ARS | Publication request: Fractional Protein Synthesis Rates Measured by An Intraperitoneal Injection of a Flooding Dose of L-[ring-2h5]phenylalanine in Pigs

Submitted to: Journal Of Nutrition
Publication Acceptance Date: July 6, 2004
Publication Date: October 1, 2004
Publisher's URL: www.nutrition.org
Citation: Bregendahl, K., Liu, L., Cant, J.P., Bayley, H.S., Mcbride, B.W., Milligan, L.P., Yen, J., Fan, M.Z. 2004. Fractional Protein Synthesis Rates Measured By An Intraperitoneal Injection Of A Flooding Dose Of L-[ring-2h5]phenylalanine In Pigs. Journal Of Nutrition. 134:2722-2728.

Interpretive Summary: Protein synthesis accounts for 20% or more of the daily energy expenditure in growing animals. It is affected by the growth or development stage, plane of nutrition, immune status, activity level, hormones, and stress of the animals. The study of protein metabolism is integral to understanding animal biology and the effects of dietary and/or medicinal treatments. Fractional protein synthesis rates can be measured by the continuous-infusion or the flooding-dose methods, both of which rely on the administration of a labeled amino acid tracer and its subsequent incorporation into protein. For the continuous-infusion method, several hours of constant tracer infusion are needed to label the precursor pool, during which the study subject must remain in steady state. Administering a substantially larger dose of a labeled amino acid to flood amino acid free pools in all tissues minimizes the difference between tracer amino acid enrichments or specific activities of intra- and extracellular free amino acid pools and the true protein precursor, amino-acyl tRNA and also shortens the time needed to reach uniform precursor tracer enrichment or specific activities from hours to minutes after administration. Although the flooding dose can be delivered intravenously using a syringe and needle, surgical placement of at least one catheter under anesthesia and an ensuing recovery period are required. The trauma inflicted by surgery affects protein metabolism, whereas the post-surgical period carries with it risks of infection and catheter occlusion. Intraperitoneal injection of a flooding dose of a labeled tracer to measure fractional protein synthesis would eliminate the need for surgery and post-surgical care of the animals. The present study investigated the time course of physiological responses, isotopic enrichment of the free and bound tracer pools in the target tissues, and fractional protein synthesis estimates after an intraperitoneal injection of a flooding dose of L-phenylalanine containing a stable-isotope phenylalanine tracer in piglets. Results of the present study showed that the intraperitoneally injected tracer phenylalanine rapidly distributed into plasma, and intra- and extracellular spaces, and was effective for use in measuring fractional protein synthesis in visceral organs of pigs.

Technical Abstract: Our objectives were to examine the effect of an intraperitoneal (ip) injection of a flooding dose of L-phenylalanine (Phe) containing L [ring-2H5]Phe on time courses of physiological responses, the tracer Phe enrichments, and fractional protein synthesis rates (FSR) in plasma, visceral organs, and muscles. In a completely randomized block design, five blocks of five littermate piglets were weaned at 16 d of age, and injected ip with a flooding dose of L-Phe (1.5 mmol/kg body weight) on d 8 post-weaning under fed conditions. Tissues were collected after killing at 15, 30, 45, 60, and 75 min post-injection. Plasma glucose concentration increased (cubic effect, P < 0.05) from 4.8 pre-injection to 5.8 mmol/L 15 min post-injection and returned to pre-injection levels thereafter. Plasma insulin concentration did not change (P > 0.05) over time. Plasma Phe concentration increased logarithmically (P < 0.05) from 85 to 711 µmol/L and reached 95% of the maximum concentration 48 min post-injection. No changes (P > 0.05) in tissue contents of free amino acids other than Phe were observed. The Phe free pools in plasma, visceral organs, and muscles were evenly enriched (32.3 ± 1.4 mol%) with L-[2H5]Phe 15 min after the ip injection. The FSR in visceral organs did not change (P > 0.05), whereas plasma, and muscle protein FSR decreased (P < 0.05) over time. It is concluded that the intraperitoneally injected tracer Phe rapidly distributed into plasma, and intra- and extracellular spaces, and was effective for use in measuring FSR in visceral organs of pigs.