Hens Produce Artificially Enriched 13C Egg Proteins for Metabolic Tracer Studies

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 13 C-labeled proteins using egg laying hens and investigated the relationship between tracer dose and method of delivery on 13 C-protein production. We enriched hens with one of two isotope tracers ( 13 C-1-leucine or a uniformly labeled 13 C-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 13 C in egg white proteins of the hen fed 13 C-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 13 C. The time required for half maximal 13 C enrichment depended chiefly on the mode of tracer administration, and ranged from 2.5 days for 13 C-leucine dissolved in water to 4.9 days for 13 C-leucine mixed in food. Relative rates of 13 C recovery in the egg protein were lowest for hens fed the uniformly 13 C labeled amino acid mixture, presumably because of the high proportion of nonessential amino acids. The time required for the 13 C-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 13 C amino acids. We conclude that delivering small amounts of 13 C amino acid tracers in the drinking water of hens is the most effective way to produce 13 C-enriched proteins to for tracer studies that do not require δ 13 C-enrichment above 200‰.

Using stable isotope analysis to answer fundamental questions in invasion ecology: Progress and prospects

“…Ideally, these controlled feedings may occur over the course of an animal's life (McCue, Amaya, et al, ; McCue, Guzman, Passement, & Davidowitz, ), but should at least include a biologically relevant period (e.g. hours and days for plasma and liver vs. weeks and months for muscle and connective tissue) that is sufficient to permit measurable enrichment of endogenous tissues (Khalilieh, McCue, & Pinshow, ; McCue, Arquisola, et al, ; Munoz‐Garcia, Aamidor, McCue, McWilliams, & Pinshow, ).…”

Carbon stable‐isotope tracking in breath for comparative studies of fuel use

“…13 C-1-l-leucine, an essential amino acid and 13 C-1palmitic acid are cost-effective, widely available, and have proven to be effective tracer molecules for isotopically enriching the protein and the lipid pools, respectively, in the bodies of a variety of animals. 59,60 The use of 13 C-glucose tracers is not recommended for long-term 13 C-enrichment studies because any of the six carbon atoms could eventually become incorporated into the lipid and protein pools during de novo synthesis of fatty acids and nonessential amino acids, respectively. 60 Accounting for the bioconversion of 13 C-glucose into other nutrient pools is possible but may not be practical for most applications.…”

“…62) Unfortunately, the practice of intrinsic labeling has not been widely adopted. 59 Intermediary metabolism and CO 2 elimination When 13 C-labeled tracers are given to animals either as part of their diets over a protracted period, infused into circulation, or fed as a single bolus, the appearance of 13 C in the breath cannot be used to accurately track the intermediary metabolism of a given tracer. This is a particularly important point for studies of the fates of biochemically labile molecules like glucose, pyruvate, or acetate.…”

“…In order to do this effectively, the selected 13 C‐labeled tracer molecules should not undergo extensive oxidation during the postprandial period, occur in relatively high amounts in the given macronutrient pool, and exhibit minimal biochemical conversion into other tissue pools. 13 C‐1‐ l ‐leucine, an essential amino acid and 13 C‐1‐palmitic acid are cost‐effective, widely available, and have proven to be effective tracer molecules for isotopically enriching the protein and the lipid pools, respectively, in the bodies of a variety of animals . The use of 13 C‐glucose tracers is not recommended for long‐term 13 C‐enrichment studies because any of the six carbon atoms could eventually become incorporated into the lipid and protein pools during de novo synthesis of fatty acids and nonessential amino acids, respectively .…”

Introduced and Native Congeners Use Different Resource Allocation Strategies to Maintain Performance during Infection

“…We expressed these d 13 C enrichments in terms of log d per gram, and these values were used as a proxy for leucine allocation (hereafter absolute leucine allocation). We also compared the relative leucine allocation to the lymphoid tissues in terms of allocation to the gonad tissue (i.e., absolute allocation to splenic or hepatic tissues divided by absolute allocation to gonadal tissue; sensu McCue et al 2013), as well as the weighted leucine allocation (i.e., the absolute leucine in a particular tissue divided by the total absolute leucine in all tissues measured); the resulting ratios were unitless.…”