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 independently.

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.

Dietary amino acids fed in free form and as protein components do not differently affect postprandial plasma insulin, glucagon, growth hormone and corticosterone responses in rats.

This study examined, whether the postprandial fate of dietary amino acids from different amino acid sources is regulated by the responses of insulin, glucagon, corticosterone and growth hormone (GH). Male Wistar rats were cannulated in the vena jugularis and assigned to dietary groups. The diets contained 21% casein or the same amino acids in free form. In the free amino acid diets, methionine level was varied between the groups. The feed was supplied in two distinct meals. In previous experiments it was established that oxidative amino acid losses of the free amino acid diets and protein diets were different. After 3 weeks on those diets, it appeared that the differences in postprandial oxidative losses had been diminished. GH was measured every 12 min, from 144 min before the start of the experimental meal over the following 144 min. Insulin and corticosterone were measured six times from the start of the meal until 270 min after the meal. No differences have been observed between the hormonal responses to both meals at day 5 and at day 26. In conclusion, it has been found that the differences in the oxidative losses between protein and free amino acid meals are not mediated by the combined action of the insulin, glucagon, corticosterone and GH. Postprandial catabolism of amino acids is most probably regulated by substrate induction.

mRNA levels of the calpain system in longissimus muscle of young pigs during prolonged feeding of a protein-free diet.

This experiment was conducted to investigate the effects of feeding a protein-free diet on mRNA levels of the calpain system in skeletal muscle of growing pigs during a 15-d feeding trial. Twenty crossbred barrows were divided into two dietary treatments: control or protein-free diet (mean initial weight for both groups: 38.3 kg). Daily diets were provided at 2.5 times energy for maintenance (twice a day). On d 0, 3, and 14, biopsies were taken from longissimus muscle between the third and fourth ribs (d 0 and 3) and between the fourth and fifth rib (d 14). On d 15, animals were slaughtered and longissimus muscles were dissected and analyzed for calpastatin, and mu- and m-calpain activity. From biopsies, mRNA level of skeletal muscle calpain, mu- and m-calpain, and calpastatin were measured using reversed transcription PCR. Subsequently, PCR products were quantified using ELISA. Feeding the protein-free diet lowered growth rate to almost zero. Only total level of mRNA of mu-calpain on d 14 was influenced by dietary treatments, being lower for the protein-free group than for the control group (P < .05). However, proteolytic activities were not different between treatments. Total RNA concentration in longissimus muscle decreased during the experiment for both treatments, but on d 14 this was more pronounced for the protein-free than for the control group (P < .05). If mRNA levels were corrected for this change, specific mRNA level on d 14 of skeletal muscle calpain and mu-calpain were lower (P < .05) for the protein-free than for the control group. These data suggest that activity of the components of the calpain system are differentially regulated.

Partitioning of fat and protein in energy retention of growing animals.

With increasing intakes the body fat content increases and that of protein decreases. It is most often assumed that this is brought about because each increment in retention contains more fat and less protein. Experimental results, however, showed that this explanation is not true. In two experiments male broiler chickens were fed at levels between 60 and 100 % of recommended energy intake. Body composition at 1500 g showed, as expected, that with increasing intakes body fat content increased and protein content decreased. Both fat and protein retention per day were linearly related to total energy retention (ER). This means that each increment in retention has the same protein and fat content. At zero fat retention only protein was retained, about 50 % of maximal retention. At zero ER protein was retained and fat mobilized. Energy and N balance experiments confirmed the constant composition of each increment in retention. The results of both experiments show that total ER consisted of two components: a basic constant daily protein retention and a variable additional ER, mainly consisting of fat. The basic protein retention is about half of maximal retention. With increasing energy intakes the basic protein retention is combined with an additional amount of protein and fat in a constant ratio.

Short-term dynamics in protein and amino acid metabolism.

Actual amounts of free amino acids in the blood are sufficient to support whole body protein synthesis for some minutes only. This indicates that the levels of free amino acids in the circulation are kept small and constant relative to the amounts of amino acids supplied by daily intake and turnover of body proteins. The clearance of the amino acids originating from either endogenous or exogenous sources is mainly due to protein synthesis and metabolic degradation. The partitioning of dietary amino acids between these processes, on the short term, is supposed to play an important role in whole body amino acid economy. Therefore whole body amino acid economy could be improved by nutritional measures that favour the clearance of dietary amino acids by protein synthesis instead of by metabolic degradation. These nutritional measures should to be focused on threshold values for metabolic degradation of individual amino acids.

Benefit of more but smaller meals at a fixed daily protein intake.

The influence of meal frequency on change of body weight and protein status, measured by level of amino acid oxidation (decarboxylation) in the postabsorptive state, was studied at a fixed daily protein intake. Growing rats (250g) were fed through gastric canula a feeding solution based on Nutrison Standard supplying 1.6g protein and 266kJ ME daily. This amount was given in either 2 large meals at the beginning and the end, or in 6 smaller meals, or by continuous infusion during entire dark period (10 hrs). After 3 weeks of feeding the mean growth rate of the rats fed continuously was nearly 20% higher than rats fed the same amount in 2 meals. The rats fed 6 meals a day had a growth rate rather similar to the rats fed continuously. The percentile recovery of label as 14CO2 in the breath after an intraperitoneal injection of [1-14C]leucine (4 hrs after last meal) was significantly higher (p.05) for the animals fed continuously (27% sd 2.6) compared to the rats fed 2 meals (21.9% sd 4.0). The value for 6 meal group was intermediate (24.5 sd 1.8). The results indicate that the metabolic utilization of a fixed daily amount of protein is clearly influenced by the way of supply. With respect to the change of body weight and protein status, animals have more benefit of the same amount of protein if the supply is more equable. It is suggested that the difference is caused by metabolic restriction for an adequate utilisation of large meals. Therefore large meals are supposed to cause a waste of amino acids in the postprandial phase. As a consequence amino acid amount that will be stored in the body to be available in the postabsorptive phase will be less.

Meal feeding and leucine utilization in pregnant rats.

During pregnancy a higher amino acid requirement may be expected, but the increase in food intake does not match the increased growth rate during pregnancy. It is hypothesized that amino acid utilization can be increased during both fasting and feeding in order to account for the increased requirement. Therefore mature female rats (20 weeks old) were investigated before and at day 18 of pregnancy. Rats were fed on a high-protein (HP) diet (210 g casein/kg diet) for 3 weeks and fasted overnight. Rats were then subjected to an 8 h constant infusion of L-[1-14C]leucine with continuous measurement of expired 14CO2 (as a percentage of the infused dose). After 3 h infusion a 5 g HP or low-protein (LP; 75 g casein/kg diet) meal was offered for 30 min. Pregnant rats had a significantly lower percentage leucine oxidation in the fasted state (12.5 (SE 0.7) v. 15.9 (SE 1.1)%; P < 0.05), which suggests improved reutilization of leucine. Meal ingestion resulted in a fast increase in 14CO2 expiration. After the LP meal the level of 14CO2 expiration decreased again after the acute response (0-1.5 h), but this was not the case after the HP meal. After the HP meal (average 1.5-5 h), no difference was observed between pregnant and non-pregnant status (36.8 (SE 1.6) v. 35.0 (SE 2.5)%). After the LP meal (average 1.5-5 h), however, the percentage leucine oxidation tended to be lower in pregnant rats but this difference did not reach statistical significance (19.7 (SE 1.1) v. 25.8 (SE 2.8)%).(ABSTRACT TRUNCATED AT 250 WORDS)

Effect of dietary energy restriction on retention of protein, fat and energy in broiler chickens.

1. Broiler chickens were fed 60-100% of recommended energy intakes to study the effects of energy restriction on protein and fat retention. 2. At an energy retention of 179 kJ/kg W0.75 d, only protein was retained. At higher energy intakes, each increment in retention had a rather constant composition: about 85% energy in fat and 15% in protein. At lower energy intakes body fat was mobilised whereas protein was deposited. 3. The efficiencies of energy retention in protein and fat were estimated to be 0.66 and 0.86 respectively. 4. The rather constant composition of additional retained energy after additional energy supply provides an explanation for a linear relationship between energy intake and energy retention.

Effects of acute and chronic level of protein supply on metabolic leucine utilization in growing and mature rats.

Effects of acute (meal) and chronic (diet) level of protein supply on metabolic leucine utilization were investigated in growing (10 weeks) and mature (> 1 year) rats. Rats were conditioned on a high-protein (HP) diet (210 g casein/kg feed) or a low-protein (LP) diet (75 g casein/kg feed) from 7 weeks of age. Overnight-fasted rats were offered a HP or LP meal during a 8 h 14CO2 breath test with a constant infusion of either L-[1-14C]leucine (carboxyl, CL) or L-[U-14C]leucine (universal, UL). Before the meal 14CO2 output was lower for overnight-fasted rats fed on LP than on HP (P < 0.001), and also lower for growing than for mature rats (P < 0.001). Meal ingestion resulted in a rapid increase in 14CO2 output. From 2 h after the start of the meal the effect of acute protein supply on 14CO2 output was significant (P < 0.001), while the effect of chronic protein supply disappeared for CL. After the meal 14CO2 output was transiently lower for growing than for mature rats (P < 0.05), especially after the LP meal. The difference in 14CO2 output between CL and UL increased transiently after the meal, indicating an increase in decarboxylation relative to total oxidation of leucine.(ABSTRACT TRUNCATED AT 250 WORDS)

[Effect of increasing protein ingestion on the nitrogen balance of mechanically ventilated critically ill patients receiving total parenteral nutrition]. / Efecto del aumento de la ingesta de proteínas sobre el balance nitrogenado en pacientes críticos ventilados mecánicamente y sometidos a nutrición parenteral total.

The amount of protein recommended to minimise N loss in critically ill patients receiving total parenteral nutrition (TPN) varies in the literature. Therefore, we studied the effect of increased protein intake on the N balance, administering TPN with either 1.2 g protein/kg/day (low N diet) or 1.8 g protein/kg/day (high N diet). Fifteen mechanically ventilated critically ill patients were studied in a surgical intensive care unit. After at least two days of standard TPN, patients were randomly assigned to either the low or the high N diet. Ten patients were studied on the low N diet and 11 on the high N diet; 6 patients were studied on both diets. Nonprotein energy was supplied according to estimated energy requirements. For five consecutive days, the N balance was measured daily. Total urinary nitrogen (TUN) was analysed using the Kjeldahl method. There was no difference in N balance between the groups. On the low N diet, N balance was -0.113 +/- 0.088 and on the high N diet -0.113 +/- 0.109 g N/kg/day. In patients studied twice, N balance was -0.087 +/- 0.054 and -0.050 +/- 0.060 g N/kd/day respectively. Results of a previous pilot study showed that in 20 similar patients the N balance became 80% less negative (from -5.7 +/- 5.1 to -1.1 +/- 8.2 g N/day) when protein intake was increased from 0.9 to 1.5 g/kg/day. Since these results are consistent with other studies, we conclude that the optimal range of protein supply in this type of critically ill patients is approximately 1.1-1.5 g protein/kg/day.

The metabolic utilization of amino acids: potentials of 14CO2 breath test measurements.

The present paper offers a dual 14CO2 breath test approach to study the metabolic utilization of free amino acids in the body. Using the carboxyl-[14C]isotopomer of an amino acid as the test substrate the percentage recovery of the isotope as 14CO2 reflects which part of the labelled amino acid flux has been decarboxylated. The residual C fragments may flow to total oxidation at least to the level recovered for the universal [14C]isotopomer. In the case that recovery for total oxidation is less than for decarboxylation, part of the [14C]fragments are retained in the body by either exchange or non-oxidative pathways. Utilization of tyrosine and leucine was measured in the post-absorptive phase in adult rats, conditioned on isoenergetic diets containing 210, 75 or 0 g protein/kg. It was shown that the level of dietary protein exerts an influence on both decarboxylation and total oxidation. Although the responses of leucine and tyrosine were not different for total oxidation, there was a difference between the amino acids in their relative rate of decarboxylation. That this dual 14CO2 breath test approach can be used as a tool to evaluate whether the protein and amino acid supply has been adequate to support actual requirements is discussed.

Gel filtration in sodium dodecyl sulphate of hydrophobic muscle proteins on Sephacryl S-400 Superfine.

Gel filtration has been applied to muscle proteins in the presence of the detergent sodium dodecyl sulphate (SDS). This detergent not only solubilizes a variety of proteins but also increases their hydrodynamic volumes, which is a disadvantage for the process of gel filtration. Nevertheless, appropriate choice of gel type, in this case Sephacryl S-400 Superfine, and optimal elution conditions make SDS gel filtration a useful tool in the separation of heterogeneous protein mixtures in the molecular weight range of 20,000-200,000.

A study on the relative turnover of muscle proteins. Comparison of turnover rates in a single step procedure.

In order to enable a direct comparison of the turnover rates of all muscle proteins we introduce a single step procedure. This procedure is based on gel filtration in the presence of a detergent SDS (sodium dodecyl sulphate) which solubilises all muscle proteins. Although the separation is still very crude, some preliminary conclusions may be drawn abut the relative turnover rates of myofibrillar proteins. In our opinion it is unlikely that the myofibril turns over as a unit.

Role of calcium in exocrine pancreatic secreation. VI. Characteristics of the paracellular pathway for divalent cations.

(1) The transepithelial permeability for Ca2+ and Mg2+ in the isolated rabbit pancreas has been studied. (2) Values for the permeability of the unstimulated pancreas were obtained either by adding radioactive tracers to the bathing medium and measuring their concentration in the secreted fluid under steady-state conditions, or by analysis of the Ca2+ and Mg2+ concentrations in the secreted fluid after correction for protein-bound divalent cations. (3) Both methods give almost the same results: 27 and 26% for Ca2+ and 21 and 18% for Mg2+, respectively; both values being expressed as the percentage of the concentrations in the bathing medium. (4) The amounts of Ca2+ and Mg2+, appearing in the secretory fluid after correction for protein-bound cations, are linearly related to the extracellular Ca2+ and Mg2+ concentrations in the bathing medium, which indicates passive permeation. The two cations appear to pass through the paracellular route in their hydrated form. (5) Stimulation with carbachol or pancreozymin causes an increase in the paracellular permeability. This increase is approximately equal for the two divalent cations. Its time dependence and magnitude depend on the concentration of the stimulant rather than on the type of stimulant.

Role of calcium in exocrine pancreatic secretion. III. Comparison of calcium and magnesium movements in rabbit pancreas.

1. Calcium and magnesium movements in the isolated rabbit pancreas and in rabbit pancreas fragments are compared in a qualitative and quantitative way. 2. At the basal secretion rate calcium and magnesium are present in the secreted fluid in concentrations of about 30% of their concentrations in the bathing medium. 3. Addition of 10(-6) M carbachol to the bathing medium results in enzyme secretion accompanied by calcium and magnesium release, in divalent cation-free medium as well as in a complete medium. 4. The secretion of each divalent cation is the sum of two components: an extracellular flux and a flux of protein-associated cations, the so-called secretory flux. 5. The extracellular flux is proportional to the concentration of the divalent cation in the bathing medium. The secretory flux is not dependent on the presence of the divalent cation in the bathing medium, but is proportional to the amount of protein secreted. About 25 nmol of each cation is secreted per mg protein. 6. Ca2+ and Mg2+ can be nearly completely separated from the digestive enzymes by gel filtration. They equilibrate completely with their radioactive isotopes added to the sample just before elution, indicating that the cations are rapidly exchangeable after secretion. 7. Efflux studies on rabbit pancreas fragments, pre-loaded with 45Ca2+, show a carbachol-stimulated 45Ca2+ efflux (the stimulatory flux) in addition to a release of amylase. Fragments pre-loaded with 28Mg2+ do not show carbachol stimulation of the tracer efflux. 8. These studies indicate that calcium and magnesium behave quite similarly with respect to the extracellular and secretory fluxes. The absence of a stimulatory flux for magnesium, suggests that the increase of the cytoplasmic calcium concentration plays a specific role in the stimulus-secretion coupling of pancreatic enzyme secretion.

Role of calcium in exocrine pancreatic secretion. II. Comparison of the effects of carbachol and the inophore A-23187 on enzyme secretion and calcium movements in rabbit pancreas.

1. The secretory effects of carbachol and the ionophore A-23187 on the isolated rabbit pancreas and rabbit pancreas fragments are compared in order to obtain more insight in the involvement of calcium in the stimulus-secretion coupling of pancreatic enzyme secretion. 2. The divalent cation ionophore A-23187 mimicks the effect of carbachol on pancreatic enzyme secretion in both preparations. 3. The action of the ionophore is dependent on the presence of extracellular calcium. The carbachol effect is much less dependent on calcium, as it occurs even in a Ca2+ -free medium containing 10(-4) M ethyleneglycol-bis(beta-aminoethylether)-N,N-tetraacetic acid. 4. Carbachol causes a marked increase in the 45Ca2+ efflux from pre-loaded pancreas fragments in both a normal Krebs-Ringer bicarbonate medium and this Ca2+ -free medium. 5. Although the tissue still contains about 50% of its original 45Ca2+ content, at the time of stimulation, the ionophore has little or no effect on the 45Ca2+ efflux. This indicates that the cytoplasmic 45Ca2+ concentration is very low, and hence that most of the 45Ca2+ must be sequestered in one or more intracellular stores. 6. It is concluded that both substances stimulate pancreatic enzyme secretion by increasing the cytoplasmic calcium concentration, through an increase in the calcium permeability of the plasma membrane in the case of the ionophore, and through a release of Ca2+ from intracellular stores in the case of carbachol.

Role of calcium in exocrine pancreatic secretion. I. Calcium movements in the rabbit pancreas.

1. Calcium movements in the isolated rabbit pancreas and in rabbit pancreas fragments have been studied with the aid of 4 5 Ca2+. 2. Addition of 4 5 Ca2+ to the incubation medium of the isolated rabbit pancreas results in an immediate appearance of isotope in the secreted fluid reaching a constant specific activity in 30 min. The absolute activity in the secreted fluid is 30-40% of that in the incubation medium. 3. Addition of 10(-5) M carbachol after 2 h preincubation with 4 5 Ca2+ results in enzyme secretion accompanied by calcium release. There is also an increase in 4 5 Ca2+ secretion, but this is maximal 10 min after the protein and total calcium peaks. 4. Partial removal of 4 5 Ca2+ from the bathing medium, before stimulation, reduces the increase in 4 5 Ca2+ secretion nearly proportionally. 5. [3H]Mannitol, added to the bathing medium, appears in the secreted fluid and behaves upon carbachol stimulation similarly to 4 5 Ca2+. 6. Upon repeated stimulation with 10(-5) M acetylcholine, a 4 5 Ca2+ peak appears, even in virtual absence of enzyme secretion. In this case the peak coincides with a small total calcium peak. 7. Efflux studies of rabbit pancreas fragments, preloaded with 4 5 Ca2+, show a carbachol-stimulated 4 5 Ca2+ efflux in addition to a release of amylase. 8. These studies indicate that there are three calcium movements in rabbit pancreas which can all be influenced by cholinergic agents: (a) an extracellular route for calcium and other small molecules and ions; (b) a calcium release across the apical membrane along with the enzymes, originating from a pool which does not freely exchange with 4 5 Ca2+ in the bath; (c) a calcium flux across the serosal membrane, which involves calcium exchanging freely with 4 5 Ca2+ from the bath. The third flux is thought to result from an increase in cytoplasmic calcium, which may be involved in the stimulus-secretion coupling of pancreatic enzyme secretion.