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Am J Physiol Endocrinol Metab (January 25, 2005). doi:10.1152/ajpendo.00580.2004
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Submitted on December 7, 2004
Accepted on January 20, 2005

Using 2H2O to study the influence of feeding on protein synthesis: Effect of isotope equilibration in vivo vs in cell culture

Danielle A. Dufner1, Ilya R. Bederman1, Daniel Z. Brunengraber1, Nadia Rachdaoui1, Faramarz Ismail-Beigi1, Brett Siegfried1, Scot R. Kimball1, and Stephen F. Previs1*

1 Department of Nutrition, Case Western Reserve University School of Medicine, Cleveland, OH, USA

* To whom correspondence should be addressed. E-mail: sxp29{at}case.edu.

We previously reported that 2H2O can be used to measure rates of protein synthesis during prolonged steady-state conditions (Am J Physiol Endo 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 media) 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.




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