Quantitative Two-dimensional Thin-layer Chromatography Research Articles

Metabolism of adenosine and deoxyadenosine by human erythrocytes and CCRF-CEM leukemia cells

Human lymphocytes lacking adenosine deaminase die and T-cell leukemias are killed by deoxycoformycin (dCf), an inhibitor of adenosine deaminase, due to impaired metabolism of dAdo. The initial metabolism of exogenous adenosine (Ado) and deoxyadenosine (dAdo) has been compared in human erythrocytes and CCRF-CEM leukemia cells and the data obtained have been simulated using kinetic constants obtained in vitro for the enzymes involved. Cells were mixed with 3H-labelled Ado or dAdo, samples were taken at 3 sec intervals and progress curves for the 3H-labelled metabolites formed were determined by quantitative two-dimensional thin layer chromatography. Erythrocytes rapidly take up Ado and the predominant metabolite after 60 sec is hypoxanthine (Hyp), while for dAdo, deoxyinosine (dIno) predominates. By contrast, leukemia cells convert Ado predominantly to AMP, while dAdo is converted first to Hyp and then to AMP. The presence of dCf had little effect upon Ado metabolism but induced accumulation of dAdo. Erythrocytes rapidly degrade Ado and dAdo to Hyp, although the phosphorolysis of dIno is relatively slow. Human CCRF-CEM leukemia cells convert most of the Ado or dAdo to AMP after 60 sec. For dAdo, the sequence of reactions would be dAdo→dIno→Hyp→IMP→sAMP→AMP. dCf does not significantly affect the conversion of Ado→AMP, but dCf blocks AMP accumulation from dAdo, consistent with the reaction sequence shown above. A computer model has been developed for the metabolism of Ado and dAdo, but some of the kinetic constants determined in vitro for this model do not pertain to intact cells.

Quantitative two-dimensional thin-layer chromatography

Two-dimensional thin-layer chromatography (TLC) is seldom used as a quantitative method in spite of its enormous potential, the main reasons being a single sample, a complicated solvent system optimizing procedure, and lack of adequative evaluation procedures. A special set of computer programs which enable scanning and evaluation TLC scanner is described. A whole TLC plate is scanned in linear mode with normal slit shape, for instance 0.2 x 8 mm, with as small as 0.5 to 1.0 mm steps between scans. Each run is processed as for a normal densitogram, the baseline is constructed, substracted, and the eventual peaks are located and integrated. In the next step a special program selects the correct positions of peaks, according to the biggest peak height, of all the scans. The selected scans are reintegrated and peak heights and areas are reported. Manual mode results were prepared in addition to the automatic mode. The operator selects the most convenient scan and integrates it using the cursors and chromatograms on the screen. The practical application of the scanning and evaluation programs is presented with quantitative determination of some phenolic components in propolis. Plates are developed with the solvent system: toluene—ethyl acetate—formic acid (75:15:3) and in the perpendicular direction with the solvent system: chloroform—methanol—formic acid (75:5:4). Identification and quantification of some characteristic flavonoids is performed with the 2-aminoethyldiphenylborinate/polyethylene glycol 4000 derivation reagent and external standards.

The polymyxin sensitivity and the phosphatidyl ethanolamine content of the Vibrio cholerae membranes.

The phospholipid composition of three different strains of Vibrio cholerae was determined by quantitative two-dimensional thin-layer chromatography. The polymyxin sensitivity of the whole organisms or of the liposomes derived from the total phospholipids of these organisms depended solely on the phosphatidyl ethanolamine content of the system concerned and could be quantitatively related by the equations Y1 = 1.074 X1 - 9.828 and Y2 = 1.22 X2 - 34.47 where Y represents the maximum lysis (%)/hr and X, the % phosphatidyl ethanolamine content of the system concerned, the suffix 1 and 2 corresponding to the liposomal and the whole organisms respectively. The analysis revealed the requirement of a threshold amount of phosphatidyl ethanolamine for the expression of polymyxin action on the system concerned.

Lipid patterns in human leukocytes maintained in long-term culture

The lipid composition of leukocytes maintained in long-term culture was examined in order to clarify the role of immaturity in previously observed differences between normal mature leukocytes and leukemic cells. Cell cultures derived from three types of leukocytes were examined: normal lymphocytes, Burkitt lymphoma, and chronic myelocytic leukemia. Lipid extracts were analyzed for total lipid weight, phospholipids, neutral lipids, and glycolipids. Distribution of individual phospholipids was determined by quantitative two-dimensional thin-layer chromatography. The main phospholipids were phosphatidylcholine (51-54%) and phosphatidylethanolamine (24-25%), with smaller amounts of phosphatidylinositol, phosphatidylserine, sphingomyelin, and cardiolipin. All three types of cultured cells showed a remarkable similarity in total phospholipid content (17-18 x 10(-15) moles/cell) as well as in phospholipid distribution. More variation was seen in neutral lipid content. Glycolipid was abundant (17-23% of total lipid weight) and was present mostly as ceramide dihexoside. Compared with normal lymphocytes or polymorphonuclear leukocytes, the cultured cells showed increased phosphatidylcholine, decreased sphingomyelin, and decreased cholesterol content, similar to the changes found in leukemic leukocytes. These findings suggest that the altered lipid patterns found in leukemic leukocytes are a reflection of cell immaturity rather than a characteristic peculiar to the leukemic state.

Comparison of the phospholipid composition of Bifidobacterium and Lactobacillus strains.

Phospholipid composition of 10 Bifidobacterium strains of human intestinal origin and of 9 Lactobacillus strains was determined by quantitative two-dimensional thin-layer chromatography. Phospholipids of three Bifidobacterium strains from honey bees and of two strains from bovine rumen liquor were qualitatively investigated. Diphosphatidylglycerol and phosphatidylglycerol were present in strains of both genera. All Bifidobacterium strains contained as specific phospholipids a new polyglycerolphospholipid, compound 15, and its lyso derivatives, earlier detected in B. bifidum var. pennsylvanicus. Also, lyso compounds of diphosphatidylglycerol and alanyl phosphatidylglycerol were only present in this genus in variable amounts. Lysyl phosphatidylglycerol was the only ninhydrin-positive phospholipid in seven Lactobacillus strains. In L. delbrückii and L. helveticus it was absent and partially replaced by an unidentified ninhydrin-negative phospholipid. The differences in phospholipid composition between bifidobacteria and lactobacilli may be another argument to differentiate these two genera.

Quantitative two-dimensional thin-layer chromatography of blood phospholipids

A detailed picture of the phospholipid composition of normal human erythrocytes and plasma was obtained by quantitative two-dimensional thin-layer chromatography on silica gel containing an alkaline component. Two extraction procedures for erythrocytes were tested and the techniques and results were compared with those of other recent investigations.

The alk-1-enyl group content of mammalian myelin phosphoglycerides by quantitative two-dimensional thin-layer chromatography

Myelin phospholipids have been examined by a separation-reaction-separation procedure for two-dimensional thin-layer chromatography on silica gel. After separation in one dimension, alk-1-enyl groups are cleaved by exposure of the plates to HCl fumes. Development in the second dimension quantitatively separates acid-labile and acid-stable phosphoglycerides as well as the aldehydes released from the acid-labile phosphoglycerides. Myelin phospholipids from the central nervous systems of the rhesus monkey, squirrel monkey, ox, and mouse contain 32-36% acid-labile ethanolamine phosphoglycerides (ethanolamine plasmalogens) and 8-14% acid-stable ethanolamine phosphoglycerides. Acid-labile choline and serine phosphoglycerides account for less than 1% of the myelin phospholipids.

Phospholipids in tissues of the eye I. Isolation, characterization and quantitative analysis by two-dimensional thin-layer chromatography of diacyl and vinyl-ether phospholipids

1. 1. Eleven phospholipids occurring in tissues of calf eye were characterized and their concentrations determined by quantitative two-dimensional thin-layer chromatography on silica gel containing alkaline magnesium silicate. Five unidentified phospholipids were present in trace amounts. 2. 2. Total phospholipid concentration in retina was relatively high, in vitreous body very low, and in iris, choroid, lens, cornea and sciera intermediate. 3. 3. Phosphatidyl choline, phosphatidyl ethanolamine, phosphatidyl serine, sphingomyelin and phosphatidyl inositol were the major phospholipids in all tissues of the eye. The concentrations of phosphatidyl glycerol, diphosphatidyl glycerol, phosphatidic acid, lysophosphatidyl choline and the unidentified phospholipids were very low. 4. 4. Vinyl-ether analogues of phosphatidyl ethanolamine and phosphatidyl choline were present in all eye tissues.

The distribution of phospholipids in some mammalian milks

Phospholipids were isolated from cow, sheep, Indian buffalo, camel, ass, pig, and human milks. The distribution of phospholipids was determined by quantitative two-dimensional thin-layer chromatography. The distribution of phospholipids was found to be remarkably constant in the milks of all the species studied, and it is concluded that the phospholipids probably fulfil similar or identical functions in these milks.

Effect of Chronic Ethanol Feeding on Rat Liver Phospholipid

Determinations of liver phospholipid concentration and composition were made in experiments to investigate the effect of chronic ethanol ingestion on liver phospholipid metabolism in the rat. Each ethanol-fed animal was grouped with a control animal pair-fed the basal diet plus sucrose and a control animal fed the basal diet ad libitum. Both male and female animals fed ethanol developed increased concentrations of liver phospholipid; the increment in the males was greater than that in the females. Quantitative two-dimensional thin-layer chromatography of the phospholipid fractions showed an increase in lysolecithin and phosphatidyl ethanolamine in the ethanol-fed animals, whether calculated on the basis of phospholipid concentration or total liver content. The increase in lysolecithin, a membrane-lysing agent, may be relevant to alterations reported in experimental and clinical chronic alcoholism.

Quantitative two-dimensional thin-layer chromatography of naturally occurring phospholipids

A method for the complete separation and essentially quantitative isolation of individual components of phospholipid mixtures is described. The mixture is chromatographed in two dimensions on thin layers of Silica Gel G and the phospholipids are extracted from the adsorbent with a solvent mixture containing formic acid.

Quantitative two-dimensional thin-layer chromatography of deoxyribonucleic acid components

In studying the various parameters of normal and malignant mammalian tissues, it became apparent that there was a need for a rapid quantitative procedure for the determination of deoxyribosides and deoxyribotides. A new technique, thin-layer chromatography (1), has shown great versatility in the separation of different types of chemical compounds. Application of this technique for the separation of various ribonucleosides and ribonucleotides has been recently reported by Randerath (2, 3), but his procedure does not give sufficient resolution for quantitative determination. We wish to report a two-dimensional thin-layer chromatographic method for the quantitative determination of deoxyribosides and their 5′-phosphates.