesis.

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.

Measurement of protein turnover rates by heavy water labeling of nonessential amino acids.

In vivo measurements of protein synthesis using isotope-labeled amino acids (AAs) are hampered by the heterogeneity of AA pools and, for slow turnover proteins, the difficulty and expense of long-term labeling. Continuous oral heavy water (2H2O) labeling can safely maintain stable body water 2H enrichments for weeks or months. 2H is metabolically incorporated into C-H bonds of nonessential AAs (NEAAs) and hence into proteins. No posttranslational label exchange occurs, so 2H incorporation into protein NEAAs, in principle, reports on protein synthesis. Here, we show by mass isotopomer distribution analysis (MIDA) of 2H2O-labeled rodent tissue proteins that metabolic 2H flux into C-H bonds of Ala, Gly, or Gln used for protein synthesis is nearly complete. By 2H2O labeling of rodents, turnover of bone and muscle mixed proteins was quantified and stimulation of liver collagen synthesis by CCl4 was detected. Kinetics of several human serum proteins were also measured, reproducing published t1/2 estimates. Plateau enrichments in Ala varied among different proteins. Moderate amounts of protein, isolated chromatographically or electrophoretically, sufficed for kinetic analyses. In conclusion, 2H2O labeling permits sensitive, quantitative, operationally simple measurements of protein turnover in vivo by the rise-to-plateau approach, especially for proteins with slow constitutive turnover.