Total Amino Acid Release Research Articles

Whey-based sports supplements: Heat damage and protein breakdown after in vitro gastrointestinal digestion

This study was aimed to evaluate the effect of heat damage on the release of total amino acids (AA), essential AA (EAA), branched-chain AA (BCAA) and bioactive peptides following in vitro static simulated gastrointestinal digestion (SGID) of four commercial whey-protein based sports supplements. The extent of protein glycation and denaturation was evaluated through the determination of the content of furosine and soluble whey proteins. The strongest protein breakdown (41.3 %) and the highest release of AA, EAA and BCAA (36.20, 27.78, and 11.30 g/100 g protein, respectively) was observed in the sports supplement characterised by the lowest (52.5 %) level of soluble whey proteins; whereas the protein glycation had a negligible impact on the studied parameters. The SGID also led to the release of several peptides with various reported bioactivities that may be beneficial to sports activity.

248 Maternal Nutrient Restriction During Mid-gestation Decreases Uteroplacental Release and Fetal Uptake of Essential Amino Acids in Sheep

Abstract To examine the effects of maternal nutrient restriction on net uteroplacental flux during mid-gestation, 14 singleton ewes (48.2 ± 4.0 kg body weight) were fed 100% (control; CON; n = 7) or 60% of nutrient requirements (restricted; RES; n = 7) from day 50–90 (mid-gestation). On day 90, uteroplacental blood flow was measured via Doppler ultrasonography and blood samples were collected from the femoral artery, uterine vein, umbilical artery, and umbilical vein. Blood vessel glucose and amino acids (AA) concentrations were measured and arterial-venous (uterine, AV; fetal, va) differences and net fluxes were calculated. Data were analyzed using the GLM procedure of SAS for effects of treatment. Nutrient restriction during mid-gestation did not influence (P ≥ 0.17) uterine or umbilical blood flows. Uterine AV and fetal va differences of total, essential, and nonessential AA were not influenced (P > 0.10) by nutrient restriction. Nutrient restriction decreased (P ≤ 0.05) uterine and uteroplacental release of total AA and tended to decrease (P = 0.07) total AA uptake by the fetus. Uteroplacental release and fetal uptake of essential AA were decreased (P = 0.03) with RES by 53.4% and 45%, respectively. Uterine and uteroplacental release of nonessential AA were decreased (P = 0.03) with RES but, fetal uptake was not affected (P = 0.14). Nutrient restriction decreased (P ≤ 0.04) fetal uptake of methionine, phenylalanine, threonine, and valine and tended to decrease (P ≤ 0.10) fetal uptake of isoleucine, leucine, and tryptophan. Umbilical artery glucose concentrations were 32% lesser (P = 0.01) with RES and RES tended to increase (P = 0.08) fetal glucose uptake. Nutrient restriction during mid-gestation altered uteroplacental and fetal flux of AA in the current study. The results may indicate that fetal metabolism shifts to adapt to reduced AA supply which results in greater glucose utilization.

Amino acids release from enriched bread with edible insect or pea protein during in vitro gastrointestinal digestion

The aim of this study was to investigate the amino acid (AA) release from breads enrichment with edible insects, Alphitobius diaperinus and Tenebrio molitor or pea protein during in vitro gastrointestinal digestion. Bread was enriched at 5 and 10% with insect flour or pea protein. Enriched and control breads were subjected to standardised static in vitro gastrointestinal digestion. The free AAs of breads before and after each phase of digestion (gastric, intestinal and at the end of digestion) were determined by HPLC. During digestion, the highest AA release from breads occurred in the intestinal phase. Using pea protein, Alphitobius diaperinus, and Tenebrio molitor powder at any level assayed presented a significantly higher value of total free AA than the control, accessible for body absorption. There is an effect of enrichment ingredient concentration (10 > 5%) in bread on total AA release after in vitro gastrointestinal digestion. Higher protein enrichment induced higher realease of AA during the digestion.

Caracterización y mejoramiento de la harina de Cannavalia ensiformis como alimento balanceado para Oreochromis niloticus

Background. The legume Cannavalia ensiformis is an excellent source of energy, protein, vitamins and minerals to be used in animal production; however, it contains antinutritional factors (ANF), which limit its use for fishes. Goals. In this study 7 flours obtained from the processes of hydration, acid extraction, decorticating, cooking, germination, autoclaving and degreasing of the seeds of C. ensiformis were evaluated as means of elimination of ANF. Methods. The nutritional values of the meals were corroborated with proximal chemical analysis and in vitro digestibility, determining the degree of hydrolysis (GH%) of the flours of C. ensiformis obtained by pH STAT, the release of total amino acids (TAAL, μg mL-1) was calculated, using multienzymatic extracts of stomach and intestine of O.niloticus juveniles. Results. The acidic/alkaline GH values for cooked C. ensiformis flour were 0.76 ± 0.01% / 6.04 ± 0.37%, being significantly higher with respect to the other flours. The values of acidic/alkaline TAAL (mg mL-1) of this cooked flour (0.02 ± 0.006 / 0.40 ± 0.02) were significantly higher in the alkaline phase in relation to the other treatments. Conclusion. We detected that cooked meal of C. ensiformis, allow to be used as a source of protein in diets for Oreochromis niloticus.

In vitro protein digestibility of different grow-out stages of spotted rose snapper (Lutjanus guttatus, Steindachner, 1869)

The aim of this study was to compare in vitro protein digestibility between two groups of fish, at early (21 g) and late stages (400 g) of spotted rose snapper Lutjanus guttatus, to evaluate the degree of hydrolysis (DH) and total amino acid release (TAAR) using crude extracts from stomach, pyloric caeca and intestine of 13 protein ingredients including marine, animal and plant meals. Degree of hydrolysis and TAAR were measured by a pH-Stat method, and the PAGE-Zymogram was also used as complementary technique. Differences in DH were found between both grow-out stages mainly in the alkaline hydrolysis phase. Fish and squid meals (marine sources) had the highest DH and TAAR, followed by porcine meat and poultry meal by-products from recycling sources, and soybean and canola meals (plant sources), which represent better protein sources for use in practical diets. Stomach zymograms showed two pepsin isoforms in both grow-out stages. Pyloric caeca and intestine zymograms showed five bands with proteolytic activity in the early grow-out stage, whereas four additional bands were found in late grow-out stage. Alkaline proteases were identified as serine and metalloproteases. Thus, L. guttatus presents an ontogenetically differentiated digestive enzyme pattern that modifies the DH and TAAR of different protein sources.

Debriding Effect of Bromelain on Firearm Wounds in Pigs

Wound excision is the standard treatment for firearm wounds. However, achieving a satisfactory curative effect is difficult because of the traumatic mechanism of high-velocity projectiles. We propose a new therapy by using topical bromelain as a supplement to wound incision for the debridement of firearm wounds. We clarified the debriding effect of bromelain on firearm wounds in pigs. In vitro, muscle tissues around the wound track and normal muscle were incubated in bromelain solutions of different concentrations. Tissue hydrolization was estimated by measuring tissue weight and the release of total amino acids. In vivo, the hind limbs of 15 pigs were wounded with high-velocity projectiles. Five groups were classified as follows: wound excision (E), wound incision (I), bromelain (B), incision + bromelain (IB), and control (C). Debriding effectiveness was estimated using bacterial content, histopathologic examination, and wound healing time. In vitro, hydrolization of wound tissue was significantly more intensive than that of normal tissue. Bromelain solution (10 mg/mL) hydrolyzed wound tissue rapidly with minimal proteolysis of normal tissue. In vivo, the wound-track bacterial content of group IB was similar to that of group E and was significantly lower than that of groups I, B, and C. The wound healing time of group IB was also shorter. Bromelain is effective in the debridement of uncomplicated firearm wounds if used as a supplement to simple wound incision. This new therapy shows notable advantages over conventional surgical debridement as it greatly simplifies the procedures.

Carbohydrate metabolism during prolonged exercise and recovery: interactions between pyruvate dehydrogenase, fatty acids, and amino acids

During prolonged exercise, carbohydrate oxidation may result from decreased pyruvate production and increased fatty acid supply and ultimately lead to reduced pyruvate dehydrogenase (PDH) activity. Pyruvate also interacts with the amino acids alanine, glutamine, and glutamate, whereby the decline in pyruvate production could affect tricarboxycylic acid cycle flux as well as gluconeogenesis. To enhance our understanding of these interactions, we studied the time course of changes in substrate utilization in six men who cycled at 44+/-1% peak oxygen consumption (mean+/-SE) until exhaustion (exhaustion at 3 h 23 min+/-11 min). Femoral arterial and venous blood, blood flow measurements, and muscle samples were obtained hourly during exercise and recovery (3 h). Carbohydrate oxidation peaked at 30 min of exercise and subsequently decreased for the remainder of the exercise bout (P<0.05). PDH activity peaked at 2 h of exercise, whereas pyruvate production peaked at 1 h of exercise and was reduced (approximately 30%) thereafter, suggesting that pyruvate availability primarily accounted for reduced carbohydrate oxidation. Increased free fatty acid uptake (P<0.05) was also associated with decreasing PDH activity (P<0.05) and increased PDH kinase 4 mRNA (P<0.05) during exercise and recovery. At 1 h of exercise, pyruvate production was greatest and was closely linked to glutamate, which was the predominant amino acid taken up during exercise and recovery. Alanine and glutamine were also associated with pyruvate metabolism, and they comprised approximately 68% of total amino-acid release during exercise and recovery. Thus reduced pyruvate production was primarily associated with reduced carbohydrate oxidation, whereas the greatest production of pyruvate was related to glutamate, glutamine, and alanine metabolism in early exercise.

TNF-alpha has no direct in vivo metabolic effect on human muscle.

Tumor necrosis factor alpha (TNF-alpha) is thought to have a key role in metabolic changes of muscle tissue during inflammatory diseases. It is unknown whether TNF-alpha affects muscle metabolism directly or whether these changes are mediated by secondary mediators. We studied 6 patients undergoing isolated limb perfusion with TNF-alpha for irresectable soft-tissue sarcoma or in-transit melanomas. Glucose, lactate, ammonia and amino-acid consumption or production were measured in the perfusate during 3 perfusion periods: before, after TNF-alpha and after the combined administration of TNF alpha and melphalan. Arterial glucose, lactate, ammonia and amino-acid concentrations were monitored to detect metabolic effects of TNF-alpha after it entered the systemic circulation. Glucose uptake and lactate release by the limb remained unchanged after the injection of TNF-alpha alone, as well as after the combination of TNF-alpha and melphalan. Furthermore, glutamine, alanine, phenylalanine, tyrosine and total amino-acid release into the perfusate did not increase during TNF-alpha and melphalan treatment, indicating that muscle metabolism was not changed. After the isolated limb perfusion, TNF-alpha entered the systemic circulation and induced metabolic changes resulting in a doubling of arterial lactate concentrations, decreased arterial glucose concentrations and decreased arterial amino-acid concentrations. Our study shows that regional administration of TNF-alpha alone or in combination with melphalan does not directly affect muscle glucose and protein metabolism. The data suggest that systemic metabolic changes induced by TNF-alpha are mediated through secondary, centrally produced, factors.

Electrical stimulation and amino acid and ammonia metabolism in the canine gastrocnemius muscle.

This study examined the effects of electrical stimulation on amino acid and ammonia (NH3) metabolism in the isolated in situ canine gastrocnemius muscle preparation. Cut sciatic nerves of 10 mongrel dogs were stimulated at either 3 or 5 twitches/s (10 V, 0.2-ms duration) for 60 min. Muscle NH3 release dramatically increased on stimulation, and over 60 min the 3- and 5-Hz groups released 86.7 +/- 24.2 vs. 160.8 +/- 17.4 mumol.min-1.100 g-1 (P < 0.05) of NH3, respectively. Similarly, the intramuscular NH3 concentration was elevated (P < 0.05) above rest for both groups throughout stimulation, and it was higher (P < 0.05) at 5 min for the 5-Hz (82.7 +/- 2.4 mumol/100 g wet wt) than for the 3-Hz (67.4 +/- 7.4 mumol/100 g wet wt) group. Stimulation was also characterized by a large release of amino acids by both groups. The total amino acid release for 60 min was 415.4 +/- 64.9 vs. 193.3 +/- 56.2 (P < 0.05) mumol/100 g for the 3- and 5-Hz groups, respectively. However, there were no shifts or differences between groups in the intramuscular total amino acid pools. Glutamine (Gln) and alanine (Ala) dominated the amino acids released by muscle and together represented 35 and 46% of the total amino acids released over 60 min for the 3- and 5-Hz groups, respectively. The total release of Gln was higher (P < 0.05) for the 3-Hz (81.1 +/- 5.6 mumol/100 g) than for the 5-Hz (49.4 +/- 10.7 mumol/100 g) group, but there were no differences between groups in total Ala release. In contrast, both groups demonstrated an uptake of branched-chain amino acids (valine, isoleucine, and leucine) after 45 min of stimulation. These data show a stimulation-dependent production of NH3 and release of amino acid by the canine gastrocnemius muscle. These data further show that the degree of net muscle NH3 production is proportional to the frequency, whereas the degree of amino acid release is an inverse function of frequency.

Leg Metabolism of Amino Acids and Ammonia in Patients with Chronic Renal Failure

Leg metabolism of amino acids and ammonia in the postabsorptive state was evaluated in 10 patients with chronic renal failure (CRF) and in 10 patients with normal renal function (controls) by measuring the arterial-femoral venous (A-FV) differences for free amino acids and ammonia. Total amino acid release from the leg and alanine and glutamine release, which accounts for the greatest amount of the total amino acid release, are similar in patients and controls. Total amino acid uptake from the arterial blood and glutamate uptake, which is the amino acid extracted at the highest rate, are comparable in both groups. Taken together these data, in addition to the similarity of A-FV differences for proteolytic markers, namely tyrosine, phenylalanine and histidine, suggest that protein breakdown in peripheral tissues is not increased in patients with CRF. In CRF selective metabolic abnormalities for some amino acids are evident. Whilst only the A-FV differences for valine, leucine and isoleucine are decreased, additional alterations are observed by relating the A-FV difference for each amino acid to that of proteolytic markers. Such a procedure demonstrates that in CRF histidine release relative to that of proteolytic markers is reduced, whereas proline and arginine release is increased. In CRF the reduced release of some amino acids, mainly branched-chain amino acids, by the leg probably affects the pattern of circulating amino acids. Finally, both in patients and in controls a significant uptake of ammonia is observed; the ammonia uptake is related to arterial levels of this metabolite, confirming the role of peripheral tissues in removing ammonia from circulation.

Total Amino Acid Release Rates of Soluble and Insoluble Protein Fractions of Concentrate Feedstuffs by Streptomyces griseus

Streptomyces griseus (5mg) protease was incubated with increasing protein concentrations of casein, soybean meal, or corn gluten meal. Rates of amino acid release were 1.04, .72, and .36 μmoles of amino acid per hour with enzyme saturation at 7.5 and 8.5mg of casein and soybean meal protein. A ratio of protein to enzyme of 1.25 gave rates of release within saturation limits for protein sources differing in degradability. Partitioning of nitrogen fractions for 11 protein and 6 energy concentrate ingredients showed dehydrated alfalfa and corn gluten feed had largest proportions of soluble nitrogen and amino acids and least amount of potentially available insoluble protein compared to other protein ingredients. Incubation of Streptomyces griseus protease with soluble and insoluble protein fractions indicated that most feedstuff soluble protein fractions have amino acid release rates two to three times faster than insoluble protein fractions. Ratio of soluble to insoluble protein amino acid release were 1.2 for corn gluten meal and peanut meal and .5 for dehydrated alfalfa and corn gluten feed. Soluble protein fractions of certain protein feedstuffs may not be as available in the rumen as insoluble protein fractions.

Effect of heat treatment on true digestibility in the rat, in vitro proteolysis and available lysine content of cottonseed meal protein.

Effects of heating cottonseed meal (CSM) protein were quantitatively assessed by determination of true digestibility (TD), in vitro proteolysis, N solubility and fluorodinitrobenzene (FDNB) available lysine. Flaked, dehulled cottonseed was extracted with hexane and desolventized at 25 C, then autoclaved (121 C, 1.1 kg/cm(2)) for 0, 15, 30, 60, 90 or 120 minutes. Free gossypol was subsequently extracted, and TD was determined in weanling rats. Metabolic fecal N (the fecal N excreted by rats fed a basal diet containing 4% casein protein) was 1.84 +/- .10 mg N/g dry matter intake. TD and FDNB-available lysine (percentage of total) were 91 and 89%, respectively, in the unheated meal. TD and FDNB-available lysine were reduced to 84 and 78% after 60 min of autoclaving, and to 71 and 44% after 120 min of autoclaving. The effect of heat treatment on TD was described by the equation: % TD = 100 - 9.28e(.0096t) (r = .998), where t = minutes of autoclaving. This indicated an accelerated decline in TD as heating time increased. No more than 40% of the loss in FDNB-available lysine was attributable to gossypol binding. In vitro release of total amino acids from autoclaved CSM samples during pepsin-pancreatin incubations was highly correlated to TD (r = .996), but N solubility in .02 N NaOH was poorly correlated to TD. In samples of solvent-extracted and screw-pressed CSM, TD (estimated from pepsin-pancreatin incubations) ranged from 80 to 85% and FDNB-available lysine ranged from 73 to 85%, and both were only slightly lower in screw-pressed than in solvent-extracted meals. Intake of FDNB-available lysine was correlated (r = .902) to weight gain in rats fed diets containing the CSM that were more severely autoclaved. Results suggest that heat treatment must be more severe than that which normally occurs in commercial CSM processing to cause substantial, selective loss in lysine availability.

The preparation of matrix-bound proteases and their use in the hydrolysis of proteins

Four proteases, crude acid protease from Aspergillus, pronase, amino-peptidase M, and prolidase, have been covalently attached to activated agarose and to amino propyl glass beads. The matrix-bound enzymes have been tested as catalysts for the complete hydrolysis of protein substrates, with the primary goal to isolate unstable amino acid derivatives present in the substrate protein. Under conditions used in the present work, the total amino acid release from the protease-catalyzed hydrolysis of four substrate proteins (pancreatic ribonuclease, egg white lysozyme, yeast enolase, and bovine insulin) was 95–103% of that observed in standard acid hydrolysis. Recovery of individual amino acids showed greater deviation from the theoretical values, but cystine was the only amino acid recovered in low yields (42–77%) from all four proteins. Derivatized amino acids, such as methionine sulfoxide, O-(butylcarbamoyl)-serine, and N-glycosyl asparagine have been obtained from chemically modified proteins or from unmodified glycoprotein in good yield, and normal amino acid constituents of proteins which cannot be quantified after acid hydrolysis (tryptophan, asparagine, and glutamine) have also been determined either directly after proteolysis or after proteolysis in conjunction with acid hydrolysis.

Alanine: key role in gluconeogenesis.

Of 20 amino acids measured, alanine is the principal amino acid released by forearm muscle of man, in accord with its being the principal amino acid extracted by liver for gluconeogenesis. This occurs in both the postabsorptive state and after 4 to 6 weeks of starvation, when total amino acid release is markedly diminished.