Bidimensional Thin-layer Chromatography Research Articles

Progesterone metabolism in adipose cells

The aim of the present study was to investigate pathways of progesterone metabolism in human adipose cells. Adipose tissue samples from the omental (OM) and subcutaneous (SC) fat compartments were surgically obtained in women. In isolated mature adipocytes, progesterone was converted to 20alpha-hydroxyprogesterone as the main metabolite, most likely through the activity of aldo-keto reductases 1C1, 2 and 3 (20alpha-HSD, 3alpha-HSD type 3 and 17beta-HSD type 5, respectively). In cultured preadipocytes, progesterone was converted to several metabolites identified using bidimensional thin layer chromatography, with or without the dual inhibitor of 5alpha-reductase type 1 and 2 (17beta-N,N-diethylcarbamoyl-4-methyl-4-aza-5alpha-androstan-3-one (4-MA)). Major metabolites identified in OM and SC preadipocytes which were incubated for 24h with (14)C-labelled progesterone were 20alpha-hydroxyprogesterone, 5alpha-pregnane-3alpha/beta-ol-20-one, 5alpha- and 5beta-pregnanedione, 5alpha- and 5beta-pregnane-20alpha-ol-3-one, 5alpha-pregnane-3alpha/beta-ol-20-one and 5beta-pregnane-3alpha/beta-20alpha-diol. Induction of preadipocyte differentiation increased expression levels of AKR1C1 and modified the pattern of progesterone metabolism substantially, leaving 20alpha-hydroxyprogesterone as the main metabolite generated. On the other hand, progesterone itself showed no consistent effect on adipocyte differentiation. In conclusion, preadipocytes and lipid-storing, mature adipocytes efficiently generate progesterone metabolites in women, which is consistent with rather modest effects progesterone on abdominal fat cell differentiation.

Fusarium crookwellense-produced zearalenone in maize stubble in the field

Waste grain and vegetative material (stems and leaves) collected from a maize field several months after harvest was analysed by bi-dimensional thin layer chromatography for the presence of aflatoxins, deoxynivalenol, ochratoxin A, sterigmatocystin, T-2 toxin and zearalenone. Deoxinivalenol (0.7 mg/kg) and T-2 toxin (4.1 mg/kg) were found in the grain and zearalenone (3.0 mg/ kg) was found in the stem and leaf. No other toxins were detected. The stubble was examined for the presence of potentially toxigenic Fusarium species, and F. poae, F. moniliforme var. subglutinans and F. crookwellense were isolated and identified. When these isolates were cultured on cracked corn, only F. crookwellense was found to produce micotoxins and then only zearalenone was detected. As corn stubble is commonly grazed in Argentina and in other countries, these findings identify a further source of mycotoxins that may adversely affect animal health and productivity.

Platelet-activating factor preferentially stimulates the phospholipase A2/cyclooxygenase cascade in the rabbit cornea.

Platelet-activating factor (PAF) is formed in the cornea after injury as well as by infiltrating inflammatory cells. We have studied the effects of PAF on the release and metabolism of arachidonic acid (AA) in the rabbit cornea. Corneal lipids were labeled in vivo by injecting [3H]AA and subsequently incubated in vitro with 100 nM PAF in the presence or absence of 10 microM BN50727, a PAF antagonist. The AA and eicosanoids released by incubated corneas were analyzed by high-performance liquid chromatography (HPLC). Tissue lipids were examined by mono- and bidimensional thin-layer chromatography (TLC). Within 5 min, PAF stimulated AA release to 76% above control levels. BN50727 inhibited the AA release elicited by PAF at all time points studied. The decreased content of [3H]AA in phosphatidylcholine (PC), phosphatidylethanolamine (PE) and phosphatidylinositol (PI) following PAF exposure and the lack of stimulation by PAF on the release of [3H] linoleic acid suggest that the cytosolic phospholipase A2 was activated. PAF also stimulated depletion of AA from the inositol lipids, phosphatidylinositol-4-phosphate (PIP) and phosphatidylinositol-4,5-biphosphate (PIP2) and increased content of [3H]AA into diacylglycerol (DAG) and phosphatidic acid (PA). This reaction indicates that PAF could also mediate activation of other phospholipases in the cornea. In addition, PAF preferentially stimulated the cyclooxygenase pathway. The PAF antagonist BN50727 mainly suppressed the PAF-stimulated release of PGE2. The antagonist did not inhibit lipoxygenase activity even after 30 min of PAF stimulation. These results suggest that PAF activate a phospholipase A2/cyclooxygenase pathway in the cornea via a PAF-receptor mechanism.(ABSTRACT TRUNCATED AT 250 WORDS)

Biochemical and ultrastructural studies on an Epstein-Barr virus-transformed lymphoid cell line from a Niemann-Pick disease type C patient

Human lymphoid cell lines established from normal subjects and from a Niemann-Pick disease type C patient were investigated from a triple point of view of enzymology, metabolism and ultrastructure: (1) Sphingomyelinase activities, isoenzyme electrofocusing profiles and properties of the major enzyme were quite similar in type C and normal lymphoid cell lines. Similarly, no significant difference was observed in non-specific phosphodiesterases hydrolysing bis(methylumbelliferyl)phosphate and bis(methylumbelliferyl) pyrophosphate. (2) The study of the lipid composition of type C cells showed no obvious accumulation of sphingomyelin or other phospholipid, but only a higher amount of glycolipids (mainly GlcCer and GbOse 3Cer), as visualized by bidimensional thin-layer chromatography. (3) Ultrastructural studies demonstrated, in type C cells, the presence of an obvious lysosomal storage of amphiphilic lipids quite similar to that observed in tissues of type C patients. These studies, which demonstrate the validity of lymphoid cell lines as an experimental model system for type C disease, agree with the current opinion that an impairment of sphingomyelin catabolism is not the primary defect in type C disease.

Turnover of phosphocholine and phosphoethanolamine in ether-phospholipids of Krebs II ascite cells.

Krebs II ascite cells suspended in Eagle medium were incubated at 37 C for up to 6 hr in the presence of [3H] glycerol or [32P] orthophosphate. After extraction, their lipids were treated with guinea pig phospholipase A1 under conditions where all diacyl-phospholipids (diacyl-PL) became hydrolyzed with 55% recovery of lyso-PL. Using a bidimensional thin layer chromatography (TLC) involving exposure to HCl fumes between the two runs, it then became possible to determine at once the specific radioactivity of the three subclasses (diacyl-, alkylacyl- and alkenylacyl-) present in choline glycerophospholipids (CGP) and ethanolamineglycerophospholipids (EGP). Compared to diacyl-PL, a lower de novo synthesis of ether subclasses was evidenced in both CGP and EGP by [3H] glycerol incorporation. Although the same profile was obtained for CGP with [32P] orthophosphate, the three EGP subclasses displayed in this case the same specific radioactivity. These data indicate a higher turnover rate of the polar head group of ether-EGP compared to either-CGP. The simple methodology used in the present study might thus prove helpful in developing enzymatic studies dealing with the mechanism of this accelerated renewal.

Ether-phospholipid composition in neutrophile and platelets

It is well documented that ether-lipids, especially the 1-O-alkyl-2-O-acyl- sn -glycero-3-phosphocholine, can serve as precursors in paf-acether (platelet-activating factor) biosynthesis. This study was undertaken to determine the amount of these compounds in two cell types which are good producers of paf-acether : human neutrophils and rabbit platelets. The method of phospholipid analysis was based on selective destruction of diacyl molecules by lipase from guinea pig pancreas and of plasmalogens by acidolysis ; phospholipids were then separated by bidimensional thin-layer chromatography. In platelets as in neutrophils, the major phospholipids were choline (37%) and ethanolamine (30 and 32 %, respectively) phosphoglycerides and sphingomyelin (18 and 16%, respectively). In rabbit platelets the 1-alkyl molecules represented 15% of the choline class (0.7 nmol/10 7 cells). Neutrophils differed strikingly from platelets by the high level of these molecules which constituted 50% of the choline class (16.3 nmol/10 7 cells). It therefore appears that in the two cell types, the amount of 1-O-alkyl-2-O-acyl- sn -glycero-3-phosphocholine largely exceeded what would be necessary for paf-acether biosynthesis.

Androgen Metabolism by Human Fetal Epiphyseal Cartilage and Its Chondrocytes in Primary Culture*

Androgen metabolism by human epiphyseal cartilage and chondrocytes in primary culture was studied during fetal life. Testosterone (T) and androstendione (delta 4) were incubated with epiphyseal cartilage and with chondrocytes in Dulbecco's medium for 4, 12, and 24 h. Androstanedione, delta 4, dihydrotestosterone (DHT), and androstanediol were separated by bidimensional thin layer chromatography and delta 4, DHT, and T recrystallized in five different solvent systems. The results showed that delta 4 was transformed into androstanedione, DHT, and T whereas T was metabolized into delta 4 and 5 alpha-reduced androgens. In both systems (epiphyseal cartilage and chondrocytes) delta 4 was the principal metabolite produced from T. There were no major differences in the metabolic patterns of delta 4 and T between epiphyseal cartilage and chondrocytes according to sex nor gestational age (11-32 weeks). In conclusion, 5 alpha-reductase activity was found in chondrocytes from human fetuses of both sexes, but T was mainly transformed into delta 4.

Studies on ether phospholipids: I. A new method of determination using phospholipase A1 from guinea pig pancreas: Application to krebs ii ascites cells

A new method for ether phospholipid analysis has been devised, based on the selective destruction of diacyl phospholipids by guinea pig phospholipase A 1 and of plasmalogens by acidolysis. The paper describes optimal conditions allowing a specific degradation of diacyl phospholipids by the enzyme(s). This requires the incubation of a total lipid extract in the presence of 2.4 mM sodium deoxycholate, at pH 8.0, at a temperature of 42° C. As shown with various radioactive markers, all the diacyl phospholipids become degraded, whereas sphingomyelin and ether phospholipids remain refractory to phospholipase A 1 attack. Phospholipids are then separated by a bidimensional thin-layer chromatography involving the exposure of the plates to HCl fumes between the two runs, in order to hydrolyse plasmalogens. Selectivity of both hydrolytic procedures is further demonstrated upon analysis of acetyl diacylglycerol derived from phospholipids. Various phospholipids can thus be determined by phosphorus measurement using sphingomyelin as an internal standard. By this way, it is shown that Krebs II cells present a very high content of ether phospholipid species (around 25% of total). Among these, about 50% are alkyl forms in ethanolamine phosphoglycerides, whereas this value reaches 70% in choline phosphoglycerides.

Analysis of mycolic acids from a group of corynebacteria by capillary gas chromatography and mass spectrometry.

The cell wall of leprosy-derived corynebacteria (a group of 'diphtheroids' isolated from human leprosy lesions and patients' blood) was previously shown to contain, in addition to peptidoglycan and arabinogalactan, mycolic acids. These alpha-branched beta-hydroxy fatty acids were attributed to the corynomycolic group, according to their RF in monodimensional thin-layer chromatography. In the present work, mycolic acids from leprosy-derived and reference corynebacteria have been fractionated by monodimensional and bidimensional thin-layer chromatography and by gas chromatography. Pyrolyzed mycolic acids have been analyzed on conventional packed columns, whereas intact methyl esters of mycolic acids with free and silylated beta-hydroxyl group have been analyzed on capillary columns, and their structure has been established by mass spectrometry. In all leprosy-derived corynebacteria, some 20 components containing 24-36 carbon atoms and 0-4 double bonds were obtained. The three major groups had 32, 34 and 36 carbons, and the frequency of unsaturated versus saturated chains increased proportionally to the molecular weight. For comparison, the main components of a reference corynebacterium. Corynebacterium diphtheriae PW8, had 30 and 32 carbons, and their hydrocarbon chains were essentially saturated. This work confirms the relative chemical homogeneity of different leprosy-derived corynebacteria and describes some peculiar traits in the chemical structure of this group of organisms. In addition, it shows the complexity of the mycolic acid fraction of corynebacterial cell wall and suggests that the mycolic acid pattern is a sort of fingerprint of each bacterial strain grown under standard conditions. Finally, the fractionation of intact corynomycolic acid methyl esters with free or silylated beta-hydroxyl group by capillary gas chromatography proved to be the best analytical procedure at present available for resolving this complex mixture of corynomycolate isomers. Structural determination of silylated samples by mass spectrometry is preferred because they have more diagnostic fragments.

In vitro methylation of yeast tRNAAspby rat brain cortical tRNA-(adenine-1) methyltransferase

Rat brain cortices from young animals contain large amounts of tRNA (adenine-1)methyltransferase(s). The enzyme(s) can methylate E. coli tRNA and to a lower degree yeast tRNA. Among yeast tRNA species which can be methylated we have selected tRNAAsp as a substrate for the brain enzyme. The digestions of in vitro methylated [Me-3H]-tRNAAsp with pancreatic and/or T1 ribonucleases followed by chromatographies on DEAE-cellulose, 7 M urea, suggested that the methylation of tRNAAsp occurred at a single position within the D-loop. Further digestion of the radioactive oligonucleotide recovered after DEAE-cellulose chromatography by phosphomonoesterase and snake venom phosphodiesterase enzymes followed by bidimensional thin layer chromatography enabled us to determine the location of the adenine residue which becomes methylated by the brain enzyme. This one resulted to be the adenine 14 in the D-loop of yeast tRNAAsp.

Improved technique for the analysis of amino acids and related compounds on thin layers of cellulose : XI. Direct, visual micro-method for investigating the primary structural sequence of collagen and other proteins

The present paper describes a new variation of the “finger printing” technique for examining the primary structure of proteins. It is based on the production of a reproducible, partial acid hydrolysate consisting of amino acids and small peptides which are separated into compact spots by bi-dimensional thin-layer chromatography. The spots are characterized by their R F values and colour reactions with a variety of reagents. The method was initially developed for collagen but has been applied to other proteins, with interesting results. It has developed logically from earlier, systematic studies on amino acids and peptides, and holds promise for the comparative investigation of normal and diseased human collagen.

Analyse et métabolisme des phospholipides du cœur. Incorporation in vivo de 32P-orthophosphate dans les lipides cardiaques du rat

Heart phospholipid metabolism was studied by intraperitoneal injection of 32 P-orthophosphate to the rat. Phospholipids were separated by silicic acid column chromatography and bidimensional thin-layer chromatography on silicagel. Phosphatidylcholine, phosphatidylethanolamine and diphosphatidylglycerol are the major phospholipid constitutents of the heart, we noted also the presence of phosphatidylglycerol and the low level of sphingomyelin. Our technique was particularly suitable for the separation of phosphatidylserine and phosphatidylinositol. Our technique was particularly suitable for the separation of phosphatidylserine and and 14 hours), showed a strong incorporation into phosphatidylinositol, phosphatidylcholine and phosphatidylglycerol. Sphingomyelin, phosphatidylethanolamine and diphosphatidylglycerol had, in contrary, very low specific activities even after 14 hours. Studying their different fatty acid composition and their different specific activities we try to explain the metabolism of phosphatidylglycerol and its conversion into diphosphatidylglycerol in rat heart. Le métabolisme des phospholipides du cœur est étudié après injection de 32 P-orthophosphate chez le rat. Les phospholipides sont séparés par chromatographie sur colonne d'acide silicique et sur couche mince de silicagel par chromatographie uni ou bidimensionnelle. Ces techniques nous permettent de confirmer la prépondérance des phosphatidylcholines, des phosphatidyléthanolamines et du diphosphatidylglycérol, le taux faible des sphingomyélines et la présence de phosphatidylglycérol. Elles nous permettent également d'obtenir une bonne séparation des phosphatidylsérines et des phosphatidylinositols. Les activités spécifiques des phospholipides, mesurées en fonction du temps (4, 6, 8 et 14 heures après l'injection de l'isotope) montrent une forte incorporation dans les phosphatidylinositols, les phosphatidylcholines et le phosphatidylglycérol. Sphingomyélines, phosphatidyléthanolamines et diphosphatidylglycérol, montrent au contraire, des activités spécifiques faibles même au bout de 14 heures. A la lumière de leur composition en acides gras, et de leurs activités spécifiques nettement différentes nous essayons d'expliquer le métabolisme du phosphatidylglycérol et sa conversion en diphosphatidylglycérol dans le cœur de rat.

A sensitive chromatographic technique for screening of amino acid metabolic defects in the newborn

A new technique for the bidimensional thin-layer chromatography of amino acids in blood and urine is described. It is especially suited to the screening of amino acid metabolic defects in the newborn, inasmuch as it requires only 10 μ1 of untreated urine and 2 drops of blood. The Chromatographie system is characterized by rapid development, great sensitivity of the spots to ninhydrin and clear separation of 28 amino acids.

Identification of bufotenin in toad brain by chromatography and mass spectrometry of its dans-derivative

In extracts of the brain of the toad (Bufo bufo) a substance was found which cochromatographed with bufotenin (N,N-dimethyl-5-hydroxytryptamine) on silica gel thin layer in three solvent systems. An undeveloped spot was scraped off the plate and dansylated. The bidimensional thin layer chromatography of the dansylation products gave one spot which cochromatographed with DANS-bufotenin. In the mass spectrographic analysis, this spot gave a mass spectrum identical with that of DANS-bufotenin. The compound in this way identified as bufotenin was detected in toad brain, retina, and skin, but was absent in toad liver and pancreas, and in rat brain stem.

Determination of 5,6-dihydrouridine in ribonucleic acid

Two simple methods to rapidly and accurately quantify 5,6-dihydrouridine in small amounts of RNA are presented. One involves a colorimetric microassay for dihydrouridine. It is performed directly on RNA in less than one hour. It requires only 25–100 μg of tRNA and is reproducible to ±5%. The other involves enzymic digestion of RNA to nucleosides followed by bidimensional thin-layer chromatography on microcrystalline cellulose. It may be used to quantify dihydrouridine in RNA labeled with a radioactive pyrimidine. It is extremely sensitive, relatively rapid, and allows simultaneous determination of other major and minor pyrimidine nucleosides. The methods have been used to quantify dihydrouridine in tRNA of Escherichia coli, Salmonella typhimurium, yeast, and rat liver, and rRNA of E. coli, with excellent agreement between methods and with literature values.

Fluorometric assay of corticosterone after purification by bidimensional thin-layer chromatography in birds

A bidimensional thin layer chromatography (B.T.L.C.) purification process was adapted for the fluorometric assay of corticosterone in bird plasma and adrenal tissue. The degree of purification obtained is such that this simple, rapid and sensitive technique may replace the double isotope dilution technique for the accurate assay of corticosterone. The main steps are the following: — Extraction of the corticosteroids. — Purification by B.T.L.C. — Evaluation [radioactive tracer] of the recovery level, and fluorometric assay. This technique was used for a physiological approach of adrenal cortical activity in birds.

Chromatographie des produits de radiolyse de la thymine

Chromatography of thymine radiolysis products [ 14C]Thymine in aqueous solution (10 −3 to 10 −4 M) was irradiated with γ rays of 60Co at doses between 10 2 and 10 6 rad. Different chromatographic techniques for separation of the radiolysis products are described. For the paper system solvents such as butanol—water (86:14) and acetonitrile—phosphate buffer (0.2 M NaH 2PO 4 and 0.2 M Na 2HPO 4) (85:15) were employed. Bidimensional thin-layer chromatography with cellulose powder was also used. For this experiment the best solvents were either butanol—methanol—water—NH 4OH (60:20:20:1) or propanol—water (10:3) in the first dimension, and isopropanol—water-saturated (NH 4) 2SO 4—1 M K 2HPO 4 (3:77:20) in the second dimension. The separation is rapid and can be obtained at 4°. We have identified a new photoproduct of thymine: 5-methyl-5-hydroxy hydantoin. R F values of this compound are given.

Thin-layer chromatography of carbohydrates on silica gel impregnated with sodium acetate, monosodium phosphate and disodium phosphate

The chromatographic behavior of carbohydrates on silica gel plates impregnated with sodium acetate, monosodium phosphate and disodium phosphate is described and discussed. The study shows that bidimensional thin-layer chromatography of carbohydrates with the above impregnants is not feasible, but that excellent monodimensional separations of up to ten sugars can be achieved with the sodium acetate and monosodium phosphate impregnants. An investigation is specifically devoted to the setting up of a monodimensional chromatogram which separates the most important carbohydrates (biologically) in an 8-hour run.

Bidimensional thin-layer chromatography of carbohydrates on silica gel impregnated with boric acid

The separation of a large number of sugars by means of bidimensional thin-layer chromatography is described. Factors which influence separation of carbohydrates on silica gel impregnated with boric acid, and the limitations if the monodimensional technique are investigated and discussed.

Separation and quantitative determination of 131-I-triiodothyronine and 131-I-thyroxine in human plasma by thin layer chromatography.

ABSTRACT A method is described for the quantitative determination of 131I-triiodothyronine and 131I-tetraiodothyronine in human plasma after oral administration of 131I-iodide. Steps in the method are: acidified n-butanol extraction of the thyroid hormones from the plasma deprived of inorganic iodine by means of an ion exchange resin; addition of stable iodo-aminoacid carriers to the butanol extract and its evaporation to dryness under reduced pressure; bidimensional thin layer chromatography of the rediluted extract on cellulose in combined organic alkaline and organic acid systems; staining of the carriers with eerie sulphate-sodium arsenite reagent and diluted methylene blue; radioactivity measurements of the cellulose scraped from the stained spots in a well type scintillation counter. The reliability of the method was established by recovery experiments and by duplicate chromatography of plasma to which mixtures of 131I-T3 and 131I-T4 and 131I-iodide were added. Its specificity was confirmed by autoradiography. Good agreement was found with the results yielded by simultaneous paper chromatography of the same extracts. The method requires smaller amounts of plasma and allows quicker determinations than paper chromatographic procedures. Relative determinations of 131I-T3 and 131I-T4 are reported in some euthyroid and hyperthyroid patients given therapeutic and diagnostic doses of 131I-iodide.