Using [sup.2][H.sub.2]O to study the influence of feeding on protein synthesis: effect of isotope equilibration in vivo vs. in cell culture

We previously reported that [sup.2][H.sub.2]O 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 [sup.2][H.sub.2]O, [sup.2]H atoms in body water rapidly equilibrate with free alanine before it is incorporated into newly synthesized proteins. We have now directly examined whether [sup.2][H.sub.2]O can be used to measure the influence of a single meal on protein synthesis. In addition, we have compared the use of [sup.2][H.sub.2]O for measuring rates of protein synthesis in vivo vs. in cell culture. Using a rat model, we observed rapid equilibration between [sup.2]H in body water and free alanine; therefore we were able to study the response of protein synthesis to a single meal. We observed that ~50% of the plasma albumin that is synthesized over the course of 24 h is made within ~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 [sup.2][H.sub.2]O for in vitro studies. In particular, since there can be slow equilibration of [sup.2]H between water and alanine in the cell culture medium, special consideration must be made to avoid underestimating the rate of protein synthesis in vitro. albumin turnover; nutritional status; stable isotopes; gas chromatography-mass spectrometry