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The Journal of Nutrition Vol. 128 No. 11 November 1998,
pp. 1969-1977
INRA, Station de Recherches Porcines, 35590 Saint Gilles, France
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ABSTRACT |
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Abstract
Introduction
Methods
Results
Discussion
References
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We assessed the influence of sampling site when using the isotope dilution method to determine ileal endogenous N losses. Three growing pigs were prepared with ileorectal anastomoses and fitted with three catheters (portal, jugular and carotid). A 15N-leucine solution was infused for 24 d, alternating between the carotid artery and the jugular vein. Blood was sampled from the portal catheter and from the systemic catheter not used for the infusion. The pigs were fed successively a protein-free diet, an isolated pea protein diet and a hydrolyzed pea protein diet according to a Latin-square design. The 15N was transferred from leucine to isoleucine, valine, alanine, glycine and proline. Free 15N alanine, glycine and valine enrichments were closer to the respective amino acid enrichments in secretory tissues in the portal vein than in the systemic blood. The enrichment of total nitrogen was higher in the trichloroacetic acid-soluble fraction of the plasma than in the ileal digesta of pigs fed the protein-free diet. Lysine enrichment was significantly different from zero in all tissues except muscle, suggesting that essential amino acids can be synthesized by microflora and used for protein synthesis in the host. We conclude that the total nitrogen isotope dilution method is inappropriate to determine the endogenous loss of amino acids. Moreover, the amino acid dilution method should be performed with portal blood sampling. The main limititation of this method is that a number of essential amino acid losses cannot be determined.
KEY WORDS: pigs · real ileal digestibility · endogenous N · 15N-leucine · isotope ratio mass spectrometry
The accurate knowledge of the ileal N-digestibility of a foodstuff is one of the tools for improving nitrogen nutrition and particularly protein efficiency with the objective of reducing environmental pollution. The direct measurement of digestibility provides the "apparent digestibility," which does not account for the effect of the endogenous secretions in the digestive tract that are not reabsorbed and are therefore excreted. Several techniques have been proposed to assess this endogenous fraction; these include the protein-free diet, the regression method (Mariscal-Landin et al. 1995) and the totally digestible N diet (Moughan and Rutherfurd 1990 The aim of this study was first to compare three methods to determine basal and total endogenous losses, i.e., the protein-free diet and the hydrolyzed protein diet in which amino acids are presumed totally digestible for determination of the basal losses, and the 15N dilution methods for the determination of the total endogenous losses. Because the N from leucine can be transferred to different amino acids, the second objective of this study was to compare the15N total N dilution technique and15N amino acid dilution technique with labeling of amino acids through [15N]-leucine infusion. Using systemic infusion (carotid or jugular), we compared portal and systemic free amino acid enrichments as a reference for the labeling of endogenous N or amino acids.
Animals: surgical preparation.
The experiment was conducted under the guidelines of the French Ministry of Agriculture for animal research. Three growing Piétrain × Large White pigs from the herd of St. Gilles at an average body weight (BW)5 of 33.7 kg were housed individually in metabolism crates that allowed total and separate collection of ileal chyme and urine. The pigs were deprived of food for 2 d and of drinking water for 12 h; pigs were then prepared with an end-to-end ileorectal, antevalvular anastomosis as described by Laplace et al. (1989) Diet and digesta collection.
A protein-free diet (PF) and two 18% protein diets using a pea isolated protein (PP) and an enzymatically hydrolyzed pea protein (HP), each as sole protein source (Nutrinov, Rennes, France), were formulated (Table 1). The animals were fed 80 g dry matter/(kg0.75 · d) at 0800 and 1530 h in two equal meals mixed with water (1:2). Three days after the beginning of the 15N leucine infusion (see below), the experimental diets were offered according to a Latin-square design. After 4 d of adaptation to an experimental diet, the ileal digesta were totally recovered in 500 mL of 0.7 mol/L H2SO4 and collected twice daily after feeding over 3 d (Mariscal-Landin et al. 1995). Urine was recovered in 90 mL of 1.9 mol/L H2SO4 and collected daily. The collection days were d 8-10, 15-17 and 22-24. The ileal digesta were freeze-dried and finely ground.
Infusions and blood sampling.
The infusion of the 15N-leucine (99% 15N-enrichment; Cambridge Isotope Laboratories, Andover, MA) started in the week after the insertion of the catheters. Leucine was dissolved aseptically in sterile saline and then sterilized through a 0.22-µm filter (Sterifix, Braun, Germany). It was infused at a level of 8.9 mg leucine/kg at a rate of 2 mL/h for 24 d. The infusion was performed with a syringe pump (Perfusor, Braun, Germany). The infusion site was alternated between the carotid artery and the jugular vein. To assess the best sampling point, systemic or portal, blood collection was performed in parallel from the portal vein and from the systemic catheter, which was not used for the infusion. The first blood sample was obtained from the systemic blood before starting the infusion to determine the basal 15N enrichment of total nitrogen and amino acids in the pigs. During the infusion, each sample of 100 mL consisted of four equal portions collected at 0800 (before the morning meal), 1000, 1200 and 1400 h on d 2, 4, 7, 9, 11, 14, 16, 18, 21 and 23. The last two (portal and systemic) were obtained at one time before slaughter at 0800 h. A total of 23 blood samples were collected for each pig. Each blood sample was collected into ice-cooled tubes that contained 50 IU heparin and centrifuged within 2 h at 2000 × g for 15 min at 2°C. Plasma was removed and stored at Samples preparations.
On the morning of d 25, after 17 h of food deprivation, the infusion was stopped and the pigs were killed by electrical stunning and bled out. Tissue samples, including liver, pancreas, parotid, spleen, longissimus dorsi, semitendinosus and trapezius muscles, were dissected. The digestive tract was removed and emptied. The small intestine was isolated and divided into three equal segments. Gastric, jejunal, duodenal and ileal mucosa samples were obtained by scraping after the lumen was flushed with ice-cooled saline. The remaining duodenal, jejunal and ileal muscle and serosa parts were kept for analysis. All samples were immediately frozen in liquid nitrogen and stored at Chemical and isotope analyses.
Nitrogen content was measured with an elementary analyzer according to the DUMAS method. For total nitrogen measurements, a Leco FP 428 analyzer (Leco, St. Joseph, MO) was used. For total 15N analysis of the remaining 8 mL TCA-soluble fractions and the diets and digesta, an elementary analyzer was used (C. E. 1500 NA, Carlo Erba, Milan, Italy) interfaced with an Isotope Ratio Mass Spectrometer (Optima, Micromass, Sheshire, UK).
Calculations.
The ileal flows of endogenous N and amino acid (AA) losses per kilogram dry matter intake (Nendo) were calculated using the following equation (de Lange et al. 1990
INTRODUCTION
Abstract
Introduction
Methods
Results
Discussion
References
). The endogenous losses determined with these methods may be termed "basal losses." Correction of digestibility values for these losses provides "true digestibility." Some dietary constituents that can be referred to as "feed-specific" losses could lead to higher than basal ileal endogenous losses (Seve and Henry 1995). To assess the total endogenous losses (basal + specific) in pigs, Souffrant et al. (1986) proposed the 15N-dilution technique, which allows the estimation of the real digestibility (Low 1982). This technique requires more evaluation because some points remain controversial, such as the reference pool for the determination of the labeling of the endogenous secretions and the validity of the 15N dilution method when applied to total nitrogen or to a particular amino acid (Lien et al. 1997).
MATERIALS AND METHODS
Abstract
Introduction
Methods
Results
Discussion
References
under general anesthesia using a gas mixture of 4% halothane. The pigs were fed daily increment of 100 g/d up to a maximum of 80 g of dry matter intake (DMI) per metabolicBW (kg0.75). A second surgery that consisted in the insertion of three catheters was performed under general anesthesia 3 wk after the anastomosis. A silicon jugular catheter (i.d., 0.85 mm; o.d., 1.60 mm; Silastic, Vermed, France) was fitted 18 cm deep through the vena jugularis externa into the vena cava cranialis. A polyvinyl chloride carotid catheter (i.d., 1.02 mm; o.d., 1.50 mm; Dural Plastic Engineering, Australia) was fitted 18 cm deep in the arteria carotis externa. A catheter similar to the one in the carotid was fitted 4 cm deep in the portal vein. The patency of the catheters was maintained by daily flushing with sterile heparinized saline [50,000 IU/560 mL (Laboratoire Leo, St Quentin-en-Yvelines, France)]. The day after surgery, the pigs were fed 400 g of a standard grower diet (Croisinra, Glon SA, Pontivy, France). They recovered their normal feeding levels in 4 d.
View this table:
Table 1.
Composition of the experimental diets
20°C until further analysis.
20°C.
), which leads to the formation of N(O)-ethoxycarbonyl ethyl ester derivatives. The chromatographic conditions were as follows: capillary column RTX1701; 0.25 mm i.d. × 30 m with 0.5-µm fitness film (Restek, Evry, France), carrier gas (He) at a flow rate of 1.2 mL/min, injection temperature 240°C. Samples (2 µL) were injected in split mode (15:1). The best resolution was obtained with a temperature program that started at 130°C (6 min), rose to 180°C by 2°C/min and then to 270°C by 10°C/min. The temperature was maintained for 14 min at 270°C. The temperature was 850°C in the combustion oven and 400°C in the reduction oven.
):
where Ndigesta is the total N (AA) in ileal juice; Ediet, Eblood, Edigesta is the N enrichment of N (AA) in the diet, the plasma TCA-soluble fraction and the ileal juice, respectively.
where Nbasendo is the basal endogenous loss determined with the protein-free diet (g/kg DM intake); Ndiet is the N (AA)/kg dry diet; and NdigDMI is the total N (AA) ileal flow/kg DM intake.
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RESULTS |
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Abstract
Introduction
Methods
Results
Discussion
References
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During the infusion, all pigs were in good health and ate all of their feed allowance except for pig 1; on d 24, this pig had to be slaughtered because of a sudden intestinal blockage caused by the portal catheter.
A continuous leucine infusion was performed for 24 d. The N from leucine can be transferred more directly to the other branched-chain amino acids (BCAA) than to nonessential amino acids. The first step of leucine metabolism, as one of the BCAA, is a reversible deamination that leads to the formation of branched-chain keto acids because of the BCAA aminotransferase (Harper et al. 1984
Manuscript received 11 March 1998. Initial reviews completed 27 April 1998. Revision accepted 16 July 1998.
We acknowledge the participation of Ralston Purina in amino acids analyses, Yves Lebreton for surgical operations, Francis Le Gouevec for animal care, and Philippe Ganier and Yolande Jaguelin-Peyraud for their technical assistance.
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Fig 1.
Time course of 15N leucine enrichment in the plasma trichloroacetic acid (TCA)-soluble fraction of the portal vein and TCA-precipitable fraction (protein-bound) in pig 1 (panel A), pig 2 (panel B) and pig 3 (panel C) expressed in atom percent excess (APE) when pigs were fed PF (protein-free diet), HP (hydrolyzed pea protein diet) or PP (isolated pea protein diet).
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Fig 2.
Enrichments of 15N free isoleucine, valine, glycine, proline, and alanine in the portal vein of pig 1 (panel A), pig 2 (panel B) and pig 3 (panel C) expressed in atom percent excess (APE).
View this table:
Table 2.
Mean 15N-enrichments of free amino acids and total nitrogen in the portal vein and systemic blood (carotid artery and jugular vein) of pigs during digesta collection and in the digesta1
View this table:
Table 3.
15N-enrichments of amino acids in tissue samples and in the third collection of ileal juice TCA-precipitable and -soluble fraction of the portal and systemic blood samples at the time of slaughter in the three pigs1
View this table:
Table 4.
Flow of amino acids at the end of the ileum in pigs after ingestion of the protein-free diet (PF),
the hydrolyzed pea protein diet (HP) and the pea protein diet (PP)1
View this table:
Table 5.
Endogenous amino acid and N losses estimated through collection of digesta from protein-derived pigs or through the isotope dilution method using either the portal or the systemic enrichment as reference1
View this table:
Table 6.
Apparent (AD), true (TD) and real (RD) ileal digestibility coefficient of amino acids and N of
the hydrolyzed pea protein (HP) and the pea isolated protein (PP)1
DISCUSSION
Abstract
Introduction
Methods
Results
Discussion
References
). The other BCAA, isoleucine and valine, are deaminated with the same enzyme, and their keto acids were most likely the first acceptors of the labeled amino group. This would explain why they were the most enriched amino acids after leucine. Lien et al. (1997) measured a higher enrichment of isoleucine than of valine but the difference was not as great as the present one. This discrepancy could be related to the analytical methods. Accurate measurements of enrichment by GC/MS made by Lien et al. (1997) require high enrichments, which might not have been achieved in their experiment. This was not a problem in our experiment with GC/C/IRMS, which is designed for the measurement of low enrichments. On the other hand, by GC/C/IRMS, the isoleucine enrichment determination might be overestimated (see results). In the muscles, the N from BCAA can also be transferred to glutamine and alanine (Felig 1975). The BCAA are the main donors of N to alanine (Ben Galim et al. 1980, Haymond and Miles 1982). Proline has been reported to be synthesized from glutamine through deamination into glutamic acid, which may also produce ornithine and citrulline in the intestinal mucosa (Wu et al. 1995). These aspects of amino acid metabolism explain the rank of labeling that was observed, confirming the important heterogeneity of N labeling from 15N-leucine.
, Schulze et al. 1995), whereas others did the opposite (Huisman et al. 1992, de Lange et al. 1990). In tracers studies, systemic plasma leucine labeling is sensitive to the systemic infusion and sampling site (Helland et al. 1988). However, in this study, no difference between jugular and carotid enrichments was measured and the data were pooled. On the other hand, during jugular infusion of labeled amino acids, it has been shown that free amino acid enrichments in the portal vein are lower than those in the arterial blood (Lobley et al. 1996), which was confirmed by the current data. Because the most important secretory organs (i.e., the intestinal mucosa and the pancreas) are drained by the portal vein and because dietary amino acids are rapidly incorporated into the endogenous secretions (Leterme et al. 1996b), the hypothesis that portal enrichment could be used as a reference pool was tested. The plasma 15N alanine, glycine and valine enrichments were closer to the tissue enrichments in the portal vein than in the systemic blood at slaughter. Therefore, these data seem to indicate that the portal vein would be adequate as a sampling site.
). These proteins are very resistant to the digestive process, which may explain the elevation of the enrichment of the ileal digesta up to a value close to that in the parotid glands. There are several possible explanations for leucine. First, this could be due to the presence of some indigestible leucine in the protein-free diet. In fact, the protein-free diet provided 800 mg nitrogen/kg DM. Second, it could be due to an overestimation of the 15N enrichment of leucine determined in the portal vein, which would be inconsistent with the mucosa data although consistent with those of the pancreas. Third, this overestimation could be related to the fact that it was the infused amino acid whose enrichment was always higher in plasma than in tissues. The infused amino acid must enter into the tissue to be incorporated into proteins. A protein-free intake induces a lower protein turnover rate, leading to an accumulation of the tracer in the plasma. As a consequence, plasma leucine enrichment increases, breaking the isotope steady state and resulting in an overestimation of the enrichment in secretory tissues. This problem is less critical for the other amino acids, which are labeled from leucine in the body tissues. The enrichment of total nitrogen in the plasma TCA-soluble fraction followed a similar pattern to that of the free amino acids other than leucine, i.e., it reached a plateau when a protein diet was fed, in agreement with previous data (de Lange et al. 1990, Huisman et al. 1992, Schulze et al. 1995, Souffrant et al. 1993), and increased to a lesser degree than leucine when the protein-free diet was offered. Indeed, the enrichment of the ileal nitrogen was higher in the digesta than in the portal plasma TCA-soluble fraction when pigs were fed the protein-free diet (Table 2). Consequently, the nitrogen endogenous losses calculated according to the isotope dilution technique were higher than the amount of nitrogen in the ileal juice of protein-deprived pigs. This was probably due to an underestimation of the labeling of endogenous protein-bound nitrogen as a consequence of the combination of heterogeneous labeling and different amino acid profiles in plasma compared with secretory proteins. These experimental data validate the amino acid dilution method, on the basis of measurements of the endogenous protein obtained from a protein-free diet, suggesting that it should be preferred to the N-isotope dilution method, as previously anticipated by de Lange et al. (1990) and Lien et al. (1997).
). Microbial activity is low in the duodenum and jejunum but increases in the ileum (Bach Knudsen et al. 1991). These microflora provide the host with lysine up to a level representing 2.6 times the maintenance requirement (Torallardona et al. 1994). Some tissues have incorporated labeled lysine in their proteins. Thus, these bacterial amino acids have been digested and used for protein synthesis by these pigs. Phenylalanine may theoretically transaminate, but it is a minor metabolism pathway (Krempf et al. 1990). The fact that phenylalanine enrichment, like lysine, was higher in digesta than in tissues suggests that the bacterial de novo synthesis rate and incorporation into the host tissue protein were greater than the transamination rate in the host. Our results support the concept of a supply of essential amino acids by the microflora, although no indication of a positive balance in favor of the host was presented.
, Mariscal-Landin et al. 1995). To avoid this, some authors tried to correct the N status by total parenteral N nutrition in parallel with the distribution of a protein-free diet (de Lange et al. 1989, Leterme et al. 1996a). The correction of the N status did not modify the amino acid losses except for proline (de Lange et al. 1989, Leterme et al. 1996a), histidine and lysine (Leterme et al. 1996a). When synthetic amino acids were added to the protein-free diet, no effect of the supplementation was observed on the ileal nitrogen and lysine flows (Butts et al. 1993, Moughan et al. 1992). These experiments did not prove that the basal ileal endogenous losses were depressed by consumption of a protein-free diet. Alternatively, to resolve the drawback of the protein-free diet, Moughan and Rutherfurd (1990) fed pigs hydrolyzed protein, and the collection of digesta was followed by an ultrafiltration. Although some endogenous materials can also be removed by ultrafiltration, this method showed an increased recovery of endogenous nitrogen when a hydrolyzed casein was added to a protein-free diet (Butts et al. 1993). This was consistent with the present data in which highly digestible protein, hydrolyzed or not, led to higher endogenous losses. Now it is a question of determining which of these values may be considered to be an estimate of basal losses. It is well known that the simple presence of protein stimulates gastrointestinal secretions (Gitler 1964). Moreover, dietary protein may protect the endogenous components from the digestive process (Snook and Meyer 1964). Therefore, we may consider that protein, similar to other dietary factors (e.g., fibers or antinutritional factors), will influence the digestive process, and the measured losses could be termed specific rather than basal. Further experiments are required to establish whether protein-specific losses occur in proportion to the dietary protein supply.
1
Reported in part at the VIIth International Symposium on Digestive Physiology in Pigs, May 26-28, St Malo, France [Hess, V., Thibault, J. N. & Seve, B. (1997)
Apparent, true and real digestibility of intact or hydrolyzed isolated pea protein. In: Proceedings of the VIIth International Symposium on Digestive Physiology in Pigs (Laplace, J. P., Fevrier, C. & Barbeau, A., eds.), pp. 587-591. EAAP Publication 88].
FOOTNOTES
2
Supported by Ralston Purina Europe (J. Van Eys).
3
The costs of publication of this article were defrayed in part
by the payment of page charges. This article must therefore be hereby marked "advertisement"
in accordance with 18 USC section 1734 solely to indicate this fact.
4
To whom correspondence should be addressed.
5
Abbreviations used: AA, amino acid; BCAA, branched-chain amino acid; BW, body weight; DMI, dry matter intake; GC-C-IRMS, gas chromatograph coupled with a combustion oven and an isotope ratio mass spectrometer; HP, enzymatically hydrolyzed pea protein diet; PF, protein-free diet; PP, pea isolated protein diet; TCA, trichloroacetic acid.
ACKNOWLEDGMENTS
LITERATURE CITED
Abstract
Introduction
Methods
Results
Discussion
References
0022-3166/98 $3.00 ©1998 American Society for Nutritional Sciences
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