Abstract

Clinicians and researchers studying protein metabolism in vivo, typically use isotopically-labeled free amino acids as metabolic tracers rather than isotopically-labeled proteins because such proteins are commercially unavailable. However, the use of free amino acids in lieu of protein tracers violates the critical assumption that tracer molecules undergo the identical biochemical reactions as the tracee molecules of interest. To address this problem we synthesized ¹³C-labeled proteins using egg laying hens and investigated the relationship between tracer dose and method of delivery on ¹³C-protein production. We enriched hens with one of two isotope tracers (¹³C-1-leucine or a uniformly labeled ¹³C-amino acid mixture) mixed in their food or dissolved in their drinking water at different dosing levels (86-432 mg day^-1). The recovery of ¹³C in egg white proteins of the hen fed ¹³C-leucine ranged from 14% to 21%; recovery rates were highest at the lowest dosing level. At the highest dosing level egg whites were enriched more than 150‰ above background levels of ¹³C. The time required for half maximal ¹³C enrichment depended chiefly on the mode of tracer administration, and ranged from 2.5 days for ¹³C-leucine dissolved in water to 4.9 days for ¹³C-leucine mixed in food. Relative rates of ¹³C recovery in the egg protein were lowest for hens fed the uniformly ¹³C labeled amino acid mixture, presumably because of the high proportion of nonessential amino acids. The time required for the ¹³C-enrichment in eggs to return to background levels at the end of the enrichment period was about twice that required to initially reach isotopic equilibrium with the diet, indicating significant biochemical discrimination of endogenous ¹³C amino acids. We conclude that delivering small amounts of ¹³C amino acid tracers in the drinking water of hens is the most effective way to produce ¹³C-enriched proteins to for tracer studies that do not require delt ¹³C-enrichment above 200‰.

This work is licensed under a Creative Commons Attribution 4.0 License.