Cellulose Thin-layer Chromatography Research Articles

Advances in analytical techniques for neutron capture therapy: thin layer chromatography matrix and track etch thin layer chromatography methods for boron-10 analysis.

A new track etch autoradiographic technique for quantitating boron-10 containing compounds used for neutron capture therapy is described. Instead of applying solutions of Cs2B12H11SH and its oxidation products directly to solid-state nuclear track detectors, diethylaminoethyl cellulose thin layer chromatography (TLC) plates are utilized as sample matrices. The plates are juxtaposed with Lexan polycarbonate detectors and irradiated in a beam of thermal neutrons. The detectors are then chemically etched, and the resultant tracks counted with an optoelectronic image analyzer. Sensitivity to boron-10 in solution reaches the 1 pg/microliter level, or 1 ppb. In heparinized blood samples, 100 pg boron-10/microliter are detected. This TLC matrix method has the advantage that sample plates can be reanalyzed under different reactor conditions to optimize detector response to the boron-10 carrier material. Track etch/TLC allows quantitation of the purity of boron neutron capture therapy compounds by utilizing the above method with TLC plates developed in solvent systems that resolve Cs2B12H11SH and its oxidative analogs. Detectors irradiated in juxtaposition to the thin layer chromatograms are chemically etched, and the tracks are counted in the sample lane from the origin of the plate to the solvent front. A graphic depiction of the number of tracks per field yields a quantitative analysis of compound purity.

Mevalonic aciduria: Family studies in mevalonate kinase deficiency, an inborn error of cholesterol biosynthesis

Mevalonic aciduria was found in a 19-month-old boy with severe failure to thrive and developmental delay (Sweetman et al., 1985; Hoffmann et al., 1986). Cardinal manifestations included recurrent anaemia, mild hepatosplenomegaly, bilateral central cataracts and dysmorphic features. Serum cholesterol was low (1.82.1mmol/L). Urinary organic ~acids were normal except for a large elevation of mevalonic acid, an intermediate in cholesterol biosynthesis. The activity of meval0nate kinase (ATP: mevalonate-5-phosphotransferase, EC 2.7.1.36), the enzyme catalysing the second committed step in cholesterol biosynthesis, was severely deficient in the patient's fibroblasts, lymphocytes and lymphoblasts. We have developed an optimized assay for mevalonate kinase in extracts of cultured human fibroblasts utilizing [2-14C]mevalonate as substrate. The enzymatic products, [2-14C]mevalonate-5-phosphate and [2-14C]mevalonate-5-pyrophosphate, were separated and quantified by cellulose thin-layer chromatography. With heterozygote detection as a goal, we undertook a study of family members of the patient with mevalonic aciduria. This report presents the activities of mevalonate kinase in lysates of lymphocytes isolated from whole blood of these individuals and of controls.

Screening complex hazardous wastes for mutagenic activity using a modified version of the TLC/Salmonella assay

10 complex hazardous wastes were tested for mutagenic activity using a modified version of the TLC/Salmonella assay developed by Bjørseth et al. (1982). This fractionation/bioassay scheme couples thin-layer chromatography (TLC) with the Salmonella/mammalian-microsome (Ames) assay for the detection of mutagenic constituents in complex mixtures. Crude (unadulterated) hazardous wastes and selected hazardous waste extracts were fractionated on commercially available cellulose TLC plates. Mutagenicity testing was performed in situ by applying a single overlay of minimal growth agar, tester strain TA98 or TA100, and the optional metabolic activation system directly onto the developed chromatogram. A mutagenic effect was indicated either by the appearance of localized clusters of revertant colonies or by an increase in total revertant growth vis-à-vis control plates. 7 of 10 hazardous wastes (including tars, emulsions, sludges, and spent acids and caustics) demonstrated mutagenic activity when tested by this method. To assess the sensitivity of the modified TLC/Salmonella assay, 14 Salmonella mutagens from a wide range of chemical classes and polarities were tested. Selected compounds included heterocyclics, aromatic amines, alkylating agents, antitumor agents, a nitrosamine and a nitroaromatic, 11 of the 14 mutagens were positive in this test system. The 3 compounds refractory to analysis included a polycyclic aromatic hydrocarbon and two volatiles.

Chromatographic characterization of adult and foetal rat insulin

Foetal rat pancreatic rudiments explanted on day 14 of gestation were grown in organ culture in medium enriched with amino acids. The size of the insulin granules was increased, resulting in an insulin granule volume fraction greater than the volume fraction measured in pancreas grown in vivo. The pancreas was extracted and the insulin compared. Serial dilution curves of extracts of adult pancreas and pancreas grown in vitro are parallel in the insulin radioimmunoassay, whereas extracts of pancreas of foetus developing in utero appear immunologically different. Adult and foetal rat insulin ( in utero) were purified using chromatography on OPTI UP C12, cellulose thin-layer chromatography plates, cellulose acetate foil electrophoresis and finally high-performance liquid chromatography. The ratio of insulin I to insulin II was found to be 1.5 for the adult and 2.7 for the foetus. These results show that there is an unequal expression of the two non-allelic genes controlling insulin biosynthesis in foetal and adult rat pancreas.

Phosphorylation of extracellular carbohydrates by intact cells. Chicken hepatocytes specifically adhere to and phosphorylate immobilized N-acetylglucosamine.

Cell-cell adhesion is a multi-step process which may be initiated by binding of cell surface carbohydrates to complementary carbohydrate receptors on apposing cell surfaces. We have modeled such interactions using polyacrylamide gels covalently derivatized with glycosides, to which intact cells specifically adhere; chicken hepatocytes adhere to gels derivatized with N-acetylglucosamine (GlcNAc). Initially adhesion is blocked (or reversed) by soluble GlcNAc, but becomes sugar-resistant rapidly at 37 degrees C, perhaps due to cellular modification of the carbohydrate-derivatized surface (Guarnaccia, S. P., Kuhlenschmidt, M. S., Slife, C. W., and Schnaar, R. L. (1982) J. Biol. Chem. 257, 14293-14299). We report here that, subsequent to recognition and adhesion, intact chicken hepatocytes transfer phosphate covalently to GlcNAc-derivatized gels. Metabolically radiolabeled cells (32Pi) were incubated on polyacrylamide gels derivatized with various aminohexyl glycosides. Noncovalently bound material was then removed from the gels by extensive washing in detergents and salt solutions. Subsequent radiochemical analysis revealed that phosphate was transferred selectively to GlcNAc-derivatized gels (up to 20-fold more than to glucose-, galactose-, or mannose-derivatized gels). Soluble GlcNAc (but not other sugars) or low temperature inhibited phosphate transfer. The phosphorylation was mediated by intact cells; cell lysate was itself incapable of specific phosphate transfer and attenuated specific transfer when added to intact cells. When GlcNAc was immobilized using a cleavable (disulfide-containing) linker arm the transferred phosphate radiolabel could be solubilized by disulfide reduction and recovered for further analysis. The released phosphorylated product migrated as a single low molecular weight species upon gel permeation chromatography, paper electrophoresis, and cellulose thin layer chromatography. Acid hydrolysis of the phosphorylated product generated a compound with the mobility of GlcNAc-6-P in five different separation systems. Treatment with alkaline phosphatase converted the radiolabel to a compound with the properties of inorganic phosphate. These data indicate that; subsequent to carbohydrate recognition and adhesion, intact hepatocytes generate phosphomonoesters of recognized carbohydrates outside of their plasma membranes.

Active-site-directed inhibition of 3-hydroxy-3-methylglutaryl coenzyme A synthase by 3-chloropropionyl coenzyme A.

3-Chloropropionyl coenzyme A (3-chloropropionyl-CoA) irreversibly inhibits avian liver 3-hydroxy-3-methylglutaryl-CoA synthase (HMG-CoA synthase). Enzyme inactivation follows pseudo-first-order kinetics and is retarded in the presence of substrates, suggesting that covalent labeling occurs at the active site. A typical rate saturation effect is observed when inactivation kinetics are measured as a function of 3-chloropropionyl-CoA concentration. These data indicate a Ki = 15 microM for the inhibitor and a limiting kinact = 0.31 min-1. [1-14C]-3-Chloropropionyl-CoA binds covalently to enzyme with a stoichiometry (0.7 per site) similar to that measured for acetylation of enzyme by acetyl-CoA. While the acetylated enzyme formed upon incubation of HMG-CoA synthase with acetyl-CoA is labile to performic acid oxidation, the adduct formed upon 3-chloropropionyl-CoA inactivation is stable to such treatment. Therefore, such an adduct cannot solely involve a thio ester linkage. Exhaustive Pronase digestion of [14C]-3-chloropropionyl-CoA-labeled enzyme produces a radioactive compound which cochromatographs with authentic carboxyethylcysteine using reverse-phase/ion-pairing high-pressure liquid chromatography and both silica and cellulose thin-layer chromatography systems. This suggests that enzyme inactivation is due to alkylation of an active-site cysteine residue.

Preparation de l'acide méso diamino‐2, 6 heptane‐dioique‐1, 7‐[3H‐3, 4, 5] (méso‐diamino pimelique‐[3H‐3, 4, 5

AbstractMeso 2, 6‐diamino [3H‐3, 4, 5] 1, 7‐heptanedioc acid‐ (meso [3H‐3, 4, 5] DAP) specific activity 30 Ci/mM was obtained by catalytic dehydrohalogenation of meso 3‐chloro and 4‐chloro DAP. LL, DD and meso DAP were analytically separated by cellulose thin layer chromatography (methanol‐water‐7 : 3).

Immune deficiency due to adenosine deaminase and purine nucleoside phosphorylase deficiency: a simple diagnostic test.

A simple method is described for diagnosing adenosine deaminase and purine nucleoside phosphorylase deficiency using urine. Cellulose thin layer chromatography of 1 microliter of urine from affected children was performed and deoxyadenosine and deoxyguanosine were easily detected by phosphorescence at the temperature of liquid nitrogen. This test is not expensive and can be done in any laboratory. It should be suitable for diagnostic screening in patients with immune deficiency.

Binding of chloroform to the cysteine of hemoglobin

The products of the covalent binding of chloroform to rat hemoglobin during microsomal metabolism were isolated and identified by gas chromatography (GC) and mass spectroscopy (MS). After isolation by Proteinase K hydrolysis, amino acid analysis and cellulose thin-layer chromatography (TLC), the major product was identified by GC/MS as N-hydroxymethyl cysteine and a minor product as 2-hydroxythiazolidine-4-carboxylic acid. N-Hydroxymethyl cysteine is proposed to be formed during isolation from the 2-oxothiazolidine-4-carboxylic acid present in the intact hemoglobin.

A method for chromatographic separation and fluorometric quantification of 2′,3′-cyclic nucleotide-3′-phosphodiesterase reaction products

A sensitive method for separation and fluorometric quantification of 2′,3′-cyclic nucleotide-3′-phosphodiesterase (EC 3.1.4.37) (CNPase) reaction products with 2′,3′-cyclic adenosine monophosphate as substrate is presented. The 2′-AMP product was separated by cellulose thin-layer chromatography in 4 m MgSO 4-0.5 m sodium acetate-2-propanol (80:18:2, v/v/v). After reaction with glyoxal dihydrate, the amount of reaction product was determined fluorometrically using excitation at 328 nm and emission at 382 nm. These results correlated well with those obtained using; Kurihara and Tsukada's [(1967) J. Neurochem. 14: 1167–1174] paper chromatographic method ( r = 0.96). With this fluorometric method, amounts as low as 0.20 nmol of 2′-AMP can be determined, and its sensitivity is comparable to that of the radiochemical method. The method is easy to use and sensitive enough for measuring CNPase activity in tissue with low enzyme activity.

Reaction of dATP with N-methyl-N-nitrosourea in vitro.

Extensive methylation was found upon reaction of N-methyl-N-nitrosourea with dATP. The products of this reaction were purified by repeated anion exchange column chromatography and were found to consist of adenine deoxyribonucleotides methylated on the base moiety, terminal phosphate, or both. Products methylated on the adenine ring were identified by co-migration of acid-hydrolyzed samples with authentic standards on reverse phase high pressure liquid chromatography. Products methylated on the sugar phosphate moiety were identified by digestion with snake venom phosphodiesterase, alkaline phosphatase, or both followed by polyethyleneimine cellulose thin layer chromatography. The results demonstrate production of gamma-phosphate-methyldATP, beta-phosphate-methyl-dADP, 1-methyldATP, and gamma-phosphate-methyl-1-methyldATP as well as the relatively unstable products 3-methyldATP and gamma-phosphate-methyl-3-methyl-dATP. The identities and amounts of products formed during this reaction in vitro are consistent with our finding that cellular deoxyribonucleotide pools are a significant target for N-methyl-N-nitrosourea (Topal, M. D., and Baker, M. S. (1982) Proc. Natl. Acad. Sci. U.S.A. 79, 2211-2215).

Reversed-phase high-performance liquid chromatographic separation of nucleosides and nucleotides

The purpose of this work was to find a suitable reversed-phase high-performance liquid chromatographic method for analysing 2′-, 3′- and 5′-isomers of nucleotides and at the same time determining the free base, nucleoside, 2′,3′-cyclic nucleotide and the total diphosphates. The procedure developed was most effective for those nucleosides and their derivatives that displayed a range of retention times, allowing sufficient resolution for quantitative analyses. The isocratic analyses were particularly suitable for the determination of individual 2′-, 3′- and 5′-ribonucleotides and for 3′- and 5′-deoxyribonucleotides, which were very difficult to quantitate by cellulose thin-layer chromatography. The reversed-phase method enabled quantitative analysis of the nucleoside base, nucleoside, nucleotides and cyclic nucleotides derived from thymidine, deoxyadenosine, adenosine or guanosine. Overlapping elutions were observed for reversed-phase separations of derivatives of uracil and cytosine, but some quantitative results were obtained. The separation method was applied to the analysis of products of phosphorylation of nucleosides and deoxynucleosides in formamide solution.

Glycosaminoglycans in Embryonic Mouse Teeth and the Dissociated Dental Constituents

The nature, amounts, and distribution of glycos-aminoglycans (GAG) before and during odontoblast terminal differentiation were studied. GAG have been isolated from intact mouse tooth germs and from dissociated dental epithelia and dental papillae after labeling with [ 3H] glucos-amine or 35SO 4 2− as precursor. The kinds and relative amounts of 3H-labeled GAG were analyzed by chromatography on a DEAE-cellulose column and cellulose thin-layer sheets. The amounts of individual GAG relative to total GAG were determined from the elution profiles, whereas their nature was identified by the selective removal of chromatographic peaks after enzymatic or chemical degradation. We found hyaluronate and probably a minute quantity of heparan sulfate in the dental epithelium, while hyaluronate, heparan sulfate, and chondroitin sulfate were the main types of GAG in the dental papilla. The chondroitin sulfate recovered was further fractionated by cellulose thin-layer chromatography into two isomers, namely chondroitin-4-sulfate (the major component) and chondroitin-6-sulfate. Changes in the elution profile from DEAE-cellulose chromatography of tooth GAG extracted from different developmental stages suggest that modifications of GAG occur during odontogenesis. Alcian blue staining localized large amounts of hyaluronate and sulfated GAG along the epithelio-mesenchymal junction. Tissue specificity and changing patterns of GAG were demonstrated during odontogenesis.

Biosynthesis of hyaluronic acid in cultures of chondrocytes from the Swarm rat chondrosarcoma.

The synthesis of hyaluronic acid by cultured chondrocytes from the Swarm rat chondrosarcoma was examined using [3H]glucosamine as a precursor. [3H]Hyaluronate in samples was estimated as the specific unsaturated disaccharide released by incubation with chondroitinase ABC under conditions where all [3H]hyaluronate was converted to disaccharide even in the presence of excess chondroitin sulfate. The products of digestion of chondroitin sulfate and hyaluronate were well separated by cellulose thin layer chromatography. A second method involving quantitation of the 3H-oligosaccharides released by digestion with Streptomyces hyaluronidase gave nearly identical results. [3H]Hyaluronate formed about 12% of the [3H]glycosaminoglycans synthesized by the chondrocytes, although levels as high as 20% were found in one set of cultures. The incorporation of [3H]glucosamine into chondroitin sulfate and hyaluronate was linear and at a constant ratio over a 24-h period. The distributions of [35S]proteoglycans, [3H]chondroitin sulfate, and [3H]hyaluronate between the medium, a 4.0 M guanidine HCl extract of the cell layer, and the cell residue, were investigated over 24 h. Both hyaluronate and proteoglycan accumulated in the medium with little retention in the cell layer. Pulse-chase experiments indicated that a greater proportion of the [3H]hyaluronate than the [3H]chondroitin sulfate was initially retained in the cells, but that subsequently [3H]hyaluronate accumulated more rapidly in the medium, suggesting differences in the transit time through the extracellular matrix for the two molecules once secreted from the cell. In cultures labeled for 6 h, 4.0 M guanidine HCl extracted 66% of the hyaluronate and 58% of the chondroitin sulfate associated with the cell layer, while 1% Zwittergent, a zwitterionic detergent, extracted 59% and 29%, respectively. The selective extraction with detergent suggests that there is a pool of hyaluronate in the cell layer which is not associated with proteoglycan aggregates. Equilibrium density gradient centrifugation of cell layer extracts and culture media, under dissociative conditions, showed that 89% of the [3H]hyaluronate banded at densities between 1.43-1.55 g/ml with a peak at 1.47 g/ml. Disaccharide analyses of the gradient fractions showed that the main proteoglycan component contained primarily chondroitin 4-sulfate but also revealed a minor proteoglycan at lower densities which was considerably enriched in chondroitin 6-sulfate.

The complete separation of the base, nucleoside, mono-, di-, and triphosphonucleosides of uracil and cytosine by polyethyleneimine cellulose thin-layer chromatography

A high-resolution thin-layer chromatography procedure for the complete separation and quantitation of the base, nucleoside, mono-, di-, and triphosphonucleosides of uracil and cytosine is presented. The procedure employs polyethyleneimine cellulose plastic-backed plates developed at 0°C in 0.55 m LiCl, 0.2% (v/v) formic acid solvent. The method is particularly useful for the separation of the products and substrates in studies of the pyrimidine salvage enzymes, the pyrimidine nucleotidases, and the pyrimidine nucleotide kinases of minimally purified enzyme preparations.

Induction of Bacillus subtilis sporulation by decoyinine and the concomitant disappearance of ppGpp in vegetative cells.

Sporulation of Bacillus subtilis, growing exponentially in the presence of rapidly metabolizable nutrients, was induced by addition of decoyinine (an antibiotic inhibitor of GMP synthesis), and intracellular amounts of ppGpp were determined after 2 M formic acid extraction by polyethyleneimine (PEI)-cellulose thin-layer chromatography. Consequently, it was found that the ppGpp in vegetative cells abruptly disappeared after the addition of decoyinine. This indicates that the disappearance of ppGpp is closely correlated to the initiation of B. subtilis sporulation.

Decapeptide luteinizing hormone releasing hormone in ovine pineal gland.

Ovine pineal gland extracts were examined throughout the year for content and molecular form of immunoreactive (IMR) LH releasing hormone (LH-RH). The content of IMR LH-RH in pineal glands collected in spring and summer was 160-2230 pg/gland, while the content in pineal glands collected in autumn and winter was lower (31-39 pg/gland). Pineal gland IMR LH-RH, purified by affinity and Sephadex G-25 chromatography, yielded displacement curves parallel to those of hypothalamic IMR LH-RH and synthetic LH-RH in radioimmunoassays employing four antisera which require different regions of the decapeptide for effective binding, suggesting considerable similarities in the structure of the molecule. The majority of pineal gland IMR LH-RH behaved identically to hypothalamic IMR LH-RH and synthetic LH-RH on gel filtration, cation exchange chromatography, high-pressure liquid chromatography and cellulose thin-layer chromatography. However, a small amount of a less positively charged LH-RH species was also present in all pineal gland extracts. Our findings indicate that hypothalamic decapeptide LH-RH occurs in the ovine pineal gland.

Microfingerprints of proteins through labeling acetylation of their proteolytic peptides

Microgram amounts of proteins applied to polyacrylamide gel electrophoresis were subjected to a fingerprinting procedure using a combined proteolysis-acetylation method with the aid of 14C-labeled acetic anhydride of high specific activity. After staining, gel slices were partially dried and were resoaked in a solution of a protease. After elution and acetylation, the resulting peptides were resolved in fingerprints on cellulose thin-layer chromatography plates and subjected to autoradiography with or without sensitization. Yields, completeness of fingerprinting, and possible artefacts were investigated.

Chromatographic Behavior of Metalloporphyrins by Cellulose Thin-layer Chromatography

Abstract Cellulose thin-layer chromatographic separation of chlorophyll a, b, and c, pheophytin a, b, and c, Zn(II) and Cu(II)-complexes of protoporphyrin-IX dimethyl ester (PPDE) was studied by using a light petroleum–acetone developing solvent system. The hRf values of metal–PPDE chelates were found to be virtually linearly related with the electronegativities-to-radius ratio (En/ri) of central-metal ions. The hRf values of metalloporphyrins become progressively greater with increase in En/ri values of the central metal. This suggests that a dominant factor influencing the hRf values of metal–porphyrins is the extent of electron transfer from the four nitrogens and/or the π-electron system of porphyrin to central metal.

Paper and Cellulose Thin Layer Chromatography of Toxic Cations

Paper and Cellulose Thin Layer Chromatography of Toxic Cations