Ketoisocaproate contamination errors in protein synthesis determinations using L-[1-14C]leucine

• Published in: Journal of biochemical and biophysical methods Vol. 27; no. 1; pp. 57 - 63
• Main Authors: Lopez Hellin, J., Schwartz, S., Garlick, P.J., Rosell, M., Arbós, M.A., Andreu, A.L., García, E.
• Format: Journal Article
• Language: English
• Published: Shannon Elsevier B.V 01.08.1993; Amsterdam Elsevier Science; New York, NY
• Subjects: Analytical, structural and metabolic biochemistry; Animals; Biological and medical sciences; Carbon Radioisotopes; Chromatography, Ion Exchange; Drug Contamination; Fundamental and applied biological sciences. Psychology; General aspects, investigation methods; Intestinal Mucosa - metabolism; Jejunum; Keto Acids - isolation & purification; Keto Acids - metabolism; Ketoisocaproate; Leucine; Leucine - isolation & purification; Leucine - metabolism; Liver - metabolism; Male; Protein Biosynthesis; Protein synthesis; Proteins; Radioisotope Dilution Technique; Rats; Rats, Sprague-Dawley; Ketoisocaproate; Protein synthesis; Leucine; Radiolabelling; Rat; Liver; Rodentia; In vitro; Contamination; Vertebrata; Mammalia; Jejunum; Investigation method; Ketoacid
• ISSN: 0165-022X; 1872-857X
• DOI: 10.1016/0165-022X(93)90068-Y

Protein synthesis rate determinations in vivo using L-[1-14C]-leucine may be underestimated because of contamination by radioactive ketoisocaproate (KIC) resulting from leucine metabolism. The aim of this work was to set up a reliable methos to determine the KIC/leucine radioactivity ratio in protein-free homogenates, and to apply it to study the extent of the protein synthesis ratio error due to KIC contamination. Cation-exchange chromatography using Dowex AG 50W-X8 resin was used to separate KIC from leucine, eluting KIC with water and leucine with 4 M ammonia. The errors found in the protein synthesis ratio were 6.20% in liver and 2.34% in jejunum.