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INNOVATIVE METHODOLOGY

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

Departments of ¹Medicine and ²Nutrition, Case Western Reserve University School of Medicine, Cleveland, Ohio; and ³Department of Cellular and Molecular Physiology, The Pennsylvania State University College of Medicine, Hershey, Pennsylvania

Submitted 7 December 2004 ; accepted in final form 20 January 2005

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 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 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.

albumin turnover; nutritional status; stable isotopes; gas chromatography-mass spectrometry

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