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PubMed2005

Comparison of molecular mobility in the glassy state between amorphous indomethacin and salicin based on spin-lattice relaxation times.

PURPOSE: The purpose of the current study was to evaluate the molecular mobility of amorphous indomethacin and salicin in the relaxed glassy state based on spin-lattice relaxation times (T(1c)) and to clarify the effects of molecular mobility on their physical stability. METHODS: Pulverized glassy amorphous indomethacin and salicin samples were completely relaxed, and the T(1c) values were investigated using solid-state (13)C-nuclear magnetic resonance (NMR) at temperatures below the glass transition temperature (T(g)). All NMR spectra were obtained using the T(1c) measurement method combined with variable-amplitude cross-polarization, the Torchia method, and total sideband suppression method. RESULTS: The T(1c) value of amorphous indomethacin indicated that 73% of carbons were in a state of monodispersive relaxation, suggesting that the amorphous state was relatively homogeneous and restricted, particularly in backbone carbons. On the other hand, 92% of carbons of amorphous salicin exhibited both fast and slow biphasic relaxation. Individual structures of the salicin molecules behaved heterogeneously, and thus the entire molecule showed relatively fast local as well as slow mobility. CONCLUSIONS: At temperatures below T(g), amorphous salicin had relatively greater molecular mobility than amorphous indomethacin. This difference in the molecular mobility of the two compounds is correlated with their crystallization behavior. Solid-state (13)C NMR provides valuable information on the physical stability of amorphous pharmaceuticals.

Benzyl Alcohols↗
PubMed1997

A rapid method for the determination of vitamin E forms in tissues and diet by high-performance liquid chromatography using a normal-phase diol column.

This paper describes a simple method for the analysis of tocopherols in tissues by which frozen tissues-70 degrees C were pulverized at dry ice temperatures (-70 degrees C) and immediately extracted with hexane. There was no need to remove the coeluting lipids from tissues by saponification, since at that level of neutral lipids in the sample, there was no reduction in fluorescence response. For the analysis of oil, in which large amounts of neutral lipids were coextracted, a 20% reduction of fluorescence response was observed, but the response was equal for all tocopherol forms, and was appropriately corrected. Saponification was used only when tocopherol esters were present, and only after an initial hexane extraction to remove the free tocopherols in order to avoid their loss by saponification, particularly non alpha-tocopherol and tocotrienols. All the tocopherols and tocotrienols were separated on a normal-phase diol (epoxide) column that gave consistent and reproducible results, without the disadvantages of nonreproducibility with silica columns, or the lack of separation with reversed-phase columns. The tocopherols were quantitated by using a tocopherol form not present in the sample as an internal tocopherol standard, or using an external tocopherol standard if all forms were present, or when the sample was saponified. Piglet heart and liver samples showed the presence of mainly alpha-tocopherol, with minor amounts of beta- and gamma-tocopherol and alpha-tocotrienol, but no delta-tocopherol. Only small amounts of tocopherol esters were present in the liver but not in the heart.

Animals↗
PubMed1997

Separation and quantitation of linoleic acid oxidation products in mammary gland tissue from mice fed low- and high-fat diets.

We have developed an assay for the isolation and quantitation by gas chromatography-mass spectrometry (GC-MS) of free 9- and 13-hydroxyoctadecadienoic acid (9-HODE, 13-HODE) in the mammary glands of female mice. Internal standards consisting of 18O2-labeled analogs of 9- and 13-HODE are added to pulverized frozen tissue prior to extraction with ethanol. Nonlipid materials are removed in a chloroform/methanol/water step. The remaining lipid material is methylated with ethereal diazomethane, and much of the nonoxygenated fatty acid methyl esters are removed via silica solid-phase extraction. Samples are either further derivatized with bis(trimethylsilyl)trifluoroacetamide to form the trimethylsilyl ethers for quantitative analysis by GC-MS or are analyzed as the methyl esters by chiral high-performance liquid chromatography to determine the enantiomeric distribution of the 9- and 13-HODE. The extraction and quantitation protocol was applied to the analysis of mammary glands for free 9- and 13-HODE from mice fed isocaloric diets containing 20% corn oil, 5% corn oil, or 20% beef tallow. Chiral analysis of the products showed higher production of 13(S)-HODE relative to 13(R)-HODE; the enantiomeric excess is most likely due to enzymatic production of 13-HODE superimposed on a background of autoxidative production of 13(R)- plus 9(S)- and 9(R)-HODE. In addition, the effect of sample handling and storage conditions on the formation of 9- and 13-HODE in the samples was assessed by exposing aliquots of a common pool of rat mammary gland tissue to specified conditions prior to analysis. This methodology will be important during investigations of the contribution of linoleate oxidation products to the enhancement of mammary tumorigenesis by dietary fat.

Analysis of Variance↗
PubMed1986

Usefulness of oral dipyridamole suspension for stress thallium imaging without exercise in the detection of coronary artery disease.

Stress thallium imaging with intravenous dipyridamole permits assessment of coronary artery disease (CAD) without the need for exercise. However, intravenous dipyridamole is available in the United States only on an experimental basis. To study the use of oral dipyridamole as a clinically available alternative to intravenous dipyridamole for this purpose, 100 patients underwent thallium imaging with oral dipyridamole. Each patient received 300 mg of pulverized tablets in a 30-ml suspension. Maximal increase in mean heart rate and decrease in mean blood pressure occurred 30 minutes after ingestion. At 45 minutes, 2 mCi of thallium was given intravenously and serial imaging was begun within 7 minutes. The serum dipyridamole level (mean +/- standard deviation) 45 minutes after 300 mg was administered orally (3.7 +/- 2.2 micrograms/ml) was similar to that 5 minutes after 0.56 mg/kg was given intravenously (4.6 +/- 1.3 micrograms/ml). Fifty-five patients had some adverse effects between 15 and 75 minutes after oral ingestion, including nausea, headache, dizziness, chest pain (25 patients) and electrocardiographic changes (14 patients). Intravenous aminophylline was used to resolve these adverse effects in 21 patients. There were no severe arrhythmias, myocardial infarctions or deaths. Of the 43 patients with angiographically documented CAD, 39 had an initial perfusion defect that redistributed on the delayed images. When the results in patients who had undergone catheterization were analyzed by individual segment, the presence of thallium redistribution was associated with normal or hypokinetic contrast left ventriculographic wall motion of that segment, whereas the presence of a persistent defect was associated with akinesia or dyskinesia (Fisher's standardized Z = 9.14).(ABSTRACT TRUNCATED AT 250 WORDS)

Administration, Oral↗
PubMed1987

Methods for DNA extraction from Candida albicans.

Three different methods are described for the extraction of total genomic DNA from the dimorphic fungus Candida albicans. One method, which enables a large number of cultures to be processed simultaneously, involves pulverizing dried cells with glass beads and then allowing the disrupted cells to break apart, autolyse, by incubation in a solution which includes sorbitol and a nonionic detergent. DNA extraction by a second method with a French pressure cell can be utilized on cultures in any phase of growth, but is not practical for processing numerous samples. The third method, which involves induction of spheroplasts, is commonly used for DNA extraction from various yeasts but is not suited for processing many samples simultaneously. The DNA extracted with the three procedures is comparable in quality; in particular, it is of high molecular size (greater than 30 kbp) and reacts readily with DNA-modifying enzymes such as restriction endonucleases.

Candida albicans↗
PubMed1989

A method for preparing freeze-clamped tissue samples for metabolite analyses.

A rapid and simple method for preparing freeze-clamped tissue samples for metabolite determinations is described. Freeze-clamped rat heart tissue samples weighing from 0.8 to 1.0 g were homogenized directly in an Ultra-Turrax homogenizer for 60 s in 3.5 ml of ice-cold 0.6 M HClO4 without pulverizing them in liquid nitrogen. After centrifugation, the pellet was rehomogenized in the Ultra-Turrax homogenizer for 30 s in 1.5 ml of HClO4. Following a further centrifugation the extracts were combined and the pH was adjusted to 7.0 by adding 5 M K2CO3. The neutralized supernatant was used for the desired assays. The analyses of the tissue extracts obtained from isolated perfused rat hearts by the present method give similar results for different kinds of metabolites than those processed according to the previous classical method. Moreover, the values of the various parameters determined from the tissue extracts prepared according to the method described here are similar to the data reported in literature. The method can be readily applied to any other freeze-clamped tissue. The greatest improvement obtained is that the homogenization procedure can be accomplished easily and conveniently in about one-tenth of the time required for the earlier classical method without the time-consuming and unpleasant tissue grinding in liquid nitrogen.

Adenosine Triphosphate↗
PubMed1991

Selective removal of molybdenum traces from growth media of N2-fixing bacteria.

A new method for the selective removal of traces of molybdenum from growth media of N2-fixing bacteria (Rhodobacter capsulatus and Klebsiella pneumoniae) was developed. This method is based on the filtration of nutrient solutions through a layer of activated carbon (pulverized charcoal). The adsorption of Mo (molybdate) to activated carbon was optimal if a charcoal suspension (50 g/liter) was degassed by boiling before use and if the pH of the solutions, which had to be purified, was adjusted to values between 1.5 and 4. In this pH region no or only negligible amounts of other metal ions were adsorbed. The activated carbon method was compared with other Mo-eliminating procedures, including 8-hydroxyquinoline/dichloromethane extraction, Chelex 100 chromatography, and treatment with Mo-starved Azotobacter vinelandii cells. The activated carbon filtration appeared to be the most effective, specific, and rapid method. Whereas the untreated Rhodobacter growth medium was contaminated with 1.2 ppb Mo, as analyzed by inductively coupled plasma mass spectrometry (ICP-MS), the activated carbon-treated medium was below the ICP-MS detection limit (less than 0.05 ppb). A similarly effective removal of Mo impurities was obtained by the Azotobacter treatment. Even at low optical densities (2-5 at 436 nm) Mo traces were removed very rapidly within 10-15 min. However, because the Mo uptake/Mo adsorption capacity of A. vinelandii depended on freshly cultivated cells and on the growth phase at which the cells were harvested, this microbiological method was generally more time-consuming and less reproducible than the activated carbon method.(ABSTRACT TRUNCATED AT 250 WORDS)

Adsorption↗
PubMed1982

Lipid composition of the matrix of human submandibular salivary gland stones.

The lyophilized stone matrix, prepared by exhaustive dialysis of pulverized stones against 10 per cent EDTA and water, yielded 9.6 +/- 0.9 mg per cent of lipids. Following fractionation of the lipid extract on silicic acid columns, 57 per cent of lipids were found in the neutral lipid fraction, 40 per cent in the glycolipid fraction and 3 per cent in the phospholipid fraction. Of the total neutral lipids, 77.3 per cent were represented by free fatty acids, 14.7 per cent by cholesterol, 4.1 per cent by triglycerides and 3.2 per cent by cholesterol esters. The glycolipid fraction was comprised of simple glycosphingolipids (11.3 per cent), mainly lactosyl- and glucosylceramides, and of neutral and sulphated glyceroglucolipids (88.7 per cent). The sulphated glyceroglucolipids consisted of tri- and tetraglucosyl glyceroglucolipids, whereas hexa- and octaglucosyl glyceroglucolipids were the predominant constituents of the neutral glyceroglucolipid fraction. The phospholipids exhibited a high content of sphingomyelin (22.3 per cent), phosphatidylcholine (12.1 per cent), phosphatidylethanolamine (11.4 per cent), phosphatidylserine (10.5 per cent) and lysophosphatidylcholine (13.3 per cent). The major fatty acids of the neutral lipid fraction were hexadecanoate, octadecanoate and octadecenoate. The glycolipids contained large quantities of hexadecanoate and tetracosanoate, while the hexadecanoate, octadecanoate and tetracosanoate were the principal fatty acids of the phospholipid fraction.

Adult↗
PubMed1992

Effects of maternal caffeine intake during lactation on molar enamel surfaces in new-born rats.

Dams were fed normal laboratory chow until delivery. At birth, the litters were combined, and eight pups were randomly assigned to each dam. Dams with the recombined litters were divided into two groups. Dams of group 1 were fed a 20% protein diet as a control; dams of group 2 were fed a 20% protein diet supplemented with caffeine (2 mg/100 g of the dam's weight). On day 22, the dams of group 2 were anaesthetized with ether. They were injected with 2 iu of oxytocin in order to collect milk. Blood was collected from pups and dams to determine its caffeine concentration. The first and second molars were removed from each pup's mandible and maxilla. Radiographs were taken of 10 randomly selected first or second molars from each group. Four randomly selected molars from each litter were placed in a specially designed chamber and bathed with a constant flow of acid solution to determine the amount of mineral dissolved from the enamel surfaces. The remaining non-acid exposed molars were pulverized in freezer mills. A small portion of this powder was then analysed for the total amount of minerals. No differences were found in the radiographic density of enamel between the groups. The amount of dissolved calcium, phosphorus and magnesium from enamel surfaces in the caffeine group was consistently greater than that of the non-caffeine group in the first molars, whereas, in the second molars, there was no difference between the groups.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1992

Physical examination of caffeine's effects on the enamel surface of first molar in new-born rats.

Samples of the first molars of offspring whose dams were fed a diet supplemented with caffeine were examined by scanning electron microscopy, X-ray diffractometry and electron microprobe analysis. Scanning microscopy of the enamel surface of the caffeine group revealed a consistently rougher surface than in the non-caffeine controls, both before and after acid exposure. X-ray diffraction analysis of the pulverized whole tooth in the caffeine group showed broader diffraction peaks for the lattice plane reflections (202) and (300), indicating smaller crystallites. Pure enamel samples of the caffeine group examined with a Gandolfi X-ray camera also revealed more diffuse diffraction lines than in the non-caffeine controls, further indicating smaller crystallites in the enamel. The calcium and phosphorus contents of the acid-exposed samples in both control and caffeine groups were lower than the non-acid exposed control and caffeine groups by electron microprobe analysis. After exposure to acid, the calcium and phosphorus contents of the outer surface of the enamel in the caffeine group were greatly reduced as compared to that of the non-caffeine controls. Thus various methods consistently indicate that caffeine ingestion during early growth affects the enamel surface of the first molars, resulting in impaired mineralization. Caffeine intake may therefore have a negative effect on amelogenesis and possibly increases susceptibility to dental caries.

Animals↗
PubMed1993

Site comparisons of dentine collagen cross-links from extracted human teeth.

Covalent intermolecular cross-links in collagen provide the dentine matrix with stability and tensile strength. The density of collagen cross-links varies depending on the site within the same tissue. This variation is probably due to factors such as the different amounts of stress and different turnover rates at the respective sites. The aim was to quantify the collagen cross-links in different tooth groups as an adaptation to functional requirements. For this purpose, the types and content of major cross-links in dentine collagen of human teeth from three different sites were measured and compared: incisors, premolar-canines and molars. After removal of cementum and pulp, 23 extracted teeth at different ages (27-69 yr) were individually pulverized and demineralized with EDTA. Collagen was reduced with standardized NaB3H4, hydrolysed, and subjected to amino acid and cross-link analyses. Each cross-link was quantified on the basis of mole per mole of collagen. The results indicated: (1) all teeth contained labile, reducible (dehydro-dihydroxylysinonorleucine and dehydro-hydroxylysinonorleucine) and stable, non-reducible (pyridinoline and its lysyl analogue) cross-links, and (2) the content of both reducible and non-reducible cross-links was least in incisors and greatest in molars. This suggests that dentine collagen matrix maybe functionally adaptive.

Adult↗
PubMed1994

An in vitro model for adhesion of bacteria to human tooth root surfaces.

A model mimicking bacterial colonization of dentine has been developed. It employs uniform particles of pulverized human tooth root tissue incubated with radioactively labelled bacteria. After incubation, the number of attaching bacteria is quantified. Attachment of Streptococcus mutans UA140, Actinomyces viscosus T14, and Lactobacillus casei 101 was found to be time dependent and complete within 1-3 h. Dissociation constants (Kd) of the interactions equalled 2.5 x 10(8) and 1.6 x 10(8) cells/ml, for Strep. mutants and A. viscosus, respectively. The Kd for L. casei could not be determined as attachment was not saturable. The putative tissue components involved in adherence were studied by determining the attachment of bacteria in the presence of competing strains. The results suggest that Strep. mutans and A. viscosus recognized and competed for the same ligand (probably collagen) in the dentine. L. casei attachment did not complete with the attachment of Strep. mutans and A. viscosus. Attachment of all strains was modified by preincubation with saliva and varied with bacterial strain and saliva donor.

Actinomyces↗
PubMed1994

Identification of tissue inhibitor of metalloproteinases-1 (TIMP-1) in human teeth and its distribution in cementum and dentine.

An inhibitory activity toward matrix metalloproteinases such as interstitial collagenase, 72-kDa gelatinase/type IV collagenase, and stromelysin-1 was detected in an EDTA extract of pulverized roots of human teeth, and identified as TIMP-1 by sodium dodecyl sulphate-polyacrylamide gel electrophoresis and immunoblotting. Distribution of TIMP-1 in human cementum and dentine was investigated by a sandwich enzyme immunoassay in combination with an abrasive microsampling technique. TIMP-1 could not be detected in cementum from some teeth but in others decreased from a fairly low value at the surface towards the cementodentinal junction. TIMP-1 concentrations in the dentine increased consistently from the cementodentinal junction toward the predentine. The average TIMP-1 concentration in the dentine (54.1 +/- 18.5 pg/mg +/- SE) was significantly (P < 0.05) higher than that (9.6 +/- 6.0 pg/mg +/- SE) in the cementum.

Adolescent↗
PubMed1984

A method for quantitative wet chemical analysis of urinary calculi.

We describe a simple method for quantitative chemical analysis of urinary calculi requiring no specialized equipment. Pulverized calculi are dried over silica gel at room temperature and dissolved in nitric acid, which was the only effective agent for complete dissolution. Calcium, magnesium, ammonium, and phosphate are then determined by conventional methods. Oxalate is determined by a method based on the quenching action of oxalate on the fluorescence of a zirconium-flavonol complex. Uric acid, when treated with nitric acid, is stoichiometrically converted to alloxan, which is determined fluorimetrically with 1,2-phenylenediamine. Similarly, cystine is oxidized by nitric acid to sulfate, which is determined turbidimetrically as barium sulfate. Protein is determined spectrophotometrically as xanthoprotein. The total mass recovery of authentic calculi was 92.2 +/- 6.7 (SD) per cent. The method permits analysis of calculi as small as 1.0 mg. Internal quality control is performed with specially designed control samples.

Cations↗
PubMed1980

Distribution of substance P in the rat gastrointestinal tract--lack of effect of capsaicin pretreatment.

A new method for extraction of immunoreactive substance P (I-SP) from rat intestine including pulverization of tissue frozen in liquid nitrogen and extraction with acid acetone is described. Using this method, amounts of I-SP in the rat intestine were found to be higher than previously reported. The highest concentrations of I-SP were found in the small intestine. Capsaicin pretreatment of newborn or adult rats had no effect on intestinal I-SP concentrations indicating that intrinsic SP neurones are capsaicin-insensitive.

Animals↗
PubMed1991

The S-phase cytotoxicity of camptothecin.

The DNA topoisomerase I inhibitor camptothecin (CAM) is selectively cytotoxic to S-phase cells of HL-60, and some other myelogenous leukemic lines. The early effects of cell exposure to 0.05-0.2 micrograms/ml CAM are seen after 2 h; at that time a progressive degradation of DNA in the chromatin of S-phase cells is initiated. The degradation manifests by "pulverization" of chromatin followed by coalescence of the fine granules and nuclear disintegration. Between 2 and 6 h of treatment, a loss of about 30-70% of DNA from S-phase nuclei is detected by flow cytometry. A 10-min pulse of CAM is adequate to trigger subsequent DNA degradation. Agarose gel electrophoresis of DNA from CAM-treated cells reveals a typical nucleosome core particles "ladder," suggestive of preferential degradation of spacer DNA. Despite extensive loss of DNA and nuclear disintegration, the cell membrane of CAM-treated S-phase cells remains intact for several hours, excluding trypan blue or propidium iodide. Mitochondria, assayed for their ability to maintain a transmembrane potential (rhodamine 123 retention), as well as the lysosomal proton pump (probed by supravital uptake of acridine orange) also remain unchanged in these cells. G1 cells are refractory to CAM under these conditions. Synchronization of cells in S phase by aphidicolin increases the sensitivity of the whole cell population to CAM. The data suggest that CAM or other topoisomerase I inhibitors may be effective in some myelogenous leukemias, especially in combination with treatments synchronizing cells in S phase.

Camptothecin↗
PubMed1992

Organic compounds in the Forest Vale, H4 ordinary chondrite.

We have analyzed the H4 ordinary chondrite Forest Vale for polycyclic aromatic hydrocarbons (PAHs) using two-step laser mass spectrometry (L2MS) and for amino acids using a standard chromatographic method. Indigenous PAHs were identified in the matrices of freshly cleaved interior faces but could not be detected in pulverized silicates and chondrules. No depth dependence of the PAHs was found in a chipped interior piece. Amino acids, taken from the entire sample, consisted of protein amino acids that were nonracemic, indicating that they are terrestrial contaminants. The presence of indigenous PAHs and absence of indigenous amino acids provides support for the contention that different processes and environments contributed to the synthesis of the organic matter in the solar system.

Amino Acids↗
PubMed1996

Polystyrene as an affinity chromatography matrix for the purification of antibodies.

Affinity chromatography is used for the purification of diagnostic polyclonal antibodies in order to ensure specificity. Most commonly, activated bead-formed agarose or its derivatives are used as gel matrices. Alternative matrix materials have been described, but as yet they do not appear to offer important advantages. In this study, pulverized polystyrene (PS 158K, BASF, Mannheim, Germany) was used as a solid phase for the immobilisation of bovine immunoglobulins (Ig). Affinity chromatography was performed using these coated polystyrene beads as the column matrix material in the purification of anti-bovine Ig. The polystyrene binding capacity for the different bovine Ig classes was compared using the Mancini single radial immunodiffusion technique, and ELISA procedures were used to monitor the antibody reactivity of purified and unpurified antibodies. The degree of purification was comparable to the most commonly used procedure using gel matrices from activated bead-formed agarose (e.g. CNBr-activated Sepharose 4B, Pharmacia/LKB Biotechnology, Uppsala, Sweden), but the antibody yield per ml column volume was distinctly lower. In order to raise the yield, such polystyrene bead columns with immobilized antigen can be re-used without loss of activity or larger column volumes can be used to raise the binding capacity. The polystyrene material is quite durable, chemically and immunologically inert and has a long shelf life. We conclude that polystyrene based affinity chromatography is efficient, simple and cheap.

Adsorption↗