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PubMed1990

Clinical evidence supporting the radical scavenger mechanism of 5-aminosalicylic acid.

5-Aminosalicylic acid, the therapeutically active metabolite of sulfasalazine, was exposed to oxygen-derived free radicals produced by the Fenton reaction in vitro, and several metabolites were detected and characterized by high performance liquid chromatography and ultraviolet spectrophotometry. The majority of these metabolites were present in methanolic extracts of feces samples from sulfasalazine-treated patients with inflammatory bowel disease but not in rheumatoid arthritis patients with normal bowel function. The presence of these metabolites, which have not been demonstrated in vivo before, provides evidence of an interaction between 5-aminosalicylic acid and oxygen-derived free radicals in sulfasalazine-treated patients with inflammatory bowel disease. Since the concentration of lipid peroxides, which is dependent on the release of oxygen-derived free radicals, was significantly increased in pretreatment rectal biopsies of the patients, and further was normalized concomitantly with a significant improvement in disease activity over the 5-wk treatment period, an important role of the radical scavenger mechanism of 5-aminosalicylic acid in sulfasalazine therapy of chronic inflammatory bowel disease is strongly suggested.

Adult↗

PubMed1979

19-Hydroxy-prostaglandin E and infertility in human males.

We measured the content of 19-hydroxy-prostaglandin E (19-OH-PGE) and prostaglandin E (PGE) in the semen of 10 infertile males by alkaline dehydration, thin layer chromatographic separation and ultraviolet spectrophotometry. 7 fertile males were also studied. In both groups the content of seminal 6-keto-prostaglandin F1 alpha (6-keto-PGF1 alpha) also was measured by thin layer chromatographic separation and highly specific radioimmunoassay. The amounts of 19-OH-PGE and PGE were significantly lower (p < .02, p < .05) in the infertile group than the fertile group. Differences in mean seminal 6-keto-F1 alpha were not significant. The uterine stimulatory actions of 19-OH-PGE demonstrable in the monkey may apply to fertility in man.

6-Ketoprostaglandin F1 alpha↗

PubMed1983

Alteration in the nucleosome and chromatin structures upon interaction with platinum coordination complexes.

The interaction of various platinum coordination complexes with nucleosomes and chromatin has been investigated by ultraviolet absorption spectrophotometry, circular and electric linear dichroism, and thermal denaturation, at low binding ratios (r less than 0.1-0.2). The general trend of the changes in these physicochemical properties is similar to that observed for the DNA-platinum complexes, which indicates that the same binding sites are involved in the platinum interaction with DNA and with its nucleoprotein complex. The cis-bidentate ligands, cis-dichlorodiammine, diaminocyclohexane and ethylenediamine platinum(II), showed a distinct behavior, with a more important destabilization of the DNA structure in the nucleoprotein than the trans-bidentate ligand, trans-dichlorodiammine-Pt(II), and monodentate ligand, diethylenetriamine-Pt(II). The drastic decrease of the negative electric dichroism in the 260 nm absorption band of the bases, observed with the five ligands, indicates a profound alteration of the DNA arrangement in chromatin and nucleosomes, attributed to a condensation of its superhelical structure. Some differences with previous observations on DNA complexes with the same platinum compounds indicate the possible formation of protein-DNA crosslinks in chromatin and nucleosomes. These could have some importance for the biological effects.

Animals↗

PubMed1995

Degradation of trans-ferulic and p-coumaric acid by Acinetobacter calcoaceticus DSM 586.

Cell suspensions of Acinetobacter calcoaceticus strain DSM 586 and DSM 590 were able to grow on benzoic, p-hydroxybenzoic and vanillic acid as sole carbon source. Testing the utilization of trans-ferulic and p-coumaric acid, we found that the sole A. calcoaceticus DSM 586 efficiently degraded the lignocellulose related monomers. Cells induced with trans-ferulic acid were able to oxidize trans-ferulic, p-coumaric, vanillic, p-hydroxybenzoic and protocatechuic acid at rates higher than the uninduced culture. The same activity was found in the p-coumaric acid induced culture. Two aromatic compounds, vanillic and p-hydroxybenzoic acid, were isolated from culture filtrates of trans-ferulic and p-coumaric acid grown cells, respectively, and further characterized by high performance liquid chromatography. 1H- and 13C-nuclear magnetic resonance and ultraviolet spectrophotometry. Cell extracts of trans-ferulic or p-coumaric acid induced cultures were shown to rapidly convert protocatechuic acid to beta-carboxymuconic acid. Moreover, A. calcoaceticus DSM 586 produced high levels of protocatechuic 3,4-dioxygenase compared to cathecol 1,2-dioxygenase and gentisate 1,2-dioxygenase in the degradation of trans-ferulic or p-coumaric acid. Based upon these results, a reaction sequence for the complete degradation of trans-ferulic and p-coumaric acid in A. calcoaceticus DSM 586 is proposed.

Acinetobacter calcoaceticus↗

PubMed1992

Determination of luxabendazole in biological fluids by high-performance liquid chromatography.

Luxabendazole, a new benzimidazole, is a highly potent broad-spectrum anthelmintic. A high-performance liquid chromatographic method has been developed for its determination in serum and urine samples. In order to optimize the clean-up of samples we compared two procedures: C18 Sep-Pak cartridges and ultrafiltration through a cellulose membrane with a 30,000 relative molecular mass cut-off. In order to obtain the most suitable mobile phase, we studied the influence of pH and acetonitrile content on the capacity factor (k'). Chromatographic separation and quantification were performed on a reversed-phase column packed with 5-microns Nucleosil C18. The mobile phase was acetonitrile-0.05 M phosphate buffer (pH 7.0), (40:60, v/v). The column effluent was monitored by ultraviolet-visible spectrophotometry at 290 nm. The method shows good recovery, precision and accuracy. The lower limit of detection for luxabendazole is 15 ng/ml in serum samples and 25 ng/ml in urine samples.

Animals↗

PubMedDate not supplied

A chromatography system for drug identification.

A classification system is described for drugs using thin-layer chromatography (TLC), gas-liquid chromatography (GLC) and ultraviolet (UV) spectrophotometry. The TLC classifications are based on division of the plate into zones relative to a set of mixed drug standards. The GLC classification is based on Kovats' retention indices. The procedure for classifying drugs is presented, together with a list of over 200 classified drugs.

Chromatography, Gas↗

PubMed2006

Antibacterial activity of a bacteriocin from Lactobacillus paracasei HL32 against Porphyromonas gingivalis.

Porphyromonas gingivalis infections cause problems in periodontal diseases and in certain systemic diseases. There is evidence that Lactobacillus spp. can control populations of P. gingivalis, but there are few data on the effects of purified bacteriocins from Lactobacillus paracasei HL32 on P. gingivalis. The objective of this study was to examine the antibacterial activity of a bacteriocin from L. paracasei HL32 and to relate this activity to its composition. A bacteriocin was purified from culture supernatants of Lactobacillus spp. using a dialysis technique followed by gel-permeation chromatography. Composition of the bacteriocin was characterised by ninhydrin tests, ultraviolet spectrophotometry, thin-layer chromatography, sodium-dodecyl sulphate-polyacrylamide gel electrophoresis, electrospray ionisation mass spectrometry and amino acid analysis. The amino acid sequence from the N-terminal of the bacteriocin was determined. Antibacterial activity was examined by the cylinder plate method, microtitre assay and scanning electron microscopy as compared with standard antibiotics. The bacteriocin had a molecular weight of approximately 56kDa, was comprised of 68% carbohydrate and 32% protein, and showed maximum peak absorbance at 214 and 254nm. The bacteriocin was found to be effective against P. gingivalis; it caused swelling and pore formation on the cell envelope at a minimum bactericidal concentration of 0.14mM, and caused death within 2h. Metronidazole killed P. gingivalis but did not affect the envelope, whereas tetracycline affected P. gingivalis with cell deformation. In conclusion, the bacteriocin from L. paracasei HL32 had the ability to kill P. gingivalis, suggesting that it could be a promising alternative chemotherapeutic agent for P. gingivalis infections.

Amino Acid Sequence↗

PubMed1980

Identification of misused drugs in the clinical laboratory. I. Tricyclics.

A systematic approach evaluating the abuse of tricyclic drugs in the hospital emergency room from the laboratory point of view is presented. This comprehensive screen involves qualitative colorimetric tests, ultraviolet (UV) spectrophotometry, thin layer chromatography (TLC), gas chromatography (GC) and gas chromatography-mass spectrometry (GC/MS). Laboratories with varying facilities and resources can adapt the present screen. Amitriptyline, doxepin, loxapine and desipramine misuse were identified and confirmed using the proposed methodology in 14 cases. Increasing misuse of tricyclic antidepressants requires that the clinical laboratory have a systematic approach to identify and confirm the presence of these drugs in emergency room patients.

Adult↗

PubMed2000

Simultaneous supercritical fluid derivatization and extraction of formaldehyde by the Hantzsch reaction.

A study where the Hantzsch reaction is used to produce the chemical derivatization of formaldehyde in a supercritical medium is presented in this paper. Pressure, temperature and other parameters such as static and dynamic extraction time must be optimized to increase the yield of this kinetically controlled reaction. A 2(5-1) (resolution V) factorial design was used to study the significant parameters affecting the supercritical process in terms of resolution and sensitivity. A subsequent central composite design was employed to find the conditions of maximum response. Ultraviolet-visible spectrophotometry was used as the detection technique. The optimum conditions were used for the determination of formaldehyde in real finger-paints by means of the previous addition of known quantities of this analyte to the paint. Results were compared with those obtained with supercritical fluid extraction and subsequent chemical derivatization and an improvement of sensitivity as well as a reduction of time of analysis, solvent waste and reagents consumption were observed.

Chromatography↗

PubMed1979

Metabolism of theophylline to caffeine in premature newborn infants.

Plasma concentrations of theophylline and caffeine in seven premature neonates receiving theophylline orally for treatment of apnea at age one to 9 days were measured by high performance liquid chromatography, ultraviolet spectrophotometry, and mass spectrometry. Plasma concentrations of caffeine increased from 1.8 mg/l (range = 0.1 to 3.7) at day one to 3.7 mg/l (1.3 to 8.0) seven days after initiation of theophylline therapy. Similarly, plasma concentrations of theophylline were 4.6 mg/l (1.5 to 7.5) and 11.0 mg/l (4.0 to 19.0) on days one and 7 of theophylline therapy, respectively. In contrast, four normal adult volunteers given theophylline orally for eight to ten days had plasma theophylline concentrations ranging from 3 to 14 mg/l but no measurable caffeine. This indicates that caffeine is a biotransformation product of theophylline in premature neonates and that the metabolic pathway followed by theophylline in premature infants includes a methylation reaction producing caffeine, whereas in adults, the major metabolic pathway involves oxidative reactions (demethylation and oxidation). Some pharmacologic effects attributed to theophylline during chronic therapy for apnea may in part be due to caffeine. Routine monitoring during theophylline therapy in premature neonates with apnea should include plasma concentrations of both theophylline and caffeine in order to assess the total methylxanthine load.

Adult↗

PubMed1997

Neutrophil activation, vascular leak toxicity, and cytolysis during interleukin-2 infusion in human cancer.

BACKGROUND: Recombinant interleukin-2 (rIL-2) therapy for advanced malignancy is usually associated with a vascular leak syndrome (VLS) similar to that seen in severe sepsis. We investigated the possibility that the IL-2-induced VLS may be associated with the presence of circulating activated polymorphonuclear (PMN) leukocytes as occurs in sepsis syndrome. METHODS: Estimation of phenotypic (CD11B/CD18) and functional (H2O2, HOCl) up-regulation of circulating neutrophil activity was made by fluorescence-activated cell sorter analysis and ultraviolet spectrophotometry. Associated systemic cytokine enhancement tumor necrosis factor-alpha by enzyme-linked immunosorbent assay for bioactivity and parallel estimation of clinical evidence of vascular leak syndrome were also studied in human subjects with advanced cancer receiving therapeutic doses of rIL-2. RESULTS: The present studies confirm previous reports that tumor necrosis factor-alpha is released into the circulation during infusional therapy with rIL-2. In addition, we have found that this is accompanied by both phenotypic (up-regulation of CD11b/CD18 adhesion receptor expression) and functional (hydrogen peroxide and hypochlorous acid production) evidence of potent PMN activation. Furthermore, patients showing disease response to treatment have significantly greater production of PMN oxidants. CONCLUSIONS: These data suggest that the VLS seen during rIL-2 infusion in human beings may be attributable to PMN mechanisms similar to those invoked during severe sepsis. Consequently, this study may provide further insights into the mechanism of rIL-2's therapeutic action in advanced malignant disease.

Antigens, CD↗

PubMed2002

Inclusional complex study between 6-p-toluidinylnaphthalene-2-sulfonate and 2-hydroxypropyl-beta-cyclodextrin.

6-p-Toluidinylnaphthalene-2-sulfonate (TNS) is used as a fluorescent probe for exploring hydrophobic regions of several biological substances, such as proteins, and studying solution state folding behaviour. The current study examines the complexation of TNS with 2-hydroxypropyl-beta-cyclodextrin (HPbetaCD) in aqueous solution, mainly by ultraviolet spectrophotometry using various concentrations of HPbetaCD. The structure of HPbetaCD was confirmed by using positive-ion electrospray ionization (ESI+) mass spectrometry. The complex was examined for its stoichiometry applying the continuous variation (Job plot) method. Also, the kinetics of the complex formation was monitored and the determination of the stability constant was calculated. For this purpose, the spectrophotometric properties of TNS were observed in the presence of increasing concentrations of HPbetaCD applying the transformed Benesi-Hildebrand linear model as well as a nonlinear one. The results suggest that TNS forms a stable complex of 1:1 molar ratio, at least at the examined concentrations. Furthermore, from the measurements of 1H nuclear magnetic resonance (1H NMR) spectra, interactions between protons of TNS with HPbetaCD were determined.

2-Hydroxypropyl-beta-cyclodextrin↗

PubMed1997

The new inhibitors of ribonucleotide reductase--comparison of some physico-chemical properties.

Amidox (AX), didox (DX) and trimidox (TX), compounds synthetized as new ribonucleotide reductase inhibitors, have been investigated by ultraviolet (UV) spectrophotometry, polarography and high performance liquid chromatography (HPLC). The experiments have been performed at various pH values. The changes in UV absorption of the compounds studied were recorded and it was demonstrated that these changes are related to the pH and to structural features of the investigated molecules. From the compounds included in our series of experiments, only amidox and trimidox are reduced during polarographic experiments in Britton-Robinson buffer. The reduction of both compounds proceeded in two one-electron steps in acidic pH. One two-electron diffuse irreversible wave was observed at basic pH. The values of the half-wave potential became more negative in accordance with the increasing pH. HPLC assay also showed changes in the retention of compounds investigated, particularly when the pH of the mobile phase was close to the dissociation constant of the particular drug. The changes of physico-chemical properties detected by the all used methods are related to different chemical structures (the most significant changes were observed in alkaline pH).

Benzamidines↗

PubMed1998

The effect of photodegradation on the fluorescent properties of norfloxacin: (photodegradation and fluorescence of norfloxacin).

Norfloxacin (NFLX), a broad spectrum antibacterial quinolone, is a very thermostable but photosensitive drug, especially in solution leading to the formation of an ethylenediamine degradate. The modification of the fluorescent properties of NFLX in acid solution after exposure to fluorescent light and the degradation mechanisms were studied. Two analytical methods were previously developed and validated for NFLX, ultraviolet spectrophotometry (UV) and spectrofluorimetry (FL). Data obtained using both methods in the analysis of remaining NFLX in terms of percent recoveries revealed that there was no statistically significant modifications of the UV signal and of the recoveries obtained by the method. However, an important increase of the fluorescent signal after light exposure of NFLX solutions appeared, which led to an increase of the average recovery up to 270% over 15 months. Using a previously validated HPLC method for the photostability studies of NFLX, a loss of 5% with respect to the initial drug amount was observed. The study of UV and fluorescence spectra evidenced the formation of the degradation product, which induced significant modification of the fluorescent properties of NFLX samples. These results clearly indicated that FL analysis definitively is the method of choice and can be used to study the photodegradation of NFLX.

Anti-Infective Agents↗

PubMed1986

Unique rearrangement of ergocalciferol side chain in vitro: production of a biologically highly active homologue of 1,25-dihydroxyvitamin D3.

In vitro incubation of 24-epi-25-hydroxyvitamin D2 with chicken kidney homogenate produced several compounds, one of which had an affinity equal to that of 1,25-dihydroxyvitamin D2 for the chick intestinal receptor. The affinity of 24-epi-1,25-dihydroxyvitamin D2 for the same receptor was found to be half that of 1,25-dihydroxyvitamin D2. The unknown compound was produced only when homogenate was prepared from pooled kidneys taken from both vitamin D deficient and replete chickens. The compound has been tentatively identified as 1,25-dihydroxy-22-dehydro-26-homovitamin D3 by ultraviolet absorption spectrophotometry and mass spectrometry. Chemical synthesis of 1,25-dihydroxy-22-dehydro-26-homovitamin D3 provided additional evidence for the structure. Administration of this 26-homologue of 1,25-dihydroxyvitamin D3 at the dose level of 650 pmol/rat stimulated bone calcium mobilization in the hypocalcemic rat equal to that of 1,25-dihydroxyvitamin D3. Thus, this paper demonstrates unique methyl migration on the side chain of 24-epi-1,25-dihydroxyvitamin D3 to form a more biologically potent analogue.

Animals↗

PubMed1990

24,25,28-trihydroxyvitamin D2 and 24,25,26-trihydroxyvitamin D2: novel metabolites of vitamin D2.

Understanding of the inactivation pathways of 25-hydroxyvitamin D2 and 24-hydroxyvitamin D2, the two physiologically significant monohydroxylated metabolites of vitamin D2, is of importance, especially during hypervitaminosis D2. In a recent study, it has been demonstrated that the inactivation of 24-hydroxyvitamin D2 occurs through its conversion into 24,26-dihydroxyvitamin D2 [Koszewski, N.J., Reinhardt, T.A., Napoli, J.L., Beitz, C.D., & Horst, R.L. (1988) Biochemistry 27, 5785]. At present, little information is available regarding the inactivation pathway of 25-hydroxyvitamin D2 except its further metabolism into 24,25-dihydroxyvitamin D2 [Jones, G., Rosenthal, A., Segev, D., Mazur, Y., Frolow, F., Halfon, Y., Rabinovich, D., & Shakked, Z. (1979) Biochemistry 18, 1094]. In our present study, we investigated the metabolic fate of 25-hydroxyvitamin D2 in the isolated perfused rat kidney and demonstrated its conversion not only into 24,25-dihydroxyvitamin D2 but also into two other new metabolites, namely, 24,25,28-trihydroxyvitamin D2 and 24,25,26-trihydroxyvitamin D2. The structure identification of the new metabolites was established by the techniques of ultraviolet absorption spectrophotometry and mass spectrometry and by the characteristic nature of each new metabolite's susceptibility to sodium metaperiodate oxidation. In order to demonstrate the physiological significance of the two new trihydroxy metabolites of vitamin D2, we induced hypervitaminosis D2 in a rat using [3 alpha-3H]vitamin D2 and analyzed its plasma for the various [3 alpha-3H]vitamin D2 metabolites on two different high-pressure liquid chromatography systems.(ABSTRACT TRUNCATED AT 250 WORDS)

25-Hydroxyvitamin D 2↗

PubMed1981

Isolation and identification of 23,25-dihydroxyvitamin D3, an in vivo metabolite of vitamin D3.

Vitamin D supplemented rats produce a metabolite of 25-hydroxy[3 alpha-3H]vitamin D3 that is easily separated from known metabolites by using high-performance liquid chromatography. The production of this metabolite in vivo as well as 1,25-dihydroxyvitamin D3, 24(R),25-dihydroxyvitamin D3, and 25-hydroxyvitamin D3 26,23-lactone is largely if not totally eliminated by nephrectomy. Kidney homogenates from vitamin D supplemented chickens incubated with 25-hydroxyvitamin D3 produce significant quantities of the new, unknown metabolite. This metabolite was isolated in pure form from such incubation mixtures by using both straight-phase and reversed-phase high-performance liquid chromatography. This metabolite has been positively identified as 23,25-dihydroxyvitamin D3 by ultraviolet absorption spectrophotometry, mass spectrometry, and derivatization. This structure was confirmed by chemical synthesis of both C-23 stereoisomers. Although the natural product exactly comigrates with one of the synthetic isomers, the exact stereochemistry of the natural product remains unknown. It is possible that this new metabolite is an intermediate in the biosynthesis of 25-hydroxyvitamin D3 26,23-lactone.

Animals↗

PubMed1981

23,24,25-Trihydroxyvitamin D3, 24,25,26-trihydroxyvitamin D3, 24-keto-25-hydroxyvitamin D3, and 23-dehydro-25-hydroxyvitamin D3: new in vivo metabolites of vitamin D3.

Four new in vivo metabolites of vitamin D3 were isolated from the blood plasma of chicks given large doses of vitamin D3. The metabolites were isolated by methanol-chloroform extraction and a series of chromatographic procedures. By use of mass spectrometry, ultraviolet absorption spectrophotometry, and specific chemical reactions, the metabolites were identified as 23,24,25-trihydroxyvitamin D3, 24,25,26-trihydroxyvitamin D3, 24-keto-25-hydroxyvitamin D3 and 23-dehydro-25-hydroxyvitamin D3.

Animals↗