Intracellular Amino Acid Pattern Research Articles

Electrospun Microfibers Modulate Intracellular Amino Acids in Liver Cells via Integrin β1.

Although numerous recent studies have shown the importance of polymeric microfibrous extracellular matrices (ECMs) in maintaining cell behaviors and functions, the mechanistic nexus between ECMs and intracellular activities is largely unknown. Nevertheless, this knowledge will be critical in understanding and treating diseases with ECM remodeling. Therefore, we present our findings that ECM microstructures could regulate intracellular amino acid levels in liver cells mechanistically through integrin β1. Amino acids were studied because they are the fundamental blocks for protein synthesis and metabolism, two vital functions of liver cells. Two ECM conditions, flat and microfibrous, were prepared and studied. In addition to characterizing cell growth, albumin production, urea synthesis, and cytochrome p450 activity, we found that the microfibrous ECM generally upregulated the intracellular amino acid levels. Further explorations showed that cells on the flat substrate expressed more integrin β1 than cells on the microfibers. Moreover, after partially blocking integrin β1 in cells on the flat substrate, the intracellular amino acid levels were restored, strongly supporting integrin β1 as the linking mechanism. This is the first study to report that a non-biological polymer matrix could regulate intracellular amino acid patterns through integrin. The results will help with future therapy development for liver diseases with ECM changes (e.g., fibrosis).

Free amino acids in plasma, red blood cells, polymorphonuclear leukocytes, and muscle in normal and uraemic children.

The aims of this study were to investigate free amino acid (AA) concentrations in plasma, red blood cells (RBC), polymorphonuclear granulocytes (PMN), and muscle, sampled at the same time, in normal and uraemic children. Twelve apparently well-nourished chronically uraemic children (five females) aged a mean of 9.4+/-4.8 (range 1.7--17.7) years and 13 age-matched normal children were studied. Venous blood and muscle samples for AA analyses were taken simultaneously after an overnight fast. The intracellular AA patterns in the three cellular compartments were qualitatively similar, but the absolute intracellular concentrations were higher in muscle than in PMN, which had higher values than in RBC. The AA patterns in plasma, RBC, PMN, and muscle in the uraemic children have many similarities; typical features being low branched-chain AA (BCAA), tyrosine, and serine concentrations and variably high concentrations of some non-essential AA. Among the individual AA, there were only few correlations between their concentrations in the three cell compartments. The lack of correlation between the concentrations in RBC, PMN, and muscle for most of the AA indicates that there is no close association in the same subject between individual free AA concentrations in various types of cells, presumably because of differences in metabolism and function. Consequently, one should be cautious in assuming that AA concentrations, determined in RBC or PMN, reflect the concentrations in muscle cells. Therefore, these preliminary observations do not support the hypothesis that RBC and PMN AA analysis can be considered as a suitable alternative to muscle AA determination.

Is α-ketoisocaproyl-glutamine a suitable glutamine precursor to sustain fibroblast growth?

Background: Glutamine is considered an essential nutrient for cellular growth. Aim: To test the suitability of α-ketoisocaproyl-Gln (Kic-Gin) as a new glutamine (Gin) precursor to sustain human fibroblast growth. Methods: [3H] thymidine uptake into cellular DNA of human fibroblasts. Extracellular and intracellular aminoacid patterns were determined with peptides and acylated compounds. Results: L-alanyl-L-glutamine (used here as a recognized Gin precursor) promoted DNA synthesis, whileN-acetyl-L-glutamine (used here as a negative control since it is known to be a poor Gin precursor) and α-ketoisocaproyl-glutamine had no effect. Alanyl-glutamine progressively gave rise to free glutamine in the growth medium. In contrast, glutaminesupplied in acylated form was poorly available and did not appear in free form in the medium. In addition, only alanyl-glutamine increased intracellular glutamine and glutamate levels. In contrast,Kic-Gln was able to sustain net protein synthesis as judged by total protein content and reduced intracellular levels of most essential amino acids. Conclusion: Kic-Gln appears to be a poor extra-cellular precursor of Gin to sustain cell growth.

Albumin and Amino Acid Levels as Markers of Adequacy in Continuous Ambulatory Peritoneal Dialysis

Similar to previous findings in HD patients, a markedly decreased serum albumin level has been found to be a strong predictor of morbidity and mortality in CAPD patients. However, a slight decrease in serum albumin levels (to about 30 g/L if measured with nephelometry or the bromcresol purple method) does not always seem to reflect impaired nutritional status or to be associated with an increased morbidity or mortality in CAPD patients. A low serum albumin level among CAPD patients is related to dialysate albumin loss, comorbidity, age, and a low dietary protein intake. The possible relation between the dialysis dose (as assessed by small solute clearances) and serum albumin levels among CAPD patients is much less established and needs further study, although serum albumin tends to increase in prospective studies of increased peritoneal dialysis dose. Although the plasma levels of amino acids seem to be lower in CAPD patients compared to HD patients, this does not reflect the intracellular amino acid pattern in muscle which is less abnormal in CAPD patients, possibly because of the sustained hyperinsulinemia during CAPD, resulting in an increased intracellular to extracellular gradient. It is at present not established to what extent the amino acid abnormalities are related to the dialysis dose. Malnourished and hypoalbuminemic CAPD patients should be recommended to increase the protein intake, and if this is not effective, the dialysis dose should be increased. Furthermore, the use of amino acid-based peritoneal dialysis solutions is a promising new tool for the treatment of malnourished CAPD patients and may become an important component of CAPD therapy in the future. On the other hand, if the nutritional status deteriorates in spite of these efforts, the patient should be transferred to hemodialysis if possible.

Plasma and muscle free amino acids in maintenance hemodialysis patients without protein malnutrition

To investigate how uremia modified by maintenance hemodialysis treatment influences the extra- and intracellular amino acid pattern, we collected muscle samples by percutaneous muscle biopsy and plasma samples for determination of free amino acids in 11 functionally anephric patients (creatinine clearance less than 1 ml/min), who had been treated with hemodialysis for greater than 6 months and had no clinical or laboratory signs of protein malnutrition. Five patients had mild acidosis (standard bicarbonate pre-dialysis 18 to 21 mmol/liter). The amino acid results were compared with data from age- and sex-matched healthy controls and with data obtained earlier from non-dialyzed patients with chronic uremia. In the hemodialysis patients threonine, serine and valine were significantly reduced in plasma compared to the controls, whereas the plasma concentrations of aspartate, glycine, citrulline, cysteine and arginine were elevated. In aspartate, glycine, citrulline, cysteine and arginine were elevated. In muscle, valine, serine and the tyrosine to phenylalanine ratio were low. Compared with the untreated uremic patients the hemodialysis patients exhibited fewer significant abnormalities, but the general pattern was similar, demonstrating that hemodialysis is unable to fully correct the amino acid abnormalities of chronic uremia. There was a significant positive correlation between both pre-dialysis and post-dialysis plasma bicarbonate and the muscle valine concentration, suggesting that mild acidosis may be causally related to the inbalance of the branched-chain amino acids in uremia. Extra- and intracellular serine depletion in the presence of high plasma glycine may reflect a defect in the metabolism of glycine to serine in hemodialysis patients, related to a lack of metabolizing renal tissue.

Effects of BCAA-enriched amino acid (AA) solutions on the nitrogen balance and the intracellular AA pattern in traumatized rats. A randomized study : [formula omitted] Holm, E., Staedt, U., Martell, J., Jacob, S., Weidler, B., Dept. of Pathophysiology, Medical Clinic I Mannheim, University of Heidelberg, Mannheim, FRG

Effects of BCAA-enriched amino acid (AA) solutions on the nitrogen balance and the intracellular AA pattern in traumatized rats. A randomized study : [formula omitted] Holm, E., Staedt, U., Martell, J., Jacob, S., Weidler, B., Dept. of Pathophysiology, Medical Clinic I Mannheim, University of Heidelberg, Mannheim, FRG

Protein Metabolism in Renal Failure: Abnormalities and Possible Mechanisms

Currently, we have only fragmentary knowledge about alterations of protein metabolism in renal failure, yet several coherent strands appear to be emerging. CRF is, in part, a state of malnutrition, as evidenced by abnormalities of body composition and alterations of both plasma and intracellular amino acid patterns. Superimposed upon this baseline are abnormalities specific to renal disease, changes in the concentration of certain amino acids, and the buildup of nitrogenous wastes and potential metabolic toxins, and the interaction of these toxins with hormones or within metabolic pathways. In acute renal failure, or when intercurrent illness is added to CRF, there is an intensification of the metabolic derangements and an acceleration of the normal catabolic response. Current research is directed at improving protein anabolism by limiting total nitrogen intake, while at the same time, providing supplemental amino or keto acids to restore and maintain nutritional state. Other approaches involve the provision of alternate pathways for nitrogen disposal and by direct stimulation of nitrogen anabolism through the administration of branched-chain amino acids or their keto acid analogues. All would agree that these modifications must be performed on a background of adequate caloric intake and careful patient monitoring. Given the complex nature of the problem, careful prospective controlled studies will be necessary before any hypothesis can be accepted. It is likely that additional improvements will require both a better understanding of the underlying metabolic defects and, probably, the combined application of several of the ideas previously noted.

Plasma amino acids, intracellular amino acid pattern of muscle, and substrates of energy metabolism in septic rats: [formula omitted] Dept.of Pathophysiology, I. Med. Clinic Mannheim, University of Heidelberg, 6800 Mannheim, FRG

Plasma amino acids, intracellular amino acid pattern of muscle, and substrates of energy metabolism in septic rats: [formula omitted] Dept.of Pathophysiology, I. Med. Clinic Mannheim, University of Heidelberg, 6800 Mannheim, FRG

Intracellular free amino acids in muscle tissue of patients with chronic uraemia: effect of peritoneal dialysis and infusion of essential amino acids.

1. Free amino acids were determined in the plasma and in the muscle tissue of 14 patients with chronic uraemia; eight were not on dialysis and six were having regular peritoneal dialysis. The concentration of each amino acid in muscle water was calculated with the chloride method. 2. In both groups of patients there were low intracellular concentrations of threonine, valine, tyrosine and carnosine, and high glycine/valine and phenylalanine/tyrosine ratios. Both groups of patients had increased amounts of 1- and 3-methyl-histidine in plasma and in muscle water. 3. The non-dialysed patients had low intracellular concentrations of lysine, and the dialysed patients had high intracellular concentrations of lysine, isoleucine, leucine and of some of the non-essential amino acids. 4. After peritoneal dialysis for 22 h, the plasma concentration of several amino acids decreased but the intracellular concentrations of most amino acids did not change significantly. 5. Intravenous administration of essential amino acids and histidine during the last 4 h of dialysis increased in muscle the total free amino acids, the ratio of essential to non-essential amino acids and the valine and phenylalanine concentrations. 6. The results demonstrated that the plasma and muscle concentrations of several amino acids are grossly abnormal in chronic uraemia. Non-dialysed and dialysed patients exhibit important differences, especially in the intracellular amino acid patterns. Infusion of essential amino acids may result in enhancement of protein synthesis.

An analysis of the free intracellular amino acids of certain strains of Neurospora

1. 1. Free intracellular amino acids were demonstrated to be present as a definite cell fraction of Neurospora crassa. 2. 2. Some 35 ninhydrin-positive substances were encountered among 28 different strains analyzed; however, none of the strains had all of these substances present in their extracts. In some mutants internal free amino acids were found to be associated in their expressions with mutant characteristics. In other mutants, certain amino acids were consistently absent. Other apparent differences disappeared in backcrosses to wild type, and new amino acids showed up. 3. 3. When certain strains were backcrossed to wild type to the F 5 generation, results showed that a characteristic free intracellular amino acid pattern is associated with the genotype of the mutant that controls the mutational deficiency. Up into the F 5 generation, differences in the amino acid pool stayed consistent and were not lost on backcrosses to wild type. This indicates that the amino acid pattern must be related to the particular phenotype brought about by a single gene mutation, 4. 4. From a cross between a methionine-less mutant and an adenine-less purple pigment mutant, all possible F 1 segregants were obtained. Several strains of each recombination of segregants were tested by chromatography for both qualitative and quantitative differences in their free intracellular amino acid pattern. Differences appeared to be peculiar to each of these types of F 1 progenies. Some minor differences existed between strains that had the same mating type and growth factor requirement, but in the general pattern this experiment proved again the conclusion that was drawn from the F 5 experiment with backcrosses to wild type. 5. 5. When the wild-type strain Em5297a was grown on different supplements, such as amino acids, purine, or vitamins, a number of changes in the free intracellular amino acid fraction were observed.