Intracellular Free Amino Acid Research Articles

Intracellular free amino acid patterns in duodenal and colonic mucosa

We report for the first time the concentrations of free amino acids in human intestinal biopsies obtained by routinely performed endoscopy. We studied 15 medical patients with no changes of the mucosa and six HIV-infected persons with duodenitis. The mean (and SD) sum of all amino acids, taurine excepted, was 61.9 (5.4) mmol/kg dry weight in duodenal biopsies of HIV-negative subjects (n = 11) and 82.9 (0.6) mmol/kg in colonic specimens: 50% (44%) of the total (minus taurine) consisted of aspartate and glutamate and 14% (12%), of the essential amino acids. The relative amino acid pattern in duodenum and colon differed completely from that for muscle: aspartate was fourfold higher; glutamate, phenylalanine, glycine, valine, leucine, and isoleucine were about twofold higher. In contrast, glutamine amounted only to 4% (duodenum) to 14% (colon) of muscle glutamine. In duodenal biopsies of the HIV-infected persons, we found significantly (P less than 0.01, except glutamine: P less than 0.025) increased concentrations of glutamate (24.1 vs 17 mmol/kg dry weight), ornithine (1.4 vs 0.4), valine (2.2 vs 1.7), and glutamine.

Changes in intracellular free amino acids in tissues of the caged mussel,Elliptio complanata, exposed to contaminated environments

Intracellular tissue concentrations of free amino acids (FAA) were monitored in caged mussels (Elliptio complanata) exposedin situ for 27–29 days and 77–79 days in the Yamaska River watershed (Quebec, Canada). Total concentrations of FAA (nmol/mg wet weight) increased in both mantle and adductor muscle tissue at several sites impacted by agricultural runoff and urban effluent from municipal sewage and light industries when compared to levels in mussels located at a site with little anthropogenic impact. Consistent changes in the per cent composition of individual FAA to the total FAA pool included decreases in serine, threonine, glycine and valine as well as increases in glutamic acid and glutamine at 27–29 days but results were not consistent with longer exposure and varied amongst sites. Few changes in total or individual FAA were observed in gill tissue. The results suggest that increases and/or decreases in total FAA in some tissues of freshwater bivalves may be indicative of generalized stress induced by a variety of environmental factors and may be useful as anin situ biochemical index of toxicity.

Metabolism of Peptides and Amino Acids During In Vitro Protein Degradation by Mixed Rumen Organisms

In vitro inoculum enriched with particle-associated organisms was prepared using rumen contents from a cow fed a 60% forage, 40% concentrate diet. Treatment of in vitro inoculum with cetyltrimethylamrnonium bromide was used to release intracellular free amino acids from mixed rumen organisms. Addition of 10mM tosylarginine methyl ester, a competitive inhibitor of trypsin, decreased degradation rate and intracellular free amino acids in incubations containing either casein or serum albumin. Extracellular peptides increased rapidly to a maximum at 60min in casein incubations but were not different from zero in albumin incubations. Accumulation of intracellular free amino acids was maximal at 60min in casein and albumin incubations; the concentration observed with albumin was about one-fourth that with casein. Ammonia production from intact casein was slightly greater than that from acid and enzymatically hydrolyzed casein and about 80% greater than that from albumin. Ammonia production and appearance of extracellular free amino acids lagged behind accumulation of intracellular free amino acids. Results suggest that formation and metabolism of extracellular peptides are important in controlling the rate of protein degradation by mixed rumen organisms.

In vivo Cerebral Protein Synthesis Rates with Leucyl-Transfer RNA Used as a Precursor Pool: Determination of Biochemical Parameters to Structure Tracer Kinetic Models for Positron Emission Tomography

Leucine oxidation and incorporation into proteins were examined in the in vivo rat brain to determine rates and compartmentation of these processes for the purpose of structuring mathematical compartmental models for the noninvasive estimation of in vivo human cerebral protein synthesis rates (CPSR) using positron emission tomography (PET). Leucine specific activity (SA) in arterial plasma and intracellular free amino acids, leucyl-tRNA, alpha-ketoisocaproic acid (KIC), and protein were determined in whole brain of the adult rat during the first 35 min after intravenous bolus injection of L-[1-14C]leucine. Incorporation of leucine into proteins accounted for 90% of total brain radioactivity at 35 min. The lack of [14C]KIC buildup indicates that leucine oxidation in brain is transaminase limited. Characteristic specific activities were maximal between 0 to 2 min after bolus injection with subsequent decline following the pattern: plasma leucine greater than or equal to leucyl-tRNA approximately KIC greater than intracellular leucine. The time integral of leucine SA in plasma was about four times that of tissue leucine and twice those of leucyl-tRNA and KIC, indicating the existence of free leucine, leucyl-tRNA, and KIC tissue compartments, communicating directly with plasma, and separate secondary free leucine, leucyl-tRNA, and KIC tissue compartments originating in unlabeled leucine from proteolysis. Therefore, a relatively simple model configuration based on the key assumptions that (a) protein incorporation and catabolism proceed from a precursor pool communicating with the plasma space, and (b) leucine catabolism is transaminase limited is justified for the in vivo assessment of CPSR from exogenous leucine sources using PET in humans.

Amino acid metabolism in uremia.

The uremic syndrome is multifactorial, and affects most tissues and organs. Disturbances in protein and amino acid metabolism may play important roles, especially in chronic uremia, either directly or by production of toxic metabolites, with resultant negative nitrogen (N) balance, muscle wasting, reduced protein synthesis, and characteristically abnormal intracellular free amino acid concentrations. There are also grossly abnormal amino acid levels in the plasma of uremic patients, e.g., increases in conjugated amino acids, high levels of several nonessential and low levels of essential amino acids. The ratios of tyrosine/phenylalanine and of valine/glycine are decreased. The low tryptophan levels may contribute to encephalopathy as a result of an imbalance in neurotransmitter synthesis. Citrulline is found in excess; the explanation is unresolved. There are elevated concentrations of the sulfur-containing amino acids: cystine, taurine, cystathionine, and homocysteine. Excess of the latter is implicated in the atherogenesis of renal failure. Disturbed metabolism and interorgan exchange of amino acids in the uremic state explains some of the abnormalities in tissue and plasma concentrations of individual amino acids. Enzymatic defects are involved in the disturbed metabolism of branched chain amino acids (BCAA), with possible antagonism among them, which impairs growth and amino acid utilization. Carbohydrate intolerance, associated with insensitivity of peripheral tissues to insulin and hyperinsulinemia, elicits decreased plasma BCAA. Protein synthesis rates in normal and pathological conditions are more closely related to the intracellular amino acid pool than to plasma amino acid levels. Concentrations of individual amino acids in the plasma pool are poor indicators of their intracellular concentrations. Muscle contains the largest pool of protein and free amino acids in the body. In chronic renal failure patients, the intracellular concentrations of valine, threonine, lysine, and carnosine are low. With low protein diets and in hemodialysis, serine, tyrosine, and taurine often are also low. The low taurine may be related to fatigue and to uremic cardiomyopathies. The commonly used amino acid supplements generally fail to correct the intracellular amino acid deficits. A "New Formula" has been developed to correct these intracellular amino acid abnormalities, and to supplement a low protein diet. It provides more valine than leucine, increased tyrosine and threonine, and less histidine, leucine, isoleucine, lysine, methionine, and phenylalanine than in formulas customarily used for patients with chronic renal failure. It is uncertain whether other ap

Amino Acid Uptake and Metabolism by Larvae of the Marine Worm Urechis caupo (Echiura), a New Species in Axenic Culture.

Axenic (bacteria-free) larval cultures of the marine echiuran worm, Urechis caupo, were reliably obtained by aseptically removing gametes directly from the gamete storage organs. Trochophore larvae only removed neutral amino acids from seawater as measured by high-performance liquid chromatography (HPLC). There was no detectable uptake, as measured by HPLC, of acidic or basic amino acids. Kinetic analysis showed that the transport system for alanine in 4-day-old larvae had a Kt of 4-6 µM and a Jmax of 9-10 pmol larva-1 h-1. Following a 50-min exposure, the majority of the radioactivity (95%) from 14C-alanine was found in the trichloroacetic acid-soluble fraction. Very little label appeared as acid-insoluble material, and there was no detectable lipid biosynthesis from 14C-alanine. Approximately 12% of the total alanine transported was released in the form of 14CO2. Thin-layer chromatography of intracellular free amino acid pools demonstrated that aspartic acid and glutamic acid were radiolabeled from the alanine precursor. A comparison of the energy acquired from the transport of alanine, with the metabolic rate of 4-day-old larvae, revealed that 51% of the metabolic demand could be provided by the transport and complete catabolism of this single amino acid at a concentration of 595 nM in seawater.

Biosynthesis and photosynthate allocation patterns of arctic ice algae

Biochemical composition of the sea ice microbial community was measured in populations of different light histories in the Canadian Arctic (Resolute, N.W.T.). The average composition of the particulate organic matter [soluble and insoluble polysaccharide, particulate protein, intracellular free amino acids (IFAA), lipid, and chlorophyll a] was within the published range for microalgae, but lipid was a relatively large (31–59%) and protein a small (20–24%) part of the total. Protein and IFAA pools apparently comprised about 50% of the particulate organic nitrogen, of which 6–10% was in the IFAA pool. Over the entire spring growth season, the net synthesis of protein, IFAA, and Chl a (relative to total cell carbon) decreased with increasing light while relative synthesis of lipid and soluble polysaccharide increased, consistent with patterns of short‐term photosynthate allocation. In the early growth season patterns of synthesis were relatively insensitive to light, and rates of lipid synthesis were large for all light histories. Photosynthate allocation in 24‐h incubations greatly underestimated actual rates of net lipid synthesis and probably overestimated protein synthesis. Microalgae of cold, low‐light environments can display rates of lipid synthesis much larger than rates normally encountered in microalgae without displaying a corresponding pattern of shorter term photosynthate allocation.

Energetics of Osmoregulation in an Intertidal Copepod: Effects of Anoxia and lipid Reserves on the Pattern of Free Amino Accumulation

The intertidal copepod Tigriopus californicus (Baker) adjusts its intracellular free amino acid (FAA) content to regulate cell volume following osmotic stress. The near absence of either proline or alanine in the medium and increase in oxygen consumption suggests that these free amino acids are oxidized during hypoosmotic stress. The theoretical costs associated with the production of typical levels of proline and alanine following hyperosmotic stress (50 to 100% seawater) were estimated to be 116% of daily energy use. However, a decline in oxygen consumption rate following hyperosmotic stress was recorded, and thus supports the view that proline synthesis can interfere with Krebs cycle turnover. The rate of alanine accumulation under anoxic conditions was nearly the same as that following hyperosmotic stress but no accumulation of proline was observed. When hyperosmotically stressed under anoxic conditions, nearly 50% more alanine was accumulated by T. californicus than when only experiencing anoxia. In contrast, T. californicus under anoxic conditions accumulated only 13% of the proline normally produced following hyperosmotic stress. Copepods having high lipid levels showed a greater reliance on proline than alanine (5:1) following hyperosmotic stress compared with copepods with little or no lipid reserves (1:1). This shift in FAA accumulation from proline to alanine under restricted energy supply (i.e. anoxia and low-lipid level) would be energetically advantageous since alanine is much less costly to synthesize. The proportion of proline and alanine produced during hyperosmotic stress may be regulated at the pyruvate branch point. * Present address: Dr E. M. Goolish, School of Natural Resources, University of Michigan, Ann Arbor, Michigan 48109, USA. Key-words: Free amino acids, osmoregulation, Tigriopus californicus, anoxia, bioenergetics, oxygen consumption, salinity, alanine, proline

Long-term influence of the postoperative state on the free intracellular amino acid concentration in human skeletal muscle : Petersson B, Wernerman J, Waller S-O, Vinnars E. Departments of Anaestesiology and Intensive Care, S:t Göran Hospital, Södertälje Hospital and Huddinge University Hospital, Sweden

Long-term influence of the postoperative state on the free intracellular amino acid concentration in human skeletal muscle : Petersson B, Wernerman J, Waller S-O, Vinnars E. Departments of Anaestesiology and Intensive Care, S:t Göran Hospital, Södertälje Hospital and Huddinge University Hospital, Sweden

HPLC analysis of the phenylisothiocyanate (PITC) derivatives of taurine from physiologic samples.

Taurine is the major free intracellular amino acid. It has become the focus of study by many as a conjugator of bile and as a neurotransmitter and intracellular messenger. In this report we document a technique for measuring taurine in physiologic samples which is rapid, reproducible, and accurate. Any physiologic sample is first derivatized with phenylisothiocyanate (PITC) and separated by reverse phase HPLC, and then taurine is detected by UV at 254 nm. The advantages of this technique for the measurement of taurine are accuracy, small sample size, and reproducibility, and with an automated system many samples can be analyzed.

Influence of hyperinsulinaemia on intracellular amino acid levels and amino acid exchange across splanchnic and leg tissues in uraemia.

1. To examine whether insulin resistance in uraemia extends to amino acid metabolism, the effect of physiological hyperinsulinaemia on plasma amino acid concentrations was studied in 17 chronically uraemic and 28 healthy subjects by using the euglycaemic insulin clamp technique. 2. In six uraemic and seven control subjects, splanchnic and leg exchange of amino acids was quantified with hepatic and femoral venous catheterization, and in five uraemic and eight control subjects intracellular free amino acid concentrations were determined in muscle tissue obtained by needle biopsy. 3. In response to hyperinsulinaemia the plasma concentrations of all amino acids except alanine decreased by 10-50% in both uraemic and control subjects. Splanchnic alanine uptake declined by 20% and leg amino acid release tended to decrease in both groups. 4. The intracellular levels of all measured amino acids except alanine fell significantly and to a similar extent in uraemic and control subjects. 5. These results indicate that insulin-mediated alterations in regional amino acid exchange and in plasma and intracellular amino acid concentrations are similar in uraemic and control subjects. 6. It is concluded that tissue insensitivity to insulin in uraemia does not extend to uptake or release of amino acids and that resistance to insulin can be selective in its effect on different metabolic functions.

The relationship between plasma proteins and intracellular free amino acids during osmotic regulation in Carcinus maenas

AbstractThe osmoregulatory behavior of the Adriatic population of the shore crab Carcinus maenas is similar to that of more northern populations. Hyperosmotic regulation of the hemolymph is initiated below about 95–85% seawater (973–871 m Osm/kg) and slight hypo‐osmotic regulation appears to occur above approximately 110% seawater (1,127 m Osm/kg). Extracellular volume is actively regulated, and changes in levels of amino acids imply effective regulation of intracellular volume. Serum protein levels increase immediately after transfer of the crabs from high to low salinity, although the changes are much smaller than those reported by other investigators. Levels of free amino acids in serum decrease after transfer from high to low salinity and labeled leucine is incorporated into hemocyanin at a greater rate after such a transfer than in the absence of a change in salinity. However, the rate of incorporation of leucine is also greater at low than at high ionic strength in vitro, in cell‐free preparations. This result suggests that the experiment does not demonstrate storage of intracellular free amino acids by the extracellular oxygen carrier, but rather reflects “passive” binding to hydrophobic sites on the hemocyanin molecule.

Inorganic 14C as a probe of growth rate-dependent variations in intracellular free amino acid and protein composition of NH +4 -limited continuous cultures of Nannochloris atomis Butcher

Growth rate-dependent variations in intracellular free amino acid and protein composition were characterized and compared to their 14C content as a function of time in NH - 4 -limited continuous cultures of the marine chlorophyte alga, Nannochloris atomis Butcher (Strain GSBNANNO). The relative abundances of cellular carbon in free amino acids and protein were positively correlated with relative growth rate ( μ μ max ). There was also a positive correlation between relative growth rate and the proportion of cellular nitrogen in free amino acids, but not in protein. Significant changes in the composition of the intracellular free amino acid pool accompanied the growth rate-related decline in pool size. The percentage of intracellular free amino acid carbon contained in glutamate increased from 9 to 53% and the glutamine/glutamate carbon pool ratio decreased as relative growth rate decreased. The percentage of intracellular free amino acid carbon contained in proline decreased from 76% to 21 %, while small increases were observed for glycine + alanine, valine, and beta-aminobutyrate as relative growth rate decreased. Equilibration of the intracellular free amino acid pool with inorganic 14C was incomplete even after incubation for 25% of the generation time. Equilibration rates of glutamate, glutamine, glycine + alanine, and valine were positively correlated with growth rate. Incomplete equilibration led to underestimates of free amino acid carbon pool sizes based on 14C content. However, because the saturation rates of glutamate and glutamine were similar, their ratio of 14C pools could be used as an index of the growth rate-dependent changes in the ratio of their cellular carbon pools. Isotope disequilibrium resulted in an initially higher relative abundance of 14C in alcohol soluble low molecular weight material than the corresponding relative abundance of cellular carbon as determined from 14C distributions in equilibrium-labeled cells. Most of the short term time-dependent variation in the distribution of 14C in low molecular weight material was associated with the free amino acids. For incubations up to 25% of the generation time, the distribution of 14C in protein was lower than the equilibrium distribution. Discrepancies between short term distributions of 14C and cellular carbon distributions diminished if free amino acids and protein were considered together as a unit of cellular material. Growth rate-dependent changes in distributions of cellular carbon in free amino acids + protein were reflected by changes in the corresponding 14C distributions. The techniques described provide the ability to specifically assay growth rate-dependent variations in algal metabolism and biochemical composition without the interference of detritus and non-algal biomass.

Evolution des acides amines libres seriques

Qualitative and quantitative variations of free amino acids have been examined in the serum of Astacus leptodactylus during progressive osmotic adaptation and hyperosmotic stress. Free amino acids hardly participate in the extracellular osmotic regulation. Their variation in the serum is correlated to the fluctuations of the intracellular free amino acids during isosmotic regulation.

Free amino acid levels in muscle and liver of a patient with glucagonoma syndrome

This study set out to measure the amino acid concentrations in the femoral artery, femoral vein, hepatic vein, muscular and hepatic tissue, and urine of a patient with the glucagonoma syndrome. The total plasma amino acid concentration was severely reduced on admission (737 μmol/L, 26% of normal), with only a slight increase during intravenous administration of 200 g of amino acids per day. The total intracellular amino acid levels in the muscle were 86%, and those of the liver were 47% of the normal range. Only 0.62% of the amino acids administered were found in the urine. Arteriovenous amino acid concentration differences across the muscle and splanchnic tissue indicated the release of amino acids (mainly glutamine, glycine, and alanine) from the muscle and the absorption of amino acids by the splanchnic bed. This study shows that the infusion of a high amount of amino acids cannot increase the subnormal plasma AA levels of patients with the glucagonoma syndrome. the low total plasma AA levels are paralleled by decreased intracellular free amino acid levels in the muscle and liver.

Branched Chain Amino Acid Uptake and Muscle Free Amino Acid Concentrations Predict Postoperative Muscle Nitrogen Balance

Amino acid solutions rich in branched chain amino acids (BCAA) are commonly utilized both clinically and in experimental protocols in an attempt to reduce skeletal muscle and whole body protein catabolism. To investigate the effectiveness of BCAA infusion, amino acid formulas containing varying concentrations of BCAA were given during operation in this study to three groups of dogs undergoing a standard laparotomy and retroperitoneal dissection. A fourth group was given saline alone. With the use of previously described hindquarter flux techniques, individual and total amino acid nitrogen exchange rates were measured and utilized in estimating skeletal muscle protein catabolism. Intracellular free amino acid concentrations were measured in percutaneous muscle biopsy samples. Although there was no relationship with the rate of BCAA infusion, there was a significant correlation between the rate of BCAA uptake by muscle and diminished total nitrogen release from hindquarter skeletal muscle after operation. There was also a significant relationship between muscle nitrogen balance and the postoperative change in the muscle concentration of either total amino acids or the single amino acid glutamine. When combined in a single equation, BCAA uptake and the change in muscle free amino acid concentration predict skeletal muscle nitrogen release with an r = 0.86. Thus, the rate of BCAA uptake and the free glutamine or total amino acid concentration in muscle appear to be independent predictors of muscle nitrogen balance. The nitrogen-sparing effect of BCAA in skeletal muscle is unrelated to infusion concentration or rate of infusion.

Resolution by high pressure liquid chromatography of intracellular and extracellular free amino acids of a nitrogen deficient marine diatom, Skeletonema costatum (Grev.) Cleve, pulsed with nitrate or ammonium

Nitrate and ammonium were added to nitrogen (N) deficient, batch cultures of Skeletonema costatum (Grev.) Cleve. The range of total internal free amino acids (IN FAAs) after N perturbations was 13.0–28.5 mM, while the total external FAAs (EX FAAs) varied from 3.9–17.5 μM. The total basic IN FAAs (and the total basic IN FAA N) predominated over the acidic and neutral IN FAAs (and acidic and neutral IN FAA N) after 50 h of N deficiency and after the N perturbations. Ornithine, a non-protein basic amino acid, was the most abundant individual IN FAA in N deficient cells, accounting for 43% of the total IN FAAs. All internal FAAs increased more quickly after the NH 4 + addition than after the NO 3 − addition. During the assimilation of either N addition, ornithine increased to a temporary peak concentration more rapidly than any other observed IN FAAs, and contributed up to 80% of the total IN FAA pool. After most of the respective N additions had been assimilated, and as free ornithine began to decrease, glutamine and glutamate increased to form a combined contribution of 30% of the total IN FAA pool. The increases of internal free glutamine and glutamate occurred more rapidly with the addition of NH 4 + than with NO 3 −, and these increases did not occur until the internal NH 4 + was depleted. The EX FAA did not undergo a distinct cycling in response to the NO 3 − or NH 4 + additions, as was observed with the IN FAAs. In contrast to the composition of the IN FAAs the most abundant EX FAAs were histidine (relatively sparse IN FAA), glutamate, aspartate, ornithine, and lysine.

Osmotic shock-induced release of low molecular weight metabolites from free-living and immobilized cyanobacteria

A procedure is described for the release of low molecular weight carbohydrates and free amino acids from the unicellular cyanobacteria Synechocystis PCC6714 and Synechococcus PCC6311. Metabolite release was induced by rapid transfer of free-living or immobilized cells grown in a medium containing 490 mol m −3 NaCl to a freshwater-based medium (BG-11); up to 50% of the low molecular weight carbohydrates and 73% of the intracellular free amino acids were released within 2 min. Cells of Synechocystis PCC6714 were subjected to repeated osmotic shock, with no evidence of long-term damage to the cells after three cycles of metabolite release over a 48 h period. Because this procedure does not involve harvesting or death of the cells, it may prove to be a useful means of recovering metabolites from viable microorganisms.

Amino acid metabolism after lambda-carrageenan injury to rat skeletal muscle.

To evaluate whether the alterations in amino acid (AA) metabolism that follow the lambda-carrageenan injury are direct consequences of wounding and are therefore independent of food intake, plasma AA, in vivo muscle intracellular free AA, and AA release during isolated hindlimb perfusions were determined in pentobarbital-anesthetized rats with lambda-carrageenan-induced hindlimb muscle wounds (W) and in pair-fed (PFC) and ad libitum-fed (ALC) non-wounded controls. Both PFC and W animals showed different plasma and muscle intracellular AA composition that ALC. The alterations observed in these compartments in W animals may not have resulted exclusively as a consequence of the wound. Wounded hindlimbs released more AA during perfusion than either control group. The marked increase in net protein catabolism of W muscle appeared to be related to wounding and not a consequence of partial starvation. The normalized release (amino acid/phenylalanine ratio) of glutamine (W less than ALC), alanine (W less than PFC and ALC), and the branched-chain amino acids (W greater than PFC and ALC) differed among groups, suggesting an alteration in the metabolism of these AA in W tissue.

Intracellular free amino acid concentrations in human circulating polymorphonuclear leukocytes (PNL) : [formula omitted], J. Mangold, G. Distler, P. Stehle, P. Fürst, J. Metcoff (Inst. for Biol. Chem. and Nutrition, Univ. of Hohenheim, Stuttgart, FRG; Health Sciences Center, Univ. of Oklahoma, Oklahoma City, USA)

Intracellular free amino acid concentrations in human circulating polymorphonuclear leukocytes (PNL) : [formula omitted], J. Mangold, G. Distler, P. Stehle, P. Fürst, J. Metcoff (Inst. for Biol. Chem. and Nutrition, Univ. of Hohenheim, Stuttgart, FRG; Health Sciences Center, Univ. of Oklahoma, Oklahoma City, USA)