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Gastrointestinal Research Center, Division of Gastroenterology, Department of Medicine, University Hospital Gasthuisberg, Katholieke Universiteit Leuven, 3000 Leuven, Belgium and * Laboratory of Physiology and Immunology of Domestic Animals, Faculty of Agricultural and Applied Biological Sciences, Department of Animal Production, 3001 Heverlee, Belgium
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGMENTS
LITERATURE CITED
ABSTRACT 
Protein assimilation and metabolism studies are hindered by the lack of an adequate oral tracer, i.e., labeled proteins. We present a new and easily reproducible methodology for producing large amounts of egg proteins labeled with L-leucine-13C1. Laying hens were fed a 0.2% leucine-deficient food supplemented with 0.2% L-leucine-13C1 (99 atom %). At plateau, eggs containing highly enriched proteins were obtained. The 13C content of egg white relative to the total C content was 1.3371 atom %, corresponding to 
= 206
. The overall tracer recovery in egg proteins was high (40.2%), making this method financially attractive as well. Accurately measurable levels of 13CO2 in breath were obtained after ingestion of a physiological load of labeled egg white proteins. Thus, egg proteins with sufficient 13C enrichment and applicable for human protein assimilation and metabolism kinetic studies were produced in an easily reproducible and highly efficient manner.
INTRODUCTION
The study of the assimilation of proteins, i.e., the process of digestion and absorption in the gastrointestinal tract, remains the subject of current, intensive research. Protein digestion involves a complex series of degradative processes which are elicited mainly by the hydrolytic enzymes originating from the stomach, pancreas and the brush border of the small intestine (Alpers 1994
, Erickson and Kim 1990, Freeman et al. 1979). The result of this proteolytic activity is a mixture of amino acids and small peptides which are absorbed by the small intestinal enterocytes. Information about the overall rate and the different factors controlling the rate of the process of protein digestion and absorption in humans is scarce. This is due primarily to the lack of a safe (nonradioactive) and representative marker, which allows adequate measurement. The introduction of stable isotopes in fundamental biological research has created a number of perspectives, especially in the field of nutrition (Halliday and Rennie 1982). A whole range of labeled amino acids is currently available. Different methodologies using labeled amino acids have been developed to study protein metabolism in vivo. 15N-labeled tracers are used in the so-called "end product" method; however, this method has some serious drawbacks (Bier 1991). The most popular is that in which the turnover of a specific amino acid is measured by isotopic dilution, either during constant administration of a labeled amino acid ("constant infusion" method) or after the injection of the labeled amino acid contained in a large bolus of unlabeled amino acids ("flooding" method) (Garlick et al. 1994, Matthews et al. 1981, Millward et al. 1991). 13C-Labeling of the amino acid has the great advantage that oxidation can be followed by measuring 13CO2 excretion in breath (Ghoos et al. 1988b, Klein 1982). L-Leucine-13C1 has been studied extensively. Only a few reports are available describing methods for producing large amounts of stable isotope-labeled proteins, which could be very helpful in studying the assimilation of proteins. In the future, these stable isotope-labeled proteins could also be of great help in the study of protein metabolism during feeding. Recently Boirie et al. (1995) developed a method for producing large amounts of sufficiently enriched milk proteins labeled with L-leucine-13C1. However, the method described is rather unique and difficult to repeat in other laboratories. We present a new and more easily reproducible methodology for producing large amounts of highly enriched egg proteins. This was possible by feeding laying hens a leucine-deficient food supplemented with L-leucine-13C1.
MATERIALS AND METHODS
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Table 1. Ingredients and calculated analysis of experimental diet |
15°C until further analysis. The isotopic enrichment of the two fractions of the 16 eggs laid from d 0 to 16 of the study was measured. Thereafter, analyses of isotopic enrichment were performed at regular intervals.
13C-value (), and thereafter converted to AP. Because the carbohydrate and lipid content of egg white is very limited (Johnson 1986), the level of enrichment obtained by the method described can be assumed to be the same as the enrichment of the protein fraction within. The isotopic enrichment of the yolk can be determined in the same manner. However, because the yolk has a high lipid content, the results obtained will considerably underestimate the specific enrichment of the protein fraction within. Nevertheless, these determinations can be indicative of the enrichment pattern. The intraassay variability of this method, assessed by the coefficient of variation, was 0.39%.
Calculation of the exact amount of L-leucine-13C1 present in enriched egg white protein.
Knowing the exact amino acid composition (Table 2) and the isotopic enrichment of the egg white (measured), it is possible to calculate the amount of L-leucine-13C1 (99 atom %) present in the protein fraction of the egg white.
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(1) |
= carbon atom fraction of position C1 within leucine in egg white protein, i.e., 0.0164; 
= carbon atom fraction of the rest of all C-atoms in egg white protein, i.e., 0.9836, [9.83% of all C-atoms in egg white originate from leucine; knowing that each leucine molecule has six C-atoms, it can be stated that 1.64% of all C-atoms originate from position C1 within leucine (). As a result, 98.36% is accounted for by the rest of all C-atoms in egg white ()]; AP
= atom % enrichment of (to be determined); and AP
= measured atom % enrichment of (i.e., AP enrichment of unlabeled egg white = 1.08749).
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(2) |
= calculated atom % enrichment of .
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Table 2. The amino acid concentration and corresponding calculated C-concentration of egg white and yolk1,2 |
This allows calculation of the ratio leucine-13C1 to total leucine in egg white as well as the efficiency of incorporation (i.e., 100 × incorporated leucine-13C1/administered leucine-13C1).
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= carbon atom fraction of 13C labeled position C1 within leucine in egg white protein when L-leucine-13C1 accounts for 25% of the total leucine in the egg white, i.e., 0.25·0.0164 = 0.00410; = carbon atom fraction of the rest of all C-atoms in egg white protein, i.e., 1 0.00410 = 0.9959; AP = atom % enrichment of , i.e., 99; and AP = measured atom % enrichment of (i.e., AP enrichment of unlabeled egg white = 1.08749).
-value: max = 345.
= 155.2) mixed with 11 g of unlabeled egg white protein ( = 21.6) and was cooked in a microwave oven. The L-leucine-13C1 (99 atom %) content was calculated as being 177 mg. The meal was ingested with 200 mL water. All test meals were consumed in less than 15 min.
20/20, IRMS, Europa Scientific). The data were expressed in percentage of administered dose of 13C recovered per hour and in cumulative percentage administered dose of 13C recovered over a 6-h period. All results were expressed as means ± SEM.
RESULTS
= 206 ± 10. The enrichment pattern of the total carbon content of the yolk was analogous apart from the fact that the asymptotic value of the time labeling curve was reached some days later. Applying the calculations, as described in Materials and Methods, the average amount of L-leucine-13C1 (99 AP) incorporated into 100 g of egg white ( = 206) was 148 mg. It is known that the leucine content of egg white averages 902 mg per 100 g egg white (Table 2); thus, the ratio of L-leucine-13C1 to total leucine in egg white was 148/902 = 0.164. At plateau, it may be assumed that the ratio of L-leucine-13C1 to total leucine in the yolk is the same as in the egg white. Because 100 g of yolk contains 1360 mg of leucine, the amount of L-leucine-13C1 incorporated into 100 g of yolk at plateau was 223 mg (i.e., 1360 × 0.164). Finally, knowing the mean weight of the yolk and egg white fraction (18 and 38 g, respectively) and assuming a maximal daily feed intake of 120 g (containing 240 mg L-leucine-13C1), the efficiency of incorporation (see Materials and Methods) was calculated as 40.2%.
) of egg white protein of eggs laid by a hen which had free access to a 0.2 g/100 g leucine-deficient diet supplemented with 0.2 g/100 g L-leucine-13C1 (99 atom %) beginning on d 0. Note that because the ovulation-oviposition cycle in the chicken is >24 h, an egg was not laid on every day of the feeding period (Johnson 1986). *no egg.
Breath test studies. Figure 2 shows the mean 13CO2 excretion in expired breath, expressed in percentage of administered dose per hour, in 10 normal volunteers. The mean maximal percentage excretion of administered dose per hour was 5.75 ± 0.27 and was attained 168 ± 15 min after the protein meal was ingested. The mean cumulative percentage recovery of administered dose of 13C after 6 h (Fig. 3) was 19.71 ± 1.03.
= 155) mixed with 11 g of unlabeled egg white protein ( = 21.6). Values are means ± SD, n = 10.
= 155) mixed with 11 g of unlabeled egg white protein ( = 22). Values are means ± SD, n = 10.
DISCUSSION
), but also to assess protein metabolism during feeding in humans. Indeed, oral administration of labeled proteins mimics the normal physiological situation in assessing protein metabolism during feeding, whereas this is not the case with free amino acids (Hoerr et al. 1993). There are few reports which describe the synthesis of stable isotope-labeled proteins in sufficient amounts for use on a large scale. Uniformly, 15N-labeled milk proteins were obtained either by adding (15NH4)2SO4 to the diet of lactating cows (Colin et al. 1994) or by infusing (15NH4)2SO4 directly into the rumen of the cow (Mahé et al. 1994). If the label is 13C, a direct determination of amino acid oxidation can be obtained by measuring 13CO2 excretion in expired breath. 13C-Labeled proteins are therefore often preferred to other tracers. Because CO2 derived from the oxidation of a given amino acid represents only a small fraction of the total CO2 production, high enrichments are a prerequisite in obtaining accurate measurements of 13CO2.
). The milk was collected during and after the infusion. Afterwards, the casein and whey protein fractions were purified by membrane separation techniques. The two fractions were sufficiently enriched to be used in human studies. Nevertheless, the described method is rather cumbersome. In contrast, the present method is simple and easily reproducible on the condition that the composition of the chicken diet has been well defined. Large amounts of highly enriched ( = 206 at plateau) and specifically labeled egg proteins can be obtained. The efficiency of incorporation (40.2%) is high, making this method financially attractive as well.
= 206 in the case of egg white), is about the same as that described by Berthold et al. (1991). The initial increase in the time labeling curve of the yolk is much slower than in the case of egg white, an observation which reflects the difference in the physiology of egg protein production of the two egg fractions. Although beyond the scope of this study, it is known that the production and accumulation of egg yolk protein is spread over several days, whereas egg white proteins are produced within hours and for each consecutive egg separately (Johnson 1986). Moreover, measured enrichment of the total carbon content of the yolk is lower than the enrichment of the corresponding egg white because of the high lipid content of yolk. Without clear proof, it is accepted, however, that the isotopic enrichments of the isolated protein fraction of the yolk and egg white are the same at plateau. The L-leucine-13C1 content of the yolk at plateau has therefore been calculated (see Results). Some day-to-day variations in 13C enrichment of the egg white may occur. These daily variations most probably result from the (de)synchronization of egg white formation with the diurnal food intake pattern. Indeed, egg white becomes relatively more 13C-enriched when the major part of the daily food is consumed during the period of egg white synthesis (i.e., 4-6 h after ovulation) and even more when the food intake is uniformly spread during that period.
). This smooth course suggests high accuracy, making this test a usable tool in protein assimilation and metabolism kinetic studies in humans.
and b). It was demonstrated that the pancreatic as well as gastric phase is important in the overall assimilation process of protein.
FOOTNOTES
Manuscript received 21 February 1996. Initial reviews completed 21 May 1996. Revision accepted 8 October 1996.
ACKNOWLEDGMENTS
D. Halliday, London, is acknowledged for constructive discussions. We also thank D. Claus, N. Gorris, S. Rutten and L. Swinnen for excellent technical assistance.
LITERATURE CITED
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