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Articles

The Influence of the Albumin Fraction on the Bioavailability and Postprandial Utilization of Pea Protein Given Selectively to Humans¹

François Mariotti², Maria E Pueyo, Daniel Tomé, Serge Bérot^*, Robert Benamouzig and Sylvain Mahé

UMR INRA-INAPG de Physiologie de la Nutrition et du Comportement Alimentaire, INAPG, 75231 Paris cedex 05, France; ^* Unité de Biochimie et de Technologie des Protéines, INRA, BP 71627, 44316 Nantes cedex 3, France; and Service d’Hépatho-gastroentérologie, Hôpital Avicenne, 93009 Bobigny, France

²To whom correspondence should be addressed. E-mail: mariotti{at}inapg.inra.fr.

	ABSTRACT

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Pulse seed proteins such as those found in peas (Pisum sativum) contain fractions of very dissimilar composition and properties, which may therefore be differently utilized by the human body. To analyze the nutritional value of the soluble protein fractions of pea seed, human volunteers ingested a mixed meal of 30 g of raw purified pea protein either as [¹⁵N]-globulins (G, n = 9) or as a mix of [¹⁵N]-globulins and [¹⁵N]-albumins (GA, n = 7) in their natural proportions (22:8). Dietary and endogenous nitrogen fluxes at the terminal ileum were assessed using a tube perfusion technique with an isotopic dilution method. Systemic dietary amino acid availability and the retention of dietary amino acids were determined using ¹⁵N enrichment in plasma amino acids and deamination products in blood and urine for 8 h postprandially. The results showed that the pea albumin fraction had the following effects: 1) significantly lowered the real ileal digestibility of pea protein (94 ± 2.5% for G vs. 89.9 ± 4% for GA), probably because of a direct effect of trypsin inhibitors; 2) did not promote acute intestinal losses of endogenous nitrogen; and 3) did not significantly improve the postprandial biological value of pea protein (76.5 ± 3.9% for G vs. 78.7 ± 3.6% for GA), despite the fact that it corrected the globulin deficiency in sulfur amino acids. We conclude that both G and GA are of good nutritional value for humans and show that cysteine-rich albumins have a far more modest effect on the efficiency of postprandial dietary protein utilization than would be expected from the amino acid scores.

KEY WORDS: • pea protein • protein utilization • globulin • albumin • humans

	INTRODUCTION

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Understanding the physiologic and nutritional roles of legume seed proteins in the human diet is of fundamental importance to improving their quality by selection, processing or biotechnology. Indeed, pulse protein, and in particular pea seed protein, is composed of very dissimilar fractions, namely, albumins and globulins, that differ fundamentally in terms of their biochemical features, composition and structure (1) . The importance of each of these components to the nutritional value of pea protein has been the subject of little study, despite the fact that it may provide an interesting framework for determining the nutritional value of proteins, and more particularly, legume proteins (2) . The globulin fraction, which represents the principal fraction in pea protein, constitutes a structural reserve of proteins that are probably highly digestible (3) but low in sulfur amino acids (SAA),³ whereas the albumin fraction is very rich in SAA but contains biologically active proteins, which include antinutritional factors, essentially protease inhibitors (4) . Thus, the albumins in pulse proteins may naturally complement the amino acid pattern of globulin, but may also be the fraction that limits the bioavailability of the protein source and promotes endogenous ileal losses of nitrogen (5 ,6) .

A major problem is the difficulty in ensuring a clear and accurate assessment of the nutritional value of dietary protein sources in humans (7 8 9 10) . It is usually accepted that the nutritional value of food protein sources depends primarily on the content and bioavailability of indispensable amino acids. In the FAO/WHO recommendations for dietary protein evaluation (11) , the Protein Digestibility-Corrected Amino Acid Score expresses the concentration of the limiting amino acid in a food protein as the percentage of the same amino acid in a reference amino acid pattern, weighted by fecal protein digestibility. Because the true physiologic situation is far more complex [e.g., (12 13 14 15 16 17) ], we chose to measure directly the ileal digestibility and retention of dietary protein nitrogen during the postprandial protein gain as an integral approach to discriminating between different protein sources in humans (18) .

The aim of this study was therefore to evaluate the relative importance of globulin and albumin fractions to the nutritional quality of pea protein in humans. For this purpose, we compared postprandial utilization of the ¹⁵N uniformly labeled pea globulin fraction alone with that of a mix of ¹⁵N uniformly labeled pea globulin and albumin fractions at natural proportions, and compared the results with amino acid scores. We studied the acute response to these protein sources in healthy humans with adequate protein and energy intakes because we are concerned with the nutritional importance of proteins as part of mixed adequate diets.

	SUBJECTS AND METHODS

TOP ABSTRACT INTRODUCTION SUBJECTS AND METHODS Calculations RESULTS DISCUSSION REFERENCES

 
Peas and extraction of protein fractions.

Pea plants (Pisum sativum cv Baccara) were sprayed with ¹⁵NH₃¹⁵NO₃ (13.7 and 13.7 atom %, Eurisotop, Saint-Aubin, France) 8 d before blossoming (URGAP, INRA, Dijon, France). From finely ground ¹⁵N-labeled pea seeds, the globulin and albumin fractions were extracted using selective solubilization and diafiltration, according to a method especially designed to suppress cross-contamination and to limit structural modifications to the extracted protein fractions (19) .

Meals.

Two experimental meals were prepared. The first meal contained 30 g (301 mmol N) of pea globulins (G meal). The second meal contained 22 g of pea globulins plus 8 g of pea albumins (299 mmol N) (GA meal). Both were added to 120 g carbohydrate (100 g maltodextrin and 20 g sucrose) and 15 g sunflower oil, and mixed with water to a final volume of 500 mL. Both meals also contained 15 g polyethylene glycol 4000 (PEG 4000), a nonabsorbable marker of the liquid phase of the meal, and 75 mg [¹³C]-glycine (L-[1-¹³C]-gly 99% enrichment, Euriso-Top, Gif-sur-Yvette, France) to evaluate the gastric emptying kinetics of the liquid phase of the meals (20) . The proportion of globulins and albumins in the GA meal was chosen to reflect the mean native proportion in soluble proteins of smooth peas (4) , which is 80% of total protein in pea seeds, and almost all of the extractable proteins. This mix could therefore be regarded as a pea protein isolate (unheated).

Trypsin inhibitors (TI) and amino acids in the pea proteins.

Trypsin inhibitor activity (TIA, as mg trypsin/g sample) was assessed using the method developed by Valdebouze et al. (21) . TI were concentrated in the albumin fraction (54.8 TIA/g protein). Thus, the G meal was virtually devoid of trypsin inhibitory activity (0.2 TIA/g protein), whereas the GA meal contained 14.8 TIA/g protein, i.e., higher than the low level of TIA usually found in Baccara seeds (7.4 TIA/g protein) and in other, similar smooth-seeded spring types. For purposes of comparison, raw soybean flours, toasted soybean flours and soy protein isolates usually include 35–123, 58 and 1.4–29 TIA/g protein, respectively (22) .

For the analysis of amino acids in pea proteins (except for tryptophan and SAA), protein samples were first hydrolyzed in vacuum tubes at 110°C for 24 h with 6 mol/L HCl. The hydrolysates were then analyzed by ion-exchange chromatography with postcolumn ninhydrin detection (AminoSystem 2500, Bio-Tek; Saint-Quentin-en-Yvelines, France). For the analysis of SAA, samples were oxidized with performic acid before hydrolysis in vacuum tubes at 110°C for 24 h with 6 mmol/L HCl and analysis by ion-exchange chromatography. For tryptophan analysis, samples were hydrolyzed in vacuum tubes at 110°C for 16 h with 5 mol/L NaOH before analysis by ion-exchange chromatography.

Subjects and clinical protocol.

The study was performed in 17 healthy volunteers. They were in good health, as determined by a thorough medical examination. Subjects of each sex were randomly allocated to two groups as follows: 9 (6 men, 3 women) were given the G meal and 8 (5 men, 3 women) were given the GA meal. The protocol had previously been approved by the Institutional Review Board for Saint-Germain-en-Laye Hospital (Saint-Germain-en-Laye, France). All subjects gave their full consent to participation in the study after the experimental protocol had been explained to them in detail. The data of one man assigned to the GA meal had to be excluded because of practical problems concerning ileal sampling. The subjects were admitted to the hospital in the morning on the day before the study. Total body water (TBW) was measured by bioimpedance analysis (Analycor 5w, Spengler; Cachan, France). An intestinal tube was passed through the nose and allowed to descend to the digestive tract, as described previously (23) . The intestinal tube was used to perfuse phenol red, a nonabsorbable intestinal marker, into the ileum and collect intestinal samples by continuous suction, 20 cm distally from the perfusion site. Subjects ate their dinner at 2000 h and then fasted overnight. On the morning of the study, after the positioning of the intestinal tube at the terminal ileum had been checked by X-ray, a catheter was inserted into a superficial forearm vein for blood sampling and another catheter into the controlateral forearm for saline infusion. The subjects then ingested the G or GA meal; the postprandial sampling period lasted for 8 h. The subjects were resting and were not allowed to ingest food or fluids during the study period, although 1 L of saline was infused from 2 to 8 h postprandially. Intestinal aspirates were collected over ice and pooled at 30-min intervals over an 8-h period, the first collection (before the meal) representing the initial period. Ileal effluents were freeze-dried and stored for future analysis. Blood samples were collected hourly during the 8-h period after ingestion of the meal, except for the first 2 h postprandial when additional samples were taken. Plasma was immediately separated from whole blood by centrifugation (1500 x g, 15 min, 4°C) and frozen at -20°C until analysis. Breath samples were collected every 30 min and stored for later determinations of ¹³C isotopic enrichment. Every 2 h (0–2, 2–4, 4–6 and 6–8 h) after meal ingestion, urine was collected under mineral oil (Paraffin) to avoid contact with air and prevent ammonia losses, treated with thymol crystals (15 mg/10 mL) as preservative and stored at +4°C for later analysis.

Analyses.

Urea and ammonia were isolated using a batch method, as previously described (24) . Briefly, for the extraction of amino acids and urea, plasma proteins were pelleted by the addition of solid 5-sulfosalicylic acid. After centrifugation (2400 x g, 25 min, 4°C), the supernatant was collected. Urinary ammonia was first extracted from the urine using the Na/K form of cation exchange resin (Dowex AG-50X8, Mesh 100–200, Bio-Rad; Marne la Coquette, France). The supernatant fraction was collected for the further extraction of urea. Urea was extracted from both the plasma supernatant fraction and the ammonia-free urine fraction by conversion into ammonium through hydrolysis with urease (Sigma; Saint-Quentin-Fallavier, France) for 2 h at 30°C on cation exchange resin. The part of the plasma fraction not retained in the resin was considered to be the plasma amino acid fraction. Ammonia and urea-derived ammonia were eluted from the resins by the addition of 2.5 mol/L KH₂SO₄. The total nitrogen content of samples was determined using an elemental nitrogen analyzer (NA 1500 series 2, Micromass; Manchester, UK) with atropine as the standard. Urea was assayed in both plasma and urine by an enzymatic method (urease/glutamate dehydrogenase) on a clinical analyzer (Dimension automate, Dupont de Nemours, Les Ulis, France). Ammonia was measured in urine using an enzymatic method on a clinical analyzer (Kone automate, Kone; Evry, France). Creatinine was measured using a direct colorimetric method, also on a clinical analyzer (Dimension automate, Dupont de Nemours). Glucose was measured in plasma using a glucose oxidase method (glucose GOD-DP kit, Kone). Insulin was measured in plasma using a RIA method (INSIK-5 Diasorin; Antony, France). The concentration of PEG-4000 in the digesta was measured using a turbidimetric method, and that of phenol red by colorimetry, as described previously (25) . Nitrogen contents of meals and ileal samples were determined by Combustion Nitrogen Analysis using an elementary analyzer (NA 1500 series 2, Micromass) with atropine as the standard. Isotopic N₂ enrichments (¹⁵N/¹⁴N) were determined by isotopic ratio mass spectrometry (IRMS). An aliquot was burned at 1020°C in an elementary analyzer (NA 1500 series 2, Micromass) interfaced with an isotope ratio mass spectrometer (Optima, Micromass). The ¹⁵N/¹⁴N ratios (m/z 28: m/z 29: m/z 30) were measured with reference to a calibrated ¹⁵N/¹⁴N nitrogen tank. Isotopic CO₂ enrichments (¹³CO₂/¹²CO₂) were determined by gas chromatography (GC) coupled with an IRMS. Samples were separated by GC (HP 5890 series II, Hewlett Packard; Les Ulis, France) on a 2.5 m x 3 mm Haysep Q column (Chrompak; Les Ulis, France) at 80°C and isotopic ratios (m/z 44: m/z 45: m/z 46) were determined by IRMS (Optima, Micromass) with reference to a calibrated ¹³CO₂-¹²CO₂ tank.

	Calculations

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Dietary and endogenous nitrogen flux at the terminal ileum—real ileal digestibility.

Assessments of phenol red dilutions between the infusion solution and samples collected using the ileal perfusion technique enabled the calculation of flow rates in the ileum (calculation of the average flux by 30 min) as previously described (26) . The fraction of exogenous and endogenous nitrogen in ileal samples was calculated from both total nitrogen and the isotopic ¹⁵N/¹⁴N ratio. Dietary nitrogen (N_diet-ileal mmol N) and endogenous nitrogen (N_endo-ileal mmol N) transiting through the terminal ileum were calculated using the following equations:

where N_tot-ileal(t) was the total nitrogen transiting through the terminal ileum at time t, E(t) is the ¹⁵N enrichment (expressed as atom %) in the ileal sample at time t, and E_meal is the ¹⁵N enrichment (expressed as atom %) in the meal.

The PEG flux for each time period was also assessed on the basis of calculated flow rates and the concentration of PEG in the samples. The total amount of PEG passed at the terminal ileum was then calculated to verify that the liquid phase had passed completely and determine the overall accuracy of flow rate estimates by comparison with the total amount ingested with the meal.

The real ileal digestibility (RID) of nitrogen was calculated using the following equation:

where N_ing is the nitrogen content of the meal and CN_diet-ileal is the cumulative amount of dietary nitrogen recovered at the terminal ileum 8 h after the meal (27) .

Systemic availability of dietary amino acids.

The proportions of plasma amino acids containing nitrogen of dietary origin (AA_diet/AA_tot) were calculated using the following equation:

where E(t) is the ¹⁵N enrichment (expressed as atom %) in the amino acid fraction sampled at time t and E_meal is the ¹⁵N enrichment (expressed as atom %) of the meal.

Dietary and endogenous nitrogen in the body urea pool.

Total body urea nitrogen (N_tot-urea) was calculated as the product of the plasma urea concentration and its volume of distribution, on the assumption that urea was distributed throughout the total body water:

where [Nurea] is the plasma urea nitrogen concentration (mmol N/L) and 0.92 is the corrective factor for the proportion of water in plasma.

Dietary and endogenous nitrogen incorporated into the body urea pool (N_diet-urea and N_endo-urea mmol N) were calculated using the following formulas:

where E_urea(t) is the ¹⁵N enrichment (expressed as atom %) in the plasma urea at time t.

Dietary and endogenous urinary nitrogen.

Dietary and endogenous nitrogen incorporated in urinary nitrogen (N_diet-urinary and N_endo-urinary mmol N) were calculated using the following formulas:

where N_tot-urinary(t) is the quantity of urinary nitrogen (in the form of either total, urea or ammonia nitrogen) at time t, and E_urin(t) is the ¹⁵N enrichment (expressed as atom %) in urinary nitrogen (in the form of either total, urea or ammonia nitrogen) at time t.

¹³C excretion.

The excretion and cumulative excretion of ¹³C in breath as a percentage of ¹³C ingested were calculated from the ¹³C enrichment in breath according to the technique developed by Maes et al. (20) . Briefly, to estimate the parameters related to ¹³C excretion, the amount of ¹³C recovered (as a percentage of dose) in breath as a function of time was fitted onto the curve of equation y = m(1 - e^-kt)^ß where t is the time in hours and m, k and ß are the regression-estimated constants. From these regression-estimated constants, two parameters characterizing the gastric emptying rate were obtained. The "recovery half-time": T_1/2 = (-1/k)ln(1 - 2^-1/ß), and the "excretory lag phase": T_lag = (lnß)/k, represent the time at which half of the asymptotic recovery of ¹³CO₂ is reached on the cumulative fitted curve and the time at the point of inflection of the cumulative fitted curve (i.e., time of maximum excretion), respectively.

Dietary and endogenous deamination.

The endogenous (and dietary) deamination fluxes were computed every 2 h for the mean periods 0–2, 2–4, 4–6 and 6–8 h by totaling the urinary endogenous (or dietary) nitrogen excreted and any variations to endogenous (or dietary) body urea nitrogen during the period. The amount of dietary nitrogen retained in the body 8 h after ingestion was calculated as the amount absorbed (i.e., RID x N ingested) minus the amount deaminated. The postprandial biological value (PBV) was then calculated as the amount of dietary nitrogen retained out of the amount absorbed, and net postprandial protein utilization (NPPU) was calculated as the amount of dietary nitrogen retained out of the amount ingested (18) .

Curve fitting and other curve estimates.

Different model curves were used during the postprandial period to fit the following experimental quantities: 1) dietary plasma amino acids, 2) dietary body urea nitrogen, 3) cumulative dietary nitrogen excreted in the urine and 4) cumulative dietary deamination. For 1) and 2), the curve took the form: y = · e^{-1/2(ln(t/t₀)/ß)2}, where t is the time, and , ß and t₀ are regression-estimated constants; for 3), the curve took the form: y = a(1 - e^-bt)^c, where t is time and a, b and c are regression-estimated constants; for 4), the curve was the sum of the curves fitted on dietary body urea nitrogen and on cumulative dietary urinary nitrogen. Curve fittings of experimental data were performed using the Sigma Plot 5.0 software (SPSS, Erkrath, Germany).

Statistical analyses.

Results were expressed as means ± SD. Single planned comparisons between meals (at 8 h) were performed using unpaired Student’s t tests (SAS/STAT Version 6.03, SAS Institute, Cary, NC). The overall effect of meal and time and the interaction (meal x time) were evaluated using a repeated-measures ANOVA (General Linear Models procedure, SAS/STAT Version 6.03, SAS Institute). For those measures for which there was a significant interaction, post-hoc testing of differences between meals at each time point was performed with unpaired t tests with the P-value adjusted by Bonferroni correction (SAS/STAT). Differences with P < 0.05 were considered significant.

	RESULTS

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Dietary and endogenous nitrogen passing the terminal ileum.

The total amount of PEG recovered at the terminal ileum was consistent with the amount ingested (+2.0 and +5.9% for G and GA, respectively). The amount of dietary nitrogen transiting at the terminal ileum is represented in Figure 1A . The amount of dietary nitrogen was significantly influenced by time and by the nature of the meal, but without any significant interaction. At 8 h after ingestion of the meals, the cumulative amounts of dietary nitrogen that had passed the terminal ileum were significantly different between subjects consuming the G and GA meals (18.1 and 30.1 mmol N, respectively). Thus, the real ileal digestibility of the globulin fraction was 94.0 ± 2.5%, significantly higher than the real ileal digestibility of mixed globulins and albumins, which reached 89.9 ± 4.0%. The endogenous nitrogen flux at the terminal ileum (Fig. 1B ) did not vary significantly with time or with the nature of the protein. The total amounts of endogenous nitrogen that passed the terminal ileum during the 8-h period after the meal were similar (58.4 ± 13.9 mmol N for G and 51.4 ± 15.2 mmol N for GA).

View larger version (37K): [in this window] [in a new window]   Figure 1. Dietary (A) and endogenous (B) nitrogen transiting at the terminal ileum after the ingestion of globulin (G, n = 9) or a mix of globulin and albumin (GA, n = 7) in humans. Values are means ± SD. Throughout the treatment period, there was a significant effect of meal (and time, without interaction with time) on dietary nitrogen (repeated-measures ANOVA, P < 0.05) and there was no effect of meal or time (or meal x time) on endogenous nitrogen.

The kinetics of the breath excretion of ¹³C originating from ¹³C-glycine did not differ significantly between groups consuming the two meals (Fig. 2A ). Excretion reached its maximum 4 h after ingestion of the meal (250 ± 55 vs. 257 ± 46 min after meals G and GA, respectively), and the times of half-asymptotic excretion (370 ± 83 vs. 373 ± 72 min after meals G and GA, respectively) also did not differ. These data show that the liquid phase of the meal was emptied from the stomach at a similar speed whether G or GA was ingested. Levels of dietary amino acids entering the systemic amino acid pool are shown in Figure 2B . There was no significant meal or meal by time effect on the level of dietary amino acids in the plasma amino acid pool. After both meals, plasma glucose concentrations (Fig. 3A ) returned to baseline at 3 h after the meal (Fig. 3B ). Plasma insulin concentrations (Fig. 3B ) returned to baseline 6 and 5 h after meals G and GA, respectively. The levels and kinetics of plasma glucose and insulin were not influenced by the nature of the protein ingested.

View larger version (22K): [in this window] [in a new window]   Figure 2. 13C excretion in the breath (A) and dietary amino acid in the total plasma amino acid pool (B) after the ingestion of globulin (G, n = 9) or a mix of globulin and albumin (GA, n = 7) in humans. Values are means ± SD. Throughout the treatment period, there was no significant meal effect or meal by time interaction (repeated-measures ANOVA, P > 0.05).

View larger version (21K): [in this window] [in a new window]   Figure 3. Plasma glucose (A) and insulin (B) after the ingestion of globulin (G, n = 9) or a mix of globulin and albumin (GA, n = 7) in humans. Values are means ± SD. Throughout the treatment period, there was no significant meal effect or meal by time interaction (repeated-measures ANOVA, P > 0.05).

Dietary nitrogen was rapidly incorporated into body urea (Fig. 4A ). Throughout the entire period, the amount of dietary urea nitrogen was significantly affected by the nature of the meal, but there was no meal by time interaction. In contrast, meal type had no effect on total and endogenous urea nitrogen (Fig. 4B ), but there was a significant meal by time interaction on endogenous urea nitrogen. The urinary excretion of dietary urea nitrogen after the G or GA meals (Fig. 4A ) did not differ (meal effect: P = 0.97; meal by time interaction: P = 0.98). Dietary ammonia, with a more rapid urinary recovery, was also excreted in a similar fashion by the two groups (data not shown). The excretion of endogenous urea (Fig. 4B ) and ammonia (data not shown) did not differ between the groups fed the G and GA meals.

View larger version (26K): [in this window] [in a new window]   Figure 4. Dietary (A) and endogenous (B) body urea nitrogen (BUN) and cumulative urinary nitrogen (cUN) after the ingestion of globulin (G, n = 9) or a mix of globulin and albumin (GA, n = 7) in humans. Values are means ± SD. Throughout the treatment period, there was a significant effect of meal (without interaction with time) on dietary BUN (repeated-measures ANOVA, P < 0.05) and there was a significant meal by time interaction on endogenous BUN (P < 0.05).

PBV, NPPU and amino acids score.

Amino acid analysis of the globulin and albumin fractions is presented in Table 1 , together with the composition of the GA meal (assessed from globulin and albumin composition), and the FAO/WHO scoring pattern (11) from which the amino acid scores were calculated. For all pea proteins, the nitrogen/protein conversion factor used was 5.44, as defined by Mossé (28) for total protein in pea seeds. The digestibility, PBV and NPPU of G and GA are shown in Table 2 . Despite the significant difference in deamination 8 h after the meal, the PBV of the meals did not differ significantly, i.e., when corrected for the amount of nitrogen absorbed, differences in dietary deamination were not significant (76.5 ± 3.9% for G and 78.7 ± 3.6% for GA). NPPU reached 72.0 ± 4.2% for G and 70.9 ± 6.0% for GA, values that did not differ.

	DISCUSSION

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Pea globulins exhibited a RID of 94%. This level is very high, given that the RID of milk and soy proteins are reported to be 95 and 91%, respectively (16 ,29) . This agrees with other studies in rats, which reported that the digestibility of globulins from various legume seeds is high (3) , and is consistent with the idea that globulins are sensitive to proteolysis (30 ,31) . When some of the globulins were replaced by albumins, the RID decreased significantly to 90%, falling within the range (89–95%) of values found previously for peas (26 ,32) . As a general rule, albumins are not supposed to be more resistant to proteolysis than globulins in vivo (31) , and the exposure times to gastrointestinal proteases were similar as shown by the similar total transit time of dietary nitrogen in subjects consuming both meals. Thus, the effect of albumin on reducing digestibility could be accounted for mainly by trypsin inhibitors (TI) because other antinutritional factors (lectins, tannins and saponins) are found at low levels in pea protein or have weak activities (33) . In addition, the difference in TI between the G and GA meals was lower than that seen in the near-isogenic pea lines used by Hedemann et al. (34) who showed that TI affected fecal digestibility in rats. For purposes of comparison, the level of TI in the GA meal was higher than that in spring-type pea flour, but within the range of activity usually found in soy protein isolates (22) . Although TI may reduce the digestibility of a meal because they may themselves be poorly digested, their major effect is to impair the activity of trypsin and chymotrypsin in the intestinal lumen, for which they have a high affinity. In this context, as our data showed that the digestion and absorption of dietary nitrogen from pea proteins occurred rapidly whichever fractions were ingested, they suggest that the in vivo TI impairment of hydrolysis in humans is immediate and irreversible. TI also selectively increase pancreatic secretions in humans (35 ,36) , independently of their protease inhibitory activity, because this increase occurs whether TI are given in free form or already complexed (37) . Moreover, the protease inhibitory effect may also reduce the hydrolysis and absorption of endogenous nitrogen secretions. These direct acute effects of TI are probably the main reason for the marked increase in endogenous nitrogen losses usually reported in animals (6) . Because the amounts of endogenous nitrogen at the terminal ileum were unchanged in the presence of albumins, we suggest that the increased secretion and/or reduced digestibility of endogenous nitrogen was insubstantial during these tests. However, despite the fact that such mechanisms have mainly a direct effect, we cannot rule out the possibility that part of the TI effect is delayed or related to long-term intake. This study, however, demonstrated that the acute ingestion of TI, in amounts usually found in pulse protein products intended for use in humans, only slightly limited the digestibility of dietary nitrogen but did not trigger any additional intestinal losses of endogenous nitrogen, which could jeopardize nitrogen balance homeostasis (6 ,38) .

The measurement of globulin deamination after the acute ingestion of a meal indicated that PBV and NPPU were 76.5 and 72%, respectively. By comparison, in a study performed under very similar conditions and in particular, using the same type of meal, we found PBV and NPPU of 80.1 and 73.3% for a soy protein isolate (Mariotti et al., unpublished data), i.e., slightly higher than for globulins in the present study. When albumins were included in the meal, we observed that the postprandial deamination of dietary nitrogen was significantly lower than with the globulin meal. This decrease compensated for the lower absorption and maintained the systemic availability of dietary amino acids but resulted in a nonsignificant (P = 0.27) increase in the postprandial biological value and a very similar overall level of net postprandial protein utilization. A further interesting outcome of the present study was that endogenous deamination did not differ in subjects consuming the two meals. Indeed, total mean endogenous deamination throughout the postprandial period decreased with albumin intake in a way that was similar to dietary deamination, but the effect was not significant (P = 0.18), probably because of a higher standard deviation. Dietary and endogenous deamination are therefore likely similarly affected by modifications to amino acid composition, when insulinemia and amino acid absorptive kinetics do not exert any differential effects (16) .

The levels of bioavailable, indispensable amino acids in a dietary protein are considered to be the main factor governing the nutritional value. On the basis of the FAO/WHO scoring pattern (11) , globulins with a marked deficiency in SAA, and also slightly deficient in tryptophan, have a low amino acid score of 0.44. In contrast, because of the high SAA and tryptophan content of albumins, the GA mixed meal exhibited only a small marginal deficiency in SAA (amino acid score: 0.94). Therefore, the efficiency of mixed pea proteins and soy protein isolate utilization in subjects with adequate nutritional status is in reasonable agreement with the amino acid scores (1.07 for soy protein isolate and 0.94 for pea proteins mix). Because the pea protein mix used in this experiment represented a naturally proportioned mix of extractable proteins in pea seeds (i.e., a pea protein isolate) and pea seed proteins are not usually found to be limited in their amino acid pattern (39 ,40) , these data indicate that the nutritional value of pea protein is close to that of soy protein. In contrast, our study also shows that albumins have a moderate effect on the efficiency of postprandial dietary nitrogen utilization compared with what could be anticipated from their corrective effect on the amino acid pattern of globulin. Three physiologic events may explain this result. First, TI are very rich in SAA and especially cysteine (41) and may be resistant to enzymatic hydrolysis particularly because they form irreversible complexes with trypsin and chymotrypsin. Because the latter are also rich in SAA, dietary and endogenous SAA (and especially cysteine) may be less available than other individual amino acids, thus reducing the dietary complementation effect. This suggestion is supported by data obtained in pigs, in which both the apparent and real ileal digestibility of cysteine in pea proteins were markedly inferior to total nitrogen digestibility (42 ,43) . Second, the SAA requirements in adults are 13–16 mg/(kg · d) i.e., 25–27 mg/g protein (7 ,44) , which is similar to the value used in the FAO/WHO scoring pattern (11) . However, SAA requirements are expressed as a combined requirement for methionine and cysteine because it is not known what proportion of the total requirement may be met by cysteine (45) . Because albumins correct the total SAA content of globulins mainly by increasing the cysteine content, the modest sparing effect of albumins on dietary and endogenous amino acid oxidation is in line with recent tracer studies, which did not evidence any whole-body sparing effect of dietary cystine on methionine oxidation (46 ,47) . Third, dietary SAA are used to synthesize nonspecific proteins but are also specifically targeted to the synthesis of sulfur metabolites such as glutathione (48) . The latter is a reservoir for cysteine (49) , and its synthesis has been shown to respond rapidly to SAA availability (50 ,51) particularly during the postprandial phase after acute feeding (52) , whereas the glutathione efflux from the liver is linked mainly to the hepatic glutathione pool (50) . These data suggest that glutathione may buffer a reduction in the SAA pool and therefore sustain postprandial protein deposition when meals low in SAA are given selectively to humans with nondepleted glutathione stores. Such potential acute accommodations may not be maintained during repeated feeding with protein fractions that are low in SAA. That different nutritional situation requires further specific studies.

We conclude from the present data that pea albumins lower the bioavailability of pea proteins, likely due to a direct effect of TI, but do not promote acute intestinal losses of endogenous nitrogen in humans. The albumin fraction only slightly improves the postprandial biological value of pea protein, failing to enhance overall net postprandial retention. The modest effect of cysteine-rich albumins on postprandial biological value, compared with the large improvement in the amino acid score, may be due to poor dietary and endogenous availability of cysteine and to a moderate sparing effect of cysteine on methionine oxidation, but also to short-term accommodation. However, when given selectively to healthy humans, pea proteins exhibit a good nutritional value, similar to that of soy protein.

	ACKNOWLEDGMENTS

 
The authors thank S. Daré and C. Luengo for their technical help and the Gastroenterology Unit and the Biochemistry Laboratory at the Avicenne Hospital for their work during the clinical procedures. We gratefully acknowledge the contribution of G. Duc and P. Marget (INRA, Unité de Recherche en Génétique et d’Amélioration des Plantes, Dijon, France) for growing ¹⁵N-labeled pea seeds

	FOOTNOTES

 
¹ Supported by a joint research grant from CANA (Ancenis, France), CETIOM (Thiverval-Grignon, France) and ONIDOL (Paris, France). CANA and the French Department of Research jointly support F.M.

³ Abbreviations used: G, pea globulin meal; GA, pea globulin and albumin mixed meal; GC, gas chromatography; IRMS, isotopic ratio mass spectrometry; NPPU, net postprandial protein utilization; PBV, postprandial biological value; PEG, polyethylene glycol 4000; RID, real ileal digestibility; SAA, sulfur amino acids; TBW, total body water; TI, trypsin inhibitors; TIA, trypsin inhibitor activity.

Manuscript received December 7, 2000. Initial review completed January 20, 2001. Revision accepted March 2, 2001.

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