Quantitation of Valdecoxib in human plasma by high-performance liquid chromatography with ultraviolet absorbance detection using liquid-liquid extraction

A simple, sensitive and specific HPLC method with UV detection (210 nm) was developed and validated for quantitation of Valdecoxib in human plasma, the newest addition to the group of non-steroidal anti-inflammatory drugs-a highly selective cyclooxygenase-2 inhibitor. The analyte and an internal standard (Rofecoxib) were extracted with diethyl ether/dichloromethane (70/30 (v/v)). The chromatographic separation was performed on reverse phase ODS-AQ column with an isocratic mobile phase of water/methanol (47/53 (v/v)). The lower limit of quantitation was 10 ng/ml, with a relative standard deviation of <20%. A linear range of 10-500 ng/ml was established. This HPLC method was validated with between-batch and within-batch precision of 1.27-7.45 and 0.79-6.12%, respectively. The between-batch and within-batch bias was 0.74-7.40 and -0.93 to 7.70%, respectively. Frequently coadministered drugs did not interfere with the described methodology. Stability of Valdecoxib in plasma was excellent, with no evidence of degradation during sample processing (autosampler) and 30 days storage in a freezer. This validated method is suitable for bioequivalence studies following single dose in healthy volunteers.     

Acetylsalicylic acid and ammonium-induced somatic embryogenesis and enhanced plumbagin production in suspension cultures of <Emphasis Type="Italic">Plumbago rosea</Emphasis> L.

Summary Somatic embryogenesis was induced from suspension cultures (derived from leaf callus) of an important medicinal plant, Plumbago rosea L. While acetylsalicylic acid (ASA) alone induced embryogenesis, indole-3-acetic acid (IAA) failed to elicit a similar response. This is the first time that ASA-induced somatic embryogenesis has been reported in cultured cells. Optimal embryogenic response per culture was observed in Murashige and Skoog’s medium containing a combination of ASA (8.32 μM) and IAA (5.06 μM). but 1-naphthaleneacetic acid and indole-3-butyric acid individually did not induce somatic embryogenesis. Increase in the concentration of ammonium enhanced the number of embryos formed per culture. Accumulation of plumbagin, an important naphthoquinone and a medicinal compound, was three times higher in embryogenic compared to non-embryogenic suspensions.     

A sensitive, robust high-throughput electrochemiluminescence assay for rat insulin

In diabetes research, mouse and rat models are used for in vivo experiments, and quantification of insulin in serum samples under different pathophysiological conditions and after treatment with compounds is essential. There are few commercial radioimmunoassay and enzyme-linked immunosorbent assay (ELISA) assay kits to determine the rat/mouse plasma levels of insulin. However, reliability in insulin measurements using the available biological assays is a great concern. The authors report a robust, extremely sensitive electrochemiluminescence (ECL) insulin assay using the Origen technology platform. The assay performance, as judged by the Z' value of 0.82+/-0.03 and the signal-to-background (S/B) ratio of 133, suggests that this is a robust and reliable assay. The intra-assay and interassay variation is less than 5%. The dynamic range of detection for insulin is 5 pg to 5 ng in the ECL assays. Recovery of insulin was about 100% when different volumes of serum were spiked with exogenous insulin. These results suggest that the ECL insulin assay is an extremely sensitive, robust, nonradioactive homogeneous assay and can be used successfully to determine the insulin levels in rodent serum samples.     

Production of plumbagin from cell cultures of Plumbago rosea L.

Callus and suspension cultures derived from leaf explants of Plumbago rosea were established and plumbagin, a naphthoquinone, was isolated from them and confirmed by ¹H NMR and electron-ionization mass spectroscopy. Maximum content of plumbagin was obtained in the stationary phase of growth (4.3 mg g^–1 dry cell wt). Media pH, phytohormones and carbon sources were optimized for biomass and plumbagin accumulation. Cell aggregates, measuring 500 m in diam, produced 8.2 g dry cell wt l^–1, but larger aggregates (above 500 m) favored plumbagin accumulation with an yield of 4.5 mg g^–1 dry cell wt.     

Substrate binding site of naphthalene 1,2-dioxygenase: functional implications of indole binding

The three-dimensional structure of the aromatic hydroxylating enzyme naphthalene dioxygenase (NDO) from Pseudomonas sp. NCIB 9816-4 was recently determined. The refinement of the structure together with cyclic averaging showed that in the active site of the enzyme there is electron density for a flat aromatic compound. This compound appears to be an indole adduct, which in Escherichia coli is derived from tryptophan present in the rich culture medium. An indole-dioxygen adduct has been built which fits the electron density convincingly. Support for this interpretation was obtained from crystals of the enzyme purified from cells grown in the absence of tryptophan which had an empty substrate pocket. These types of crystals were soaked in indole solutions and the position of indole in this complex was similar to the corresponding part in the modelled indole-oxygen adduct. This suggests that a peroxide bound to iron end-on attacks the substrate and forms this intermediate. The substrate position has implications for the substrate specificity of the enzyme. Docking studies with indole, naphthalene and biphenyl inside the substrate pocket of NDO suggest the presence of subpockets where the one close to the active site iron is reserved for the binding of the aromatic ring which is hydroxylated upon catalysis. The plausible location for the binding of dioxygen is between this pocket and the catalytic iron. This is in accordance with the enantiospecificity of the products.     

Imprint cytology of core needle biopsy specimens of breast lesions. A rapid approach to detecting malignancies, with comparison of cytologic and histopathologic analyses of 173 cases

OBJECTIVE: To investigate whether imprint cytology of core needle biopsy (CNB) specimens from breast lesions is a useful method of rapidly obtaining additional diagnostic information and potentially can be used to reduce the number of biopsies needed. STUDY DESIGN: Cytologic analysis was performed on 173 breast lesions and compared with their histopathologic diagnoses (143 malignant and 30 benign). For imprint cytology, one CNB specimen was rolled between two slides and stained with Diff-Quik and Papanicolaou stain. RESULTS: The diagnostic overall accuracy of Diff-Quik stain (Papanicolaou stain) was 95.4% (95.9%), with a sensitivity of 96.5% (97.2%), specificity of 90% (90%), positive predictive value of 97.8% (97.8%) and negative predictive value of 84.3% (87.0%). There was no statistically significant difference between the stains. Histopathologic analysis had an overall accuracy of 97.7%, with a sensitivity of 97.2%, specificity and positive predictive value of 100% and a negative predictive value of 88.2%. CONCLUSION: Imprint cytology of CNBs is a sensitive method of detecting malignancies in breast tumors. Diff-Quik is a rapid and reliable approach that can reduce the number of biopsies. Inadequate and suspicious cases should be evaluated based on complementary diagnostic procedures for breast lesions.     

The reduction of the Rieske iron-sulfur cluster in naphthalene dioxygenase by X-rays

Naphthalene 1,2 dioxygenase (NDO) displays characteristic UV-Vis spectra depending on the oxidation state of the Rieske center. Investigations on crystals of NDO grown for X-ray diffraction experiments showed spectra characteristic of the oxidized form. Crystals reduced in an anaerobic glovebox using sodium-dithionite showed a characteristic reduced spectrum. Spectra of crystals (cooled to 100 K) after being exposed to X-rays for data collection showed spectra corresponding to a reduced Rieske iron center, demonstrating the ability of X-rays to change the oxidation state of the Rieske iron-sulfur cluster in NDO.     

Effect of ATP sensitive potassium channel modifiers on antinociceptive effect of metoclopramide

Metoclopramide, a prokinetic drug, has been documented to produce antinociceptive response in animal models through opioid pathways. Morphine has been shown to act through ATP sensitive potassium channels (KATP) to produce antinociceptive response. However, such a possibility has not been examined for metoclopramide. The present study investigated this using pharmacological tools. Acetic acid induced abdominal constriction assay procedure was utilized to assess antinociception. The results confirmed that metoclopramide has antinociceptive response. Glibenclamide, a KATP channel blocker, pretreatment antagonized this response. Where as, in minoxidil pretreated animals, metoclopramide elicited an enhanced antinociceptive response. Glibenclamide and minoxidil, which are known KATP channel blocker and opener respectively, interfered with metoclopramide antinociception. These finding are suggestive of a role for KATP channels in metoclopramide antinociception in mice.