Mixture Of Free Amino Acids Research Articles

Separation of enantiomers in thin-layer chromatography

Up to now in thin-layer chromatography a series of retention mechanisms for the determination of enantiomeric compositions have been applied. Subject of this paper is a corresponding survey with a focal point on ligand exchange chromatography. In connection with this retention mechanism most successfully used in TLC for the separation of optical antipodes, some additional findings of the authors are included. Retention mechanisms in question are: - Formation of inclusion complexes within the cavities of cyclodextrins. Substance classes separated by this system are e.g. dansyl amino acids or metallocenes. - Interaction with so-called “brush type” phases. In this case, chiral groups with π-acceptor of π-donor properties are chemically or physically bonded to an aminomodified silica gel matrix. Chiral compounds, like aromatic alcohols, were separated on these stationary phases. - Differences in retention of diastereomers formed previously or during the chromatographic process on an achiral stationary phase. Some examples for the manifold applicability of this principle are the separation of chiral amines from biological matrices, hydroxy-fatty acids, and amino acids. - Selective interactions with the helical structures of cellulose or cellulose triacetate. Referring to this technique, separations of several amino acids and of the analgesic agent Oxidanac in the R-and S-species are described. - Enantioselective ligand exchange, based on reversed phase silica gels impregnated with copper salts and optically active amino acid derivatives. In thin-layer chromatography this is the most important principle used mainly for the separation of racemic mixtures of free amino acids and amino acid derivatives. The recently developed HPTLC pre-coated plate CHIR with concentrating zone is also based on this retention mechanism. Aside from the applications mentioned above, dipeptides, hydroxy-carboxylic acids, α-halogenated carboxylic acids, amino lactones, and thiazolidine derivatives have been separated on this plate up to now. Furthermore, this new pre-coated layer is particularly suitable for an optical in situ evaluation and quantitative determinations of optical antipodes.

Folate Deficiency in Rats Fed Diets Containing Free Amino Acids or Intact Proteins

Development of folate deficiency (FD) was evaluated in weanling rats fed diets containing mixtures of free amino acids or of vitamin-free casein and gelatin as sources of dietary nitrogen. FD could be produced in 21 d with amino acid diets that promoted maximum growth rate, were completely devoid of folate and contained 1% succinylsulfathiazole. Growth retardation and blood dyscrasia associated with FD could not be demonstrated in rats fed diets containing casein and gelatin as nitrogen sources because the vitamin-free casein contained low but measurable levels of folate. The most effective protocol to produce experimental FD in rats is to feed a folate-free diet that otherwise supports maximum growth in young animals. Additional modifications such as use of methotrexate or amino acid-imbalanced or protein-deficient diets are unnecessary.

Amino acid absorption and production of pancreatic hormones in non-anaesthetized pigs after duodenal infusions of a milk enzymic hydrolysate or of free amino acids.

1. Six non-anaesthetized pigs (mean body-weight 57.0 kg) were used to study the intestinal absorption of amino acids (AA) from either an enzymic hydrolysate of milk (PEP) containing a large percentage of small peptides (about 50% with less than five AA residues) and very few free AA (8%), or from a mixture of free AA with an identical pattern (AAL) infused intraduodenally in one of two amounts (55 or 110 g). Concomitant insulin and glucagon production rates were estimated. 2. Each pig was previously fitted, under anaesthesia, with an electromagnetic flow probe around the portal vein, with permanent catheters in the portal vein, the carotid artery and the duodenum. Each infusion was performed after an 18 h fasting period and each pig received each infusion. The observation period lasted for 5 h. 3. The absorption of AA was greater, more rapid and more homogeneous after PEP infusion than after AAL infusion, independent of the amount infused. 4. For the majority of AA considered individually, the absorption coefficient was higher after infusion of PEP than after that of AAL. The exceptions were methionine with a higher absorption coefficient after AAL infusion, and isoleucine, aspartic acid + asparagine and glutamic acid + glutamine with identical coefficients for both infusions. 5. Some AA, such as asparagine, ornithine, citrulline and taurine, while absent in the infusates, appeared in the portal vein in appreciable amounts after the infusion of both solutions. While a small proportion of taurine may arise from recycling of taurine-containing bile salts, it seems that the gut wall is able to synthesize all four AA. 6. Insulin production did not differ according to the nature or amount of solutions infused. Glucagon production was greater after PEP infusion.

New results concerning the breakdown of wool by moth and beetle larvae

Some insect larvae have a remarkable ability to digest and utilise keratin, i.e. highly cross‐linked protein material. Extensive investigations about the digestive mechanism of these insects have already been carried out but the actual course of keratin digestion by the insect pests is still under discussion. Investigations of moth and beetle excreta by amino acid analysis and polyacrylamide gel electrophoresis should shed more light on this subject. The present paper describes such comparative studies of larval faeces excreted after feeding with non‐mothproofed and mothproofed wool, using low concentrations of two stomach poisons as moth‐and beetle‐proofing agents. It has been found by amino acid analysis that all excreta examined contained a mixture of peptides and free amino acids. Most of the moth excreta were rich in free cystine and histidine whereas the beetle excreta contained much free cystine and arginine. Only the brown house moth (Hofmannophila) proved exceptional in this respect. Gel electrophoretic analysis of excreta proteins showed a different pattern of breakdown of wool proteins by the principal wool pests. The electrophoretic patterns permitted a differentiation between the types of larvae.

Effects of a Protein-Free Diet on the Rat Stomach

The effects of a protein-free diet containing a mixture of free amino acids on the stomach were studied using male Wistar strain rats. In the forestomach, thickening of mucosa and hyperkeratosis appeared and papillomas frequently developed, but they did not occur in the control rats fed a semipurified diet. Mucous neck cells increased and parietal cells and chief cells decreased in the fundus. Goblet cells occurred in the antral glands, and intestinal metaplastic glands developed in the late stage of the experiment. The number of deoxyribonucleic acid-synthesizing cells in the glandular stomach was significantly higher than that of the control. Moreover, in the antrum, somatostatin-producing cells increased in the early stage and gastrin-producing cells decreased in the late stage. These results suggest that dietary protein might play a crucial role in the differentiation of gastric epithelium and the development of intestinal metaplasia in the stomach.

卵白酵素分解物の栄養価に関する研究 Ｉ 卵白食，卵白酵素分解物または遊離アミノ酸混合食を投与した幼若ラットの成長の比較

卵白酵素分解物の栄養価に関する研究 Ｉ 卵白食，卵白酵素分解物または遊離アミノ酸混合食を投与した幼若ラットの成長の比較

Peptic hydrolysis of bovine .BETA.-lactoglobulin to produce a low-phenylalanine peptide foodstuff for phenylketonuria.

The peptic hydrolysis of bovine β-lactoglobulin (β-Lg) was performed to establish the basis for producing a low-phenylalanine peptide rather than a free amino acid mixture for use in the dietetics of phenylketonuria. A 1% β-Lg solution (pH 1.5) was incubated with 0.01% pepsin at 37°C for 24 hr. The peptides produced were fractionated by high-performance liquid chromatograhy to analyze their constituent amino acids. Most of the major peptides were identified in the light of the primary structure of α-Lg to assign 31 cutting points in their protein molecule. These included cutting points at the carboxyl side of Phe-82, Phe-105 and Phe-136. This result suggests that further hydrolysis of the peptic hydrolysate of β-Lg with an exopeptidase, particularly with a carboxypeptidase, would be effective in liberating phenylalanine to produce a low-phenylalanine peptide mixture.

Influence of dietary protein concentration on the oxidation of phenylalanine by the young pig.

1. Piglets were weaned at 3 d of age and reared to 2.5 kg on a liquid diet in which the protein was supplied by dried skim milk and a mixture of free amino acids. The oxidation of L-[1-14C]phenylalanine was measured as an indication of the partition of amino acids between retention and catabolism in pigs (2.5 kg) offered meals containing varied concentrations of crude protein (nitrogen x 6.25). 2. The dietary protein concentration was varied either by increasing the inclusion of a mixture of free amino acids in a series of diets containing 100 g protein/kg from skim milk, or by increasing the level of inclusion of the skim milk in a series of diets containing the equivalent of 100 g protein/kg from the free amino acid mixture. 3. The oxidation of phenylalanine was minimized by dietary protein concentrations of 240 and 258 g/kg for the diets containing increasing concentrations of free amino acids or skim milk respectively. 4. These results show that a mixture of free amino acids is used more effectively than intact protein for promoting retention of essential amino acids. 5. The recovery of radioactivity in expired carbon dioxide was inversely related to the recovery of radioactivity in liver tissue when the concentration of dietary crude protein was increased from deficient to adequate, demonstrating that the fractional oxidation of the indicator amino acid was inversely related to protein synthesis.

Proline as an essential amino acid for the young pig.

1. The catabolism of L-[1-14C]phenylalanine was used to indicate the effects of single amino acid supplementation of an inadequate protein diet (200 g crude protein (nitrogen x 6.25)/kg) on the utilization of dietary amino acids in pigs of 2.5 kg body-weight reared on an adequate protein diet (240 g crude protein/kg) containing skim milk and a mixture of free amino acids. 2. The oxidation of phenylalanine was decreased by the addition of proline or arginine to the inadequate protein diet but not by the addition of threonine, methionine, lysine or a mixture of essential amino acids, indicating that proline and arginine were limiting the utilization of dietary amino acids in the inadequate protein diet. 3. Dietary proline concentrations of 13.9 and 14.2 g/kg minimized phenylalanine oxidation in diets containing 200 or 260 g protein/kg. This indicates a dietary proline requirement of 14 g/kg. 4. Increasing the dietary arginine concentration in a diet containing 240 g protein/kg showed that an arginine concentration of 5.1 g/kg minimized phenylalanine oxidation. However, increasing the arginine concentration in a diet containing 200 g protein/kg increased phenylalanine oxidation, suggesting an amino acid imbalance involving arginine at this lower level of protein.

Kinetics and Characteristics of Absorption From an Equimolar Mixture of 12 Glycyl-Dipeptides in Human Jejunum

We have investigated rates of dipeptide disappearance during jejunal perfusion with an equimolar mixture of 12 glycyl-dipeptides and compared amino acid absorption rates from this mixture with those from the corresponding free amino acid mixture in healthy volunteers. All dipeptides contained glycine in the N-terminal position, which was designed to favor peptide absorption rather than hydrolysis. At 2 mM concentration there was little difference in disappearance among dipeptides, but at 6 mM there was some selectivity: Gly-Phe and Gly-Met exhibited the fastest, and Gly-Arg and Gly-His the slowest rates of disappearance. However, between these ends of the spectrum there were only modest differences in disappearance among dipeptides (Gly-Ala, Gly-Thr, Gly-Leu, Gly-Ile, Gly-Val, Gly-Lys, Gly-Pro, Gly-Trp). The amino acid absorption rates were generally smaller but more selective from the free amino acid than from the dipeptide mixture. We conclude that (a) the C-terminal amino acid residue influences intestinal assimilation of glycyl-dipeptides and (b) the considerably greater absorption rate of amino acids from the dipeptide than from the amino acid mixture appears to be the result of uptake by a system that has a greater transport capacity than amino acid carrier systems, thus minimizing competition among its substrates.

Influence of protein composition and hydrolysis method on intestinal absorption of protein in man.

An intestinal perfusion technique has been used in normal human subjects to investigate the influence that starter protein composition and hydrolysis procedure have on absorption of amino acid residues from partial enzymic hydrolysates of whole protein. Five starter proteins were studied. Three (egg albumin, casein/soy/lactalbumin, and lactalbumin) were hydrolysed by papain, a second lactalbumin starter protein, and a meat/soy/lactalbumin blend were hydrolysed by a porcine pancreatic enzyme system. Irrespective of starter protein composition or hydrolysis method used, four amino acid residues (threonine, glutamic acid, phenylalanine, and histidine) were absorbed significantly faster from all hydrolysates compared with absorption from their equivalent free amino acid mixtures. In contrast, both starter protein composition and hydrolysis method influenced absorption characteristics of up to nine other amino acid residues.

Relative nutritional value of whole protein, hydrolysed protein and free amino acids in man.

To compare their effects on nitrogen balance, diets containing either lactalbumin whole protein, its peptide-rich enzymic hydrolysate or an equivalent mixture of free amino acids as the sole source of dietary nitrogen were fed to two healthy subjects, each studied for 38 days on two separate occasions. The nitrogen intake (47 mg/kg body wt/day) induced a state of negative nitrogen balance, stimulating nitrogen conservation. Net daily nitrogen balance (mean +/- SD) in subject 1 was -0.23 +/- 0.72 g (amino acids) vs + 0.05 +/- 0.52 g (protein) and -0.21 +/- 0.58 g (amino acids) vs -0.05 +/- 0.57 g (hydrolysate), and in subject 2, -0.19 +/- 0.60 g (amino acids) vs -0.16 +/- 0.51 g (protein) and -0.42 +/- 0.35 g (amino acids) vs -0.62 +/- 0.34 g (hydrolysate). Analysis of these results by the cumulative sum technique showed no significant differences in the effect of the three nitrogen sources on nitrogen balance. This study indicates that there is no nutritional evidence to support the current practice of prescribing expensive enteral diets containing peptides or amino acids rather than the much cheaper whole protein to patients with normal gastrointestinal function.

Intestinal absorption of end products from digestion of carbohydrates and proteins in the pig.

The kinetics of appearance of various nutrients in the portal vein during the postprandial period was studied in conscious pigs by means of a technique based on measurement of the porto-arterial differences in nutrient concentrations simultaneously with that of the portal blood flow rate. The rate and level of appearance of sugars in the portal vein varied with the carbohydrate ingested. It was very rapid after intake of glucose and sucrose, slower after that of maize starch and very slow after that of lactose. The absorption of the latter became very rapid again if it was hydrolysed prior to its ingestion. During absorption, some sugars (fructose or galactose) released from the corresponding sucrose and lactose, respectively during digestion, were partly metabolized into glucose by the enterocyte. The rate of absorption of amino acids released in the digestive tract varied according to the origin of the food ingested, i.e. it was more rapid after intake of wheat or fish proteins than after that of barley. In the case of barley the absorption rate of amino acids differed from that of glucose of the starch. The profile of the amino acid mixtures appearing in the portal vein during absorption differed a little from the profiles of those present in the ingested proteins in the case of essential amino acids and differed much in the case of non essential amino acids. Some essential amino acids (histidine, aromatic amino acids) appeared more rapidly and others more slowly, (lysine, sulphur amino acids, arginine). Because of transaminations, only small amounts of glutamic acid occurred in the portal vein whereas the amounts of alanine as compared to those ingested, were very large. The hierarchy of amino acid absorption was the same whatever the protein studied (fish, wheat, barley). The appearance in the portal vein of alpha-amino nitrogen from enzyme hydrolysates perfused through the duodenum was more rapid than after perfusion of a mixture of free amino acids. During digestion of diets containing 6% cellulose, large amounts of volatile fatty acids (about 2200 mmol/24 h, i.e. 2500 kJ/24h) and lactic acid (60-85 g/24 h) appeared in the portal vein.

How can quantum mechanics of material evolution be possible?: Symmetry and symmetry-breaking in protobiological evolution

Material self-assembly as a self-organizing process is always accompanied by symmetry-breaking in the material configuration. Self-sequencing of amino acids during their thermal polymerization has lost a certain property of permutation symmetry that was observed in the mixture of free amino acids. The evolutionary precursor state is more symmetrical about its internal material configuration and more degenerate due to the multitude of the indistinguishable individuals. The evolution proceeds in the direction along which the degeneracy in the internal states dissolves owing to the symmetry-breaking originating in material flow equilibrium of open material aggregates. Protobiological information is latent in the material system which is highly symmetrical and highly degenerate in its internal states. Evolution of matter is an endogenous process in which the earlier symmetric property is lost and less degenerate states are approached. Quantum-mechanically, the generation of protobiological information is due to the symmetry-breaking of the Hamiltonian originating in the interaction with the exterior through material flow, in contrast to the Schrödinger equation which preserves a symmetry and the associated invariants.

PARTIAL REPLACEMENT OF SKIM MILK WITH FREE AMINO ACIDS IN DIETS FOR THE PIGLET

A semi-synthetic diet in which 60% of the nitrogen from dried skim milk was replaced with a mixture of free amino acids supported gains of 236 g/day between 3 and 18 days of age, as compared with a gain of 282 g/day for a control diet in which skim milk provided all the protein. Neutralizing the acidity of the free amino acid diet increased its palatability. All of the indispensible amino acids may be made deficient in the experimental diet, thus providing the opportunity to evaluate the amino acid requirements of the piglet. Key words: Piglets, growth, amino acids, semi-synthetic diet

Amino acid and dipeptide absorption in rats fed chemically defined diets of differing fiber composition.

In rats chronically fed a fiber-free diet or one of the three nutritionally equivalent fiber-containing diets, in vivo jejunal absorption of L-leucine from a free amino acid mixture of L-leucine and glycine as well as equimolar solutions of the dipeptides, L-leucyl-glycine and glycyl-L-leucine, were compared. In addition, total and brush border amino peptidase activities for the two dipeptide substrates were examined in small bowel segments. With two of three fiber diet groups, absorption of L-leucine from the free amino acid solution was reduced without a detectable change in peptide transport or aminopeptidase activities. This investigation provides evidence that peptide transport mechanisms are relatively spared with long-term feeding of fiber-containing diets similar to observations recorded in disease states associated with protein-energy malnutrition.

Studies on the absorption of ‘Small peptides’ and free amino acids in elemental diets

Previous studies have indicated that small peptides (di- or tri-peptides) are more rapidly absorbed from the small intestines than free amino acid mixtures. We made a practical elemental diet (ED) in which small peptides were used as the nitrogen source, and studied the absorption of nitrogen using canine Thiry-Vella loop in comparison with ED prepared with a mixture of amino acids with the same composition, as the nitrogen source. The absorption of nitrogen was significantly high in ED with small peptides (36.8±2.2%) compared to ED with free amino acids (27.3±2.5%). The result suggested that small peptides are suitable for use as the nitrogen source for ED.

Amino acid oxidation by young pigs receiving diets with varying levels of sulphur amino acids.

Piglets were weaned at 3 d of age and were introduced to liquid diets in which 400 g/kg protein was supplied as skim-milk powder and the balance as a mixture of free amino acids. The skim milk contributed 2.3 g methionine and 1.4 g cystine/kg diet; the experimental diets were made by supplementing these levels with free amino acids. The adequacy of the test level of the amino acid in the diet was assessed by measuring the oxidation of [1-14C]phenylalanine as an indicator of the partition of the essential amino acids between incorporation into protein and degradation. Radioactivity recovered as carbon dioxide was used as a measure of catabolism. Addition of 0.2, 0.4, 0.7, 1.2 and 1.7 g L-methionine/kg to a diet containing 5 g cystine and 3 g choline chloride/kg showed that phenylalanine catabolism was minimal for the diet with 2.7 g methionine/kg indicating that this is the dietary requirement for methionine to serve as a source of methionine residues for protein synthesis. Addition of D-methionine to produce a series of diets with graded levels of methionine showed that the D-isomer was less effective than the L-isomer in reducing phenylalanine catabolism: the addition of 0.8 g D-methionine/kg diet was needed to produce the same effect as 0.4 g L-methionine/kg diet showing that the replacement value of D-methionine for L-methionine was 50% in the young pig.(ABSTRACT TRUNCATED AT 250 WORDS)

Oxidation of an indicator amino acid by young pigs receiving diets with varying levels of lysine or threonine, and an assessment of amino acid requirements.

The catabolism of [14C]phenylalanine was used to indicate the effects of varying the dietary level of lysine and threonine on the retention of dietary amino acids by 2-week-old pigs receiving diets containing skim milk and a mixture of free amino acids. Reducing the dietary level of lysine from 16 to 12 g/kg had no influence on phenylalanine oxidation, reducing the lysine level from 12 to 11 then to 10 g/kg caused an almost linear increase in phenylalanine oxidation whereas further reduction to 9 or 8 g/kg resulted in a less-marked increase in phenylalanine oxidation. This showed that 12 g lysine/kg was required to maximize amino acid retention and indicated that lysine was conserved more effectively at low dietary concentrations than at dietary concentrations approaching the requirement. Reducing the dietary level of threonine from 8 to 6 g/kg had no influence on phenylalanine oxidation, whereas further reduction to 4 g/kg caused a linear increase in phenylalanine catabolism showing that 6 g threonine/kg was required to maximize amino acid retention. Reduction of the levels of lysine, threonine and methionine from the generous levels characteristic of a diet containing 240 g protein from skim milk/kg, to the requirement levels determined separately in the presence of the generous levels of all the other amino acids, resulted in a twofold increase in phenylalanine catabolism. This shows that the pig seems able to conserve limiting intakes of a single amino acid, but not if the intakes of two or three amino acids are limiting.

Determination of amino acid requirements of young pigs using an indicator amino acid.

Mixtures of skim milk and free amino acids were compared as diets for pigs which would allow manipulation of dietary amino acid levels. Piglets gained 208 g/d between 3 and 14 d of age on the skim-milk diet, but replacement of 600 g/kg of the dietary nitrogen with free amino acids reduced growth rate to 148 g/d. Supplementation of a lysine-deficient diet with lysine reduced the catabolism of [14C]phenylalanine showing that phenylalanine catabolism could be used as an indicator of the adequacy of diet with respect to another essential amino acid. The dietary level of phenylalanine which would provide an excess for catabolism by the piglet was estimated directly by measuring the influence of dietary phenylalanine level on [14C]phenylalanine oxidation. Reduction of the dietary phenylalanine level below 7 g/kg had no effect on phenylalanine oxidation, whereas increasing the dietary phenylalanine level above 7 g/kg resulted in a linear increase in phenylalanine oxidation. An indirect estimate of histidine requirement was made by examining the influence of dietary histidine level on [14C]phenylalanine oxidation. In diets containing more than 4 g histidine/kg, phenylalanine oxidation was minimal. In diets containing less than 4 g histidine/kg, [14C]phenylalanine oxidation increased as the level of dietary histidine was reduced. This showed that the utilization of the essential amino acid phenylalanine, for protein synthesis, was not limited by histidine supply in diets containing more than 4 g histidine/kg. A direct estimate of histidine requirement was made by examining the influence of dietary histidine level on [14C]histidine oxidation. Diets with more than 4 g histidine/kg contained an excess which was catabolized: there was a linear increase in histidine oxidation in response to dietary histidine levels greater than 4 g/kg. This confirmed the previous indirect estimate of histidine requirement.