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© 2008 American Society for Nutrition J. Nutr. 138:556-561, March 2008

Nutrient Requirements and Optimal Nutrition

A Casein Hydrolysate Does Not Enhance Gut Endogenous Protein Flows Compared with Intact Casein When Fed to Growing Rats1,2

Amélie Deglaire3,4, Paul J. Moughan3,*, Cécile Bos4, Klaus Petzke5, Shane M. Rutherfurd3 and Daniel Tomé4

3 Riddet Centre, Massey University, Palmerston North, New Zealand 4474; 4 INRA, AgroParisTech, UMR914 Nutrition Physiology and Ingestive Behavior, CRNH-IdF, F-75005 Paris, France; and 5 German Institute of Human Nutrition Potsdam-Rehbruecke, 14558 Nuthetal, Germany

* To whom correspondence should be addressed. E-mail: p.j.moughan{at}massey.ac.nz.


   ABSTRACT
TOP
 ABSTRACT
Introduction
 Materials and Methods
 Results
 Discussion
 LITERATURE CITED
 
We studied the effect of dietary free peptides vs. peptides released naturally during digestion on gut endogenous nitrogen (N) flow (ENFL) and amino acid (AA) flow (EAAFL). Semisynthetic diets containing 110 g/kg diet of the same casein, intact (C) or hydrolyzed (HC), were formulated with TiO2 as a dietary marker. Sprague-Dawley rats were fed the diets hourly (0800–1500 h) for 10 min each hour for 7 d. Rats received unlabeled diets for 6 d and 15N-labeled diets on d 7, whereby they were killed and digesta sampled (6 observations per group) along the intestinal tract. EAAFL and ENFL were determined by 15N-isotope dilution (ID) for C or by ID or after centrifugation and ultrafiltration (UF) for HC. Ileal EAAFL and ENFL (ID) were not enhanced with diet HC compared with diet C. The AA compositions (g/16 g N) of ileal ENFL did not differ between rats fed HC and C except for Asp, Phe, Tyr, and Ser, for which contributions were relatively lower (P < 0.05) for rats fed C. Ileal EAAFL and ENFL (HC) were considerably lower (P < 0.05) with ID than with UF, but flows of Gly, Phe, and His were similar. There was no stimulatory effect of dietary peptides from HC on endogenous ileal protein flow compared with C, but the result is tentative given the high degree of dietary N recycled within endogenous protein and which could have occurred at a differential rate between rats fed diets C and HC.


   Introduction
TOP
 ABSTRACT
 Introduction
Materials and Methods
 Results
 Discussion
 LITERATURE CITED
 
The amount of ileal digesta protein of nondietary origin, commonly referred to as endogenous protein, is a fundamental measure in the study of protein digestion. Estimates of ileal endogenous amino acids (AA)6 are required to determine true coefficients of ileal AA digestibility (1,2) and the loss of endogenous AA from the small bowel is an important component of the daily AA requirement (3,4).

Although protein-free diets have traditionally been used to determine endogenous protein in digesta (5), such an approach has been criticized as leading to a physiologically abnormal metabolism (6), resulting in lower ileal endogenous protein losses compared with protein-containing diets (4,7–9). The hydrolyzed protein ultrafiltration (UF) technique (10) was developed as an alternative approach, whereby the gut is directly supplied with AA and peptides, assumed to be similar to those arising during digestion in the gut, to allow the determination of endogenous AA flows (EAAFL). After feeding enzyme-hydrolyzed protein [usually casein, molecular weight (MW) <5 kDa] to animals or human subjects, ileal digesta are centrifuged and ultrafiltered (10 kDa MW cut-off). Proteins and peptides larger than 10 kDa in size are assumed to be of endogenous origin and any undigested small peptides and unabsorbed AA are discarded in the ultrafiltrate. This method allows a direct simultaneous determination of endogenous flows for all AA and nitrogen (N) and is of practical interest for the development of routine digestion assays. Questions have been raised, however, as to whether potentially bioactive peptides present in the hydrolysate may enhance the loss of AA from the small bowel (8,11–14) over and above that which may be found with the corresponding intact protein.

To our knowledge, no carefully controlled study has been conducted to date to specifically compare gut endogenous protein flow for a hydrolyzed dietary protein (various sized oligopeptides) with its parent intact protein, whereby peptides are released naturally during digestion. The primary aim of this work was to assess whether hydrolyzed protein enhances gut endogenous protein flows compared with the parent intact protein. Semisynthetic diets based on casein in either the intact or hydrolyzed form were fed to growing rats. Both forms of casein originated from the same source of 15N-labeled milk so that endogenous protein flows could be determined and compared for both diets using the isotope dilution (ID) technique. Although the 15N-labeling of diets may lead to an underestimation of gut endogenous N loss, the method is considered a valid technique for making relative comparisons (3,15,16). Additionally, endogenous ileal protein flows were determined using the enzyme-hydrolyzed protein UF technique to enable a comparison with the ID method.


   Materials and Methods
TOP
 ABSTRACT
 Introduction
 Materials and Methods
Results
 Discussion
 LITERATURE CITED
 
This study was conducted as part of a larger study (7).

    Rats and housing. Twenty-four Sprague-Dawley male rats (219 ± 14 g body weight) were housed individually in raised stainless steel cages with wire mesh floors in a room maintained at 21 ± 2°C and with a 12-h-light/-dark cycle. Rats were fed during the light cycle and water was continuously available. Ethics approval was received from the Massey University Animal Ethics Committee (protocol 05/04).

    Diets. Two semisynthetic isonitrogenous (17.7 g N/kg dry matter) test diets (Table 1) were prepared containing as a sole source of N either uniformly 15N-labeled native phosphocaseinate, referred to as intact casein (diet C) or a 15N-labeled casein-derived (derived from the former native phosphocaseinate) hydrolysate (diet HC). Adaptation diets had the same composition as the test diets except that the proteins, obtained from New Zealand Pharmaceuticals, were unlabeled. Sodium bicarbonate was added to diet C as previously described (7) to equalize the dietary electrolyte balances. TiO2 was included in the diets as an indigestible marker. A preliminary diet was also prepared (Table 1). Diets were formulated to meet the nutrient requirements of the growing rat (17).


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  		TABLE 1 Ingredient compositions of the diets given to growing rats

 
15N-labeling of milk and dietary protein purification were performed as described previously (18). Briefly, 3 lactating cows were perfused ruminally with (15NH4)S04 (Eurisotop) for 5 d. 15N-labeled milk was collected 5 times over 3 d and then pooled. 15N-labeled native phosphocaseinate was extracted by microfiltration and then purified through water diafiltration. All processes were carried out at the National Institute of Agronomic Research (Rennes, France). The resulting casein was freeze-dried. Its global 15N isotopic enrichment was 0.5450 atom %, and the 15N enrichments for individual AA are shown in Table 2. An aliquot of 15N-casein was hydrolyzed with pig pancreatin as described earlier (7). The MW profile, determined by HPLC gel filtration (7), indicated that 21% of the peptides were between 1 and 5 kDa in size and 79% were <1 kDa. In comparison, the MW profile of the commercial hydrolyzed casein used in the adaptation diet, which was similar to that previously used in our work (4,9,12), showed that 12% of the peptides were between 1 and 5 kDa and 88% were <1 kDa.


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  		TABLE 2 AA composition of casein and 15N-enrichment of individual AA

 
    Experimental design. On each day of the study, the rats received 8 meals at hourly intervals commencing at 0800 h, each meal lasting 10 min. Food intake was recorded daily. The rats were acclimatized to the cages and to the feeding regimen from d 0 to 9 while receiving the preliminary diet. They were then randomly and equally allocated to the adaptation (unlabeled) diets from d 10 to 16. On d 17, they received the test (labeled) diets and were killed as described previously (7). The entire digestive tract was dissected and digesta from the stomach, proximal intestine, medial intestine, the last 20 cm of the ileum, and cecum/colon were collected as described previously (7). Digesta were freeze-dried, ground, and pooled across randomly selected pairs of rats within the same diet for each intestinal section such that there were 6 observations per treatment. Pooled digesta were analyzed for TiO2, total N, and 15N-enrichment. Ileal digesta samples were analyzed for AA content and 15N-enrichment of AA. Ileal digesta from rats fed diet HC were divided in 2 portions and analyzed "as is" or after centrifugation and ultrafiltration with a 10-kDa MW cutoff (UF digesta) as previously detailed (7). The UF digesta were analyzed for TiO2, total N, AA, and total 15N-enrichment (7).

    Chemical analysis. TiO2 was determined by a colorimetric assay after ashing the sample and digestion of the minerals (19). AA were determined after acid hydrolysis (6 mol/L HCl) of the digesta samples and were separated and quantified using a Waters ion exchange HPLC system (20). Cys, Met, and Trp, destroyed during acid hydrolysis, were not determined. The 15N-enrichment and total N were determined on an isotopic ratio mass spectrometer (Optima, Fisons Instruments) coupled to an elemental N analyzer (NA 1500 series 2, Fisons Instruments) as described previously (21).

The 15N-enrichments of individual AA were determined by GC combustion isotope ratio MS (Finnigan Delta S; Thermo Fisher Scientific) as described previously (22–24). Briefly, digesta samples were hydrolyzed in 2 mL of 6 mol/L HCl (24 h, 110°C), dried under N at 60°C, and dissolved in 0.1 mol/L HCl. AA were derivatized to their N-pivaloyl-i-propyl AA esters (23) before being analyzed for their individual 15N-enrichments.

    Data analysis. Total N and AA flows (TNFL, TAAFL) in µg/g dry matter intake (DMI) were calculated as follows:

Formula 1(Eq. 1)

Dietary and endogenous N flows (DNFL, ENFL) determined according to the ID method were calculated as follows (16):

Formula 2(Eq. 2)


Formula 3(Eq. 3)

where Ediet is the 15N-enrichment in the diet (expressed as atom %), Es is the 15N-enrichment in digesta sample, and E0 is the basal enrichment. Dietary and endogenous flows of single AA determined using the ID method were calculated as described above for N.

ENFL and EAAFL determined in the UF digesta (diet HC) were determined as follows:

Formula 4(Eq. 4)

with UF digesta = precipitate + retentate (>10 kDa).

15N recovered in the digesta was assumed to be trace unabsorbed dietary 15N. However, some 15N was detected in the >10-kDa fraction of digesta, suggesting that some dietary 15N had been absorbed, incorporated into protein, and then recycled into the gut lumen. The latter 15N would be falsely considered as unabsorbed dietary N tracer. 15N-labeled endogenous protein due to tracer recycling was calculated based on the 15N-measurements in the endogenous N as determined in the UF digesta and expressed as a proportion of endogenous N (MW > 10 kDa, R %) as follows:

Formula 4

where EUFS is the 15N-enrichment in the UF digesta (diet HC).

Ileal N digestibility (%) was calculated as follows:

Formula 5(Eq. 5)


Formula 6(Eq. 6)

where dietary N intake is expressed as µg/g DMI. The terminology standardized digestibility was used as defined by Stein et al. (13) and relates to the previously used term "true digestibility."

Data were tested for homogeneity of variance using Bartlett's test, and if they were not homogenous they were log10-transformed. The daily food intake data were subjected to a 1-way ANOVA for repeated measures. N and AA flows and apparent and standardized ileal N digestibility coefficients from rats fed different diets were subjected to a 1-way ANOVA (SAS, version 8.2). Flows and standardized ileal N digestibility from rats fed diet HC and obtained either by the ID or UF method were compared using a paired t test (SAS, version 8.2). Differences were considered significant at P < 0.05. Results are given as means ± SE.


   Results
TOP
 ABSTRACT
 Introduction
 Materials and Methods
 Results
Discussion
 LITERATURE CITED
 
The rats consumed the diets readily and remained healthy during the experiment. The body weight gains (d 9–16) were similar for rats fed diets HC (17.4 ± 1.5 g) and C (20.3 ± 1.6 g). Daily food intake was similar for rats fed diets HC (15.9 ± 0.7 g/d) and C (16.6 ± 0.8 g/d) from d 10 to 16 but was higher (P < 0.05) on d 17 for those fed diet HC (13.3 ± 0.7 g/d) than for those fed diet C (11.2 ± 0.6 g/d).

DNFLID was lower (P < 0.05) for rats fed diet HC, in the stomach, proximal intestine, and terminal ileum and ENFLID was lower (P < 0.05) for rats fed diet HC, in the proximal and medial intestine (Fig. 1). The proportion of digesta endogenous N to total N (Table 3) was similar for rats fed diets HC and C along the entire digestive tract except in the proximal intestine and cecum/colon, where it was 12 and 4% as units higher, respectively (P < 0.05) for rats fed diet C.


Figure 1
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  		FIGURE 1  DNFL (A) and ENFL (B), determined using the ID method, along the digestive tract of rats fed diets C and HC. Values are means ± SE, n = 4–6. Statistical analyses were performed on transformed data (log10). *C and HC differ, P < 0.05. S, Stomach; PI, proximal intestine; MI, medial intestine; TI, terminal ileum; and Ce, cecum + colon.

 

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  		TABLE 3 Endogenous N (ID) expressed as a percentage of total N along the digestive tract of rats fed diets C and HC1

 
Ileal TAAFL and TNFL were lower (P < 0.05) for rats fed diet HC than for those fed diet C (Table 4), except for Ala, Leu, Tyr, Phe, His, Lys, and Arg, for which there was no significant difference. Ileal EAAFL and ENFL, when determined using the ID technique, did not differ significantly between rats fed diets HC and C (Table 5) except for Gly flow, which was lower (P = 0.05) for rats fed diet HC. The AA compositions of these endogenous protein flows did not differ significantly between rats fed diets HC and C, except for Asp, Phe, Tyr, and Ser, for which the relative contributions tended to be lower (P < 0.05) for rats fed diet C (data not shown).


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  		TABLE 4 Ileal TAAFL and TNFL determined in rats fed diets C and HC1

 

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  		TABLE 5 Ileal EAAFL and ENFL determined using the ID method in rats fed diets C and HC1

 
Ileal EAAFL and ENFL were lower (P < 0.05) when determined using the ID method compared with the UF method (Table 6), except for Gly, Phe, and His, which did not differ. The AA composition of endogenous ileal protein flows from rats fed diet HC differed (P < 0.05) when determined using the UF method compared with that determined using the ID method, except for Asp, Ala, and Leu, which did not differ (data not shown).


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  		TABLE 6 Ileal EAAFL determined using the ID or UF methods in rats fed diet HC1

 
The portion of dietary N recycled (R) within endogenous protein, i.e. present in the >10-kDa fraction from rats fed diet HC (n = 5), was 64.7 ± 2.9% of the N collected in this fraction.

Apparent ileal N digestibilities tended to be lower (P < 0.1) for diet C than for diet HC (Table 7). Standardized ileal N digestibility, when corrected for ENFL determined using the ID method, was lower (P < 0.05) for rats fed diet C than for those fed diet HC. Standardized ileal N digestibility for rats fed diet HC, when corrected for ENFL determined using the UF method, was 99.9 ± 1.0% and was higher (P < 0.05) than that corrected for ENFL determined using the ID method.


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  		TABLE 7 AID and SID of N in diets C and HC fed to growing rats1

 

   Discussion
TOP
 ABSTRACT
 Introduction
 Materials and Methods
 Results
 Discussion
LITERATURE CITED
 
The enzyme-hydrolyzed protein method allows for the determination of ileal endogenous protein losses under peptide alimentation, with an array of dietary peptides assumed to be similar to that arising naturally during digestion of the parent protein. However, the concern of a possible artificial stimulatory effect of dietary peptides on gut protein secretions with this method has been raised (11,13). Most studies (4,7–9,25) have assessed only the impact of dietary peptides vs. protein-free or synthetic AA-based diets. Yin et al. (12) compared dietary casein peptides vs. casein, but the comparison was somewhat biased by methodological factors as discussed below. The objective of our work, therefore, was to determine within a controlled study the impact of 15N-labeled casein either in its intact form or after hydrolysis on ileal endogenous protein flows. Although the ID method is known (26) to lead to some degree of underestimation of absolute gut endogenous protein flows, it was considered a useful tool to allow a comparison to be made using the same protein source.

There was no evidence of a heightened ileal endogenous protein loss due to the dietary peptides per se. This was the case along the entire digestive tract from the stomach to the colon. To the contrary, Yin et al. (12) reported significantly higher ileal endogenous protein flows in growing pigs given hydrolyzed casein compared with an intact casein. However, the intact and hydrolyzed caseins used were not from the same source. Additionally, endogenous flows were determined using either the hydrolyzed protein-UF method with the indigestible marker TiO2 for the hydrolyzed-casein diet or the homoarginine method and the marker Cr2O3 for casein (12). Different dietary markers behave differently (27) and the UF method has been reported to give higher estimates of endogenous protein flows than the homoarginine method (2). Endogenous ileal protein flows have been reported to be similar for pigs fed intact and hydrolyzed pea protein when determined using the 15N-infusion method (15N-labeling of endogenous protein) (28), a finding that supports our results.

The relative contribution of endogenous N to total N in ileal digesta was similar for rats fed diets C and HC and a similar value was found in an identical study in our laboratory in which rats were fed a 15N-labeled acid casein (mean 46%; A. Deglaire, P. Moughan, unpublished data). Also, the AA compositions of the endogenous protein flows were similar for rats fed diets HC and C. These results show that the ileal endogenous protein losses were qualitatively similar regardless of the dietary treatment. The AA composition of endogenous protein determined using ID was similar to that determined previously using a protein-free diet (25,29,30), suggesting that the presently derived estimates of endogenous protein losses were qualitatively similar to minimal endogenous losses (13). The exceptions were Ser, Val, and Gly, for which the relative amounts were lower in this study. The AA composition of endogenous protein flows determined using the hydrolyzed protein-UF method exhibited a similar pattern to that previously reported using the same technique (2,30,31).

ENFL and DNFL determined along the upper digestive tract were consistently lower for rats fed diet HC than for those fed diet C, suggesting a higher degree of absorption and reabsorption and/or faster digesta transit rates along the intestinal tract.

The ileal ENFL and EAAFL for rats fed diet HC (UF method) were in the range of previously comparable data (2,30,32). Ileal ENFL and EAAFL (ID method) were 53% lower than with the UF method. A similar difference has been observed in pigs (46%) and in rats (35%) (2). The UF method, however, has been shown to give similar estimates of ENFL or EAAFL compared with the 15N-infusion method (33) or to a diet based on zein, a maize protein naturally deficient in Lys, allowing a direct estimation of its endogenous flow (8). It is likely that rapid recycling of 15N within the gut (16,26,34,35) led to a degree of underestimation of endogenous N with the ID method. This is highlighted here by the high proportion of 15N recovered in the endogenous protein fraction after UF (MW > 10 kDa) for the HC-fed rats. It is unlikely that the 15N could arise from incomplete separation of the unabsorbed dietary 15N-peptides, as dietary peptides have been reported to represent only 2% of the endogenous N (MW > 10 kDa) (36). The recycled 15N might result from the luminal dietary 15N used for gut protein synthesis (37,38) and subsequently reexcreted. 15N was found in pancreatic enzymes within 50 min following the ingestion of a labeled meal, in bile secretion after 90 min, and in mucins after 4 h (26). The recycled dietary 15N may also arise due to incorporation of dietary AA into microbial proteins. Although not strictly endogenous, microbial proteins have been traditionally considered as part of the endogenous component. Microbial N has been reported to contribute from 15 to 50% of the total ileal N (39,40) and from 30 to 45% of the ileal endogenous N (41). Evidence for a possible substantial use of dietary N for microbial N synthesis has been found in recent studies (39,42).

Our data do not provide any information regarding the extent of the recycling in the upper parts of the intestine. It is also possible that different recycling rates occurred between diets HC and C. A degree of caution should be exercised, therefore, in our comparison of endogenous flows between the HC and C diets. The comparison is only truly valid if the relative rates of recycling were similar between the 2 diets and this remains unknown.

The present value for the standardized (true) ileal N digestibility of intact casein was similar to values reported previously (3,43) but lower than values reported in other studies (12,44). Differences across studies are unlikely due to the different casein forms used, which were either the micellar form (native phosphocaseinate) (3,43), such as in the present study, or acid-precipitated casein (12,44); rather, they probably reflect differences in methodology. In a similar study conducted within our laboratory (A. Deglaire and P. J. Moughan, unpublished data), the ileal N digestibility of 15N-acid-precipitated casein (87.0 ± 0.2% AID, 92.9 ± 0.2% SID) was similar to the value for the micellar (intact) casein reported here. The ileal ENFL for rats fed the acid casein in the latter unpublished work (939 µg/g DMI) was also similar to that for rats fed the intact casein in this study.

Our study has demonstrated that there was no specific effect of dietary peptides from hydrolyzed dietary casein on endogenous ileal protein flow in rats compared with intact dietary casein. The latter conclusion is tentative, however, because the ID method (with 15N-labeled dietary protein) led to substantially lower estimates of endogenous ileal protein flows compared with the UF method. The lower ENFL with ID were assumed to be due to a recycling of 15N within the splanchnic bed and may have been maximized by the experimental design used (i.e. sampling of the ileal digesta samples after 8 h of continuous feeding). In contrast, following single 15N-meals (which we frequently used in the past in humans) (3,45,46), we calculated that the recycling would account for only a 4–9% error in the calculation of ileal ENFL and a subsequent 0.6–1.6% error in the determination of true ileal protein digestibilities. It appears that the amount of gut protein recycled under the conditions of our study was considerable. The present results were obtained with growing rats using a frequent feeding regimen. This work needs to be extended to other species of animals.


   ACKNOWLEDGMENTS
 
We thank Anne Singh and Jacques Fauquant for providing the food ingredients. We thank Carla Bond Smith and Petra Albrecht for sample analysis.


   FOOTNOTES
 
1 Supported by grants from the Riddet Centre (Massey University, Palmerston North, New Zealand) and National Institute of Agronomic Research (Paris, France). Back

2 Author disclosures: A. Deglaire, P. J. Moughan, C. Bos, K. Petzke, S. M. Rutherfurd, and D. Tomé, no conflicts of interest. Back

6 Abbreviations used: AA, amino acid; AID, apparent ileal digestibility; C, casein; DNFL, dietary nitrogen flow; DMI, dry matter intake; EAAFL, endogenous amino acid flow; ENFL, endogenous nitrogen flow; HC, hydrolyzed casein; ID, isotope dilution; MW, molecular weight; R, dietary N recycled; SID, standardized ileal digestibility; TAAFL, total amino acid flow; TNFL, total nitrogen flow; UF, centrifugation and ultrafiltration. Back

Manuscript received 16 October 2007. Initial review completed 28 November 2007. Revision accepted 17 December 2007.


   LITERATURE CITED
TOP
 ABSTRACT
 Introduction
 Materials and Methods
 Results
 Discussion
 LITERATURE CITED
 

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