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PubMed1977

Extraction and analytic procedures for cytosine arabinoside and 1-beta-D-arabinofuranosyluracil and their 5'-mono-, di-, and tri-phosphates.

A qualitative and quantitative comparison of perchloric acid (PCA) and trichloroacetic acid (TCA) extraction procedures of biologic material containing cytosine arabinoside (Ara-C) and 1-beta-D-arabinofuranosyluracil (Ara-U) and their biologic 5'-phosphate derivatives revealed definite superiority of the PCA procedure over the TCA procedure. High-pressure liquid chromatography provides rapid and accurate quantitative and qualitative separation of both Ara-C and Ara-U and their 5'-mono-, di-, and tri-phosphates (mu Bondapak NH2 column), or their 5'-mono-, di-, and tri-phosphates without nucleosides (ABX Permaphase columns).

Animals↗

PubMed1994

Assay of ornithine aminotransferase with ninhydrin.

We developed an assay system for ornithine aminotransferase (EC 2.6.1.13) using ninhydrin. Pyrroline 5-carboxylate, a product of enzymatic transamination, reacts with ninhydrin under hot acidic conditions to form a reddish pigment soluble in ethanol. The millimolar extinction coefficient of reaction product dissolved in ethanol was 16.5 at 510 nm. Acidification with perchloric acid effectively abolished the interfering color development by L-ornithine and L-glutamate. The paired activity measurement in mouse tissues by ninhydrin and o-aminobenzaldehyde methods showed a good correlation (gamma = 0.985). In our ninhydrin method, stable ninhydrin replaced unstable o-aminobenzaldehyde, and sensitivity was much higher than that with the conventional o-aminobenzaldehyde method.

Animals↗

PubMed1986

A modified ferrozine method for the measurement of enzyme-bound iron.

A general procedure for the determination of the iron content of enzymes by digestion with methanesulfonic acid to release protein-bound iron has been developed. This procedure replaces the tedious and potentially hazardous method of wet ashing with concentrated nitric-sulfuric-perchloric acids. The method has been used to determine the stoichiometry of iron for nanomole quantities of heme-iron proteins, iron-sulfur proteins, complex iron-sulfur proteins, as well as in phenylalanine hydroxylase, an enzyme with iron in an undetermined coordination.

Catalase↗

PubMed2006

Determination of CYP4A11-catalyzed lauric acid 12-hydroxylation by high-performance liquid chromatography with radiometric detection.

Lauric acid serves as an endogenous substrate for the cytochrome P450 enzyme CYP4A11. A reverse-phase, high-performance liquid chromatography method is described for the quantification of 12-hydroxylauric acid formed enzymatically by incubation of 14C-labeled lauric acid with cDNA-expressed CYP4A11 or human liver microsomes. Analytical separation is achieved using a C18 column and a gradient of 30% acetonitrile and 2 mM perchloric acid to 100% methanol, using a detection scintillation counter. This method is applicable to enzymatic studies for determination of lauric acid 12-hydroxylation activity.

Catalysis↗

PubMed1986

Relationship between DNA acid-solubility and frequency of single-strand breaks near apurinic sites.

Using [32P]DNA alkylated with [3H]methyl methanesulfonate, depurinated by heating at 50 degrees C for various periods, then treated with sodium hydroxide, a table was constructed giving the DNA fraction soluble in 5% perchloric acid at 0 degree C as a function of the frequency of strand breaks. The alkaline treatment placed a break near each apurinic site; the apurinic sites were counted in two ways which gave consonant results: by the loss of [3H]methyl groups and by reaction with [14C]methoxyamine. The 32P label of DNA was used to measure the acid-solubility.

DNA, Bacterial↗

PubMed1977

Explanation of the observation of pancreatic ribonuclease activity at pH 4.5.

A recent conclusion that beef pancreas contained a molecular species of ribonuclease with intrinsically high activity at pH 4.5 has been found to be incorrect. The particular assay used in the earlier experiments gives anomalous results at acid pH in the presence of low concentrations of ions such as phosphate which was used during the fractionation. By turning to the more widely employed form of the perchloric acid precipitation assay, interference is avoided and the ribonuclease in beef pancreas is confirmed as consisting almost completely of the molecular species well-characterized as ribonuclease A. The clarification of the assay question permits a clear interpretation of the results of each step of the chromatographic purification procedure that led to the initial conclusion, including an artifact that arose when gel filtration was attempted with distilled water rather than with buffer.

Animals↗

PubMed1975

Stability and dissociation of P3H4-1 Burkitt's lymphoma cell soluble complement-fixing antigen identified with human serum.

The soluble complement-fixing antigen of the P3HR-1 Burkitt's lymphoma cell line, identified with human serum containing antibody against Epstein-Barr virus viral capsid antigen, loses activity under a variety of conditions. The major characteristic reported here is retention of activity on exposure to specific physical or chemical conditions followed by loss of activity after subsequent neutral dialysis. Antigen of untreated cell lysates, which retains activity after dialysis against large volumes of neutral buffers or 0.14 M NaCl, will lose activity if the lysate is first heated to 56 degrees or if the lysate is exposed to acid perchlorate, and the resulting precipitate and supernatant are redialyzed separately to neutral pH. The P3HR-1 soluble complement-fixing antigen appears to be an aggregate including a labile, dissociable component.

Antibodies, Viral↗

PubMed1985

Quantitative liquid chromatographic determination of bromadoline and its N-demethylated metabolites in blood, plasma, serum, and urine samples.

Bromadoline and its two N-demethylated metabolites were extracted into ether:butyl chloride after the addition of internal standard and basification of the various biological fluids (blood, plasma, serum, and urine). These compounds were then extracted into dilute phosphoric acid from the organic phase and separated on a reversed-phase chromatographic system using a mobile phase containing acetonitrile and a buffer of 1,4-dimethylpiperazine and perchloric acid. The overall absolute extraction recoveries of these compounds were approximately 50-80%. The background interferences from the biological fluids were negligible and allowed quantitative determination of bromadoline and the metabolites at levels as low as 2-5 ng/mL. At mobile phase flow rate of 1 mL/min, the sample components and the internal standard were eluted at the retention times within approximately 7-12 min. The drug- and metabolite-to-internal standard peak height ratios showed excellent linear relationships with their corresponding concentrations. The analytical method showed satisfactory within- and between-run assay precision and accuracy, and has been utilized in the simultaneous determination of bromadoline and its two N-demethylated metabolites in biological fluids collected from humans and from dogs after administration of bromadoline maleate.

Benzamides↗

PubMed1996

Finger printing of Mycobacterium tuberculosis in patients with intracranial tuberculomas by using in vivo, ex vivo, and in vitro magnetic resonance spectroscopy.

In vivo, ex vivo, and in vitro proton magnetic resonance spectroscopy was performed in 12 patients with intracranial tuberculomas with an aim of detecting the biochemical constituents of Mycobacterium tuberculosis in a granuloma. One dimensional (1D) single pulse and spin-echo sequences and 2D correlative spectroscopy were used for the ex vivo study to confirm the resonances seen on in vivo study. Spectroscopic studies of the perchloric acid and lipid extract of granuloma and M. tuberculosis were performed to look for similarity of resonance. In vivo study showed the presence of lipids at 0.9, 1.3, 2.0, 2.8 ppm, and phosphoserine at 3.7 ppm. All these resonances were confirmed on ex vivo study. In addition, distinct resonances of serine and phenolic lipids were seen on ex vivo and in vitro study of tuberculous granuloma, which have not been observed in other intracranial tumors. Lipid extract of granuloma and M. tuberculosis showed phenolic lipids at 7.1 and 7.4 ppm, a constituent of the cell wall of the bacteria in a tuberculoma. It appears that it may be possible to finger print the biochemicals of the cell wall of M. tuberculosis in a tuberculous granuloma and thus may help in detection and diagnosis of such lesions.

Amino Acids↗

PubMed1997

31P NMR identification of metabolites and pH determination in the cyanobacterium Synechocystis sp. PCC 6308.

The identity of a number of phosphorus-containing metabolites present in Synechocystis sp. PCC 6308 has been confirmed by 31P NMR spectroscopy. The presence of D-ribulose 1,5-bisphosphate (RuBP); DL-glyceraldehyde 3-phosphate (GlyP); D(-)3-phosphoglyceric acid (3PGA); D-ribulose 5-phosphate (Ru5P);6-phosphogluconic acid (6PGA); phosphoenolpyruvate (PEP); inorganic phosphate (Pi); uridine diphosphoglucose (UDPG); ADP and ATP were demonstrated by the pH dependence of their 31P NMR chemical shifts in spectra of perchloric acid cell extracts. Intracellular pH of cells was determined to be 7.5-7.7.

Adenosine Diphosphate↗

PubMed2004

Structure of the hydrated, hydrolysed and solvated zirconium(IV) and hafnium(IV) ions in water and aprotic oxygen donor solvents. A crystallographic, EXAFS spectroscopic and large angle X-ray scattering study.

The tetrameric hydrolysis products of zirconium(IV) and hafnium(IV), the zirconyl(IV) and hafnyl(IV) ions, [M(4)(OH)(8)(OH(2))(16)(8+)], often labelled MO(2+).5H(2)O, are in principle the only zirconium(IV) and hafnium(IV) species present in aqueous solution without stabilising ligands and pH larger than zero. These complexes are furthermore kinetically very stable and do not become protonated even after refluxing in concentrated acid for at least a week. The structures of these complexes have been determined in both solid state and aqueous solution by means of crystallography, EXAFS and large angle X-ray scattering (LAXS). Each metal ion in the [M(4)(OH)(8)(OH(2))(16)](8+) complex binds four hydroxide ions in double hydroxo bridges, and four water molecules terminally. The M-O bond distance to the hydroxide ions are markedly shorter, ca. 0.12 A, than to the water molecules. The hydrated zirconium(IV) and hafnium(IV) ions only exist in extremely acidic aqueous solution due to their very strong tendency to hydrolyse. The structure of the hydrated zirconium(IV) and hafnium(IV) ions has been determined in concentrated aqueous perchloric acid by means of EXAFS, with both ions being eight-coordinated, most probably in square antiprismatic fashion, with mean Zr-O and Hf-O bond distances of 2.187(3) and 2.160(12) A, respectively. The dimethyl sulfoxide solvated zirconium(IV) and hafnium(IV) ions are square antiprismatic in both solid state and solution, with mean Zr-O and Hf-O bond distances of 2.193(1) and 2.181(6) A, respectively, in the solid state. Hafnium(IV) chloride does not dissociate in N,N'-dimethylpropyleneurea, dmpu, a solvent with good solvating properties but with a somewhat lower permittivity (epsilon= 36.1) than dimethyl sulfoxide (epsilon= 46.4), and an octahedral HfCl(4)(dmpu)(2) complex is formed.

Journal Article↗

PubMed1970

The effects of oestradiol on the acid-soluble nucleotides of rat uterus.

1. Techniques have been developed to measure the concentrations of the ribonucleotides of the immature rat uterus in vivo. Tissue was frozen rapidly in liquid nitrogen, ground to a fine powder, dispersed in frozen perchloric acid and thawed slowly. Nucleotides were separated from other acid-soluble constituents on short columns of polyethyleneimine-cellulose and the mixture was resolved into individual nucleotides by two-dimensional thin-layer chromatography on polyethyl-eneimine-cellulose plates. 2. The nucleotides of immature rat uterus consisted of approximately 75% of ATP-ADP, 10-12% each of GTP-GDP and UTP-UDP and less than 2% of CTP. 3. Injection of oestradiol (5mug) promoted a linear decrease in the amounts of purine nucleotides to approximately 60% of control values in 4-5h, followed by a return to greater than control values in 8-10h. Concentrations of the pyrimidine nucleotides remained constant for 4-6h and then increased to 200% of control at 12h after hormone treatment.

Adenine Nucleotides↗

PubMed1994

Changes in regional protein synthesis in rat brain and pituitary after systemic interleukin-1 beta administration.

A convenient and sensitive method has been developed for measuring changes in protein synthesis in discrete areas of the brain and pituitary of conscious freely moving rats. A single injection of high-concentration low-specific activity L-[35S]methionine is given to flood amino acid precursor pools, thereby equalizing the specific activity of the L-[35S]methionine throughout the tissue. Unincorporated L-[35S]methionine is removed from cryostat sections by treatment with perchloric acid (2%) before quantitative autoradiography. The sensitivity of this technique is demonstrated by the detection of changes in protein synthesis in regions of the brain and pituitary after systemic administration of interleukin-1 beta, a cytokine that has centrally mediated effects but which is not thought to cross the blood-brain barrier. Areas of the brain found to exhibit significant increases in protein synthesis were the subfornical organ, the choroid plexus, the medial habenular, the dentate gyrus, and the anterior and posterior lobes of the pituitary. In the brain, the cingulate cortex and the pineal gland showed significant decreases in the rate of protein synthesis.

Animals↗

PubMed1990

Analysis of tricarboxylic acid cycle of the heart using 13C isotope isomers.

13C-nuclear magnetic resonance (NMR) spectroscopy provides a new approach to the analysis of metabolic pathways, because it detects an interaction between adjacent 13C nuclei. Previous models of isotope distribution in the tricarboxylic acid cycle were designed for analysis of radioisotope data and did not consider the information provided by 13C-13C coupling. A mathematical model of the tricarboxylic acid cycle was developed that preserves all isotope isomer (isotopomer) information and yields simple relationships between 13C-NMR spectra of glutamate and metabolic parameters under steady-state conditions. With the use of relative peak areas measured from the spectra of tissues supplied with 13C-enriched substrate(s), the relative fluxes through both oxidative (acetyl-CoA utilization) and nonoxidative (anaplerotic) pathways of the tricarboxylic acid cycle can be determined. Furthermore, with the judicious selection of 13C-labeling patterns in a mixture of substrates, direct substrate competition experiments can be performed. The perchloric acid extracts of Langendorff and working rat hearts oxidizing 13C-enriched fatty acids or carbohydrates are analyzed to illustrate this approach, and the importance of measuring the fractional enrichment of the available substrate is demonstrated. The technique can of course be used with all tissues, not just heart, and is applicable to the analysis of in vivo 13C-NMR spectra.

Animals↗

PubMed1977

Determination of solasodine in fruits of Solanum species.

A potentiometric nonaqueous titration procedure was developed for the quantitative determination of solasodine in fruits of the Solanum species. The steroid glycoalkaloids were extracted from freshly harvested fruits with 2% acetic acid and methanol. After hydrolysis, the common aglycone solasodine was extracted with benzene. An aliquot was mixed with an equal volume of acetone and titrated potentiometrically with 0.005 N perchloric acid in dioxane, using glass and silver electrodes for the determination.

Fruit↗

PubMed1995

Determination of extracellular and intracellular thiopurines and methylthiopurines by high-performance liquid chromatography.

The thiopurine antimetabolites 6-thioguanine and 6-mercaptopurine are important chemotherapeutic drugs in the treatment of childhood acute lymphoblastic leukaemia. Measurement of metabolites of these thiopurines is important because correlations exist between levels of these metabolites and the prognosis in childhood acute lymphoblastic leukaemia. The reversed-phase method for the determination of extracellular thiopurine nucleosides and bases was previously developed and has been modified such that methylthiopurine nucleosides, bases, thioxanthine and thiouric acid can be measured also. The anion-exchange method enables the determination of intracellular mono-, di- and triphosphate (methyl)thiopurine nucleotides in one run. Extraction on ice with perchloric acid and dipotassium hydrogenphosphate results in good recoveries for (methyl)thiopurine nucleotides in lymphoblasts and peripheral mononuclear cells and for methylthioinosine nucleotides in red blood cells. Measurement of the low concentrations of mono-, di- and triphosphate thioguanine nucleotides in red blood cells (detection limit 20 pmol/10(9) cells) is possible after extraction with methanol and methylene chloride, followed by oxidation of thioguanine nucleotides with permanganate and fluorimetric detection.

Calibration↗

PubMed2003

Square wave adsorptive stripping voltammetric determination of piromidic acid. Application in urine.

A simple procedure for the determination of piromidic acid by square wave adsorptive stripping voltammetry (SW-AdSV) at a hanging mercury drop electrode has been developed. The variables affecting to accumulation process such as concentration of perchloric acid, accumulation potential and accumulation time have been optimised (0.025 mol L(-1), -0.25 V and 140 s, respectively) by using response surface methodology. A linear relationship between concentration of piromidic acid and peak intensity has been found in the range 2.22 x 10(-9) to 3.33 x 10(-8) mol L(-1). The detection limit (1.65 x 10(-9) mol L(-1)) has been calculated by the method proposed by Clayton et al. so that protection against both false positive and false negative errors is assured. The procedure was successfully applied to determine piromidic acid in spiked urine samples. The obtained recovery values were in the range 97.3-103.3% at different levels of concentration of piromidic acid.

Electrochemistry↗

PubMed2002

Determination of donepezil, an acetylcholinesterase inhibitor, in human plasma by high-performance liquid chromatography with ultraviolet absorbance detection.

A simple and sensitive high-performance liquid chromatographic (HPLC) method with UV absorbance detection is described for the quantification of donepezil, a centrally and selectively acting acetyleholinesterase inhibitor, in human plasma. After sample alkalinization with 0.5 ml of NaOH (0.1 M), the test compound was extracted from I ml of plasma using isopropanol-hexane (3:97, v/v). The organic phase was back-extracted with 75 microl of HCl (0.1 M) and 50 microl of the acid solution was injected into a C18 STR ODS-II analytical column (5 microm, 150x4.6 mm I.D.). The mobile phase consisted of phosphate buffer (0.02 M, pH 4.6), perchloric acid (6 M) and acetonitrile (59.5:0.5:40, v/v) and was delivered at a flow-rate of 1.0 ml/min at 40 degrees C. The peak was detected using a UV detector set at 315 nm, and the total time for a chromatographic separation was approximately 8 min. The method was validated for the concentration range 3-90 ng/ml. Mean recoveries were 89-98%. Intra- and inter-day relative standard deviations were less than 7.3 and 7.6%, respectively, at the concentrations ranging from 3 to 90 ng/ml. The method shows good specificity with respect to commonly prescribed psychotropic drugs, and it could be successfully applied for pharmacokinetic studies and therapeutic drug monitoring.

Cholinesterase Inhibitors↗