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‰.
Highlights
1.1 Isotopes and Breath Testing13CO2-breath testing is increasingly used in nutritional research and as a supplement to diagnose various medical conditions including bacterial infections, digestive disorders, and metabolic dysfunction (Amarri & Weaver, 1995; Anania et al, 2008; Braden, 2007, 2010; Koletzko et al, 1988; Parra & Martinez, 2006; Romagnuolo et al, 2002; Weaver, 1998)
Most research exploring the oxidative fates of exogenous proteins in vivo does not employ isotopically labeled proteins, but rather purified 13C-labeled amino acid tracers under the assumption that these free amino acids ‘behave’ exactly like those amino acids that are biochemically integrated into proteins (Braden, 2009; Fischer & Wetzel, 2002; Ghoos & Beaufrere, 1998; McCue, 2011)
We evaluated the possibility that prolonged exposure to artificially enriched 13C-leucine in the diet could cause gradual, long-term enrichment in egg products using paired t-tests to compare the δ13C of egg whites and egg yolks at the same dosing level (i.e. 200 mg day-1) at different points in the experiment
Summary
This technique relies on the assumption that the 13C-labeled tracer molecules introduced into the body (usually orally) are eventually oxidized and exhaled as 13CO2. These individuals justify their actions by citing the fact that large amounts (> 100 grams) of nutritionally complete, artificially enriched, 13C-labeled proteins remain commercially unavailable (Berthold et al, 2011; Braden, 2010; Fromentin et al, 2011a; Jonderko et al, 2005)
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