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,2
Department of Gastroenterology;
* Department of Nephrology, University Hospital Gasthuisberg; and
Laboratory of Physiology and Immunology of Domestic Animals, Faculty of Agricultural and Applied Biological Sciences, Catholic University of Leuven, 3000 Leuven, Belgium
1To whom correspondence should be addressed. E-mail: karen.geboes{at}uz.kuleuven.ac.be.
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ABSTRACT |
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 TOP ABSTRACT SUBJECTS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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KEY WORDS: • breath test • protein digestion • stable isotopes
The nutritional value of protein is related both to its digestibility and to the subsequent metabolism of the absorbed amino acids. Tracer techniques using stable isotopes are attractive and safe methods for the in vivo study of various aspects of protein assimilation and metabolism.
Studies based on the use of 15N-labeled proteins or naso-ileal catheters provide information concerning the digestibility of proteins, postprandial oxidation, and retention (1–3).
When amino acids are labeled with 13C at the 
-COOH position, their oxidation following digestion and absorption can be evaluated by measuring 13CO2 excretion in the breath (4,5). Labeled amino acids must be incorporated into the protein to adequately represent the fate of ingested proteins (4,6,7). Studies of the kinetics of amino acid metabolism often use [13C-leucine], because leucine is rapidly metabolized, has small plasma and intracellular pools, and has high turnover rates in these pools (8).
An excellent correlation was found between 13CO2 excretion in the breath and duodenal trypsin output after the ingestion of proteins intrinsically labeled with 13C-leucine (9). Intraluminal pancreatic digestion, small bowel transit time, and the absorptive capacity of the gut are very important in the overall process of protein assimilation (10). The breath test may be a valuable tool to evaluate protein assimilation in adults and children with various diseases (e.g., pancreatic disease, celiac sprue, radio enteritis, short bowel syndrome, and motility disorders) and to monitor the effects of pharmaceuticals on protein assimilation (9,11,12).
The test meals used in earlier studies of protein assimilation were almost exclusively composed of proteins (4,6,9). However, the kinetics of protein assimilation after the ingestion of a single-protein meal may differ from those after the consumption of a complex, physiologically normal meal containing carbohydrates, fat, and protein (4,6).
In this study, a complex pancake meal containing 12 g of fat, 27 g of carbohydrate, and 19 g of protein was developed, and overall protein assimilation parameters in subjects that consumed the meal were evaluated.
Only a few techniques for the production of proteins intrinsically labeled with stable isotopes are described in the literature (1,5,13). In particular, it was difficult to obtain sufficiently high enrichment levels of protein with adequate labeling patterns. Previously, we developed a methodology for producing large amounts of highly enriched egg proteins labeled with 13C-leucine, by feeding laying hens a leucine-deficient diet supplemented with 2 g/kg of 13C-leucine (13). Because of technical problems with the production of the leucine-deficient diet, in the present study we also evaluated the use of a leucine-sufficient chicken diet supplemented with 3g/kg of 13C-leucine. The use of a leucine-sufficient chicken diet supplemented with 3 g/kg of 13C-leucine may affect the isotopic enrichment patterns of the egg white and yolk and the efficiency of tracer incorporation.
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SUBJECTS AND METHODS |
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TOPABSTRACTSUBJECTS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Pancake test meal. A pancake batter was prepared using 66 g of labeled and 33 g of unlabeled egg white, 34 g of labeled egg yolk, 7 g of sugar, 17 g of wheat flour, 3 g of milk powder, and 25 mL of water. The pancake was prepared with 5 g of butter, and 5 g of sugar was poured on top of the finished pancake. The meal was consumed together with 1 glass of water (200 mL). The total energy content of the test meal was 1.37 MJ, and the meal contained 19 g of protein, 6 g of fat, and 27 g of carbohydrate.
Production of 13C-enriched eggs.
A chicken diet that met the nutrient requirements for laying hens [8 g/kg of leucine, NRC requirements (13,14)] was supplemented with 3 g/kg of 13C-leucine (99%, mol:mol; Euriso-top). During peak egg production, 6 laying hens (Hisex Brown, body wt 
2 kg) were given free access to the 13C-leucine–supplemented diet. The mean daily food intake of the hens was 100 g, with a maximum of 120 g. Eggs were collected daily and dated. Each egg was opened under sterile conditions and separated into white and yolk fractions. The fractions were freeze-dried and stored until further analysis. The isotopic enrichment of the 2 fractions of each egg laid from d 0 to d 20 was measured. Thereafter, isotopic enrichment was assayed at regular intervals.
Administration of the breath tests. To establish the normal range of the parameters of protein assimilation, the 13C-leucine pancake breath test was administered to 26 healthy volunteers (5 men, 21 women; age range 21 to 50 y).
Gastric emptying was measured in a subgroup of 22 volunteers (5 men, 17 women), by adding 74 kBq of sodium 14C-octanoate (ARC) to the meal (15).
To measure the orocecal transit time using the hydrogen breath test, 5 g of Raftilin HP (Orafti) was added to the pancake batter (16). This did not affect the energy content of the meal.
The parameters of protein assimilation and gastric emptying were compared with those of subjects that consumed a single-protein meal containing 100 g of labeled egg white, 100 g of unlabeled egg white, and 17 g of egg yolk. The parameters of protein assimilation were assessed using 48 subjects, and the parameters of gastric emptying were assessed using 16 other healthy volunteers (9,12).
After the subjects fasted overnight, breath samples were taken to establish the basal values of 13CO2. After the ingestion of the test meal, samples were taken at 15-min intervals for 6 h.
When both gastric emptying and orocecal transit time were measured, additional samples were taken before ingestion of the meal to establish the basal values of 14CO2 and hydrogen. Thereafter, additional samples were taken at 15-min intervals for 4 and 10 h to assess gastric emptying and orocecal transit time, respectively.
Analysis of the 13C-enriched eggs. The 13C-enrichment of egg white, egg yolk, and fat was measured using an elemental analyzer (ANCA-SL; PDZ) coupled with a Stable Isotope Ratio Mass Spectrometer (IRMS; PDZ). The lipids were extracted from freeze-dried samples of egg yolk using continuous (Soxhlet) extraction (Soxtec Avanti 2050 Automatic Extraction System;Foss Tecator).
Analysis of the breath samples. For the 13CO2 and hydrogen measurements, breath samples were collected in Exetainers (PDZ). The breath 13C content was analyzed using an Isotope Ratio Mass Spectrometer (PDZ). Hydrogen was measured using an Exhaled Hydrogen Monitor (GMI Medical) that contained an electrochemical cell and provided a digital readout of hydrogen concentration in ppm.
For the 14CO2 measurement, each subject blew through a pipette into a vial containing 2 mmol of hyamine hydroxide until the thymolphtaleine indicator became discolored, corresponding to the capture of 2 mmol of CO2. Hionic fluor (Perkin Elmer) (10 mL) was then added to the sample, and the 14CO2 content was measured by ß-scintillation counting (Packard Tricarb Liquid Scintillation Spectrometer, Model 3375; Packard Instruments).
Calculation of the administered dose of 13C-leucine.
To simplify calculations, we assumed that all 13C atoms were present in the C1 position of leucine. This means that the mole percentage (MP)3 of 13C-leucine in egg protein equals the atom percentage (AP) of 13C in the carbon atom fraction of C1 in leucine in egg protein, which is represented as AP.
Because the carbohydrate and lipid contents of egg white are very low, we assumed that the enrichment of egg white obtained by the method described above was the same as the enrichment of the protein fraction within. Knowing the mean amino acid composition of eggs laid by Hisex Brown hens (Tables 1, and 2) and the measured isotopic enrichment of the egg white, the amount of 13C-leucine present in the protein fraction of the egg white can be calculated (13).
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 | (1) |
 | (2) |
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where ' = fraction of all carbon atoms in egg yolk in protein = 0.240 (Table 1)
AP' = 13C atom percentage of ' (i.e., AP enrichment of the protein fraction of egg yolk)
= fraction of carbon atoms in position C1 in leucine in egg yolk protein = 0.018
ß = fraction of all other carbon atoms in egg yolk protein =0.982
APß = measured 13C atom percentage of ß (i.e., AP enrichment of unlabeled egg yolk protein) = 1.088
AP = 13C atom percentage of (i.e., of the fraction of carbon atoms in position C1 in leucine)
and
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where 
= fraction of all carbon atoms in egg yolk in lipid = 0.760 (Table 1)
AP = measured 13C atom percentage of
AP = enrichment of lipid fraction of egg yolk = 1.086
Analogously to egg white, the amount of 13C-leucine (99%, mol:mol) incorporated in egg yolk protein can be calculated from the following equation:
 | (3) |
where m = mg of 13C-leucine (99%, mol:mol) to be incorporated into y mg of egg yolk protein
0.085 = mg leucine/mg egg yolk protein (Table 2)
MP13C-leu administered = enrichment of 13C-leucine used for supplementation of food = 99% (mol:mol)
AP = calculated 13C atom percentage of (i.e., the fraction of carbon atoms in position C1 in leucine in egg yolk protein, which, assuming that all 13C atoms are in the C1 position, equals the MP enrichment of leucine)
Breath test data analysis. We assumed human CO2 production to be 300 mmol/m2 body surface area per hour (10,13,15). The body surface area was calculated by the weight-height formula of Haycock et al. (17).
Breath test results for protein assimilation were expressed as the percentage of the administered dose of 13C excreted per hour and the cumulative percentage of the administered dose of 13C excreted over 6 h. The maximum excretion rate of 13C and the time of maximum excretion rate of 13C were also estimated (10). Background enrichment of breath after administration of an unlabeled test meal was previously evaluated with 10 healthy volunteers and was found to be negligible.
The 14CO2 excretion data were further analyzed by nonlinear regression to obtain curve fitting and the calculation of gastric emptying parameters, i.e., the gastric emptying coefficient and the half-emptying time. The details were as previously published (15).
Hydrogen excretion was expressed in ppm. A consistent rise in hydrogen excretion of 10 ppm above baseline was defined as a cutoff value for the orocecal transit time (16).
Statistical analysis.
Results are expressed as means ± SD. The nonparametric Mann-Whitney test was used to compare the enrichment of egg white and egg yolk, because of the low number of hens (n = 6) used in the experiment. The unpaired Student’s t test was used to compare the parameters of protein assimilation and gastric emptying obtained after subjects consumed the different meals ( = 0.05). Statistica 6.0 software (Statsoft) was used for the analysis.
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RESULTS |
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TOPABSTRACTSUBJECTS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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The variability in egg white enrichment at plateau among hens (n = 6) was small (CV = 2.67%). A mean egg white enrichment of 1.333% was calculated (mean APegg white at plateau, n = 6).
The enrichment pattern of the egg yolk protein was similar, although the plateau was reached somewhat later, after 14 to 18 d. The within-hen CV, describing the variability of egg yolk enrichment at plateau in 1 hen, ranged from 0.10 to 1.95%, with a median of 0.51%. The CV in egg yolk enrichment among the 6 hens was 0.35%. A mean egg yolk enrichment of 1.144% was measured (mean APegg yolk at plateau, n = 6).
Calculation of the exact amount of 13C-leucine present in enriched egg protein. The 13C-leucine:leucine ratio in the white and yolk of eggs that reached plateau levels of enrichment was calculated using the measured values for APegg white and APegg yolk and the previously described equations.
For example, in an egg white with APegg white = 1.337% and APegg yolk = 1.153%, the 13C-leucine:leucine ratio (mol:mol) was 16.271:100 in egg white and 16.486:100 in egg yolk.
The mean MP of 13C-leucine in egg white was 16.085 ± 2.168%, and the mean MP of 13C-leucine in egg yolk was 14.112 ± 0.914%. Because these means did not differ significantly, the 13C-leucine:leucine ratios at plateau in the protein fractions of the white and yolk of a labeled egg were equal, allowing the calculation of the total amount of 13C-leucine present in the egg on the basis of the measurement of APegg white.
By application of the calculations as described, the amount of 13C-leucine in 1 egg was 81 mg.
Efficiency of tracer incorporation. Assuming a maximal daily feed intake by a laying hen of 120 g, containing 360 mg 13C-leucine (3 g/kg), and assuming a daily production of 1 egg [which is a slight overestimation, because the ovulation-oviposition cycle in chickens is slightly longer than 24 h (13)], the efficiency of incorporation was calculated to be 22.5%.
Mole percentage excess of 13C-leucine in the new test meal.
An unlabeled egg contained 6 mg of 13C-leucine [assuming all naturally occurring 13C-atoms to be present in the -COOH position of leucine; Eqs. 1 to 3 (13)]. The unlabeled meal contained 16 mg of 13C-leucine (12 mg in 2 whole eggs + 3 mg in an extra egg white + 1 mg in milk powder). Using 2 labeled eggs in the test meal (162 mg in 2 labeled whole eggs + 3 mg in an extra egg white + 1 mg in milk powder), a total amount of 166 mg of 13C-leucine was administered.
The total amount of leucine in the meal was calculated to be 2.4 g, using Table 2 and information provided by the supplier (Nestlé) of the milk powder (351g leucine/kg milk powder).
Therefore, the 13C-leucine:total leucine ratio of the test meal was 0.068, meaning that the MP of labeled leucine in the test meal was 6.9%, making the mole percentage excess (MPE) of 13C-leucine in the test meal 6.3%.
Assessment of protein assimilation, gastric emptying, and orocecal transit time. The mean maximal percentage of the administered dose excreted per hour was 5.65 ± 1.48% (Fig. 2A), which was attained 145 ± 25 min after the ingestion of the meal. The mean cumulative percentage of the administered dose of 13C recovered after 6 h was 17.22 ± 4.74% (Fig. 2B).
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The administration of the three differently labeled markers in the test meal allowed the simultaneous evaluation of gastric emptying, parameters of protein digestion, and orocecal transit time (Fig. 3).
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DISCUSSION |
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TOPABSTRACTSUBJECTS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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When calculating the enrichment of the eggs, we assumed that all 13C-leucine was incorporated as such into the egg protein and that no 13C was incorporated into other amino acids through CO2 fixation.
Because the mole fractions of 13C-leucine in egg white and yolk appeared to be equal, APegg yolk could be calculated using the measured value for APegg white. As a consequence, the whole egg could be used in the preparation of the test meal without measuring the enrichment of the white and yolk separately.
We prepared the pancake meal by adding wheat flour, milk powder, and sugar. The test meal contained 19 g total protein. The calculated MPE of labeled leucine was 6.3%, comparable with that of other meals used to study protein assimilation [minimum MPE = 3.42% (4,11)].
Aside from easy availability and palatability, the most important advantage of this test meal was the fact that pancakes with exactly the same composition could be used in the evaluation of gastric emptying with 14C-octanoic acid as a marker and in the evaluation of orocecal transit time using inulin as a substrate in a hydrogen breath test (15,16). The incorporation of differently labeled substrates in the same test meal enabled the assessment of the effect of various gastrointestinal processes on the overall assimilation of proteins (Fig. 3). The effects of administration of other nutrients or medication on protein digestion and transit parameters could be studied simultaneously.
When we compared the protein assimilation parameters obtained after administration of the newly developed test meal with the values obtained using a pure protein test meal (9), we found that the incorporation of the labeled proteins into a complex meal did not affect the main parameters of gastric emptying and overall protein assimilation (Tables 3, and 4). Carbohydrates and lipids may affect both intraluminal digestion of proteins (gastric emptying, mixing of nutrients, or small bowel transit) and postprandial metabolism (insulin response). The rate-limiting step in the overall process of protein assimilation is intraluminal proteolysis (9). Because protein assimilation parameters in subjects that consumed the pancake meal did not differ from those obtained in volunteers that consumed a single-protein meal, we concluded that the inclusion of carbohydrates and lipids in the test meal did not influence intraluminal protein digestion (including gastric emptying and small bowel transit).
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In conclusion, this study showed that highly enriched, specifically labeled egg proteins can be obtained from hens fed a normal chicken diet substituted with 3 g/kg of 13C-leucine and that both egg white and yolk can be used in a breath test.
The newly developed pancake test meal is a tasty, physiologically normal meal, part of the normal western diet. The main parameters of overall protein assimilation did not differ from the parameters previously established using a meal containing almost exclusively egg white protein. It is important to note that the same test meal has been used in breath tests for the evaluation of gastric emptying and orocecal transit time. The incorporation of various labeled substrates enables the simultaneous assessment of different gastrointestinal processes.
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FOOTNOTES |
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3 Abbreviations used: AP, atom percentage; MP, mole percentage; MPE, mole percentage excess.
Manuscript received 25 September 2003. Initial review completed 28 October 2003. Revision accepted 12 January 2004.
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LITERATURE CITED |
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TOPABSTRACTSUBJECTS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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1. Bos, C., Mahé, S., Gaudichon, C., Benamouzig, R., Gausserès, N., Luengo, C., Ferrière, F., Rautureau, J. & Tomé, D. (1999) Assesment of net postprandial protein utilization of 15N-labelled milk nitrogen in human subjects. Br. J. Nutr. 81:221-226.[Medline]
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3. Gaudichon, C., Bos, C., Morens, C., Petzke, K. J., Mariotti, F., Everwand, J., Benamouzig, R., Dare, S., Tomé, D. & Metges, C. C. (2002) Ileal losses of nitrogen and amino acids in humans and their importance to the assesment of amino acid requirements. Gastroenterology 123:50-59.[Medline]
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6. Metges, C. C., El-Khoury, A. E., Selvaraj, A. B., Tsay, R. H., Atkinson, A., Regan, M. M., Bequette, B. J. & Young, V. R. (2000) Kinetics of L-[1-13C]leucine when ingested with free amino acids, unlabelled or intrinsically labelled casein. Am. J. Physiol. Endocrinol. Metab. 278:E1000-E1009.
7. Daenzer, M., Petzke, K. J., Bequette, B. J. & Metges, C. C. (2001) Whole-body nitrogen and splanchnic amino acid metabolism differs in rats fed mixed diets containing casein or its corresponding amino acid mixture. J. Nutr. 131:1965-1972.
8. Wootton, R. (1985) Non-steady state modelling of glucose and leucine kinetics. Halliday, D. Garrow, J. S. eds. Substrate and Energy Metabolism 1985:16-26 John Libbey London, UK. .
9. Evenepoel, P., Hiele, M., Geypens, B., Geboes, K. P., Rutgeerts, P. & Ghoos, Y. (2000) 13C-egg white breath test: a non-invasive test of pancreatic trypsin activity in the small intestine. Gut 46:52-57.
10. Ghoos, Y., Rutgeerts, P. & Vantrappen, G. (1985) 13CO2-breath tests in nutritional diagnosis: present applications and future possibilities. Dietze, G. eds. Clinical Nutrition and Metabolic Research 1985:192-207 Karger Basel, Switzerland. .
11. Ghoos, Y. & Beaufrère, B. (1998) 13C protein breath tests. Gut 43:S23-S24.
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13. Evenepoel, P., Hiele, M., Luypaerts, A., Geypens, B., Buyse, J., Decuypere, E., Rutgeerts, P. & Ghoos, Y. (1997) Production of egg proteins enriched with L-leucine-13C1, for the study of protein assimilation in humans using the breath test technique. J. Nutr. 127:327-331.
14. National Research Council (1994) Nutrient Requirements of Poultry 1994 National Academic Press Washington, DC.
15. Ghoos, Y., Maes, B., Geypens, B., Mijs, G., Hiele, M., Rutgeerts, P. & Vantrappen, G. (1993) Measurement of gastric emptying rate of solids by means of a carbon labelled octanoic acid breath test. Gastroenterology 104:1640-1647.[Medline]
16. Geboes, K. P., Luypaerts, A., Rutgeerts, P. & Verbeke, K. (2003) Inulin is an ideal substrate for a hydrogen breath test to measure the orocaecal transit time. Aliment. Pharmacol. Ther. 18:721-729.[Medline]
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