Trichloroacetic Acid Method Research Articles

Total, and chemical fractions, of nitrogen and phosphorus in Eucalyptus seedling leaves: Effects of species, nursery fertiliser management and transplanting

Total and nitrogen (N)- and phosphorus (P)-containing chemical fractions extracted in trichloroacetic acid (TCA) were assessed after nursery production (pre-planting) and during establishment of E. globulus Labill. and E. nitens (Deane and Maiden) Maiden seedlings in the field and following transfer of E. nitens seedlings from warm temperatures in a nursery to cool temperatures in a growth chamber. In field experiments, seedlings of both species were transplanted in spring onto a site at 350 m above sea level (asl) and E. nitens in early winter onto a site at 700 m asl. E. nitens seedlings received either high- or low-nutrient, treatments in the nursery before planting at 700 m asl or before transfer to the growth chamber. E. globulus had greater foliar concentrations of total N and P at planting than E. nitens after nursery production under the same fertiliser regime. In both field trials and the growth chamber experiment a decrease in N, and generally P, concentration in the leaves was observed between pre-planting and the first post-planting measurement. Decreased N was observed after a period of one week and 15 weeks at 350 and 700 m asl, respectively. E. globulus seedlings had higher concentrations of insoluble P complexes and lower concentrations of inorganic P than E. nitens. Increased levels of soluble N across treatments were associated with warm spring temperatures in both field experiments. N and P concentrations with time during establishment were similar in all experiments. This may be related to retranslocation associated with growth after transplanting. Despite differences in N and P concentrations, partitioning of total N to soluble (nitrate, ammonia and amino acid), nucleic acid and protein N and of total P to nucleic acid, sugar, inorganic and insoluble P were similar between E. nitens high- and low-nutrient treatments, and with time during establishment. The TCA method yielded insights additional to those of total N and P. This study showed that the plant nutrition associated with eucalypt seedling transplanting is highly dynamic and complex.

Proteolytic enzymes as a limitation for pulmonary absorption of insulin: in vitro and in vivo investigations

In vitro biodegradation of insulin in lung cytosol and subcellular pellets of normal and diabetic rats was investigated. Rat lung was homogenized and subcellular fractions were isolated by ultracentrifugation. Degradations of [ 125I]-insulin after incubation with lung cytosol or subcellular pellets was determined using the trichloroacetic acid method. The results show that insulin is highly degraded in cytosol and subcellular pellets. Cytosolic insulin degradation was strongly inhibited by bacitracin or sodium cholate. The degradation of insulin in the lung cytosol from diabetic rats was significantly less than from normal rat. The lung protease activity reached a maximum at pH 7.4. Enzyme inhibitors like bacitracin and sodium cholate noticeably enhanced the relative pharmacological bioavailability of insulin when given intratracheally with insulin to normal rats. Acidic insulin solutions (pH 3.0) had more pronounced hypoglycaemic effects than neutrol solution (pH 7.0). These in vitro and in vivo results suggest that the proteolytic enzymes in the lung limit pulmonary delivery of insulin. The coadministration of protease inhibitors would be a useful approach for improving the pulmonary absorption of insulin.

Development of a novel method for determination of acetyl-CoA:1-alkyl- sn-glycero-3-phosphocholine acetyltransferase activity and its application to screening for acetyltransferase inhibitors : Inhibition by magnolol and honokiol from Magnoliae cortex

A method was developed for determining the activity of acetyl-CoA:1-alkyl- sn-glycero-3-phosphocholine acetyltrabsferase (EC 2.3.1.67), a key enzyme in the biosynthesis of platelet-activating factor (PAF,1-alkyl-2-acetyl- sn-glycero-3-phosphocholine). The assay involves measurement of the radioactivity in the trichloroacetic acid (TCA)-precipitated complex of radioactive product and albumin after incubation of 1-alky- sn-glycero-3-phosphocholine and [ 3H]acetyl-CoA with rat spleen microsomes or membrane fractions of human polymophonuclear leukocytes (PMNs). The radioactive product associated with the precipitate was identified as PAF using an ultrahigh-sensitivity TV camera system after extraction and separation by TLC. This TCA method was then used to screen the components of crude preparations that inhibited acetyltransferase activity. Major components from the cortex of Magnoliae (magnolol and honokiol), which have anti-inflammatory and anti-bacterial actions, inhibited the acetyltransferase activity in rats spleen microsomes ( ic 50 150 and 150 μM, respectively) and membrane fractions of human PMNs ( ic 50, 70 and 60 μM, respectively). The inhibitory action of magnolol and honokiol was reversible, and similar to or higher than that of nordihydroguaiaretic acid. PAF production in human PMNs stimulated by the ionophore A23187 was also suppressed dose dependently by magnolol and honokiol. These activities may be relevant to the claimed therapeutic effects of the extract from Magnoliae cortex.

Nucleotide preparation from cells and determination of nucleotides by ion-pair high-performance liquid chromatography

Procedures for the analysis of cellular purine and pyrimidine nucleotides are described. The commonly used perchloric acid and especially the trichloroacetic acid methods for nucleotide extraction interfere with ion-pair high-performance liquid chromatography, but we have developed such a system for the separation and determination of major cellular nucleotides in biological matrices, including tri-, di-, monophosphates, cAMP, cGMP, NAD, NADP, UDP-glucose and UDP-galactose. Compared with perchloric acid extraction, no degradation of the nucleotide standards used was observed with respect to triphosphates and other relatively unstable nucleotides. Cellular nucleotides were extracted by lysing cells in a hypotonic buffer containing an ion-pair reagent (tetrabutylammonium hydrogen-sulphate) to decrease enzymic degradation of nucleotides in combination with ultrafiltration of the cell lysate to remove compounds of higher molecular mass, for example enzymes. This method is a simple and reproducible procedure for investigating nucleotide pools in cells.

Degradation of 125I-Glucagon, -Pancreatic Polypeptide and -Insulin by Acid Saline Extract of Rat Submaxillary Gland and Their Protection by Proteinase Inhibitors.

The acid saline extract (ASE) of rat submaxillary gland exerts a powerful degrading effect on 125I-glucagon. In order to study the degradation of other 125I-peptides by ASE and the effects of their inhibitors, 125I-pancreatic polypeptide (PP) and 125I-insulin were used together with 125I-glucagon. The degradation studies were done by the trichloroacetic acid (TCA) method or gel filtration. Besides 125I-glucagon, 125I-PP was found to be destroyed by ASE in the ordinary immunoassay system using the TCA method, but 125I-insulin was intact in the presence of ASE. Leupeptin, and to a lesser extent p-chloromercuriphenyl-sulfonic acid (PCMS) and N-ethylmaleimide, inhibited the destruction of 125I-glucagon or -PP under the TCA method. PCMS was especially protective at high concentrations, for example 16 mM. These findings were confirmed by gel filtration of the assay mixture. In the presence of leupeptin (0.4 mM) and PCMS (16 mM), no shift in the peak of labelled glucagon or PP occurred. Thus ASE degrades not only 125I-glucagon but -PP, and thiol proteinase inhibitors have a strong inhibitory action on them.

Protein kinase assay by paper-trichloroacetic acid method: High performance using phosphocellulose paper and washing an ensemble of samples on flat sheets

Phosphocellulose paper has been found to be the paper of choice in assaying protein kinase activities using [γ- 32P]ATP by the trichloroacetic acid method of precipitation and washing. A study of binding of ATP of increasing concentrations at constant specific activity with Whatman 3MM or ATP-coated Whatman 3 MM papers (in vogue) versus phosphocellulose paper (proposed here) has shown that the latter has the least affinity for ATP when washing is done with either trichloroacetic acid or trichloroacetic acid containing pyrophosphate. In an experiment where the placental cytosolic protein kinase was serially diluted, the phosphocellulose paper was found to give higher signal/noise ratios at all dilutions studied compared to the other two papers. With regard to the technological side of washing the papers, we have found that the traditional method of cutting papers into small squares before loading the samples is perhaps not the best. Instead, we propose the use of a flat sheet matrix for loading the samples because this method ensures uniformity of washing among the samples while shaking is performed on a simple shaker. In addition, the whole paper matrix can provide an almost instantaneous autoradiogram of hundreds of samples facilitating biochemical experimentation with protein kinases.

Assessment of binding of l-cystine and l-lysine by rat renal brush-border membranes

Cystine and lysine bind to isolated rat renal brush-border vesicles. Three methods to determine the extent of amino acid binding to the membranes have been compared, one relying on the osmotic reactivity of the vesicle, a second by trichloroacetic acid precipitation of membrane-bound material and a third by initial rate analysis. For cystine, all methods yield comparable results at early time points, indicating the trichloroacetic acid method is a simple and valuable tool for binding estimation under initial-rate or near initial-rate conditions. For lysine, initial rate analysis and osmotic perturbation are the methods of choice since lysine co-precipitates with trichloroacetic acid.

A modification of the Lowry method for detecting protein in media containing lignocellulosic substrates

Clostridium thermocellum culture media containing crude cellulosic substrates, prepared by the steam-explosion of wood or straw, were found to interfere strongly with the Lowry method of protein determination. Investigations into the mode of interferences demonstrated that either the intercept (blank value) and/or the slope (intensity of color development) were affected. The efficiencies of precipitation procedures using the trichloroacetic acid method and the deoxycholate-trichloroacetic acid method were compared.

Assay of cerebrospinal fluid protein: a rate biuret method evaluated.

We evaluated a rate colorimetric method (Beckman) for measuring total protein in cerebrospinal fluid. The automated instrument we used was Beckman's ASTRA TM. A 100-microL sample of spinal fluid is introduced into the biuret reagent in the reaction cell and the increase in absorbance at 545 nm is monitored for 20.5 s. Solid-state circuits determine the rate of alkaline biuret-protein chelate formation, which is directly proportional to the total protein concentration in the sample. The linear range of measurement is 120 to 7500 mg/L. Day-to-day precision (CV) over the range of 150 to 1200 mg/L ranged from 15.2 to 2.3%. The method was unaffected by radical alteration of the albumin/globulin ratio, but there is a positive interference in the presence of hemoglobin, a suppression in the presence of bilirubin, and no effect by xanthochromia. The method is precise, accurate, rapid, and convenient. The method was compared with the trichloroacetic acid method as performed on the Du Pont aca III, giving a correlation coefficient (r2) of 0.9693. The method is precise, accurate, rapid, and convenient.

Sensitive assay, based on hydroxy fatty acids from lipopolysaccharide lipid A, for Gram-negative bacteria in sediments

Biochemical measures have provided insight into the biomass and community structure of sedimentary microbiota without the requirement of selection by growth or quantitative removal from the sediment grains. This study used the assay of the hydroxy fatty acids released from the lipid A of the lipopolysaccharide in sediments to provide an estimate of the gram-negative bacteria. The method was sensitive to picomolar amounts of hydroxy fatty acids. The recovery of lipopolysaccharide hydroxy fatty acids from organisms added to sediments was quantitative. The lipids were extracted from the sediments with single-phase chloroform-methanol extraction. The lipid-extraction residue was hydrolyzed in 1 N HCl, and the hydroxy fatty acids of the lipopolysaccharide were recovered in chloroform for analysis by gas-liquid chromatography. This method proved to be about fivefold more sensitive than the classical phenol-water or trichloroacetic acid methods when applied to marine sediments. By examination of the patterns of hydroxy fatty acids, it was also possible to help define the community structure of the sedimentary gram-negative bacteria.

Endotoxins of gram-negative anaerobic bacteria.

The author reviews the data available on endotoxins of Fusobacterium, Sphaerophorus, Leptotrichia, and Veillonella species, and reports his own findings with lipopolysaccharides extracted from Bacteroides fragilis strains by the trichloroacetic acid method. The latter extracts showed a very low toxicity for mice after i.v. injection of dosages of less than or equal to 500 micrograms and a slight leucopenic effect in mice after intraperitoneal injection of 50 micrograms.

24] Colorimetric estimation of vitamin A with trichloroacetic acid

Publisher Summary This chapter discusses the use of trichloroacetic acid method for colorific estimation of vitamin A in serum and liver tissue. In this method, trichloroacetic acid crystals (50 g), freshly washed with solvent, are dissolved in alcohol-free distilled chloroform (25 ml). This gives about 50 ml of reagent that is stable for 5 h with daylight excluded. Optical density readings tend to decrease with storage for longer periods, up to 24 h. No color is given by vitamin A with reagent exposed to bright diffuse sunlight for several hours. It is observed with this method that the colors produced from vitamin A, its acetate, and palmitate with the Carr-Price antimony trichloride reagent and trichloroacetic acid reach the same intensity for any given amount of vitamin. The concentration of trichloroacetic acid controls the rate of formation and stability of anhydrovitamin, which may be correlated with loss of vitamin A or its acetate. It is important to observe that change occurs without appearance of blue color in the weaker acid medium. Formation of anhydrovitamin with fine-structure absorption follows protonation of vitamin A, and the slower formation from vitamin A acetate reflects differences in the rates of dehydration and deacetoxylation.

Fragmentation of human albumin with proteolytic enzymes and its antigenicity with special reference to human-specificity.

Human albumin prepared by the trichloroacetic acid method was treated with bromelin, ficin, papain, pronase or trypsin. Pronase- or trypsin-treated albumin showed four fragments on polyacrylamide disc electrophoresis. When rabbit antiserum to pronase-treated albumin was adsorbed with monkey albumin, antibody with human specificity was completely abolished, whereas anti-albumin serum adsorbed with pronase-treated albumin retained the antibody to human albumin. It was considered that antigenic site with human specificity in albumin structure was inactivated by pronase.

QUANTITATIVE ANALYSIS OF VITAMIN A IN CEREAL PRODUCTS BY HIGH SPEED LIQUID CHROMATOGRAPHY

ABSTRACTA high speed liquid chromatographic method is described for the determination of vitamin A. Retinol is separated isocratically at ambient temperature via high pressure liquid adsorption chromatography on silica from the nonsaponifiable fraction of the sample. Quantitative determination is accomplished by monitoring the ultraviolet absorbance of retinol at 313 nm. Recovery of vitamin A as retinol in cereal products was 94.3%× 2.01. The primary advantages of this procedure are the use of an isocratic solvent system for the separation of retinol and the elimination of carotenoid interference and toxic chromophore‐producing reagents associated with the Cur‐Price, trifluoro‐ and trichloroacetic acid methods. The method offers improved precision for the determination of vitamin A, increases the number of vitamin A assays that can be performed per day, and shows applicability to other food products.

Massive Pseudoproteinuria Caused by Nafcillin

RECENTLY, we saw two patients in whom a heavy concentration of urinary protein reported from the laboratory was eventually proved to be a false-positive result, representing not protein but nafcillin excreted in the urine. <h3>Report of Cases</h3><h3>Case 1.—</h3> A 13-year-old boy with staphylococcal osteomyelitis of the left femur was treated intravenously with 12 gm of nafcillin sodium daily. Shortly thereafter his urine showed 4+ protein reaction on routine analysis with sulfosalicylic acid. Renal function, as measured by blood urea nitrogen (BUN) value, serum creatinine level, and creatinine clearance, was within normal limits. Protein level in a 24-hour urine sample (measured by the standard trichloroacetic acid method) was 3.9 gm. Results of urine-sediment examination were unremarkable, showing no free fat, oval fat bodies, or fatty casts. Subsequent protein testing on several occasions with sulfosalicylic acid again revealed 3+ to 4 + protein reaction, but a dipstick specific for albumin gave a

Massive pseudoproteinuria caused by nafcillin

RECENTLY, we saw two patients in whom a heavy concentration of urinary protein reported from the laboratory was eventually proved to be a false-positive result, representing not protein but nafcillin excreted in the urine. <h3>Report of Cases</h3><h3>Case 1.—</h3> A 13-year-old boy with staphylococcal osteomyelitis of the left femur was treated intravenously with 12 gm of nafcillin sodium daily. Shortly thereafter his urine showed 4+ protein reaction on routine analysis with sulfosalicylic acid. Renal function, as measured by blood urea nitrogen (BUN) value, serum creatinine level, and creatinine clearance, was within normal limits. Protein level in a 24-hour urine sample (measured by the standard trichloroacetic acid method) was 3.9 gm. Results of urine-sediment examination were unremarkable, showing no free fat, oval fat bodies, or fatty casts. Subsequent protein testing on several occasions with sulfosalicylic acid again revealed 3+ to 4 + protein reaction, but a dipstick specific for albumin gave a

Comparison of TCA and collagenase in the isolation of tissue collagen

Studies on collagen metabolism often require the separation of collagen from other noncollagen proteins. The two major methods of isolating collagen are hot (90°C) trichloroacetic acid (TCA) extraction and/or collagenase solubilization. Though both procedures are widely used, a direct comparison and evaluation of their specificity and efficiency has not been investigated. In this study the specificity of both methods was examined using [ 14C]tryptophan labeled proteins and the efficiency of collagen isolation was examined using a [ 14C]glycine labeled rat aortic homogenate. Protease free collagenase was highly specific in solubilizing collagen, while the hot TCA extract contained up to 14% of [ 14C]tryptophan-labeled noncollagen proteins. The hot TCA method consistently removed greater than 88% of total collagen from aortic tissue while the collagenase solubilization efficiency was less than 65%. Both procedures produced pure collagen fractions based on proline/hydroxyproline ratios. These data indicate that hot TCA yields a more complete extraction than does collagenase solubilization, but collagenase is more specific in its solubilization of collagen tissue proteins.

Degradation of 131I-Insulins by Rat Liver: STUDIES IN VITRO

Abstract The time courses of degradation of 131I-insulins, with average incorporations of 0.5 to 6.0 atoms of iodine per molecule (molecular weight 6000), by supernatant fractions of rat liver homogenates were investigated. Degradation of 131I-insulin was determined by precipitability of 131I radioactivity in the incubation mixtures with 10% trichloroacetic acid and by isolation and quantitation of 131I-insulin and 131I-labeled degradation products through the use of electrophoresis on paper. As determined by both methods, the rate and extent of degradation of the 131I-insulins studied were inversely proportional to the amount of iodine attached to the hormone; but by the electrophoretic method, the differences among the iodoinsulins were more sharply defined. The degradation of 131I-insulin containing less than 1 atom of iodine per molecule was shown to proceed rapidly to completion by the electrophoretic method. However, as measured by the trichloroacetic acid method, degradation appeared to level off when approximately 60% of the 131I-insulin had been degraded. This was due to the fact that a portion of the trichloroacetic acid-soluble degradation products, such as iodotyrosines, was carried down with the protein which was precipitated in the incubation mixtures by trichloroacetic acid. 131I-Labeled degradation products migrated as a major peak toward the cathode at a slower rate than 131I-insulin. The major degradation product of a 131I-insulin preparation containing 0.8 atom of iodine per molecule was shown to have a mobility on Sephadex G-25 and an Rf value on paper chromatography characteristic of [131I]monoiodotyrosine. Tests for correspondence to a single reaction order revealed that all of the iodoinsulins studied by the electrophoretic method approximated second order kinetic behavior. The rate constants of the iodoinsulins containing less than 1 atom of iodine per molecule were almost 10-fold greater than the rate constant of iodoinsulin containing 6 atoms of iodine per molecule. No correspondence to any specific reaction order could be shown for any of the iodoinsulins by the trichloroacetic acid method. The decrease in reaction rate constant paralleled the decrease in biological activity resulting from increasing the average number of iodine atoms incorporated into the insulin molecule. The results indicate that 131I-insulins are rapidly degraded by proteolysis to simple amino acid constituents by the liver. Since neither labeled A nor B chains were identified at any time during degradation, the results neither confirm nor deny that reductive cleavage of interchain —S—S— bonds of insulins is an initial step in the degradation of insulin. The results also indicate that the trichloroacetic acid method has serious limitations when used for studying 131I-insulin degradation in vitro.

The relationship between mucosal iron and iron absorption in the guinea pig.

The non‐haeme iron content of intestinal mucosa has been proposed as responsible for controlling iron absorption. In these experiments guinea pigs bled to produce a six‐fold increase in iron absorption showed no change in mucosal non‐haeme iron content.Earlier conflicting results may be partially explained by the inclusion of a fraction of the haemoglobin iron when using the trichloroacetic acid method for extracting ‘non‐haeme’ iron.It is suggested that the iron content of intestinal mucosa cannot be assigned a general role in regulating iron absorption.

Electron Microscopy of Bacterial Endotoxin-Antibody Complexes by the Use of Ferritin-Conjugated Antibody

The molecular shape of bacterial endotoxin extracted from the cell walls with phenol-water or trichloroacetic acid (TCA) method has been observed in the electron microscope. Electron micrographs of negatively stained macromolecules of highly toxic endotoxin extracted from the cell walls of Escherichia coli strain 0113 with phenol-water revealed filamentous structures with a diameter of 100 Å in width. On the other hand, the micrographs of endotoxin extracted from the whole cells with cold TCA revealed a mixture of filaments and discs. In a precipitate formed by a reaction mixture of filamentous lipo-polysaccharides and ferritin-conjugated antibodies, a specific combination of lipopolysaccharides and antibody molecules has been observed.