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Institut National de la Recherche Agronomique, Unité de Nutrition Humaine et de Physiologie Intestinale, Faculté des Sciences Pharmaceutiques et Biologiques, 75270 Paris Cédex 06, France, and * Service de Gastro-entérologie and Service de Biochimie, Hôpital Avicenne, 93000 Bobigny, France
ABSTRACT
INTRODUCTION
SUBJECTS AND METHODS
RESULTS
DISCUSSION
ACKNOWLEDGMENTS
FOOTNOTES
LITERATURE CITED
ABSTRACT 
The aim of the present study was to evaluate postprandial absorption of pea protein as well as exogenous nitrogen retention in humans. For this purpose, after fasting overnight, seven healthy adults (4 males and 3 females) ingested [15N]-labeled pea protein (195 mmol N). Ileal effluents were collected for 8 h at 30-min intervals using a nasointestinal intubation technique. Urine and plasma samples were collected for 24 h. The [15N]-enrichment was determined in the intestinal samples, in the plasma amino acids and urea as well as in the urinary urea and ammonia fractions. The true gastroileal absorption of pea protein was 89.4 ± 1.1%. This absorption was correlated with a significant increase (P < 0.05) in [15N]-enrichment in the plasma amino acids and in the nitrogen incorporated into the body urea pool for 1 h following pea ingestion. The enrichment remained significantly higher than the basal values in these pools 24 h after pea ingestion. The recovery of total urinary exogenous nitrogen after 22 h was 31.1 ± 9.3 mmol N. Moreover, the kinetics of [15N]-labeled pea amino acids deamination reached a plateau of 39 mmol. Under these conditions, pea nitrogen retention represented 78% of the absorbed dietary nitrogen in healthy humans. The present results demonstrate the good true nitrogen digestibility and retention of pea protein in humans.
KEY WORDS: vegetable protein · stable isotope · urea · nitrogen retention · humansINTRODUCTION
The nutritional value of dietary proteins depends both on their digestibility and on the metabolism of the absorbed amino acids, which are either used for body protein synthesis or degraded to supply energy. According to current studies, these processes are highly adaptive, and the amount of oxidative losses in amino acids, as well as the rate of protein synthesis and breakdown, depends on the level and profile of nitrogen and indispensable amino acid fluxes (Waterlow 1996
, Young and Marchini 1990).
Legume proteins, such as soy and pea from either grain seeds or their protein isolates, have been suspected of having reduced nutritional efficiency in comparison with other more balanced protein sources (Friedman 1996, Young and Pellet 1994). Different reports have suggested that vegetable proteins in animals have an impaired digestibility because of the presence of both protein fractions with low digestibility and antinutritional factors (such as trypsin inhibitors and lectins) that affect overall digestion processes (Friedman 1996, Thompson 1993). An inefficient utilization of these vegetable proteins has also been reported in animals when using either heat-treated or purified protein products with reduced antinutritional activities. This inefficient utilization results not only from the impaired digestibility but also from the harmful systemic effects of the absorbed amino acids that lead to a higher nitrogen excretion in vegetable-protein-fed animals compared with that of animals fed proteins from other sources (Grant et al. 1995, Rubio et al. 1995, Van Berneveld et al. 1995). Despite these observations, soy and pea proteins have been shown to have good digestibility in humans (Baglieri et al. 1994, Gausserès et al. 1995). In addition, soy proteins have been found capable of supporting nitrogen equilibrium in humans receiving sufficient amounts of this protein as the sole source of dietary nitrogen (Young 1991, Young and Pellet 1994). Little data are available on the kinetics and metabolic fate of dietary nitrogen in humans fed vegetable proteins.
The aim of this study was to determine the gastroileal exogenous protein absorption and dietary nitrogen distribution in humans who were administered heat-treated [15N]-labeled pea flour orally. The use of a double intestinal lumen tube and [15N]-labeled pea protein allowed the precise determination of the amount of exogenous protein that is not absorbed at the ileal level. The measurement of [15N] incorporation in different nitrogen pools into the plasma and urine was used to determine the metabolic behavior of pea dietary nitrogen.
SUBJECTS AND METHODS
). They had dinner at 2000 h and then fasted overnight. On the morning of the study the position of the intestinal tube at the ileum was checked under radioscopic control. Subjects were given the test meal (195 mmol N), each subject serving as his own control, and the intestinal sampling period lasted for 8 h. Starting before meal ingestion and continuing throughout the test period, a saline solution (130 mmol/L NaCl, 30 mmol/L mannitol, 5 mmol/L KCl) containing phenolsulfonphthalein (PSP, 400 mg/L) was perfused into the intestine at the rate of 1 mL/min to calculate intestinal flow rate (Modigliani et al. 1973). Intestinal samples were collected over ice and pooled at 30-min intervals. The 30 min before meal ingestion comprised the initial period (basal). Subjects were not allowed to ingest food or fluids during the remainder of the intestinal collection period. A small catheter (Jelco, Johnson-Johnson, Chatenay-Malabry, France) was placed in a forearm vein for blood sampling. Blood samples were collected on heparin every half hour during the first 2 h and every hour during the following 6 h. A final blood sample was collected 24 h after the first one. The plasma was immediately separated from whole blood by centrifugation at 2500 × g for 20 min at 4°C and frozen at 
20°C until analysis. Urine was collected for 24 h (every 2 h during the first 8 h), treated with a thymol cristal and parafine as preservatives and stored at 4°C until later analysis.
Extraction of amino acid, urea and ammonia in plasma and urine.
Urea and ammonia were isolated by the method described by Preston and McMillan (1988) using a Na/K form of the cation exchange resin (BioRad Dowex AG-50X8, mesh 100-200, Interchim, Montluçon, France) in batch. The Na/K form of the resin was prepared as follows: 100 g of resin in the H+ form was stirred for 15 min with three 600-mL batches of 0.1 mol/L NaOH. The resin was then washed to neutrality with distilled water. The moist resin was stirred for 15 min with each of the three 600-mL batches of 0.2 mol/L sodium potassium phosphate buffer, pH 7.4. After a second washing to neutrality, the resulting suspension was stored at 4°C. For amino acid and urea extraction, 2 mL of plasma were added to 100 mg of solid 5-sulfo-salicylic acid (Prolabo, Paris, France). After mixing and then standing for 1 h at 4°C, the protein was pelleted at the bottom of the tube by centrifugation at 2400 × g for 25 min at 4°C. The supernatant was collected. From the urine, ammonia was first extracted using the prepared Na/K form of the cation exchange resin in batch. Excess fluid was collected for further urea extraction. The resin was washed three times with distilled water and stored at 4°C. The urea in an aliquot of both plasma extract and ammonia-free urine extract was converted into ammonium by hydrolysis with urease (Sigma, Saint-Quentin-Fallavier, France) for 2 h at 30°C and extracted using the cation exchange resin. The fraction not retained in the resin from the plasma extract was considered to be the plasma amino acid fraction. The resin was washed three times with distilled water and stored at 4°C. Before isotopic determination, ammonia and urea-derived ammonia were eluted from the washed resins by treatment with 2.5 mmol/L KHSO4 .
Analytical methods.
The digesta samples were treated with the protease inhibitor 0.1 mmol/L diisopropylfluorophosphate (Sigma), then frozen at 20°C and freeze-dried. The PEG-4000 and the PSP were measured by a turbidimetric method (Hyden 1955) and a spectrophotometric method (Schedl 1966), respectively. Total nitrogen content was determined by an elemental nitrogen analyzer (NA 1500 series 2, Fisons Instruments, Manchester, UK) with atropina (Carlo Erba Instruments, Fisons, Arceuil, France) as the standard. Urea was measured in both plasma and urine by an enzymatic method on a Dimension automate (Dupont de Nemours, Les Ulis, France). Ammonia was measured in the urine by an enzymatic method on a Kone automate (Kone, Evry, France). The isotopic ratio of 15N/14N was determined by isotope ratio mass spectrometry (IRMS). An aliquot (freeze-dried ileal samples, liquid plasma or urine samples) was burned in an elementary analyzer (NA 1500 series 2, Fisons Instruments) at 1020°C coupled with an isotope ratio mass spectrometer (Optima, Fisons Instruments). The isotope ratio of N2 was measured in reference to a calibrated 15N/14N nitrogen tank.
Calculations and statistical analysis.
The exogenous nitrogen (Nexo mmol N) in the digesta was calculated from the total nitrogen (Ntot mmol N) and the isotopic ratio 15N/14N determined in both the digesta (Edig atom %) and the [15N]-pea (Epea atom %) according to the formula Nexo = Ntot × Edig/Epea . Exogenous nitrogen incorporated into the urea pool of the body was calculated according to the formula Nexo-urea = Nurea × Eurea/Epea where Nurea (mmol N) is the urea pool of the body and Eurea (atom %) the isotopic ratio 15N/14N in the plasma urea. The urea pool of the body (Ntot-urea) was calculated as the sum of the plasma urea concentration and its volume of distribution with the assumption that urea was distributed throughout the total body water, which was estimated using the equation of Watson et al. (1980). Exogenous nitrogen incorporated into the urinary nitrogen was calculated according to the formula Uexo = U × Ei/Epea where U (mmol N) is the quantity of urinary nitrogen (in the form of either total, urea or ammonia nitrogen) and Ei (atom %) the isotopic ratio 15N/14N in urinary nitrogen (in the form of either total, urea or ammonia nitrogen).
RESULTS
a)]} + d, in which t is time, and a, b, c and d are parameters calculated from the model; a represents the inflexion point, b + d represents the value of the plateau and c is related to the intestinal transit of the meal. For total nitrogen a = 2.2, b = 75.2, c = 0.6 and d = 16.6 and for exogenous nitrogen a = 2.6, b = 22.9, c = 0.9 and d = 2.9. Each value represents the mean ± SD of 7 subjects.
Plasma [15N]-pea nitrogen distribution. The absorption of [15N]-labeled dietary nitrogen was correlated with a significant increase (P < 0.05) in the [15N]-enrichment of the amino acid nitrogen pool in the plasma starting 1 h after pea ingestion (Fig. 2). The enrichment peaked 3 h after pea ingestion and then progressively decreased but remained significantly higher than the basal value 24 h after pea ingestion. In the same way, the exogenous nitrogen incorporated into the urea nitrogen pool of the body significantly (P < 0.05) increased starting 1 h after pea ingestion and peaked at 19.8 ± 7.4 mmol 4 h after pea ingestion (Fig. 3). A significant fraction (8.5 ± 4.2 mmol exogenous nitrogen) was still present in the body urea nitrogen pool after 24 h. This appearance was accompanied by a significant increase in the body total urea nitrogen pool 1.5-2 h after meal ingestion.
Urinary [15N]-pea nitrogen excretion. The exogenous nitrogen was detected in total, urea and ammonia urinary nitrogen pools after pea ingestion (Fig. 4). A significant fraction (P < 0.05) of exogenous nitrogen appeared in the urine 60 min after the meal ingestion and increased progressively during several hours. The cumulative quantities of exogenous nitrogen excreted in the urine in the form of total nitrogen, urea and ammonia, fitted according to a model curve y = b/{1 + exp[c(t
a)]} + d, reached a plateau value (mmol N) at 37.35 for total, 31.86 for urea and 0.41 for ammonia exogenous nitrogen. The experimental recovery of total, urea and ammonia exogenous nitrogen after 22 h was 31.1 ± 9.3, 26.9 ± 9.9 and 0.4 ± 0.1 mmol N, respectively. Both total and urea exogenous nitrogen excretions did not reach a plateau after 22 h, in contrast to ammonia exogenous nitrogen. Urea exogenous nitrogen represented 84.5 ± 8.6% of the exogenous nitrogen recovered in the urine, whereas ammonia exogenous nitrogen represented only 1.3 ± 0.3%.
a)]} + d in which t is time, and a, b, c and d are parameters calculated from the model; a represents the inflexion point, b + d represents the value of the plateau and c is related to the urinary exogenous nitrogen excretion. For total nitrogen a = 4.9, b = 58.8, c = 0.1 and d = 21.4; for urea a = 5.8, b = 47.6, c = 0.1 and d = 15.7; and for ammonia, a = 4.05, b = 0.56, c = 0.27 and d = 0.15. Each value represents the mean ± SD of 7 subjects. Mean exogenous values were all significantly different than the basal (0 h) value (P < 0.05, Tukey's studentized range test).
[15N]-Pea nitrogen retention. The kinetics of [15N]-labeled pea protein deamination was evaluated from the sum of the exogenous nitrogen present in both the urea nitrogen pool of the body and the total exogenous nitrogen pool in the urine (Fig. 5). The quantity of exogenous nitrogen arising from [15N]-labeled pea protein deamination reached a plateau of ~39 mmol N starting 14 h after pea ingestion. Under these conditions, 20% of the exogenous pea nitrogen ingested entered the nitrogen pool arising from amino acid deamination.
exp(bt)] in which t is time, and a and b are parameters calculated from the model; a = 38.9 represents the value of the plateau; b = 0.2 is related to protein deamination. Each value represents the mean of 7 subjects.
DISCUSSION
, Rubio et al. 1995). This poor nitrogen retention was thought to be partly the result of high nitrogen excretion in the urine. High urea excretion is a well-known effect associated with unbalanced protein consumption (Eggum 1970). However, the results of the present study indicate that pea protein given in a single dose to human volunteers presents a satisfactory level of both ileal digestibility and body nitrogen retention.
). It is now well established that dietary nitrogen digestibility should be determined at the ileum rather than throughout the entire digestive tract because the undigested proteins and unabsorbed peptides and amino acids that enter the colon are subjected to metabolism by the microflora and are not of substantial nutritional value (Rowan et al. 1994, Van Barnefeld et al. 1994). As a result of [15N] recycling and subsequent reappearance in the intestine, exogenous nitrogen absorption may be underestimated. [14C]-Labeled amino acids have been shown not to reappear in the intestinal lumen of pigs via pancreatic secreta until 3-4 h after oral ingestion (Simon et al. 1983). De Lange et al. (1990) stated that dietary amino acids were not incorporated into pancreatic enzymes within 6 h postprandially. In humans, we showed that [15N]leucine, when directly and continuously infused into the plasma, did not significantly appear in digestive proteins for 2-3 h and reached a plateau after 10 h (Gaudichon et al. 1994). In return, nitrogen can be directly recycled in the enterocyte and in the digestive secretions without entering into the venous circulation (Leterme et al. 1996); little is known about this way of recycling, and it should be of interest to quantify the consequent perturbation. Although there is [15N] recycling and subsequent reappearance in the intestine, it could thus be assumed that the ileal digesta retained for digestibility determination (8 h postprandially) contained substantial amounts of [15N]-contaminated endogenous nitrogen (Leterme et al. 1996). All of these results suggest that in an 8-h study period taking into account the time the amino acids needed to appear in the plasma and to be incorporated into synthetized intestinal proteins, the perturbation should be very slight. Moreover, in miniature pigs, the overestimation of the total exogenous nitrogen from [15N] dilution was <5% after 6 h compared with reference values obtained with guanidinated casein (Roos et al. 1994). Our results indicate a true ileal digestibility of 90% for pea protein nitrogen. This high digestibility of pea protein is in agreement with previous studies on pea digestibility abstracted from balance studies in humans (FAO/WHO 1990) and in rats (Savage and Deo 1989) as well as from studies with [15N]-labeled protein in pigs (Huisman et al. 1992).
). The essential branched-chain amino acids are not metabolized in the liver but are catabolized mainly in peripheral tissues. Under these conditions, the main part of the peripheral plasma [15N] pool is probably made up of the exogenous nitrogen in the branched-chain amino acid nitrogen pool. Nevertheless, both the deamination process of amino acids and peripheral blood delivery of noncatabolized amino acids by the liver are fast, and the plasmatic [15N]-enrichment kinetics could be assumed to reflect the dietary amino acid absorption kinetics. This [15N]-enrichment in the plasma amino acid nitrogen pool was detected in the first 30 min after meal ingestion. The rapid increase of [15N]-enrichment in this pool mirrors the increase in blood concentration of amino nitrogen described in pigs after meal ingestion (Galibois et al. 1990, Rérat et al. 1988). In the present study, the maximum [15N]-enrichment of the plasma amino acid nitrogen pool was reached 4 h after meal ingestion because gastric emptying delayed the appearance of [15N] in the intestine. Taking into account both the ileal recovery of exogenous nitrogen and the kinetics of [15N] plasma amino acid nitrogen pool enrichment, it appeared that, under the present experimental conditions, the absorption of pea protein in the small intestine in humans took place in the 1- to 4-h period following ingestion and was completed after 4 h.
). The gastrointestinal tract contributes similarly to the urea pool with the delivery of ammonia to the liver via portal venous circulation. Ammonia is produced by the gastrointestinal mucosa from the hydrolysis of glutamine used in the energy metabolism of the intestinal cells (Huizenga et al. 1996). At the distal level, some products of the microbial metabolism may be either absorbed in the colon or excreted in the feces (Rowan et al. 1994). The major product of this distal metabolism is thought to be ammonia, which is either absorbed and introduced further into the urea cycle in the liver or directly incorporated into the bacterial metabolism in the colon (Jackson 1995, Patterson et al. 1995). Jackson (1995) reported a huge flux (3600 mg N/d) of systemic urea through the colon. In our study, the 280 mg of exogenous nitrogen that passed through the ileocecal valve accounted for <10% of this flux. Thus, this exogenous nitrogen contribution to the formation of urea in the liver was probably low, particularly over a short period after labeled nitrogen ingestion. Under these conditions, we can neglect this fraction and assume that the deamination-derived exogenous nitrogen originates almost entirely from the deamination of the absorbed dietary amino acid nitrogen fraction.
either alone or with the concomitant determination of the amount of exogenous nitrogen in the urea pool of the body. Both methods led to the same amount of exogenous nitrogen excretion (i.e., 37.3 and 38.9 mmol N, respectively). However, the former method required at least a 24-h urine collection and, in this case, the model curve indicated that the plateau value was reached after 32 h. On the other hand, in the latter method, the plateau value was reached after only 14 h of urine collection but required a blood sample as well. In these conditions, the pea nitrogen retention represented at least 78% of the absorbed dietary nitrogen in healthy humans. These values represented minimum values for pea nitrogen, given the fact that the distal contribution to metabolic ammonia and urea production both reduces the part of deamination of the absorbed dietary amino acids and concomitantly increases the value of exogenous nitrogen retention. For comparison, apparent biological values reported in the literature for growing animals were 69% for raw and 72% for heated pea protein in pigs (Van Barneveld et al. 1995), 48-64% for different pea seeds in rats (Savage and Deo 1989) and 68 and 63% for faba bean globulins and lupin globulins in rats, respectively (Rubio et al. 1995). The present results indicate a higher retention of pea nitrogen in humans than in rats and pigs and confirm that growing animals are a poor model for dietary protein quality evaluation in adult humans (Millward and Pacy 1995, Young 1991, Young and Pellet 1991). Millward et al. (1989) reported biological values of different protein sources in humans measured by the nitrogen balance method. These biological values were close to 50% but corresponded to a diurnal cycling of the nitrogen metabolism, i.e., including the postprandial and the postabsorptive periods. In the present study our value corresponds only to the postprandial period in which the protein retention is maximal.
ACKNOWLEDGMENTS
FOOTNOTES
Manuscript received 5 July 1996. Initial reviews completed 30 October 1996. Revision accepted 5 February 1997.
LITERATURE CITED
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