Abstract
The speed of absorption of dietary amino acids by the gut varies according to the type of ingested dietary protein. This could affect postprandial protein synthesis, breakdown, and deposition. To test this hypothesis, two intrinsically 13C-leucine-labeled milk proteins, casein (CAS) and whey protein (WP), of different physicochemical properties were ingested as one single meal by healthy adults. Postprandial whole body leucine kinetics were assessed by using a dual tracer methodology. WP induced a dramatic but short increase of plasma amino acids. CAS induced a prolonged plateau of moderate hyperaminoacidemia, probably because of a slow gastric emptying. Whole body protein breakdown was inhibited by 34% after CAS ingestion but not after WP ingestion. Postprandial protein synthesis was stimulated by 68% with the WP meal and to a lesser extent (+31%) with the CAS meal. Postprandial whole body leucine oxidation over 7 h was lower with CAS (272 ± 91 μmol⋅kg−1) than with WP (373 ± 56 μmol⋅kg−1). Leucine intake was identical in both meals (380 μmol⋅kg−1). Therefore, net leucine balance over the 7 h after the meal was more positive with CAS than with WP (P < 0.05, WP vs. CAS). In conclusion, the speed of protein digestion and amino acid absorption from the gut has a major effect on whole body protein anabolism after one single meal. By analogy with carbohydrate metabolism, slow and fast proteins modulate the postprandial metabolic response, a concept to be applied to wasting situations.
Dietary carbohydrates are commonly classified as slow and fast because it now is well recognized that their structure affects their speed of absorption, which in turn has a major impact on the metabolic and hormonal response to a meal. On the other hand, little is known about whether postprandial protein kinetics are affected by the speed of absorption of dietary amino acids; the latter is very variable, depending on gastric and intestinal motility, luminal digestion, and finally mucosal absorption. This lack of data is due to the fact that postprandial amino acid kinetics have been studied almost exclusively during continuous feeding, obtained either by a nasogastric infusion or by small repeated meals. Measurements are done 2–4 h after initiation of feeding, once isotopic and substrate steady-state is achieved. Under these conditions, any difference related to the speed of dietary amino acid absorption is blunted.
There is, however, indirect evidence that this parameter might be of importance. Indeed, the postprandial amino acid levels differ a lot depending on the mode of administration of a dietary protein; a single protein meal results in an acute but transient peak of amino acids whereas the same amount of the same protein given in a continuous manner, which mimics a slow absorption, induces a smaller but prolonged increase. Amino acids are potent modulators of protein synthesis, breakdown, and oxidation, so such different patterns of postprandial amino acidemia might well result in different postprandial protein kinetics and gain. Of interest, whole body leucine balance, an index of protein deposition, was shown recently to differ under these two circumstances.
Therefore, our hypothesis was that the speed of absorption by the gut of amino acids derived from dietary proteins might affect whole body protein synthesis, breakdown, and oxidation, which in turn control protein deposition. To test this hypothesis, we compared those parameters, assessed by leucine kinetics, after ingestion of a single meal containing either whey protein (WP) or casein (CAS), taken as paradigms for “fast” and “slow” proteins, respectively. Indeed, WP is a soluble protein whereas CAS clots into the stomach, which delays its gastric emptying and thus probably results in a slower release of amino acids. Speed of amino acid absorption was directly assessed by using a newly developed tracer, i.e., milk protein fractions intrinsically labeled with l-[1-13C]leucine. Leucine kinetics were modelized by using non-steady-state equations as recently described. Our results demonstrate that amino acids derived from CAS are indeed slowly released from the gut and that slow and fast proteins differently modulate postprandial changes of whole body protein synthesis, breakdown, oxidation, and deposition.
MATERIALS AND METHODS
Materials.
l-[1-13C]leucine (99 mol percent excess) and l-[5,5,5-2H3]leucine (97 mol percent excess) were supplied by Mass Trace (Woburn, MA). Isotopic and chemical purity of leucine was checked by gas chromatography–mass spectrometry. Solutions of tracers were tested for sterility and pyrogenicity, prepared in sterile pyrogen-free water, and membrane-filtered through 0.22-μm filters.
Two milk protein fractions, CAS and WP, intrinsically labeled with l-[1-13C]leucine were obtained by infusing lactating cows as described. Labeled milk proteins were mixed with unlabeled proteins to obtain sufficient amounts of protein of appropriate enrichments. Those mixtures then were referred to as 13C-WP for 13C leucine-intrinsically labeled WP and 13C-CAS for 13C leucine-intrinsically labeled CAS. Their final leucine enrichments and contents were checked by gas chromatography–mass spectrometry after hydrolysis.
Subjects.
Sixteen young healthy subjects participated in the different studies. All subjects had a normal physical examination (mean ± SD: age 24 ± 4 years; body mass index 21.9 ± 1.8 kg/m2). They maintained their usual physical activity, with an usual protein–energy intake of ≈38 kcal⋅kg−1⋅d−1 (16% protein) during the 3 days before the study. A written informed consent was obtained from each participant. The experimental protocol was approved by the Ethical Committee of Clermont–Ferrand.
DISCUSSION
The impact on postprandial protein metabolism of the speed of amino acid absorption by the gut has not been estimated previously in humans. Under the conditions of this study, i.e., a single protein meal with no energy added, two dietary proteins have different metabolic fates and uses. After WP ingestion, the plasma appearance of dietary amino acids is fast, high, and transient. This amino acid pattern is associated with an increased protein synthesis and oxidation and no change in protein breakdown. By contrast, the plasma appearance of dietary amino acids after a CAS meal is slower, lower, and prolonged with a different whole body metabolic response: Protein synthesis slightly increases, oxidation is moderately stimulated, but protein breakdown is markedly inhibited. The latter metabolic profile results in a better leucine balance.
The two proteins differed by their amino acid composition. In particular, leucine content was higher in WP (11% wt/wt) than in CAS (8% wt/wt). Therefore, it was not possible to have two meals of identical leucine and nitrogen content. Because nitrogen content could affect postprandial balance, we performed an additional study during which nitrogen content was matched for WP and CAS. Under these conditions, and even though the leucine intake from the CAS was lower, leucine balance remained better with CAS than with WP; this makes it highly unlikely that the better leucine balance obtained with 43 g of CAS was caused by the larger nitrogen intake. More generally, amino acid composition of the meal could affect postprandial protein metabolism, if an essential amino acid is rate limiting for protein synthesis. This possibility is unlikely because CAS and WP were given in large amounts and have a high digestibility and a well balanced amino acid score.
Studying amino acid kinetics after a single meal raises specific methodological questions. Among those, the use of nonsteady-state equations and, in particular, the problems of distribution volume, have been discussed in detail in our previous work. Also, we had demonstrated that only a tracer incorporated into the protein adequately represents the fate of the ingested amino acids by comparing labeled WP and WP added with free [13C]leucine. This is even more true for CAS: In preliminary studies (data not shown), we assessed leucine kinetics after ingestion of CAS with free labeled leucine added. The pattern of tracer appearance was completely different with a very fast and transient appearance of the oral tracer; as expected, this lead to aberrant results for leucine kinetics. Thus, intrinsically labeled proteins constitute an unique tool for assessing the speed of absorption of a protein.
A classic methodological issue is the CO2 recovery factor to be used for leucine oxidation calculations. The CO2 recovery factor that we used in this study was a constant factor of 0.8 throughout the study protocol because it is a usual factor value in the fed studies and because the CO2 recovery in nonsteady-state has not been explored in our conditions. Because the CO2 recovery is proportionally related to CO2 production, it could be expected that the recovery factor would have been slightly higher immediately after the meal. When the recovery factor was calculated from CO2 production in our study, values between 0.72 and 0.76 for CAS and between 0.72 and 0.78 for WP were found, and the consequences on leucine oxidation results were negligible. Also, Total Leu Ox was not back to baseline at the end of the study, particularly for the CAS, which might interfere with the calculations of the area under the curve of oxidation and hence leucine balance. However, when extrapolating the decay curve to baseline (which was reached at a theoretical time of 660 min), our estimate of Total Leu Ox after the CAS meal increased by only 8% (+23 μmol⋅kg−1), and this does not affect our conclusions.
We measured Total Leu Ox during an i.v. tracer infusion, as do most authors. This raises some uncertainties concerning the fate of dietary amino acids that are oxidized in the splanchnic area. Indeed, the extent to which the meal-derived leucine is oxidized in this area is questionable because the i.v. infused labeled leucine must be fully equilibrated with whole body leucine and KIC pools. In the fasted state, liver contribution to leucine oxidation is very small because only 10% of the first-pass leucine uptake is oxidized. This figure could be different after the ingestion of one single meal but, in any case, would probably be higher with WP than with CAS because of the higher initial splanchnic load of amino acids, thus leading to a larger underestimate of Total Leu Ox with WP than with CAS.
This study demonstrates that dietary amino acid absorption is faster with WP than with CAS. Our methodology does not allow identification of the rate limiting step(s) that might be gastric emptying and/or luminal hydrolysis and/or amino acid mucosal absorption. It is very likely, however, that a slower gastric emptying was mostly responsible for the slower appearance of amino acids into the plasma. Indeed, CAS clots into the stomach whereas WP is rapidly emptied from the stomach into the duodenum. Thus, after WP ingestion, large amounts of dietary amino acids (≈25 mmol of leucine) flood the small body pool (≈5 mmol) in a short time, resulting in a dramatic increase in amino acid concentrations. This is probably responsible for the stimulation of leucine oxidation and protein synthesis, as recently suggested by Giordano et al., who artificially elevated plasma amino acid concentrations by i.v. infusions of amino acids. This dramatic stimulation of protein synthesis and absence of protein breakdown inhibition is quite different from the pattern observed with classic feeding studies. By contrast, with CAS, plasma amino acid concentrations are lower, resulting in a lower oxidation and in a lesser increase of protein synthesis but also in an inhibition of protein breakdown. This metabolic response is similar to what is usually demonstrated during steady-state studies, and it is noticeable that postprandial plasma amino acid concentrations were actually at a near steady state. Explanation for the difference of inhibition of protein breakdown between WP and CAS is unclear; protein breakdown is classically inhibited by insulin, but plasma insulin concentrations were not different between the two meals. Although their effects are less powerful than insulin, amino acids also inhibit protein breakdown. The absence of any change of protein breakdown with WP, despite very high amino acid concentrations, suggests that a prolonged enough time of hyperaminoacidemia would be needed to obtain a significant protein breakdown inhibition. Finally, with respect to the issue of leucine balance, it is noticeable that Young’s group recently reported a 24-h leucine balance, which was better with three discrete meals than with multiple small repeated meals, the latter circumstance somewhat mimicking a slow protein absorption. This study and ours are not, however, directly comparable in numerous respects, such as the meal composition, which included carbohydrate in Young’s study.
In conclusion, we demonstrate that the speed of amino acid absorption after protein ingestion has a major impact on the postprandial metabolic response to a single protein meal. The slowly absorbed CAS promotes postprandial protein deposition by an inhibition of protein breakdown without excessive increase in amino acid concentration; by contrast, a fast dietary protein stimulates protein synthesis but also oxidation. This impact of amino acid absorption speed on protein metabolism is true when proteins are given alone, but as for carbohydrate, this might be blunted in more complex meals that could affect gastric emptying (lipids) and/or insulin response (carbohydrate); thus, further studies are needed to confirm the specific roles of nonprotein substrates on whole body protein metabolism. This concept of slow and fast proteins could be applied to circumstances in which protein deposition has to be improved (i.e., protein–energy malnutrition) and in which excessive protein intakes have to be avoided (elderlies, renal diseases).
ABBREVIATIONS
KIC - ketoisocaproate
CAS - casein
WP - whey protein
13C-CAS - 13C-leucine-labeled CAS
13C-WP - 13C-leucine-labeled WP
UL-CAS - unlabeled CAS
UL-WP - unlabeled WP
Ra - rate of appearance
Rd - rate of disappearance
Ox - oxidation
NOLD - nonoxidative leucine disposal
Acknowledgments
We thank Michel Genest, Paulette Rousset, and Liliane Morin for their technical help, Sandrine Corny for tracer preparations, and Olivier Ballèvre for helpful discussion. This study was supported by Grant 94G0079 from the Ministère de l’Education Nationale, de l’Enseignement Supérieur et de la Recherche, and Grants from Région Auvergne and Institut National de la Recherche Agronomique.