I heard the opposite - the bioavailability is higher when raw - you don't see foetus's cranking up the BBQ do you?
If you have some info to back up your claim would be good.
The Journal of Nutrition Vol. 128 No. 10 October 1998, pp. 1716-1722
Digestibility of Cooked and Raw Egg Protein in Humans as Assessed by Stable Isotope Techniques1,2,3
Pieter Evenepoel, Benny Geypens, Anja Luypaerts, Martin Hiele, Yvo Ghoos4, and Paul Rutgeerts
Department of Medicine, Division of Gastroenterology and Gastrointestinal Research Centre, University Hospital Leuven, B-3000 Leuven, Belgium
Egg proteins contribute substantially to the daily nitrogen allowances in Western countries and are generally considered to be highly digestible. However, information is lacking on the true ileal digestibility of either raw or cooked egg protein. The recent availability of stable isotope-labeled egg protein allowed determination of the true ileal digestibility of egg protein by means of noninvasive tracer techniques. Five ileostomy patients were studied, once after ingestion of a test meal consisting of 25 g of cooked 13C- and 15N-labeled egg protein, and once after ingestion of the same test meal in raw form. Ileal effluents and breath samples were collected at regular intervals after consumption of the test meal and analyzed for 15N- and 13C-content, respectively. The true ileal digestibility of cooked and raw egg protein amounted to 90.9 ± 0.8 and 51.3 ± 9.8%, respectively. A significant negative correlation (r = 0.92, P < 0.001) was found between the 13C-recovery in breath and the recovery of exogenous N in the ileal effluents. In summary, using the 15N-dilution technique we demonstrated that the assimilation of cooked egg protein is efficient, albeit incomplete, and that the true ileal digestibility of egg protein is significantly enhanced by heat-pretreatment. A simple 13C-breath test technique furthermore proved to be a suitable alternative for the evaluation of the true ileal digestibility of egg protein.
Other approaches therefore have been developed to distinguish the exogenous from the endogenous protein fraction in the intestinal contents including the use of dietary protein labeled with homoarginine (Siriwan et al. 1994) and the evaluation of the endogenous/exogenous ratio of effluents, which is extrapolated by comparing the amino acid composition of both the chyme and the meal (Baglieri et al. 1995, Mahé et al. 1994b). Primarily for safety reasons, however, the tracer dilution technique using stable isotopes is the most appropriate for studies involving human subjects. The widespread use of this technique for the study of protein digestibility has been hindered for a long time by the lack of an adequate substrate, i.e., stable isotope-labeled protein. In recent years, different proteins labeled with 15N have become available (e.g., milk protein, soy protein or pea protein) (Gausserès et al. 1996, Kayser et al. 1992, Mahé et al. 1994b). Very recently, we succeeded in producing both 15N- and 13C-labeled egg protein in an easily reproducible and highly efficient manner (Evenepoel et al. 1997).
The digestibility of protein is affected by its state of processing before ingestion. Both an enhanced and a reduced protein digestibility have been observed after food processing (e.g., boiling, drying, deep-freezing or microwave-heating) (Öste 1991). Using the "ileostomy model," this study attempted the following: 1) to determine the amount of nitrogen escaping digestion and absorption after ingestion of a physiologic load of egg protein and to distinguish the exogenous from the endogenous fraction; 2) to validate the 13C-egg protein breath test as a tool for the evaluation of protein digestibility in vivo; and 3) to evaluate the influence of heat-pretreatment on the efficiency of egg protein assimilation. Protein assimilation is defined as the overall process of digestion and absorption finally resulting in the appearance of free amino acids in the portal circulation.
Protein test meal. The protein test meal consisted of 100 g of 13C-labeled egg white, 100 g of 15N-labeled egg white (markers of protein assimilation) and the yolk of one egg doped with 74 kBq of [1-14C]octanoic acid (Dupont, NEN Research, Boston, MA) (marker of gastric emptying) (Ghoos et al. 1993). Five microcurie 3H-polyethylene glycol (3H-PEC) 4000 was added to the test meal as a nonabsorbable radiolabeled transit marker. All constituents were homogenized before ingestion. The test meal had to be consumed either raw or after being cooked in a microwave oven (see Experimental design). The methodology for obtaining large amounts of highly enriched egg proteins labeled with stable isotopes was extensively described elsewhere (Evenepoel et al. 1997). Briefly, 13C- or 15N-labeled proteins were produced by giving laying hens free access to a food containing 25% of the (NRC required) leucine content as free [1-13C]leucine (99 mol%, Euriso-top, Saint-Aubin, France) and [15N]leucine (99 mol%, Euriso-top), respectively. The yolk and egg white fractions of the enriched eggs were separated and pooled. The isotopic enrichment of both pools was determined using a continuous flow elemental analyzer isotope ratio mass spectrometer (IRMS)5 (ANCA-SL, Europa Scientific, Crewe, UK). Knowing the exact amino acid composition and the isotopic enrichment of the egg white, the amount of [1-13C]leucine (99 mol%) incorporated could be calculated. This value was taken into account in the calculation procedures, outlined below. Because "redistribution" of the 15N-label is likely to occur in the hen via transamination, the 15N-labeled egg protein can be assumed to be uniformly labeled. Total caloric content of the test meal was 624 kJ (25 g protein, 5.56 g fat and a negligible amount of carbohydrate).
Experimental design. Each subject was studied in two different randomly applied test situations as follows: 1) after ingestion of the cooked egg protein test meal ("cooked"), and 2) after ingestion of the same but raw protein test meal ("raw"). The test situations were separated from each other by a time span of at least 1 wk. Measurements of gastric emptying (breath test technique), small intestinal transit time (nonabsorbable transit marker) and protein assimilation (both by breath test technique and analysis of ileostomy effluents) were performed on both occasions.
All subjects were studied after an overnight fast of at least 12 h. At 0845 h on the test day, they were asked to consume the meal together with 200 mL of water within 15 min. Sodium chloride seasoning was allowed. No further food was eaten until 1500 h when the patients ingested 200 mL of a formula food, based on whey, casein and lactalbumin (Nutridrink, Nutricia, Zoetermeer, The Netherlands). Five milligrams of phenol red was added to this formula food as a nonabsorbable transit marker. Drinking of water was permitted from 1200 h on.
The patients emptied their ileostomy bag immediately after ingestion of the meal (i.e., at 0900 h), at 1-h intervals throughout the day until 1900 h and at 0900 h the next morning. Breath samples for 13CO2 and 14CO2 were collected before ingestion of the meal and every 15 min thereafter for 6 h. The experimental design is schematically represented in Figure 1.
Breath test results. As shown in Figure 2, the course of both the 13CO2 (panel A) and 14CO2 (panel B) excretion rate was influenced by the state of the test meal, whether cooked or raw. The parameters of protein assimilation and gastric emptying obtained in both test situations are given in Table 1. Significant differences between the two test situations were found for several variables.
Fig 2. Mean 13CO2 (A, representing protein assimilation) and 14CO2 (B, representing gastric emptying) excretion rates, expressed as percentage of the administered dose of 13/14C excreted per h (% dose/h) in ileostomy patients after ingestion of a test meal consisting of 25 g of either cooked or raw egg protein. Values are means ± SEM, n = 5.
Table 1. Protein assimilation, gastric emptying and ileal emptying and the small bowel transit time in ileostomy patients after ingestion of cooked and raw test meals consisting of 25 g egg protein1
Ileostomy effluents.
Validation of use of 3H-PEG as transit marker. There was a significant correlation (r2 > 0.97, P < 0.0001) between the delivery of the radioactive marker in the ileostomy effluent and the delivery of exogenous protein in each test condition (Fig. 3).
Profile of delivery of meal residues from the ileum. Ileal emptying of the test meal began in the first hour after ingestion of the cooked and raw test meals (Fig. 4). The emptying rate accelerated temporarily in the 1- to 2-h period and clearly peaked in the 6- to 7-h period. Fifty percent of the meal had emptied from the ileostomy (ileal t1/2) by 5.33 ± 0.76 and 5.29 ± 0.50 h after the ingestion of the cooked meal and raw test meal, respectively (Table 1).
Fig 4. Ileal emptying profile of the cooked and raw protein meal, expressed in percentage of administered dose of 3H-polyethyle glycol (PEG) recovered in the effluents of ileostomy patients per hour (% dose 3H-PEG/h). Values are means ± SEM, n = 5.
Exogenous and endogenous nitrogen in ileostomy effluents. Figure 5 shows the endogenous and exogenous nitrogen fraction profiles after 15N-egg protein ingestion in the two test conditions. The cumulative quantities of exogenous and endogenous nitrogen, recovered in the ileostomy effluent over 24 h are given in Table 2. A significantly greater amount of exogenous nitrogen was recovered in the ileal effluent over 24 h after ingestion of the raw test meal compared with the cooked test meal (1949.4 ± 390.3 m vs. 360.6 ± 30.6 mg, P < 0.05). Taking into account these data and the amount of N ingested, it was possible to calculate the true ileal digestibility of egg protein, i.e., the percentage of exogenous egg protein assimilated in the small intestine. The true ileal digestibility of raw egg protein was significantly impaired compared with that of cooked egg protein (51.3 ± 9.8 vs. 90.9 ± 0.8%, P < 0.05).
Fig 5. Delivery of endogenous (Nendo), exogenous (Nexo) and total (Ntot) nitrogen in ileal effluents of ileostomy patients after ingestion of cooked and raw test meals consisting of 25 g egg protein (=4000 mg N). Values are means ± SEM, n = 5.
View this table:
Table 2. Exogenous and endogenous nitrogen yield over 24 h in ileal effluents of ileostomy patients after ingestion of 25 g of either cooked or raw 15N-labeled egg protein1,2
Correlations. A significant negative correlation was found between the amount of exogenous N recovered in the ileostomy effluent and the cumulative percentage administered dose of 13C recovered in breath over 6 h (Fig. 6). There were no significant correlations between the gastric emptying half time (gastric t1/2) and the small bowel transit time (defined as ileal t1/2 gastric t1/2), nor between the small bowel transit time and the true ileal digestibility of egg protein in each of the test conditions.
Fig 6. Correlation between the cumulative amount of exogenous nitrogen, recovered in the ileal effluents over 24 h (Nexo 24 h cum) and the cumulative percentage of administered dose of 13C, recovered in breath over 6 h (% dose 13C cum 6 h) in ileostomy patients.
individual values obtained after ingestion of 25 g of raw egg protein; : individual values obtained after ingestion of 25 g of cooked egg protein). Twenty-five grams of protein corresponds to 4 g of nitrogen.
Dietary protein provides 10-15% of the total energy intake of healthy subjects consuming a standard Western diet and supplies the essential amino acids required for protein synthesis. The nutritional value of dietary protein for humans is usually determined from a "chemical score" or more recently from the "protein digestibility-corrected amino acid score" in which digestibility represents an important aspect (Young and Pellett 1991). According to results obtained from nitrogen balance studies in healthy subjects, the true (fecal) digestibility of (spray dried whole) egg protein has been estimated to be as high as 92-97% (Bodwell et al. 1980). However, because nitrogen is intensely metabolized, absorbed and secreted in the colon, fecal digestibility values do not necessarily equal ileal digestibility values. This has been demonstrated in an animal study, in which fecal and ileal digestibility were measured simultaneously (Mosenthin et al. 1994). Because the ileal digestibility represents the efficiency of protein assimilation, ileal digestibility values are more relevant from a nutritional point of view than are fecal digestibility values. The ileal digestibility of different protein sources has already been assessed in healthy volunteers with the use of an intestinal perfusion technique (Chung et al. 1979, Gausserès et al. 1996, Mahé et al. 1994a). This technique, however, is time-consuming and invasive and, moreover, appears to delay gastric emptying and to shorten transit time in the small intestine (Read et al. 1983). Shortening of the transit time may compromise the assimilation of macronutrients (Chapman et al. 1985, Holgate and Read 1993). Because ileal effluents are easily collectible in ileostomy patients, nitrogen balance studies in these subjects are an attractive alternative for the evaluation of ileal digestibility (Chapman et al. 1985, Fuller et al. 1994, Gibson et al. 1976, Rowan et al. 1994, Sandström et al. 1986). A common problem encountered in studies designed to determine the ileal digestibility is the differentiation between the exogenous and endogenous origin of nitrogen recovered in ileal effluents. Because we recently obtained large amounts of highly enriched 15N-labeled egg proteins (Evenepoel et al. 1997), we were able to apply the isotope dilution technique for the assessment of the true ileal digestibility of egg protein.
Five healthy ileostomy patients were studied after ingestion of a physiologic load (25 g) of egg protein, labeled with 13C and 15N. The double labeling of the test meal allowed the evaluation of egg protein assimilation by both 13C-breath test technique and direct analysis of the ileostomy effluents (15N-dilution technique). The subjects were studied in different experimental conditions to assess the influence of heat-pretreatment on the assimilation efficiency of egg protein.
After ingestion of the cooked egg protein meal, a substantial quantity of nitrogen was recovered in the ileal effluent over 24 h. The calculated yield of endogenous nitrogen (i.e., 0.40 g N) was close to the yield of 0.55 g N obtained by other researchers after ingestion of 17 g of pea protein (Gausserès et al. 1994). The calculated true ileal digestibility of cooked egg protein amounted to 91%. This finding demonstrates that even cooked egg protein, which has generally been considered to be easily digestible, is malabsorbed to some extent after ingestion of a physiologic load. Incomplete assimilation of dietary protein may have important consequences not only from a nutritional point of view, but also from a gastrointestinal point of view. Indeed, some metabolites resulting from bacterial fermentation of malabsorbed proteins in the colon have been implicated in the ethiopathogenesis of diseases such as colonic cancer and ulcerative colitis (Macfarlane and Cummings 1991, Pitcher and Cummings 1994, Visek 1978). It has already been reported extensively that food processing can influence protein digestibility both beneficially and detrimentally (Öste 1991). Egg white protein is generally considered to be less digestible than heat-pretreated egg white protein. However, no data are available concerning the magnitude of this impairment in vivo. In this study, it was shown that after ingestion of 25 g of raw egg protein, almost 50% is malabsorbed over 24 h. The higher digestibility of cooked egg protein presumably results from structural changes in the protein molecule induced by heating, thereby enabling the digestive enzymes to gain broader access to the peptide bonds. It has been suggested that the reduced digestibility of raw egg white is at least partially related to the presence of trypsin inhibitors in raw egg white (Matthews 1990). Ovomucoid is quantitatively the most important trypsin inhibitor (Gilbert 1971, Kassell 1970). Ovomucoid, however, does not react with human trypsin and, moreover, is relatively heat stable (Kasell 1970). Whether other egg trypsin inhibitors (e.g., ovoinhibitor or papain inhibitor) interfere with the digestibility of unprocessed egg white protein is unknown.
Interestingly, the yield of endogenous nitrogen after ingestion of the raw protein meal (i.e., 0.2 g N) was significantly lower compared with the cooked protein meal. This finding is in accordance with a recent study in which it was demonstrated that undigested protein, in contrast to digested protein, only weakly stimulates gallbladder emptying and pancreatic enzyme secretions (Thimister et al. 1996).
Labeling of the test meal with 14C-octanoic acid allowed us to follow gastric emptying simultaneously. The raw test meal was emptied significantly more quickly than the cooked test meal, most probably because of its liquid consistency. Small intestinal transit of the raw test meal, on the other hand, tended to be slower than that of the cooked test meal. A shortened transit time therefore cannot account for the observed decreased digestibility of raw egg protein.
In this study, a significant negative correlation was observed between the percentage of administered dose 13C recovered in breath over 6 h and the amount of exogenous nitrogen recovered in the ileostomy effluent over 24 h. This finding implies that the 13C-egg protein breath test may be regarded as an accurate technique for the evaluation of protein digestibility. Although the 13C-egg protein breath test is only semiquantitative (i.e., it reveals adequacy of function compared with a normal standard), it has many advantages (i.e., it is noninvasive, simple and reproducible), by which it yet might be an attractive alternative for the evaluation of the efficiency of protein assimilation in healthy volunteers in different experimental conditions as well as in patients with digestive diseases. Moreover, the information obtained with breath tests represents a dynamic evaluation, rather than a static estimation.
No significant correlation was found between the gastric t1/2 and the small bowel transit time, which is in agreement with other studies (Read et al. 1982 and 1986). These results, however, should be interpreted with caution because of the small number of subjects studied.
In conclusion, with the use of stable isotope techniques, we were able to determine the amounts of egg protein escaping digestion and absorption in the small intestine after ingestion of a physiologic load. Native egg protein is malabsorbed to an important extent. The assimilation of egg protein is facilitated by heat-pretreatment, but remains incomplete. The excellent correlation found between digestibility values and 13C-breath test data validated the 13C-egg protein breath test as an accurate alternative for the evaluation of protein digestibility. Because of its many advantages, the 13C-egg protein breath test might be very interesting for the study of protein digestibility under different experimental conditions.
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