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

Area of Science:

• Biochemistry
• Metabolic research
• Analytical chemistry

Background:

• In vivo protein synthesis rate determination using L-[1-14C]-leucine is a common technique.
• Leucine metabolism can produce radioactive ketoisocaproate (KIC), potentially contaminating measurements.
• This contamination may lead to an underestimation of actual protein synthesis rates.

Purpose of the Study:

• To develop a reliable method for quantifying the radioactivity ratio of ketoisocaproate (KIC) to leucine in protein-free homogenates.
• To assess the extent of error in protein synthesis rate measurements caused by KIC contamination.
• To investigate tissue-specific differences in KIC contamination effects.

Main Methods:

• Utilized cation-exchange chromatography with Dowex AG 50W-X8 resin for separation.
• Eluted KIC using water and leucine using 4 M ammonia.
• Applied the method to protein-free homogenates from liver and jejunum tissues.

Main Results:

• Successfully separated KIC from leucine using the developed chromatographic method.
• Quantified KIC contamination in protein synthesis rate determinations.
• Observed significant errors: 6.20% in liver and 2.34% in jejunum.

Conclusions:

• The developed method provides accurate quantification of KIC contamination in leucine-based protein synthesis studies.
• KIC contamination introduces notable underestimation of protein synthesis rates, particularly in liver tissue.
• Accurate measurement of KIC/leucine radioactivity ratio is crucial for reliable in vivo protein synthesis rate determination.