An improved assay of gangliosides separated by thin-layer chromatography

There were no differences in the recoveries of ganglioside sialic acid from silica gel G thin-layer chromatograms when they were sprayed with resorcinol reagent varying in normality between 3 and 10. However, both the temperature at and the time for which the plates were heated after spraying did affect the recoveries, which can reach 100% when the plates are heated for 6 min at 135degreesC.     

A simple determination of steroidal alkaloid glycosides by thin-layer chromatography immunostaining using monoclonal antibody against solamargine

A method of determination for solasodine glycosides by using thin-layer chromatography (TLC) immunostaining was investigated. Solasodine glycosides separated by silica gel TLC were transferred to a polyvinylidene difluoride membrane. The membrane was treated with sodium periodate solution followed by bovine serum albumin (BSA), resulting in a solasodine glycoside-BSA conjugate. Conjugation was confirmed by matrix-assisted laser desorption/ionization mass spectrometry. Individual spots were stained by monoclonal antibody against solamargine. Immunostaining of solasodine glycosides was more sensitive compared to other stainings. The newly established immunostaining method can be extended to analysis of the distribution of solasodine glycoside in the plant body.     

Quantitation of radiolabeled antibody binding to cells by thin-layer chromatography

Thin layer chromatography (TLC) was used to monitor binding of radiolabeled antibodies to cells. Labeled antibodies were reacted with cells and aliquots chromatographed on serum-blocked, ITLC strips. The cell-antibody complexes remain at the origin and unbound antibody migrates with the solvent front. The antibody binding was estimated from the ratio of radioactivity at the origin compared to the total applied. Separations are completed in about 10 min. This method does not use centrifugation or wash steps, and provides an inexpensive and self-contained system to evaluate radioligand binding. Cell binding assay results using this method are approximately the same as those obtained using bead- or cell-type assays.     

Determination of 3,4-dihydroxyphenylacetic acid and 5-hydroxyindoleacetic acid in human plasma by a simple and rapid high-performance liquid chromatography assay

In order to study the pharmacodynamic effects of drugs on dopamine and serotonin metabolism, a reversed-phase HPLC assay coupled with electrochemical detection (ECD) for measuring plasma concentrations of 3,4-dihydroxyphenylacetic acid (DOPAC) and 5-hydroxyindoleacetic acid (5-HIAA) was developed. The system was operated isocratically using a mobile phase of aqueous 0.03 M KH2PO4 buffer containing 0.15 mM EDTA in methanol (8.75:1.25), with a final pH of 4.0. The flow rate was set at 1.5 mL/min, and potentials at +450 mV. Using a signal to noise ratio of > 3, the minimum detection limit assessed by direct on-column injection of a standard solution for DOPAC and 5-HIAA was < 1 pg. The assays were linear from basal concentrations (1-10 ng/mL) to 100 ng/mL. The intra- and interassay variations were < 10% and < 20%, respectively.     

Separation of brain phosphatidylserines according to degree of unsaturation by thin-layer chromatography

Stability constants for the molecular complexes formed by protoporphyrin IX dimethyl ester and its metal chelates with Co2+, Ni2+, Cu2+, Zn2+, Mg2+ and Fe3+ with nitroarene acceptors are determined in non-aqueous solvents. The stability of the complexes is found to be largely insensitive to variations in the central metal ions provided they are in the same oxidation state. Solvent-solute interactions are seen as being important in the formation of such complexes giving rise to large variations in their apparent stability. Some of the other factors that affect the stability of the molecular complexes are also evaluated. No evidence has been found for the appearance of any intermolecular charge-transfer absorption bands. Infrared studies, however, have provided some evidence for a substantial loss of electron density from the porphyrin ring upon interactions with the acceptors.     

Quantitative determination of cardiac glycosides in Digitalis lanata leaves by reversed-phase thin-layer chromatography

We have recently chosen to undertake a comprehensive evaluation of the modulation of gene expression by oxidative stress, using mRNA as a marker. Our model system is HA-1 hamster fibroblasts, using conditions under which we observe an adaptive response. Under these conditions, the HA-1 cells respond to a minimally toxic "pretreatment" dose of hydrogen peroxide by synthesizing RNAs and proteins that protect them against subsequent exposure to a highly cytotoxic concentration of hydrogen peroxide. Using the differential display technique to screen for modulated RNAs, we have recently reported an RNA species, adapt15/gadd7, whose steady-state level is significantly induced by a pretreatment dose of hydrogen peroxide (D. R. Crawford, G. P. Schools, S. L. Salmon, and K. J. A. Davies (1996) Arch. Biochem. Biophys. 325, 256-264). Here we report a second induced mRNA, designated adapt33. Two homologous adapt33 mRNAs were revealed by Northern blot hybridization. Both of these species were inducible by hydrogen peroxide, and they were sized at 1.46 and 0.99 kb. These inductions appeared to be dependent upon calcium, occurred as early as 90 min, and were maximal at 5 h. Cell fractionation revealed that a significant proportion of adapt33 RNA is associated with active translation. adapt33 is a novel sequence, as determined by cloning, sequencing, and GenBank analysis. adapt33 represents a new oxidant-inducible RNA and marker of cellular oxidative stress and a potential aid in the study, detection, and possible therapy of oxidant-related disorders.     

Ring hydroxylation of [o-3H]methoxychlor as a probe for liver microsomal CYP2B activity: potential for in vivo CYP2B assay

An in vitro radiometric assay selective for inducible CYP2B activity is described. The assay is based on the quantification of 3H2O release that occurs during o-ring hydroxylation of [o-3H]methoxychlor by liver microsomes in the presence of NADPH. 3H2O is isolated by removing > 99.9% of the parent compound and organic metabolites by facile charcoal extraction and filtration. There was no evidence for an NIH shift during ring hydroxylation, and there was little or no isotope effect. Selectivity for CYP2B was demonstrated using liver microsomes prepared from rats and mice treated with inducers of different CYP isoforms. Ring hydroxylation of [o-3H]methoxychlor was elevated 11.4-fold over control values in liver microsomes from male rats treated with phenobarbital. With mice, phenobarbital treatment elevated liver microsomal ring hydroxylation 7.1-fold. Clofibrate, 3-methylcholanthrene, or beta-naphthoflavone treatment of male rats or pyridine treatment of female rats did not elevate liver microsomal ring-hydroxylase activity, indicating that CYP4A, 1A, and 2E1 do not support this reaction. In female rats, dexamethasone and pregnenolone-16 alpha-carbonitrile treatment elevated ring hydroxylation up to 5.5- and 3.2-fold, respectively, an activity that may be attributed to CYP2B induction in those animals. Incubation of liver microsomes from phenobarbital-treated males with monospecific anti-CYP2B monoclonal antibodies (Mab) inhibited ring-hydroxylase activity up to 86%, demonstrating predominantly CYP2B-mediated catalysis. An 86% inhibition by these Mabs was also observed using liver microsomes from male mice treated with phenobarbital, indicating the assay is not limited to rats. The CYP2B mechanism-based inhibitor orphenadrine caused a 76% decline in activity, providing further evidence for CYP2B involvement. Unlike other CYP2B-selective assays, this method may be readily adapted to in vivo studies, by measuring urinary excretion of 3H2O as an indication of total body CYP2B activity.     

Detection of opiates in the urine using thin-layer chromatography

The determination of opiates in urine by thin layer chromatography may be difficult for the low concentrations of opiates and their mixtures. The result of the analysis is influenced by the isolation recovery, sensitivity of detection and by the choice of the chromatographic conditions for the separation of opiate mixtures. The study of these factors resulted in several modifications of routinely used method, so that the results are reliable for the concentration of opiates in urine 1 mg/l. Nevertheless, for the analysis of opiates in drug abuse control the comparison of thin layer chromatography findings with other chromatographic methods or immunoassays is important for the improvement of the laboratory performance and for the interpretation of the results.     

Determination of codeine and its metabolites in microsomal incubates by high-performance liquid chromatography

One hundred seventy-eight of 326 patients had surgery for Hirschsprung's disease at Red Cross Children's Hospital (1957 to 1990) agreed to participate in a recall follow-up study. Assessment of the postoperative outcome of the Swenson, Duhamel, and Soave procedures included manometric evaluation in 43 patients by perfused-tube and balloon-series techniques. Rectal suction biopsies were performed for patients who had persistent problems with stool evaluation. One hundred fifteen of the 178 patients were more than 4 years of age, and long-term functional outcome could be assessed. Although good results were obtained in 94% overall, 16 had clinical evidence of a degree of persisting obstruction. Results of manometric assessment of anorectal function in these patients were not significantly different from those of 28 patients who had normal stool evacuation. A biopsy was performed in 14 of the 16 patients who had symptoms of obstruction postoperatively, and abnormal histological features were noted. There was aganglionosis in four, features of neuronal intestinal dysplasia in nine, ganglioneuromatosis of the colon in one. The results of two biopsies were entirely normal. Implications of postoperative dysfunction after surgery for Hirschsprung's disease are discussed, and a protocol for investigation and management is proposed.     

Improved method for studying skin lipid samples from cyanoacrylate strips by high-performance thin-layer chromatography

A non-invasive method for sampling skin surface lipids is cyanoacrylate stripping (CAC-TS). It was the purpose of our study to improve the method for sampling of skin surface lipids and the separation of epidermal lipid fractions by a modification of the methods described by Melnik and by Imokawa et al. Briefly, lipids on the glass slide sampled by CAC-TS from the forearm of 75 volunteers and from the forehead of 60 volunteers were eluted in hexane/ethanol under ultrasonication. Identification of the diluted total superficial sebaceous and epidermal skin lipids was performed by sequential high-performance thin-layer chromatography. For quantification of the lipids we used densitometric methods. By this modified method we were able to show a clear and complete separation of all relevant lipids from a cyanoacrylate strip that represented 1-2 mg stratum corneum only.     

Fluorogenic labeling of organophosphate pesticides with dansyl chloride. Application to residue analysis by high-pressure liquid chromatography and thin-layer chromatography

The analysis of some organophosphorus pesticides by fluorogenic labeling with dansyl chloride (5-dimethylaminonaphthalene--l-sulfonyl chloride) was investigated. The pesticides were hydrolysed in sodium hydroxide to the corresponding phenols. The reaction of dansyl chloride with the phenols was accomplished in a two-phase system. The resulting fluorescent derivatives were separated and analysed quantitatively by in situ thin-layer chromatography (TLC) and high-pressure liquid chromatography (HPLC). As little as 10-25 ng/spot of pesticide was detected by both TLC and HPLC.