Increase in soluble CD95L during subacute phases after human spinal cord injury: a potential therapeutic target.

STUDY DESIGN: A pilot study measuring the levels of serum-soluble CD95 ligand (CD95L) in eight spinal cord-injured patients. OBJECTIVES: To determine the soluble concentration of CD95L in spinal cord injury (SCI) patients after trauma. METHODS: We collected blood samples from eight patients with acute traumatic SCI. Soluble CD95L serum levels were determined using an enzyme-linked immunosorbent assay. American Spinal Injury Association (ASIA) was determined according to ASIA classification. The patients were monitored, and venous blood was drawn after arrival at the hospital on the 1st and 3rd day and during the 1st, 2nd, 4th, 8th and 12th weeks after trauma. RESULTS: The average patient age was 48.1 years (18-86 years). Three patients were paraplegic (two incomplete, one complete), five were quadriplegic (one complete, four incomplete). The serum concentration of soluble CD95L (sCD95L) decreased during the 1st week (41 ng(- l)) and increased after the 2nd week in all eight patients. It peaked during the 4th week (68.5 ng (- l)) and reached a plateau during the 12th week (76.2 ng (- l)). There are many possible explanations for not being able to detect a statistical significance, one of course being the small sample size. CONCLUSION: Promising results for anti-CD95L therapy have already been documented in lab studies with rodents. Anti-CD95L blocks the pro-apoptotic and proinflammatory activity of membrane-bound CD95L during the acute phase of SCI. We observed that sCD95L levels are elevated during the subacute and intermediate phases of SCI. It would be of great interest to study a larger group of patients to determine whether higher sCD95 levels are correlated with improved or impaired neurological outcome or with increasing levels of autoimmune components in peripheral blood.

Diagnostic quality in endoscopic retrograde cholangiopancreatography: comparison between lodixanol and lopromide.

BACKGROUND AND STUDY AIMS: The contrast medium for endoscopic retrograde cholangiopancreatography (ERCP) must produce adequate images, whilst keeping the risk of complications to a minimum. There is a theoretical reason to suppose that a dimeric isosmolar medium may be superior to a monomeric hyperosmolar medium. We have compared two such media in a triple-centre double-blind randomized study on patients referred with a clinical indication for ERCP. PATIENTS AND METHODS: A total of 160 patients were randomly allocated to either lodixanol or lopromide. Assessments were made of the quality of images and the complications encountered. RESULTS: A total of 151 patients were evaluated. Apart from a trend towards better diagnostic quality for main pancreatic duct imaging with lodixanol, there were no differences between the two groups. The overall frequencies of adverse events were similar CONCLUSIONS: lodixanol is a safe and effective water-soluble contrast medium for ERCP, comparable to lopromide, but despite theoretical advantages lodixanol has no clear practical benefit.

Structural basis for the insensitivity of a serine enzyme (palmitoyl-protein thioesterase) to phenylmethylsulfonyl fluoride.

Palmitoyl-protein thioesterase-1 (PPT1) is a newly described lysosomal enzyme that hydrolyzes long chain fatty acids from lipid-modified cysteine residues in proteins. Deficiency in this enzyme results in a severe neurodegenerative storage disorder, infantile neuronal ceroid lipofuscinosis. Although the primary structure of PPT1 contains a serine lipase consensus sequence, the enzyme is insensitive to commonly used serine-modifying reagents phenylmethylsulfonyl fluoride (PMSF) and diisopropylfluorophosphate. In the current paper, we show that the active site serine in PPT1 is modified by a substrate analog of PMSF, hexadecylsulfonylfluoride (HDSF) in a specific and site-directed manner. The apparent K(i) of the inhibition was 125 micrometer (in the presence of 1.5 mm Triton X-100), and the catalytic rate constant for sulfonylation (k(2)) was 3.3/min, a value similar to previously described sulfonylation reactions. PPT1 was crystallized after inactivation with HDSF, and the structure of the inactive form was determined to 2.4 A resolution. The hexadecylsulfonyl was found to modify serine 115 and to snake through a narrow hydrophobic channel that would not accommodate an aromatic sulfonyl fluoride. Therefore, the geometry of the active site accounts for the reactivity of PPT1 with HDSF but not PMSF. These observations suggest a structural explanation as to why certain serine lipases are resistant to modification by commonly used serine-modifying reagents.

High pressure liquid chromatographic assay for prednisone in bulk drug substances and tablets.

A high pressure liquid chromatographic (HPLC) method was developed for the assay of prednisone in bulk drug substances and tablets. The sample was dissolved in water-methanol and an aliquot was analyzed by using HPLC. The average recovery of prednisone added to a prednisone tablet composite was 99.5% with a coefficient of variation of 1.07%. Prednisone was determined in 46 tablets (1-50 mg prednisone/tablet) formulated by 22 manufacturers, using the HPLC method and the USP blue tetrazolium assay. The results show that the HPLC method is more specific and faster than the USP method.

Secretory/excretory antigens produced by tetrathyridia of Mesocestoides corti defined by SDS--PAGE and HPLC.

Antigens derived from culture medium in which metacestodes of Mesocestoides corti Hoeppli, 1925, had been maintained were studied by sodium-dodecyl-sulfate--polyacrylamide gel electrophoresis (SDS--PAGE) and high performance liquid chromatography (HPLC). By SDS--PAGE a minimum of 18 Coomassie-staining bands were discerned, of which 4 major bands and 4 major peaks by HPLC of similar molecular weights were observed. The HPLC eluate peaks were analyzed for antigenic activity in vitro by double diffusion in two dimensions, and in vivo in a rabbit. The rabbit had been artificially sensitized with a dialyzable leukocyte extract, showing transfer-factor-like activity, from peritoneal exudate cells removed from mice infected with tetrathyridia. All of the HPLC fractions reacted with rabbit antibody prepared against secretory/excretory antigens, but the sensitized rabbit responded only to two fractions. It is now possible by HPLC to fractionate complex antigens without denaturing them and to elucidate further the role they play during infection.

The reaction of nitro-blue tetrazolium with d,l-L-tetrahydrofolate: The effect of pH, oxygen, formaldehyde and palladium (II).

d,l-L-Tetrahydrofolate (d,l-L-FH4) transfers two electrons to nitro-blue tetrazolium (NBT) in oxygen-free buffers to form the highly coloured nitro-blue formazan and oxidized folate. Both the rate and extent of this reaction are affected by the pH, the nature of the buffer and the oxygen concentration. Inhibition of both the rate and extent of this reaction in air-saturated solutions by superoxide dismutase (SOD) indicates that the superoxide anion is an intermediate in the reaction so that formazan can be produced by both superoxide independent and superoxide dependent routes in air-saturated solutions. In oxygen-free solutions several lines of evidence can be interpreted to mean that the reduced pteridine ring of tetrahydrofolate is the electron donor in the reaction with NBT. Ionization of the amide hydrogen of the pteridine ring and subsequent increase in electron density of that ring might explain the large increases observed in the rate and extent of the reaction of tetrahydrofolate with NBT as the pH increases. Formaldehyde reacts non-enzymatically with tetrahydrofolate to form a methylene bridge between nitrogens 5 and 10 of methylenetetrahydrofolate. This molecule is much less reactive with nitro-blue tetrazolium than tetrahydrofolate. Complexes formed between tetrahydrofolate and palladium (II) ions are also less reactive with NBT than the tetrahydrofolate alone. This result provides added evidence than palladium (II) ions interact with tetrahydrofolate at the nitrogen 5, nitrogen 10 site of the molecule.

Gas chromatographic-mass spectrometric characterization of an alteration product of malathion detected in stored rice.

Four alteration products of malathion have been observed in crop extracts of rice, which may not be readily identifiable in a typical gas chromatographic-mass spectrometric (GC-MS) analysis of the residues. Extracts of rice containing malathion and several related compounds were analyzed on gas-liquid chromatographic (GLC) columns of different polarities. The methyl ethyl succinate ester of malathion co-elutes with malaoxon from a nonpolar GLC column. Retention data and GC-MS analysis indicate the presence of 3 previously reported compounds. Evidence was obtained for a fourth compound, O-methyl O-ethyl S-(1,2-bis-carbethoxy) ethyl phosphorodithioate, apparently resulting from the environmental alteration of malathion.

Effect of age and metabolic rate on lipid peroxidation in the housefly, Musca domestica L.

The objective of this study was to explore the mechanism by which metabolic rate may affect life span by determining the relationship between lipid peroxidation potential and experimentally altered life spans in the adult housefly. Lipid peroxidation potential, measured in vitro, provides an indication of the relative conditions within the tissues affecting the susceptibility of polyunsaturated fatty acids to undergo non-enzymic peroxidation. Lipid peroxidation potential increased with age at a significantly faster rate in houseflies raised at 30 degrees C as compared to those kept at 25 degrees C. The life span of flies at 25 degrees C was longer and metabolic rate lower than at 30 degrees C. The effect of physical activity on lipid peroxidation potential was studied by comparing flies maintained under high and low physical activity regimes. Lipid peroxidation potential increased at a relatively greater rate with age in high-activity flies. The results suggest that accelerated senescence associated with higher metabolic rates, increases the rate of age-associated decline in the ability of houseflies to counteract lipid peroxidation in vitro.

High-pressure liquid chromatographic determination of corticosteroids in topical pharmaceuticals.

Cortisone, hydrocortisone, prednisolone, and prednisone were separated on a reversed-phase microparticulate high-pressure liquid chromatographic (HPLC) column with a ternary mobile solvent containing tetrahydrofuran, methanol, and water. Cortisone acetate, hydrocortisone acetate, and prednisolone acetate were separated on the same reversed-phase column using acetonitrile-water. Various commercial topical formulations of these corticosteroids, except cortisone and prednisone, were prepared by both simple dilution and by extraction for analysis by the proposed HPLC procedure, by the blue tetrazolium procedure, and by the isoniazid procedure and/or phenylhydrazine method. Retention data are given for some common degradation products (C-17 ketones and C-17 carboxylic acid derivatives) and for methyl-, propyl-, and butylparabens with these mobile solvents.

Separation and determination of testosterone and testosterone esters in selected pharmaceutical formulations.

A rapid quantitative procedure is presented for the separation of testosterone esters from their hydrolysis products through the use of the acetonitrile-infusorial earth column. The method was applied to testosterone cypionate, testosterone enanthate, and testosterone propionate. Recovery and replication of reference standard testosterone and its three esters through the proposed method ranged from 99.1 to 100.3%, and the percent relative standard deviation ranged from 0.6 to 1.0%. Two samples can be separated into testosterone and testosterone ester fractions in about 1.5 hr. The analyses of 20 injectable and one buccal tablet formulations made by 12 different manufacturers are reported.

High pressure liquid chromatographic determination of polynuclear aromatic hydrocarbons in oysters.

A high pressure liquid chromatographic (HPLC) procedure is described for determining 13 polynuclear aromatic hydrocarbon (PNA) compounds in oysters at the 2 ppb level. These compounds are extracted from shellfish with acetonitrile and partitioned into petroleum ether; the petroleum ether is removed and the residue is saponified. The aromatic compounds are isolated by passing the saponifeid residue through silica gel and further purified and fractionated by muStyragel gel permeation chromatography. The in-ividual PNAs are then quantitatively determined by using a reverse phase HPLC column coupled to fluorescence, spectrophotometric, and 254 nm absorbance detectors in series. Recoveries from spiked samples generally were greater than 80%.

Kinetic and mechanistic studies of blue tetrazolium reaction with phenylhydrazines.

The reaction kinetics of blue tetrazolium with selected arylhydrazines were investigated under pseudo-first-order conditions. The reaction rate constants were obtained at various temperatures, and the enthalpy (8.4-11.2 kcal/mole) and entropy (-38--45 eu) of activations were calculated. A Hammett plot yielded a straight line with a slope of 0.52. The reaction was inhibited by atmospheric oxygen and iodine. A free radical mechanism is presented.

Rapid blue tetrazolium procedure for analysis of corticosteroids in pharmaceutical preparations.

A rapid quantitative analysis of nine selected corticosteroids and corticosteroid esters at room temperature is described. The procedure is similar to the official blue tetrazolium reaction for corticosteroids, except that methylene chloride instead of alcohol USP is used as a solvent and the reagents are dissolved in or diluted with nonaqueous solvents. These two modifications reduce the medium polarity, which increases the reaction rate. The reactions are complete in 7-18 min, and the formazans are stable for at least 90 min. The results from 15 different pharmaceutical formulations, 12 containing hydrocortisone and three containing prednisolone acetate, show that the proposed method gives results that compare favorably with those obtained by the official blue tetrazolium, isoniazid, and phenylhydrazine procedures.

Analysis of betamethasone and its organic esters in pharmaceutical products.

A rapid, quantitative analysis for betamethasone and its organic esters at room temperature is described. The method is similar to the official blue tetrazolium reaction for corticosteroids, except that methylene chloride is used as the solvent. The reaction is complete in 27-69 min, and the formazans produced are stable for at least 90 min after the addition of tetramethylammonium hydroxide. The results of the analysis of 13 different pharmaceutical formulations by the proposed method are reported. The degradation of betamethasone and its esters caused by strong bases is a pseudo-first-order reaction in methylene chloride. The average half-life of the corticosteroids studied is 56 min under the basic conditions described.

Kinetics and mechanism of blue tetrazolium reaction with corticosteroids.

The kinetics of the reaction of blue tetrazolium with corticosteroids were investigated under pseudo-first-order conditions. The reaction rates for various corticosteroids were determined at various temperatures, and the enthalpy and entropy of activation for these compounds were determined. A mechanism is proposed in which an electron pair and a proton are transferred to blue tetrazolium from the anion formed by the action of tetramethylammonium hydroxide on the alpha-carbonyl moiety of the corticosteroid. The proposed mechanism is consistent with previous experimental results.

High-pressure liquid chromatography of benzo(a) pyrene and benzo (ghi) perylene in oil-contaminated shellfish.

A high-pressure liquid chromatographic procedure is described for the determination of benzo(a) pyrene and benzo(ghi) perylene. These polynuclear aromatics are extracted with acetonitrile and partitioned into petroleum ether, the petroleum ether is removed, and the residue is saponified. The compounds are purified and isolated by passing the residue through a silica gel column and a high-pressure liquid chromatographic column, and detected by their ultraviolet absorption. Recoveries of standards through the procedure averaged 104%.

Effect of solvent on tetrazolium reaction.

The rate of color development of a tetrazolium formazan is shown to be inversely proportional to the dielectric constant of the solvent medium and directly proportional to the hydrogen-bonding capability of solvent mixtures having the same dielectric constant. The geometric isomers of the formazans have different absorbance maxima, and the wavelength of maximum absorbance of a mixture of formazans in different solvents depends upon which isomer predominates in that solvent. The trans-syn-isomer (blue form) of blue tetrazolium has a maximum absorbance at 625 nm in dimethylformamide while the trans-anti-isomer (red form) absorbs at 517 nm in methanol. The absorbance maxima of the corresponding isomers of the formazans of triphenyltetrazolium occur at 535 and 485 nm, respectively. Water and/or methanol (to a lesser extent) are important in the stabilization of the trans-anti-isomer, since the small size of these two substances allows them to form strong intermolecular hydrogen bonds with one or both nitrogen atoms of the azo linkage, thereby preventing the formation of the intramolecular hydrogen bonding exhibited by the trans-syn-isomer. The formazan produced by the reaction of corticosteroids with tetrazolium in strongly basic media can lose a reduction unit and be reoxidized to the tetrazolium. This reaction is solvent dependent and occurs at a much faster rate in chloroform than in alcohol USP.

Determination of iodochlorhydroxyquin and corticosteroids in pharmaceutical formulations.

Iodochlorhydroxyquin was separated from various corticosteroids using an acetonitrile-diatomaceous earth column. Iodochlorhydroxyquin was eluted with cyclohexane, and the corticosteroid was eluted with chloroform. Iodochlorhydroxyquin was determined by both a UV absorbance method and a new compleximetric method using the nickel chelate of iodochlorhydroxyquin. The corticosteroid was determined by the blue tetrazolium and isoniazid procedures. The average percent recovery for these four methods was 100.8, 99.4,100.7, and 99.9, respectively, for 10 known mixtures. The standard deviation for the absorbance for 10 determinations of the nickel complex was 0.002 absorbance unit (0.31%). Various characteristics of the nickel and other complexes were evaluated, including the sensitivity, solubility, and wavelength of maximum absorbance in 14 different solvents. The analyses of 23 typical products are reported, for which the standard deviation, expressed as a percentage of the amount declared, was 1.31% for the UV, 1.34% for the compleximetric, 1.49% for the blue tetrazolium, and 1.22% for the isoniazid procedures. Methods of determination in the presence of interferences are discussed.