Electrophoretic study of perchloric acid-soluble, phosphotungstic-precipitable fraction in some pathologic conditions.
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Arthritis↗
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Protein kinase C substrates from bovine brain. Purification and characterization of neuromodulin, a neuron-specific calmodulin-binding protein.
Although such solubility is uncommon among proteins generally, several bovine brain proteins were found to be soluble in 2.5% perchloric acid, and many of them were in vitro substrates for protein kinase C (Ca2+/phospholipid-dependent enzyme). Two of the perchloric acid-soluble brain proteins were purified, p43 and p17. P43 and p17 could be phosphorylated by protein kinase C only in the presence of Ca2+ and phospholipids and neither was a substrate for protein kinase II. P43 was subsequently identified as the neurospecific, calmodulin-binding protein, neuromodulin (also designated P-57, GAP43, B50, or F1) (Alexander, K. H., Wakim, B. T., Doyle, G. S., Walsh, K. A., and Storm, D. R. (1988) J. Biol. Chem. 263, 7544-7549). A rapid purification method for neuromodulin was developed taking advantage of its newly discovered property, solubility in 2.5% perchloric acid, and of its previously recognized calmodulin-binding property. Evidence was obtained that neuromodulin isolated from cytosolic extract exists as a mixture of molecular forms and that the Ca2+-binding S100 protein-beta discriminates among the different neuromodulin isoforms in forming covalent complexes via disulfide bridges; this discrimination may be explained by analogous differences observed between the NH2-terminal amino acid sequences of p57 and F1. Solubility in 2.5% perchloric acid was demonstrated for another rat brain protein kinase C substrate, p87. We suggest that perchloric acid solubility might be a common property of protein kinase C substrates.
Animals↗
[Alkylation of nucleic acid components by ethyleneimine derivatives. I. Alkylation of bases].
In the course of investigating the reaction conditions of the nucleic acid components alcylation, the interaction of thioTEPA (N,N',N''-triethylenethiophosphoamide) with hydrochloric and perchloric acids was studied, perchloric acid increasing the alkylation products yield. HPLC and UV spectroscopy were used to isolate and identify products of nucleic bases alkylation by ethylenimine and its derivatives (thioTEPA and monoaziridinediethylphosphate). It is shown that under neutral conditions phosphoaminoethylation takes place, whereas under slightly acidic conditions products of aminoethylation are formed.
Adenine↗
Characterization of a heat-shock process for reduction of the nucleic acid content of Candida utilis.
A process for reducing the nucleic acid content of Candida utilis NRRL Y900 has been developed. The optimal process consists of heating the cells suspended in spent medium initially at pH 4.0 for various times at three different temperatures. Initially a heat-shock at 68 C for 1 to 3 sec is performed followed by incubation for 1 hr at 45 to 50 C and for a 2nd hr at 52 to 55 C. The distribution of degradation products has been characterized. Initially 90% of the nucleic acids were in a polymerized form (extractable by hot perchloric acid). After 30 min, much of this material was hydrolyzed but remained within the cell (extractable by cold perchloric acid). After 2 hr, most of the hydrolysis products leaked into the surrounding medium with only a small amount of low-molecular-weight material remaining within the membrane. Predominantly 3'-mononucleotides accumulated within the cell and eventually leaked from the cell.
Candida↗
Determination of low-abundant metabolites in plant extracts by NAD(P)H fluorescence with a microtiter plate reader.
This article describes a method for the enzymatic detection of low-abundant metabolic intermediates in plant extracts via NAD(P)H fluorescence using a microtiter plate reader. The detection of changes in NAD(P)H fluorescence (excitation 340 nm, emission 465 nm) exhibits a high signal-to-noise ratio and is as sensitive (> or = 20 pmol per well) as absorbance measurements with dual-wavelength photometers. Since up to 96 reactions can be initiated, monitored, and evaluated simultaneously, this method might be suitable for high-throughput screening programs on metabolite profiles. However, in contrast to absorbance measurements, fluorescence detection of NAD(P)H yields relative data, which can be impaired by the quench characteristics and the basic fluorescence of the extracts. Hence, extensive calibration is required to gain reproducible results. Calibration of the assay system was performed using leaf or root material (equivalent to 2-35 mg of fresh weight per well) extracted with perchloric acid, chloroform/water/methanol, or hot ethanol. Extraction with perchloric acid was found to be superior for metabolite quantification. Examples of the kinetics of individual metabolite determinations are presented and the contents of 3-phosphoglycerate, hexose phosphates, triose phosphates, pyruvate, and phosphoenolpyruvate in illuminated and darkened spinach leaves as well as leaf rosettes of Arabidopsis thaliana and leaf segments of the inducible crassulacean acid metabolism plant Mesembryanthemum crystallinum were measured via NAD(P)H fluorescence and, where possible, compared to reported data determined with dual-wavelength photometers.
Arabidopsis↗
Evaluation of different staining procedures for the quantification of fibroblasts cultured in 96-well plates.
Comparison of published methods for the quantification of adherent cell numbers by the measurement of absorbance of bound stain indicates a wide variation in their sensitivity. This study aimed at comparing the sensitivities of five different staining procedures (Coomassie brilliant blue G in perchloric acid, Coomassie brilliant blue G in phosphoric acid, methylene blue, crystal violet, and toluidine blue) applied to three separate types of cultured fibroblasts (3T3 cells, Vero cells, and human gingival fibroblasts) at concentrations from 0.125 x 10(4) to 10 x 10(4) per well in 96-well microplates. Absorbance values of Coomassie blue-stained cells were measured in situ. Those of the remaining cells were measured after solubilization of the dye with 1% sodium dodecyl sulfate. All absorbance values were measured using an Elisa reader at 620 or 570 nm for crystal violet. The relationship between cell number and absorbance over the entire cell concentration range was best fitted with quadratic regression analysis, in contrast with the linear relationship described elsewhere. The order of sensitivity of the staining procedures was the same for each cell type: Coomassie blue in perchloric acid less than Coomassie blue in phosphoric acid less than methylene blue less than crystal violet less than toluidine blue. With the latter two stains absorbance values began to plateau at approximately 8 x 10(4) cells per well. However, staining with Coomassie blue in perchloric acid and methylene blue resulted in an almost linear relationship between cell number and absorbance over the entire concentration range tested.(ABSTRACT TRUNCATED AT 250 WORDS)
Cell Count↗
Formation of protoporphyrin from haemoglobin in vitro.
1. The formation of protoporphyrin from red blood cells or purified haemoglobin in aqueous perchloric acid media without the prior isolation of haemin is described. The reaction is carried out in the absence of oxygen and in red light. Even traces of oxygen inhibit the reaction by oxidative destruction of protoporphyrin and by the oxidation of haem to haematin. 2. Perchloric acid releases iron and protoporphyrin from haemoglobin at similar rates, but the amount of protoporphyrin in the filtrate varies with the solubility of protoporphyrin in the concentration of perchloric acid used. The yield of protoporphyrin may reach 50-60%. Less than 5mug. of haemoglobin/ml. can be detected by measuring the fluorescence of the porphyrin released. 3. A porphyrin other than protoporphyrin is obtained in small amounts. Its possible identity is discussed. 4. If sodium sulphite is present as a reducing agent the exclusion of oxygen is not required, but the porphyrin formed is more polar and more soluble in water than protoporphyrin. The presence of oxygen appears to be necessary for the formation of this polar porphyrin.
Journal Article↗
An improved flow injection method for determination of lactate during exercise studies.
An improved enzymatic fluorometric method for automated analysis of lactate in perchloric acid extracts of capillary blood with flow injection (FIA) is described. The concentrations of lactate can be measured in as little as 20 microliter capillary blood from an exercising person. Within-day and between-day coefficients of variation are about 2%. The recovery of lactate from whole blood is 101%. Lactate is stable in perchloric acid extracts for at least 15 days at room temperature, and at +4 degrees C at least 30 days. Blank fluorescence in perchloric acid extracts of blood is low with the improved method. The FIA system is simple to use, inexpensive to operate, and is recommended for any laboratory with a high throughput of samples.
Autoanalysis↗
Sources of error in determinations of carnitine and acylcarnitine in plasma.
Radioactive and nonradioactive L-carnitine and acyl-L-carnitine were used to evaluate the washing procedures used during the determination of free, total, short-chain, and long-chain acylcarnitine in human and sheep plasma. The volume of fluid trapped by the protein precipitated by perchloric acid is approximately 24% of the total fluid volume and thus contains 24% of free carnitine and short-chain acylcarnitine. Washing twice with distilled water removes about 25% of the long-chain acylcarnitine along with the trapped free carnitine and short-chain acylcarnitines. Washing the pellet twice with a 60 g/L solution of perchloric acid completely removes the trapped free carnitine and short-chain acylcarnitine but does not remove the bound long-chain acylcarnitines. Thus washing with perchloric acid is essential for accurate measurement of long-chain acylcarnitines in plasma samples.
Acetylcarnitine↗
Studies on the mechanisms of 6-hydroxydopamine cytotoxicity.
The uptake-accumulation and binding of radioactivity in mouse heart after administration of the catecholamine neurotoxin [3H]6-hydroxydopamine (6-OH-DA, 1 or 3 mg/kg, i.v.) has been investigated. It was confirmed that a substantial portion (8--20%) of the radioactivity taken up and retained by the heart could not be extracted with perchloric acid, in all probability representing covalently bound oxidation products of 6-OH-DA to tissue proteins. Pharmacological analysis showed that a large part of this fraction was associated with the adrenergic nerves. The time-course of the perchloric acid resistant binding to the adrenergic nerves was found to parallel that of the neurotoxic action of 6-hydroxydopamine as evaluated by monitoring the change in [3H]noradrenaline uptake. Calculation of the intranelronal 6-hydroxydopamine concentration (average) needed to induce degeneration showed it to be in the order of 50 mM. The binding ratio for tritium deriving from [3H]6-OH-DA between the intraneuronal and extraneuronal compartments was found to be 10,000 to 30,000, pointing to a very high neuronal specificity for 6-hydroxydopamine. The 'covalent' binding of oxidation products of [3H]6-OH-DA was considerably reduced after desipramine or 1-phenyl-3(2-thiazolyl)-2-thiourea administration, treatments both known to protect the adrenergic nerves from undergoing degeneration. Conversely it was found that the binding increased during conditions known to potentiate the neurotoxic action of 6-hydroxydopamine, e.g., after monoamine oxidase inhibition with nialamide. Subcellular fractionation studies indicated that the predominant site of interaction between 6-hydroxydopamine oxidation products and neuronal proteins is the cytoplasm and the axonal membrane. Analysis of the effect in vivo administration of 6-hydroxydopamine on the field-stimulated induced release of [3H]noradrenaline previously taken up in the adrenergic nerves showed a 6-hydroxydopamine indiced reduction in [3H]noradrenaline release which was approximately proportional to the reduction in the number of nerve terminals. These findings further support the view that 6-hydroxydopamine acts largely in an "all-or-none' fashion with respect to the neurodegenerative action. Administration of [3H]dopamine also resulted in a fraction which was not extractable with perchloric acid, although this fraction was very small compared to that found after an equal dose of [3H]6-hydroxydopamine. These data may indicate that oxidation products of dopamine can interact with tissue proteins. From the present results it can be concluded that there is a close relationship between 'covalent' binding of 6-hydroxydopamine oxidation products to neuronal elements and the cytotoxic action of 6-hydroxydopamine, indicating that this binding may play an important role in the neurodegenerative action of 6-hydroxydopamine on catecholamine neurons.
Adrenergic Fibers↗
A simplified quick method for determination of sialic acid in serum.
A modification of the method reported by R. J. Shamberger (J. Clin. Chem. Clin. Biochem. 22, 647 (1984) for detection of serum sialic acid is described. The whole procedure takes less than 1 h. It is based on the release of bound sialic acid by heating with 5% perchloric acid. After cooling and brief centrifugation, the supernatant is heated for 15 min with Ehrlich's reagent at 100 degrees C. Thereafter the absorbance of the color developed in the sample is measured at 525 nm.
Humans↗
Increased vigabatrin entry into the brain by polysorbate 80 and sodium caprate.
The effects of a non-ionic surfactant, polysorbate 80, and the sodium salt of the saturated fatty acid, sodium caprate (C10), as potential brain absorption enhancers for vigabatrin were studied. Vigabatrin is an enzyme-activated irreversible inhibitor of gamma-aminobutyric acid (GABA) transaminase that increases brain and cerebrospinal GABA concentrations in animals and man. Before intravenous administration, a range of concentrations of the surfactants were tested using erythrocyte lysis or the red blood cell lysis test to establish the non-toxic concentration range. Vigabatrin was dissolved in 0.1% polysorbate 80 and 0.1% sodium caprate and administered intravenously in doses of 4 mL kg(-1) to male Wistar rats (230-250 g; n = 3). Rats were killed 2 h after drug and surfactant administration and the brains were immediately removed and homogenized in 0.4 M perchloric acid. Selected ion monitoring electrospray mass spectrometry was used to determine the concentration of vigabatrin and GABA directly from the perchloric acid extract of the rat brain. This method was developed to increase the speed and efficiency of the analysis by removing the need for complex extraction and derivatization procedures while retaining the specificity of the mass spectrometer as a detector. The stability of both vigabatrin and GABA in perchloric acid was established by monitoring their pseudo molecular ions in standard solutions at timed intervals over 24 h. Although the detection level for vigabatrin and GABA was at least 50 pg, only GABA was detected in rat brain. Vigabatrin caused a small increase in whole brain GABA. However, GABA levels were higher in the samples with vigabatrin + enhancer than in the samples where vigabatrin alone was administered. One-way analysis of variance indicated a significant effect of the surfactants on GABA levels (F (5,17) = 11.86, P < 0.01) and vigabatrin absorption was presumed. The rectal temperature of the rats is lowered by the presence of vigabatrin in the brain. Vigabatrin alone decreased rectal temperature by 6%. When given with either polysorbate 80 or sodium caprate, the extent of temperature lowering was significantly greater (P < 0.001). There was no significant difference after 2 h between polysorbate 80 + vigabatrin, and sodium caprate + vigabatrin.
Animals↗
A 31P nuclear magnetic resonance study of Crithidia luciliae.
31P NMR has been used to observe phosphorus-containing compounds in both perchloric acid and KOH extracts and in whole cell suspensions of Crithidia luciliae. Cells were grown in Bone and Steinert medium, or in a modified RPMI culture medium and harvested after about 72 h in mid- to late log phase. 31P NMR spectra of the perchloric acid extracts indicated that 3-phosphoglycerate, which is normally at low concentrations in most cells, was the dominant phosphorus-containing compound in the sugar phosphate region. 3-Phosphoglycerate is the end product of glycosomal glycolysis and our finding is consistent with previous observations of the failure to detect prior glycolytic intermediates. Other metabolites observed were ATP, ADP, NAD(P)+, phosphoenolpyruvate and low molecular weight polyphosphates (PPn, n less than 20). The presence of high-molecular-weight polyphosphates was established by spectra recorded on extracts obtained through subsequent treatment of the insoluble fraction with KOH. 31P NMR experiments on whole cells indicated that the average main internal pH of cells in late-log growth phase was approx. pH 7.2 +/- 0.1, using the orthophosphate resonance as an indicator. The cells responded to the addition of glucose (final concentration approx. 35 mM) with a decrease in pH, both internal (delta pH = -0.9 (55 min)-1) and external (delta pH = -1.3 (15 min)-1). Polyphosphates and ATP could not be observed in whole cell experiments, although perchloric acid extracts of identically treated cells showed no significant depletion of these compounds.
Animals↗
Chromogenic endotoxin assay in plasma. Selection of plasma pretreatment and production of standard curves.
The aim of this study was to define the optimal conditions for the plasma pretreatment and to improve the production of standard curves for plasma endotoxin determination by a chromogenic substrate assay. Endotoxin standard from E. coli O 111:B 4 (0-50 ng/l) was added to pyrogen-free water or to plasma samples from 12 healthy subjects and 24 alcoholics, before pretreatment by heating (75 degrees C, 5 minutes) or with perchloric acid (0.32 mol/l). When endotoxin standard curves were determined using a microprocessor-controlled reader, the slopes of the curves obtained with plasma differed from those with pyrogen-free water. The slope of the standard curve prepared with plasma samples from different patients exhibited marked interindividual variations. Compared with the heating method, the perchloric acid method gave more variable results and a lower recovery of added endotoxin, especially in plasma from alcoholics. The results permit the following conclusion: 1. For plasma endotoxin determination, a standard curve should be prepared for each individual plasma sample. 2. The endotoxin standard should be added before pretreatment of the plasma. 3. Pretreatment of the plasma by heating at 75 degrees C for 5 minutes provides more reliable results than pretreatment with perchloric acid.
Adult↗
Extraction of cadmium and iodocadmat species by di(2-ethylhexyl) phosphoric acid from perchloric and phosphoric media.
We have investigated the extraction of cadmium with di(2-ethylhexyl) phosphoric acid (HDEHP) in various solvents from perchloric and phosphoric solutions. The distribution coefficient D obtained is not very dependent on the nature and the polarity of the diluent used. The extraction of Cd(II) by HDEHP dissolved in benzene, from sulfophosphoric solutions shows the absence of sulfuric or mixed sulfophosphoric complexes of the metal. However, the presence of iodide anions in aqueous perchloric or phosphoric media results in enhanced extraction coefficient of cadmium with HDEHP (HX) in benzene. The extracted species were found to be CdIX,3(HX)2 and CdI2,3(HX)2 and their formation constants are 0.033 and 0.036, respectively. The recovery of cadmium from phosphoric medium by tributylphosphate (TBP), trioctyl phosphine oxide (TOPO), triphenyl phosphine oxide (TPPO), diphenylamine or their mixtures with HDEHP is lesser than with HDEHP alone.
Benzene↗
In vivo and ex vivo study of metabolic and cellular effects of 5-fluorouracil chemotherapy in a mouse mammary carcinoma.
The effect of 5-fluorouracil (5FU) on the 31P nuclear magnetic resonance (NMR) profile of a mouse mammary carcinoma, implanted on the foot of CH3/He mice, was studied both in vivo and in perchloric acid extracts. In vivo, significant increases in the ratios, nucleotide triphosphate:inorganic phosphate (Pi) (p < 0.02) and phosphocreatine:Pi (p < 0.005), were observed 48 h after 5FU, relative to control. Two readily resolvable peaks were observed in the phosphomonoester region of the in vivo NMR spectrum, phosphocholine (PC) and a peak (denoted PME') comprised of mainly phosphoethanolamine (PE). PME':PC was significantly elevated relative to control from 24 h to 168 h (p < 0.0001 at 48 h). Perchloric acid extract data indicate that the change in this ratio was due to an increase in the PE concentration rather than a decrease in PC. PE increased from 0.56 +/- 0.11 micromol/g tissue in controls to 0.95 +/- 0.29 micromol/g tissue 48 h after 5FU (p < 0.006). Perchloric acid extracts also revealed a significant increase in phosphodiesters. Glycerophosphocholine increased from 0.82 +/- 0.24 micromol/g tissue in controls to 1.82 +/- 0.61 micromol/g tissue in 5FU treated tumors after 48 h (p < 0.002), and glycerophosphoethanolamine increased from 0.25 +/- 0.06 micromol/g tissue in controls to 0.36 +/- 0.10 micromol/g tissue in treated tumors (p < 0.004). These changes suggest that ethanolamine and choline containing metabolites in this tumor may be metabolized via different pathways. Cell cycle analysis showed only relatively small changes in cell cycle distribution and apoptotic fraction following 5FU.
Adenocarcinoma↗
Enzymic fluorometric continuous-flow assays for blood glucose, lactate, pyruvate, alanine, glycerol, and 3-hydroxybutyrate.
We describe enzymic fluorometric methods of automated analysis for glucose, lactate, pyruvate, 3-hydroxybutyrate, glycerol, and alanine in perchloric acid extracts of blood. Unmodified Technicon AutoAnalyzer II apparatus is used. The usual concentrations of all these metabolites can be measured in as little as 0.1 ml of blood from a fasting subject. Within-batch and between-batch coefficients of variation ranged from 0.4 to 4.4% for all metabolites except 3-hydroxybutyrate, for which CV's were higher for low concentrations. Analytical recovery of added metabolites ranged from 92 to 98%. Glucose, lactate, alanine, and 3-hydroxybutyrate are stable in perchloric acid extracts for at least 13 days at room temperature, and a year at -20 degrees C; pyruvate shows a 6--8% loss after 3 days and 52% by one year at -20 degrees C; glycerol concentrations were stable at -20 degrees C for at least 13 days. Blank fluorescence is found in perchloric acid extracts of blood, necessitating blank runs for pyruvate, 3-hydroxybutyrate, glycerol, and alanine. The systems are simple to use, relatively inexpensive to operate, and are recommended for any laboratory with high throughput of samples.
Alanine↗
Thymine in the acid-soluble fraction of Arbacia eggs.
Mature Arbacia eggs were extracted with cold dilute perchloric acid, the extract concentrated, and the concentrate digested in hot perchloric acid. Thymine was recovered from the digest by paper chromatography, and the amount per egg found to be about 5 times the amount per sperm. This was the amount expected from previous experiments and is believed to represent all or almost all of the thymine in the egg. The result supports previous observations that DNA is absent from the mature egg although present in the nucleus of the egg in the germinal vesicle stage. No thymine could be recovered from a similar extract of 5,000 times as many sperm of the same species. The observations are consistent with the theory that DNA and its derivatives act as metabolic antagonists of the corresponding ribose compounds.
Animals↗