Two-dimensional Thin-layer Chromatography Research Articles

Structure and antigenicity of the major glycolipid from Taenia solium cysticerci

Lipids were extracted from cysticerci of the human tapeworm Taenia solium isolated from various infected pigs and analysed by two-dimensional thin-layer chromatography. These consisted of both alkali-labile and alkali-stable glycolipids, and phosphorylated non-glycosylated lipids. Because abundant and immunogenic glycolipids of parasites have been implicated in host–parasite interactions, the major lipid, an alkali-stable glycolipid, was purified by chromatography and its structure and antigenicity were determined. The structure of the major glycolipid of T. solium, GSL-I, was elucidated through a combination of chemical degradative methods, gas chromatography/mass spectrometry analyses of the degradative products, matrix-assisted-laser desorption/ionisation time of flight mass spectrometry and nuclear magnetic resonance spectroscopy. This analytical strategy led to the identification of a family of β-galactosylceramides composed mainly of phytosphinganine (2-hydroxylated sphinganine) N-acylated by C16–C24 fatty acids, with the predominance of 2-hydroxylated homologues. Enzyme-linked immunosorbent assay showed no correlation between the antibody titres directed against GSL-I in the human sera and the infective status; in contrast, a very high specific immunoreactivity and a sensitivity above 50% were observed when GSL-I was tested with cerebrospinal fluids from well characterised infected humans. Thus, although these results do not support the use of GSL-I alone as an antigen for the detection of neurocysticercosis, its use as part of an antigen cocktail for the diagnosis of the disease in cerebrospinal fluids merits further investigations.

Insulin receptor-mediated p62dok tyrosine phosphorylation at residues 362 and 398 plays distinct roles for binding GTPase-activating protein and Nck and is essential for inhibiting insulin-stimulated activation of Ras and Akt.

A GTPase-activating protein (GAP)-associated 60-kDa protein has been found to undergo rapid tyrosine phosphorylation in response to insulin stimulation. However, whether this protein is a direct in vivo substrate for the insulin receptor (IR) tyrosine kinase and whether the tyrosine phosphorylation plays a role in insulin signaling remain to be established. Here we show that the insulin-stimulated tyrosine phosphorylation of the GAP-associated protein, now identified as p62(dok), is inhibited by Grb10, an adaptor protein that binds directly to the kinase domain of the IR, both in vitro and in cells. Replacing Tyr(362) and Tyr(398) with phenylalanine greatly decreased the IR-catalyzed p62(dok) tyrosine phosphorylation in vitro, suggesting that these two residues are the major IR-mediated phosphorylation sites. However, mutations at Tyr(362) and Tyr(398) only partially blocked insulin-stimulated p62(dok) tyrosine phosphorylation in cells, indicating that p62(dok) is also a target for other cellular tyrosine kinase(s) in addition to the IR. Replacing Tyr(362) with phenylalanine abolished the interaction between p62(dok) and Nck. Mutations at Tyr(362/398) of p62(dok) disrupted the interaction between p62(dok) and GAP and decreased the inhibitory effect of p62(dok) on the insulin-stimulated activation of Ras and Akt, but not mitogen-activated protein kinase. Furthermore, the inhibitory effect of p62(dok) on Akt phosphorylation could be blocked by coexpression of a constitutively active Ras. Taken together, our findings indicate that p62(dok) is a direct substrate for the IR tyrosine kinase and that phosphorylation at Tyr(362) and Tyr(398) plays an essential role for p62(dok) to interact with its effectors and negatively regulate the insulin signaling pathway.

Triterpene Glycosides Analyzed by Two-Dimensional Thin-Layer Chromatography

Two-dimensional TLC with judiciously chosen solvent systems that differ in pH values is proposed for identification of triterpene glycosides in plant extracts (mono- and bisdesmosidic, acidic, sulfated, acylated)

Two-dimensional thin-layer chromatography of plant ecdysteroids

Two-dimesional thin-layer chromatography has been used to screen plant samples for ecdysteroids. Satisfactory separations were obtained even in the presence of substantial impurities and for mixtures containing many ecdysteroids. Normal-phase silica and octadecyl silica plates were used to differntiate apolar and polar ecdysteroids, respectively. The major advantages of thin-layer chromatography are multiple detection, which enables specific detection of the ecdysteroids. Use of a small amount of water in the mobile phase in the fist dimension, the a water-free mobile phase in the second dimension, can afford favorable separations.

Identification of Thermus thermophilus HB8 tRNA (Gm18) methyltransferase gene.

For the purpose of identification of the gene for Thermus thermophilus tRNA (Gm18) methyltransferase [tRNA (guanosine-2'-)-methyltransferase, EC 2.1.1.34], the purified enzyme from native source was analyzed by the peptide-mass mapping. The target gene encoded the amino acid sequences of the obtained peptides was searched in data from Thermus thermophilus HB8 genome-sequencing project. We found the target gene AB05130, which was expected to encode a protein composed of 194 amino acid residues and the molecular mass of this protein was calculated as 22083. The recombinant protein was expressed in E. coli as an active form. The Gm18 formation activity of the purified recombinant protein was confirmed by in vitro methylation followed by two-dimensional thin layer chromatography and Liquid Chromatography/Mass Spectrum analysis of substrate tRNA.

Kidney lipids in galactosylceramide synthase-deficient mice: absence of galactosylsulfatide and compensatory increase in more polar sulfoglycolipids

UDP-galactose:ceramide galactosyltransferase (CGT) catalyzes the final step in the synthesis of galactosylceramide (GalCer). It has previously been shown that CGT-deficient mice do not synthesize GalCer and its sulfated derivative GalCer I(3)-sulfate (galactosylsulfatide, SM4s) but form myelin containing glucosylceramide (GlcCer) and sphingomyelin with 2-hydroxy fatty acids. Because relatively high concentrations of GalCer and SM4s are present also in mammalian kidney, we analyzed the composition of lipids in the kidney of Cgt(-/-) and, as a control, Cgt(+/-) and wild-type mice. The homozygous mutant mice lacked GalCer, galabiaosylceramide (Ga(2)Cer), and SM4s. Yet, they did not show any major morphological or functional defects in the kidney. A slight increase in GlcCer containing 4-hydroxysphinganine was evident among neutral glycolipids. Intriguingly, more polar sulfoglycolipids, that is, lactosylceramide II(3)-sulfate (SM3) and gangliotetraosylceramide II(3),IV(3)-bis-sulfate (SB1a), were expressed at 2 to 3 times the normal levels in Cgt(-/-) mice, indicating upregulation of biosynthesis of SB1a from GlcCer via SM3. Given that SM4s is a major polar glycolipid constituting renal tubular membrane, the increase in SM3 and SB1a in the mice deficient in CGT and thus SM4s appears to be a compensatory process, which could partly restore kidney function in the knockout mice.

Concurrent measures of protein kinase C and phosphoinositides in lithium-treated bipolar patients and healthy individuals: a preliminary study

This study examined the hypothesis that lithium inhibits the PI signaling pathway in humans during in vivo administration by concurrently measuring PKC isozymes and platelet membrane phosphoinositides in lithium-treated patients and healthy individuals. The platelet membrane and cytosolic levels of PKC alpha, beta I, beta II, delta, and epsilon were measured using Western blotting. The relative platelet membrane contents of phosphatidylinositol (PI), phosphatidylinositol-4-phosphate (PIP), and phosphatidylinositol-4,5-bisphosphate (PIP 2) were measured with two-dimensional thin-layer chromatography. Nine euthymic lithium-treated bipolar subjects and 11 healthy control subjects were studied. Compared to control subjects, lithium-treated bipolar patients had significantly lower levels of cytosolic PKC alpha isozyme ( t-test=−3.24, d.f.=17, P=0.01) and PIP 2 platelet membrane levels ( t-test=−2.51, d.f.=18, P=0.02), and a trend toward reduced levels of cytosolic PKC beta II isozyme ( t=−2.17, d.f.=17, P=0.05). There was no significant correlation between PIP 2 and any of the PKC isozymes. These preliminary findings suggest that chronic lithium treatment may decrease the levels of both cytosolic PKC alpha isozyme and membrane PIP 2 in platelets of bipolar disorder patients.

Cloning and Characterization of the Gene for Phosphatidylcholine Synthase

Phosphatidylcholine (PC) is the major membrane-forming phospholipid in eukaryotes and can be synthesized by either of two pathways, the CDP-choline pathway or the methylation pathway. In prokaryotes only the methylation pathway was thought to occur. Recently, however, we could demonstrate (de Rudder, K. E. E., Sohlenkamp, C., and Geiger, O. (1999) J. Biol. Chem. 274, 20011-20016) that a second pathway for phosphatidylcholine biosynthesis exists in Sinorhizobium (Rhizobium) meliloti involving a novel enzymatic activity, phosphatidylcholine synthase, that condenses choline and CDP-diacylglyceride in one step to form PC and CMP. Using a colony autoradiography method we have isolated mutants of S. meliloti deficient in phosphatidylcholine synthase and which are no longer able to incorporate radiolabeled choline into PC. Complementation of such mutants with a sinorhizobial cosmid gene bank, subcloning of the complementing fragment, and sequencing of the subclone led to the identification of a gene coding for a presumptive CDP-alcohol phosphatidyltransferase. Amplification of this gene and its expression in Escherichia coli demonstrates that it codes for phosphatidylcholine synthase. Genomes of some pathogens (Pseudomonas aeruginosa and Borrelia burgdorferi) contain genes similar to the sinorhizobial gene (pcs) for phosphatidylcholine synthase. Although pcs-deficient S. meliloti knock-out mutants show wild type-like growth and lipid composition, they are unable to perform rapid PC biosynthesis that normally is achieved via the phosphatidylcholine synthase pathway in S. meliloti wild type.

Comparison of the fatty acid composition of total lipids and phospholipids in breast milk from Japanese women

Fatty acid (FA) composition of total lipids (TL) and phospholipids (PL) in breast milk obtained from 20 normal delivery healthy women in Tokyo, Japan was analyzed. Total lipids were extracted from the samples and then PL, consisting of phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylserine (PS), sphingomyelin (Sph) and phosphatidylinositol (PI), were separated by two-dimensional thin-layer chromatography. The FA composition of TL and PL was analyzed by gas liquid-chromatography. Compared with previous reports, the contents of eicosapentaenoic acid (EPA; 20:5n-3) and docosahexaenoic acid (DHA; 22:6n-3) in TL from Japanese women were higher than those from Chinese and Canadian women, which may be caused by different dietary habits and food types consumed by those populations. The contents of arachidonic acid (AA; 20:4n-6), EPA and DHA in PE and PI were much higher than those in PC. In addition, no significant correlation of EPA or DHA content was found between TL and PL. The findings indicate that PL especially PE and PI in human milk may be a source of EPA and DHA for infants in the rapid developmental stage. These results should be considered in infant formula production.

Cisplatin DNA Adduct Detection and Depurination Measured by 32P DNA Radiolabeling and Two-Dimensional Thin-Layer Chromatography: A Time and Concentration Study

Platinum-based chemotherapies cause the formation of DNA adducts and have profound effects on DNA. This study measured cis-diamminedichloroplatinum II (cisplatin) DNA adducts by 32P-radiolabeling DNA, enzymatically digesting radiolabeled DNA, separating the formed adducts on two-dimensional thin-layer chromatography, and quantitating the adducts with autoradiography and densitometry. HeLa DNA was incubated with cisplatin at varying concentrations (6.25–325 nM) and times (0 min to 72 hr). Cisplatin rapidly depurinated dGMP and dAMP (90%, 15–min incubation with 325 nM cisplatin). Partial depurination of dGMP (15%) and dAMP (25%) occurred with lower cisplatin concentrations at equal incubation times. a minimum of four new adducts, with relatively rapid migratory patterns, were detected at high cisplatin concentrations with short incubation times. These results indicate that the depurination of DNA correlates with DNA adduct formation and that the quantification of these adducts may be applicable to monitoring tumor and host cell response to cisplatin chemotherapy.

Simultaneous separation of lysophospholipids from the total lipid fraction of crude biological samples using two-dimensional thin-layer chromatography

A novel solvent system for two-dimensional thin-layer chromatography was shown to simultaneously separate lysophospholipid standards, including lysophosphatidylcholine, lysophosphatidylethanolamine, lysophosphatidylserine, lysophosphatidylinositol, lysophosphatidylglycerol, lysophosphatidic acid, lysosphingomyelin (sphingosylphosphorylcholine), and sphingosine-1-phosphate from diradylphospholipids, glycosphingolipids, and neutral lipids. Lysophospholipids contained in the total lipid fraction of activated platelets were also well separated by the same system. The present system is a useful tool for the metabolic and structural analysis of lysophospholipids in biological samples. —Yokoyama, K., F. Shimizu, and M. Setaka. Simultaneous separation of lysophospholipids from the total lipid fraction of crude biological samples using two-dimensional thin-layer chromatography.

Multiresidue screen for cardiotoxins by two-dimensional thin-layer chromatography.

A two-dimensional thin-layer chromatographic method was developed for the qualitative determination of the cardiotoxins oleandrin, gitoxin, digitoxin, gitoxigenin, and grayanotoxins I, II, and III in gastrointestinal contents (stomach, rumen, colon, and cecum contents), feces, and plant material. The cardiotoxins were extracted with dichloromethane. The extract was cleaned up by charcoal and reverse phase solid-phase extraction columns. Analysis was performed by two-dimensional thin-layer chromatography on silica gel plates and visualized by aluminum chloride followed by chloramine T spray. The method detection limits were 0.05 microg/g for oleandrin, 0.1 microg/g for gitoxin, and 0.2 microg/g for the other toxicants in gastrointestinal contents and feces and were 5 times higher in plant material. Four replicate fortifications of bovine rumen contents, bovine feces, and alfalfa at these levels were all well recovered. The diagnostic utility of the method was tested by analyzing samples submitted to the veterinary toxicology laboratory.

Differences in flower color and pigment composition among white carnation ( Dianthus caryophyllus L.) cultivars

Measurement of flower colors with a color analyzer showed significant differences among 13 white carnation ( Dianthus caryophyllus L.) cultivars. Petals of three cultivars, `White Mind', `Kaly' and `White Barbara', were nearly pure white, compared with the other 10 cultivars. Analysis of pigment composition by two-dimensional thin-layer chromatography classified 13 white cultivars into three types. The nearly pure-white cultivar, `White Mind' lacked flavonoid compounds in the petals. `Kaly' and `White Barbara' accumulated a large amount of naringenin derivatives. The other normal white cultivars contained kaempferol derivatives as the major flavonoid. We concluded that `White Mind', `Kaly' and `White Barbara' were nearly pure white because they lack flavonols, such as kaempferol derivatives. Our results suggest that in `White Mind', the flavonoid biosynthesis pathway is blocked before chalcone synthesis, whereas in `Kaly' and `White Barbara' it is blocked at the flavanone-3-hydroxylase step.

Variation in the Deoxynucleotide Composition between Organic and Nonorganic Strawberries

The differences in xenobiotic chemical content between organic and nonorganic products can be chemically measured. In this study, the deoxynucleotide composition of strawberry samples is used to demonstrate chromatographic methods of quantifying the differences between pesticide- and toxic-exposed strawberries. The samples were analyzed by 32P labeling and two-dimensional thin-layer chromatography. This technique is sensitive for detecting rare (1/1010) deoxynucleotide adducts and analogues (minor bases) in DNA. The results indicate differences in the amount and type of adduct formation between organic and nonorganic strawberry samples and within different parts of the same type of sample. The elucidation of the content and effects of pesticides and toxics in foods is critical in differentiating organic from common produce. It can also aid the agricultural industry in improving the application of chemicals in pest management. Furthermore, it helps to enhance the understanding of long-term epidemiology in nutrition research, especially in susceptible populations. These findings are of particular application in the pediatric population, where dietary habits are restricted to specific food groups.

Acylation of monolysocardiolipin in rat heart

Cardiolipin is a major mitochondrial membrane glycerophospholipid in the mammalian heart. In this study, the ability of the isolated intact rat heart to remodel cardiolipin and the mitochondrial enzyme activities that reacylate monolysocardiolipin to cardiolipin in vitro were characterized. Adult rat heart cardiolipin was found to contain primarily linoleic and oleic acids. Perfusion of the isolated intact rat heart in the Langendorff mode with various radioactive fatty acids, followed by analysis of radioactivity incorporated into cardiolipin and its immediate precursor phosphatidylglycerol, indicated that unsaturated fatty acids entered into cardiolipin mainly by deacylation followed by reacylation. The in vitro mitochondrial acylation of monolysocardiolipin to cardiolipin was coenzyme A-dependent with a pH optimum in the alkaline range. Significant activity was also present at physiological pH. With oleoyl-coenzyme A as substrate, the apparent K(m) for oleoyl-coenzyme A and monolysocardiolipin were 12.5 microm and 138.9 microm, respectively. With linoleoyl-coenzyme A as substrate, the apparent K(m) for linoleoyl-coenzyme A and monolysocardiolipin were 6.7 microm and 59.9 microm, respectively. Pre-incubation at 50 degrees C resulted in different profiles of enzyme inactivation for the two activities. Both activities were affected similarly by phospholipids, triacsin C, and various lipid binding proteins but were affected differently by various detergents and myristoyl-coenzyme A. [(3)H]cardiolipin was not formed from monolyso[(3)H]cardiolipin in the absence of acyl-coenzyme A. Monolysocardiolipin acyltransferase activities were observed in mitochondria prepared from various other rat tissues. In summary, the data suggest that the isolated intact rat heart has the ability to rapidly remodel cardiolipin and that rat heart mitochondria contain coenzyme A-dependent acyltransferase(s) for the acylation of monolysocardiolipin to cardiolipin. A simple and reproducible in vitro assay for the determination of acyl-coenzyme A- dependent monolysocardiolipin acyltransferase activity in mammalian tissues with exogenous monolysocardiolipin substrate is also presented.

Non-HPLC separation of water-soluble choline metabolites by two-dimensional high voltage electrophoresis and thin layer chromatography

In cholinergic neurons choline is directed to three main pathways; (1) conversion to phosphorylcholine (PCh) and cytidine diphosphate choline (CDP-choline) for the synthesis of phosphatidylcholine, (2) acylation to the neurotransmitter acetylcholine and (3) oxidation to betaine for the formation of methionine. Thus, the distribution of choline among the different metabolites is important for a better understanding of the regulation of these pathways in neurons. A non-HPLC method for the simultaneous separation of five choline metabolites found in neurons is described. High voltage electrophoresis (HVE) was combined with thin layer chromatography (TLC) to separate choline, PCh, CDP-choline, acetylcholine and betaine. This method is useful in studying the distribution of choline among its different metabolites in radiotracer experiments. Aqueous metabolites from leukemia inhibitory factor treated LA-N-2 cells labeled with [methyl- 3H]choline were separated by HVE followed by TLC in the same dimension. Although the separation appeared to be complete, some ‘tailing’ by PCh significantly elevated the radioactivity measured in CDP-choline. This tailing of PCh was confirmed by subjecting radiolabeled PCh alone to this multiple separation method. Contamination of CDP-choline by PCh was eliminated by subjecting the samples to HVE followed by TLC in the second dimension. This two-dimensional approach was consistently reproducible and achieved excellent resolution of all five metabolites. In addition, this technique also resolved a sixth choline-containing metabolite, glycerophosphorylcholine (GPC), a breakdown product of phosphatidylcholine.

Plasma membrane phospholipid integrity and orientation during hypoxic and toxic proximal tubular attack

Acute cell injury can activate intracellular phospholipase A2 (PLA2) and can inhibit plasma membrane aminophospholipid translocase(s). The latter maintains inner/outer plasma membrane phospholipid (PL) asymmetry. The mechanistic importance of PLA2-mediated PL breakdown and possible PL redistribution ("flip flop") to lethal tubule injury has not been well defined. This study was performed to help clarify these issues. Proximal tubule segments (PTS) from normal CD-1 mice were subjected to either 30 minutes of hypoxia, Ca2+ ionophore (50 microM A23187), or oxidant attack (50 microM Fe). Lethal cell injury [the percentage of lactate dehydrogenase (LDH) release], plasma membrane PL expression [two-dimensional thin layer chromatography (TLC)], and free fatty acid (FFA) levels were then assessed. "Flip flop" was gauged by preferential decrements in phosphatidylserine (PS) versus phosphatidylcholine (PC; PS/PC ratios) in response to extracellular (Naja) PLA2 exposure. Hypoxia induced approximately 60% LDH release, but no PL losses were observed. FFA increments suggested, at most 3% or less PL hydrolysis. Naja PLA2 reduced PLs in hypoxic tubules, but paradoxically, mild cytoprotection resulted. In contrast to hypoxia, Ca2+ ionophore and Fe each induced significant PL losses (6 to 15%) despite minimal FFA accumulation or cell death (26 to 27% LDH release). Arachidonic acid markedly inhibited PLA2 activity, potentially explaining an inverse correlation (r = -0.91) between tubule FFA accumulation and PL decrements. No evidence for plasma membrane "flip flop" was observed. In vivo ischemia reperfusion and oxidant injury (myohemoglobinuria) induced 0 and 24% cortical PL depletion, respectively, validating these in vitro data. (a) Plasma membrane PLs are well preserved during acute hypoxic/ischemic injury, possibly because FFA accumulation (caused by mitochondrial inhibition) creates a negative feedback loop, inhibiting intracellular PLA2. (b) Exogenous PLA2 induces PL losses during hypoxia, but decreased cell injury can result. Together these findings suggest that PL loss may not be essential to hypoxic cell death. (c) Oxidant/Ca2+ overload injury induces early PL losses, perhaps facilitated by ongoing mitochondrial FFA metabolism, and (d) membrane "flip flop" does not appear to be an immediate mediator of acute necrotic tubular cell death.

Phosphoamino Acid Analysis

Proteins involved in signal transduction are often phosphorylated. Determination of the specific amino acid residue(s) involved is used in characterizing the particular pathway. Partial acid hydrolysis of phosphorylated proteins followed by two-dimensional thin layer chromatography is used to identify the phosphorylated residues of the protein as phosphoserine, phosphothreonine, or phosphotyrosine. Mild alkaline hydrolysis is used to enhance detection of phosphothreonine and phosphotyrosine.

Fatty acid composition of brain glycerophospholipids in peroxisomal disorders.

This paper shows for the first time the differential fatty acid composition of ethanolamine plasmalogens (EP) and phosphatidylethanolamine (PE) in the brains of 12 patients with disorders of peroxisomal biogenesis and compares the results to normal values for the age. Other important glycerophospholipids (GPL), such as phosphatidylserine (PS) and phosphatidylcholine (PC), are also included in this study. GPL were separated by two-dimensional thin-layer chromatography, and their fatty acid composition was determined by capillary column gas-liquid chromatography. Total brain GPL were slightly decreased in peroxisomal disorders (27.98+/-2.95 micromol/g in the patients against 34.5+/-6.21 micromol/g in age-matched controls, P = 0.005), and the distribution of the different GPL classes was much altered. In confirmation of known data, EP were very much decreased (2.18+/-1.3 micromol/g in the patients against 6.9+/-2.3 micromol/g in controls) at the expense of PE, which was increased (8.58+/-2.17 micromol/g in the patients against 5.97+/-0.58 micromol/g in controls, P<0.005). PS and PC were both significantly decreased (P = 0.0001 and P = 0.037, respectively). The polyunsaturated fatty acid (PUFA) composition of all the GPL fractions was markedly abnormal. In absolute terms, docosahexaenoic acid (22:6n-3) was drastically decreased in all GPL classes (always at the P<0.0001 level) while arachidonic acid (20:4n-6) was increased in PE and PS (P<0.001 in both cases). In the alkenyl acyl form, EP, 22:6n-3, and 20:4n-6 were both very significantly decreased (P<0.0001), although the former was always the most affected. The myelin PUFA adrenic acid (22:4n-6) was decreased in EP (P<0.0001) and slightly increased in PS (P<0.05). The changes found confirm that 22:6n-3 deficiency is a predominant defect in the brain in peroxisomal disorders.

Direct enantiomeric resolution of some 2‐arylpropionic acids using (−)‐brucine‐impregnated thin‐layer chromatography

Direct enantioseparation of (±)-ibuprofen and (±)-flurbiprofen was achieved by two-dimensional thin-layer chromatography on silica gel plates impregnated with optically pure (−)-brucine as chiral selector. The solvent systems that were successful in resolving both the compounds were acetonitrile–methanol (16:3 v/v) for the first dimension and acetonitrile–methanol–water (16:3:0.4, v/v) for the second dimension. Iodine vapour was used for detection; the detection limit for both (±)-ibuprofen and (±)-flurbiprofen was 0.1 µg. Copyright © 1999 John Wiley & Sons, Ltd.