Postprandial fate of amino acids: adaptation to molecular forms

During the postprandial phase dietary proteins are digested to peptides and amino acids and absorbed. Once absorbed the peptides are further hydrolyzed to amino acids and transported to the tissues. These amino acids are largely incorporated into body proteins. Not all amino acids are, however, incorporated into body proteins part of these amino acids are oxidized, and can, thus, no longer be utilized to support protein metabolism in the body. The objective of this thesis was to increase the understanding of those processes that determine the utilization efficiency of dietary proteins. The studies described in this thesis, focused on the appearance rate of dietary amino acids in the free amino acid pool of the body. The rate of appearance of dietary amino acids in this pool has been shown to modulate the postprandial oxidation of amino acids and thereby also their utilization efficiency for physiological purposes. As a consequence postprandial oxidative losses influence the nutritional protein status of the body. This thesis aimed to elucidate whether the body is able to cope with diets in which the amino acid appearance rate is high and what mechanisms are involved in this process.First an effort was made to establish the metabolic consequences of amino acid sources with a high appearance rate in a rat and a human model. Postprandial oxidation of free or protein derived [1-13C]- leucine was determined in a [13CO2]-breath test, using both a diet consisting of only free amino acids including [1-13C]-leucine and a diet consisting of proteins in which [1-13C]-leucine was incorporated, and 1:1 mixtures of both diets. In those mixed diets either the protein part or the free amino acid part was labeled. The postprandial oxidative losses of dietary leucine after 5 days being fed these diets (short-term adaptation) appeared to be significantly higher for the free amino acid diet compared to the protein diet. These differences between dietary free amino acids and dietary protein persisted in the mixed diets, as measured by the [13CO2] breath-test. It was concluded that amino acids derived either from a free amino acid or a protein diet, were handled independently even when ingested simultaneously during the same meal. Results obtained in rats were comparable to the results obtained in humans.The differences in oxidation between a free amino acid and a protein diet had largely disappeared after long-term adaptation (after 26 to 30 days on the diet). An adaptive decrease in the oxidation of free amino acids was observed. In the second study it was examined to which extent increasing levels of methionine supplements in a diet (50, 100 or 200% methionine supplement relative to casein) were retained in body protein. This was thus far not clear since a higher appearance rate in the free pool has been reported to have a negative influence on the efficiency of utilization of amino acids from the diet. Moreover, only specific patterns of amino acids are supposed to be deposited in body protein. Higher dietary methionine levels resulted in higher postprandial oxidative losses of methionine. The groups, which were fed the diets with the highest methionine levels, showed the lowest methionine retention as part of intake but the highest retention in absolute terms. After long term adaptation, however, to the free amino acid diets, methionine retention was increased in all groups. It was concluded that postprandial retention of dietary amino acids is, at least in part, driven by the amino acid composition of the diet.In third study it was examined whether the postprandial fate of different dietary amino acid was regulated by hormonal responses to the diet. It has been observed that the differences in oxidative losses between diets consisting of free amino acids or protein were not mediated by the combined action of insulin, glucagon, corticosterone and GH. Hence, postprandial catabolism of amino acids is probably regulated by other mechanisms. As stated above, the amino acid appearance rate plays a crucial role in determining the postprandial utilization of amino acids. In the fourth study it was, therefore, investigated, whether the amino acid absorption rate can adapt to dietary free amino acids. Rats were kept on a free amino acid diet for 0 (nonadapted), 5, or 26 to 30 days (long-term adaptation). The methionine absorption of long-term adapted rats was lower than that of the non-adapted rats. It was concluded that the absorption of amino acid by the intestine plays a crucial role in minimizing the postprandial oxidative losses

Post prandial oxidative losses for free amino acids in the diet: studies on interactions with dietary protein and on long term adaptation

In this study we have tried to elucidate differences in postprandial oxidation between free and protein bound amino acids both after short and long term exposure. By labeling the different dietary forms their fate of the different dietary forms could be traced independently. By this approach we were also able to study potential interactions between the oxidation of free and protein-bound amino acids when both are mixed in one mea

Postprandial oxidative losses of free and protein-bound amino acids in the diet: interactions and adaptation

Postprandial oxidation of dietary free amino acids or egg white protein was studied using the [13CO2] breath test in rats, as well as in humans. Thirty-eight male rats were assigned to four dietary test groups. Two diets only differed in their protein fraction. Diet I contained 21% egg white protein. For the breath test egg white protein, intrinsically labelled with [1-13C]-leucine, was used as a substrate. Diet II contained the same amino acids as diet I, though not as egg white protein but in free form. Free [1-13C]-leucine was used to label this diet. In addition, two 1:1 mixtures of both diets were used. During the breath test either the free amino acid or the protein fraction was labelled as in diets I or II. The animals were breath-tested following short-term (day 5) and long-term adaptation (day 20) to their experimental diet. For all diets, including the mixed diets, postprandial oxidative losses on day 5 were significantly higher for the free leucine compared with the protein-derived leucine. Differences between free and protein-derived leucine oxidation had, however, largely disappeared on day 20. The human subjects were breath-tested without any adaptation period to the diets. The oxidative losses of free leucine were also higher than those of protein-derived leucine. None of the studies showed any indication for an interaction between the oxidation of protein-derived amino acids and free amino acids. It is concluded that free and protein-derived amino acids in the diet are mainly metabolized independentl

Application of a [13CO2] breath test to study short-term amino acid catabolism during the postprandial phase of a meal

A [13CO2] breath test was applied as a non-invasive method to study the catabolism of ingested amino acids shortly after a meal. This test requires the ingestion of a [1-13C]-labelled amino acid and the analysis of expired air for [13C] enrichment and CO2. The recovery of label as [13CO2] reflects the catabolism of the [1-13C]-labelled substrate. Such a non-steady state approach provides information that is complementary to the information obtained by steady-state methods using a primed continuous infusion of tracer amino acids during the fed state. In a model study with twenty adult male rats, two groups of animals were fed twice a day with one of two semi-synthetic iso-energetic diets. One diet contained egg white protein (EW) as the sole amino acid source. The second diet contained a mixture of free amino acids with a pattern similar to that of the EW diet. On day 5 of the dietary treatment, l-[1-13C]leucine, either bound in EW protein or in free form, was ingested as part of the morning meal. The expired air was sampled at 30 min intervals for 5 h. The rate of recovery ranged from 0 % to 6 % of the dose/h. Up to 120 min after the onset of the meal, the recovery values for the free amino acid diet were higher than those for the EW diet. Differences in recovery reflect differences in postprandial utilisation. The differences in label recovery were mainly determined by the [13C] enrichment of the expired air. As a consequence, CO2 measurements are not mandatory when CO2 production is comparable

Dietary amino acids fed in free form or as protein do differently affect amino acid absorption in a rat everted sac model

In the present study, the effect of free amino acid (FAA) diets on the intestinal absorption rate of methionine and leucine was studied 'ex vivo' with rats adapted for different periods of time to the diets, using the everted sac method. The adaptation period to the 21% FAA diet with an amino acid content based on casein was either, 0 (no adaptation, N-ADA), 5 (short-term adaptation, ST-ADA), or 26-33 days (long-term adaptation, LT-ADA). Within the ST-ADA and the LT-ADA groups, three different levels of methionine were included: 50%, 100% and 200% of the level normally present in casein. All diets were iso-nitrogenous and iso-caloric. After the adaptation period (0, 5, or 26-33 days), intestinal everted sacs were prepared. Methionine or leucine was added to the medium as transport substrate. The methionine absorption rate of the rats of the LT-ADA groups was higher than that of the N-ADA groups. Furthermore, adaptation to 200% dietary methionine levels caused a significantly slower leucine absorption compared to the 100%, and 50% group. Methionine absorption was similar in the 100% and 200% groups, but the absorption of methionine in the 50% group was enhanced in the distal part of the intestines. We concluded that in response diets with 21% FAAs as only amino acid source, amino acid absorption is decreased to avoid toxic effects of high levels of methionine in the circulation

Encapsulation of GFP in complex coacervate core micelles

Protein encapsulation with polymers has a high potential for drug delivery, enzyme protection and stabilization. Formation of such structures can be achieved by the use of polyelectrolytes to generate so-called complex coacervate core micelles (C3Ms). Here, encapsulation of enhanced green fluorescent protein (EGFP) was investigated using a cationic-neutral diblock copolymer of two different sizes: poly(2-methyl-vinyl-pyridinium)41-b-poly(ethylene-oxide)205 and poly(2-methyl-vinyl-pyridinium)128-b-poly(ethylene-oxide)477. Dynamic light scattering and fluorescence correlation spectroscopy (FCS) revealed a preferred micellar composition (PMC) with a positive charge composition of 0.65 for both diblock copolymers and micellar hydrodynamic radii of approximately 34 nm. FCS data show that at the PMC, C3Ms are formed above 100 nM EGFP, independent of polymer length. Mixtures of EGFP and nonfluorescent GFP were used to quantify the amount of GFP molecules per C3M, resulting in approximately 450 GFPs encapsulated per micelle. This study shows that FCS can be successfully applied for the characterization of protein-containing C3Ms