We previously reported that 2H2O can be used to measure rates of protein synthesis during prolonged steady-state conditions (Previs SF, Fatica R, Chandramouli V, Alexander JC, Brunengraber H, and Landau BR. Am J Physiol Endocrinol Metab 286: E665-E672, 2004). The underlying premise of our method is that following the administration of 2H2O, 2H atoms in body water rapidly equilibrate with free alanine before it is incorporated into newly synthesized proteins. We have now directly examined whether 2H2O can be used to measure the influence of a single meal on protein synthesis. In addition, we have compared the use of 2H2O for measuring rates of protein synthesis in vivo vs. in cell culture. Using a rat model, we observed rapid equilibration between 2H in body water and free alanine; therefore we were able to study the response of protein synthesis to a single meal. We observed that approximately 50% of the plasma albumin that is synthesized over the course of 24 h is made within approximately 5 h after eating (in rats trained to eat a complete 24-h ration of food in a single meal). Contrary to what we observed in vivo, feeding (the replenishment of cell culture medium) does influence the use of 2H2O for in vitro studies. In particular, since there can be slow equilibration of 2H between water and alanine in the cell culture medium, special consideration must be made to avoid underestimating the rate of protein synthesis in vitro.

The partitioning of whole body carbon flux between fat and lean compartments affects body composition. We hypothesized that it is possible to simultaneously determine whole body carbon (energy) balance and the dynamics of lipids and proteins in specific tissues in vivo. Growing C57BL/6J mice fed a high-fat low-carbohydrate diet were injected with a bolus of "doubly labeled" water (i.e., 2 H2O and H2 18 O). The rate of CO2 production was determined from the difference between the elimination rates of 2 H and 18 O from body water. The rates of synthesis and degradation of triglycerides extracted from epididymal fat pads and of proteins extracted from heart muscle were determined by mathematically modeling the 2 H labeling of triglyceride-bound glycerol and proteinbound alanine, respectively. We found that mice were in positive carbon balance (ϳ20% retention per day) and accumulated lipid in epididymal fat pads (ϳ9 mol triglyceride accumulated per day). This is consistent with the fact that mice were studied during a period of growth. Modeling the 2 H labeling of triglycerides revealed a substantial rate of lipid breakdown during this anabolic state (equivalent to ϳ25% of the newly synthesized triglyceride). We found equal rates of protein synthesis and breakdown in heart muscle (ϳ10% of the pool per day), consistent with the fact that the heart muscle mass did not change. In total, these findings demonstrate a novel application of the doubly labeled water method. Utilization of this approach, especially in unique rodent models, should facilitate studies aimed at quantifying the efficacy of interventions that modulate whole body carbon balance and lipid flux while in parallel determining their impact on (cardiac) muscle protein turnover. Last, the simplicity of administering doubly labeled water and collecting samples allows this method to be used in virtually any laboratory setting. metabolic regulation; carbon-energy balance; triglyceride turnover; protein turnover; stable isotope tracer kinetics OBESITY PRESENTS A GLOBAL HEALTH CHALLENGE (20,25), and many attempts are being made to determine the influence of environmental vs. genetic factors on the modeling of body composition. The application of isotope tracers in novel animal models offers a means of dissecting the importance of specific reactions on the pathogenesis and treatment of disease (12,15).We recently demonstrated the use of 2 H 2 O for measuring the turnover rates of triglycerides and proteins in vivo (3,10,24). Briefly, following the administration of 2 H 2 O, 2 H in body water equilibrates with the carbon-bound hydrogens of glycerol 3-phosphate and alanine. The rates of triglyceride and protein syntheses are determined by measuring the incorporation of the respective 2 H-labeled precursors into the appropriate end products. This simple and powerful method can be used in free-living subjects and complements other tracer methods for studying intermediary metabolism (12,15,18,22,36).In our previous studies (3, 10, 24), rates of biochemical flux were ...

Soy isoflavones may impede atherogenic processes associated with cardiovascular disease. Research suggests that the postprandial generation of TG-rich remnants contributes to the development of atherosclerosis. The purpose of the current study was to determine if 39 g soy (85 mg aglycone isoflavones, treatment) compared with 40 g milk protein (0 mg aglycone isoflavones, control) in combination with a high-fat meal can modify postprandial, atherogenic-associated events and biomarkers for oxidative stress, inflammation, and thrombosis. Fifteen healthy men (20-47 yr) participated in a double-blind cross-over meal-challenge study occurring on two nonconsecutive days. The study meals consisted of two high-fat apple muffins consumed with either a soy or milk shake (229 mL, 41% fat, 41% carbohydrate, and 18% protein). Blood samples were obtained at baseline (fasted) and hours two, four, and six postprandial. Plasma TG significantly increased in both treatment and control meal challenges compared with baseline. There were no significant differences (P > 0.05) between treatment (soy) and control (milk) for ex vivo copper-induced LDL oxidation, serum C-reactive protein, serum interleukin-6 (IL-6), serum fibrinogen, or plasma lipids (total cholesterol, HDL, LDL, TG). IL-6-concentrations significantly decreased as a function of time during either meal challenge (P = 0.005). These data suggest that consumption of soy or milk protein in conjunction with a high-fat meal does not acutely modify postprandial oxidative stress, inflammation, or plasma lipid concentrations in young, healthy men.

As the use of 2H(2)O requires few assumptions, the application of this tracer should yield sound information regarding the regulation of biochemical reactions in vivo.