Measurement of the Rate of Protein Synthesis in Chickens by HPLC/MS

  • Seo, S.S. (Department of Natural Science, Albany State University) ;
  • Coon, C. (Department of Poultry Science, University of Arkansas)
  • Published : 2004.09.01

Abstract

The fractional synthesis rates(FSR) were measured with 2l-wk and 3l-wk-old broiler breeder pullets and hens to investigate the effect of sexual maturity on FSR. The FSR were obtained from chicken tissues and blood samples using High-Performance Liquid Chromatography/Mass Spectrometry(HPLC/MS). A L-l-13C, 15N -leucine saline solution was infused by bolus injection as a tracer into broiler breeder pullets in the experiment. A rapid HPLC/MS method was developed to measure the isotopic enrichments of leucine in plasma, tissue samples, and eggs. The enrichments of stable isotope leucine incorporated into protein and the enrichments of the stable isotope free leucine were measured in liver, breast muscle and blood samples. Two sets of experiments were conducted. In experiment one, 2l-wk-old, sexually immature broiler breeder pullets were divided into groups of three and blood samples were collected at 20 or 30 min intervals until 1.5 h from initial injection. The pullets were sacrificed in groups of three at varying time intervals for 7 h after injection. The liver, breast muscle and blood samples were removed for analysis. The FSR were estimated to be 8.7l%/day for liver, 4.06%/day for breast muscle, and 5.08%/day for blood samples in 30 minutes after injection from the enrichment ratios. In experiment two, sexually matured 3l-wk-old broiler breeder hens were assorted into groups of three and blood samples were obtained at 20 or 30 min intervals for 2 h. The FSR for blood samples were determined. The broiler breeder hens were sacrificed in groups of three at various time intervals until 7 h after injection and liver, breast muscle and blood samples were removed for analysis. The FSR were calculated to be 5.96%/day for liver. Eggs were collected from five chickens daily for 10 days after large bolus injection. The average of total enrichments of stable isotope in egg albumin was increased by 0.064% at 4 days after injection and was back to normal in 7 days.

Keywords

References

  1. Antoine E. El-Khoury 1999 Methods for investigation of amino acid and protein metabolism. CRC press LLC
  2. Attaix D, Manghebati A, Grizard J, Arnal M 1986 ssessment of in vivo protein synthesis in lamb tissues with [3H] valine flooding doses. Biochimica and Biophysica Acta 882:389-397
  3. Baumann P, Stirewalt S, O'rourke B, Howard D, Nair K 1994 Precursor pools of protein synthesis: A stable isotope study in a swine model, Am J Physiol 267(Endocrinol. Metab. 30): E203-E209
  4. Cheng K, Dworzak F, Ford G, Rennie M, Hallidy D 1985 Direct determination of leucine metabolism and protein breakdown in humans using the forearm model. European Journal of Climical Inrestigation 15:349-354
  5. Cheng K, Pacy P, Dworzak F, Ford G, Halliday D 1987 Influence of fasting on leucine and muscle protein metabolism across the human forearm determined using L-[1-13C, 15N] leucine as the tracer. Clinical Science 73:241-246
  6. Chinkes D, Klein S, Zhang X, Wolfe R 1996 Infusion of labeled KIC is more accurate than labeled leucine to determine human muscle protein synthesis. Am J Physiol 270(Endocrinol. Metab. 33): E67-E71
  7. El-Khoury A, Sanchez M, Fukagawa N, Young V 1995a Whole body protein synthesis in healthy adult humans: 13CO2 technique vs. plasma precursor approach. Am J Physiol 268(Endocrinol. Metab. 31): E174 -E184
  8. El-Khoury A, Sanchez M, Fukagawa N, Gleason R, Tsay R, Young V 1995b The 24-h kinetics of leucine oxidation in healthy adults receiving a generous leucine intake via three discrete meals. Am J Clin Nutr 62: 579-590
  9. Garlick P, Millward D, James W 1973 The diurnal response of muscle and liver protein synthesis in vivo in meal-fed rats. Biochem J 136: 935-945
  10. Garlick P, McNurlan M, Preedy V 1980 A rapid and convenient technique for measuring the rate of protein synthesis in tissues by injection of [3H] phenylalanine. Biochem J 192:719-723
  11. Garlick P, Wernerman J, McNurlan M, Essen P, Lobley G, Milne E, Calder G, Vinnars E 1989 Measurement of the rate of protein synthesis in muscle of postabsorptive young men by injection of a 'flooding dose' of [1-13C] leucine. Clinical Science 77:329-336
  12. Hiramoto K, Muramatsu T, Okumura J 1990 Protein synthesis in tissues and in the whole body of laying hens during egg formation. Poultry Sci 69:264-269
  13. Holecek M, Sprongl L, Tichy M, Pecka M 1988 Leucine metabolism in rat liver after a bolus injection of endotoxin. Metabolism 47(6): 681-685 https://doi.org/10.1016/S0026-0495(98)90030-0
  14. Jahoor F, Zhang X, Baba H, Sakurai Y, Wolfe R 1992 Comparison of constant infusion and flooding dose techniques to measure muscle protein synthesis rate in dogs. J Nutr 122:878-887
  15. Katz J 1989 The determination of mass of metabolites with tracers. Metabolism 38(8): 728-733
  16. Lobley G, Harris P, Skene P, Brown D, Milne E, Calder A, Anderson S, Garlick P, Nevison I, Connell A 1992 Responses in tissue protein synthesis to sub- and supramaintenance intake in young growing sheep: Comparison of large-dose and continuous-infusion techniques. British Journal of Nutrition 68:373-388
  17. Maria GB, Reid S 1975 A possible regulator of protein turnover in muscle. J Clinical Investigation 1250-1261
  18. MacDonald ML, Swick RW 1981 The effect of protein depletion and repletion on muscle protein turnover in the chick. Biochem J 194:811-819
  19. Matthews D, Motil K, Rohrbaugh D, Burke J, Young V, Bier D 1980 Measurement of leucine metabolism in man from a primed, continuous infusion of L-[1-13C] leucine. Am J Physiol 238(Endocrinol. Metab. 1): E473-E479
  20. Pomposelli J, Palombo J, Hamawy K, Bistrian B, Blackburn G, Moldawer L 1985 Comparison of different techniques for estimating rates of protein synthesis in vivo in healthy and bacteraemic rats. Biochem J 226: 37-47
  21. Rennie M, Smith K, Watt P: Measurement of Human Tissue Protein Synthesis, 1994 An optimal approach. Am J Physiol 266(Endocrinol. Metab. 29): E298-E307
  22. Smith K, Barua J, Watt P, Scrimgeour, Rennie M 1992 Flooding with L-[1-13C] leucine stimulates human muscle protein incorporation of continuously infused L-[1-13C] valine. Am J Physiol 262(Endocrinol. Metab. 25): E372-E376
  23. Southorn B, Kelly J, McBride B 1992 Phenylalanine flooding dose procedure is effective in measuring intestinal and liver protein synthesis in sheep. J Nutr 122:2398-2407
  24. Van Eijk H, Rooyakkers D, Soeters P, Deutz N 1999 Determination of amino acid isotope enrichment using liquid chromatography-mass spectrometry. Analytical Biochemistry 271:8-17
  25. Waldhausl W, Bratusch-Marrain P, Vierhapper H, Nowotny P 1983 Insulin pharmacokinetics following continuous infusion and bolus injection of regular porcine and human insulin in healthy man. Metabolism 32(5):478-486
  26. Wolfe R, Wolfe M, Nadel E, Shaw J 1982 Isotopic determination of amino acid-urea interactions in exercise in humans. J Appl Physiol : Respirat. Environ. Exercise Physiol. 52: 458-466