Using High Performance Thin Layer Chromatography-Densitometry to Study the Influence of the Prion [RNQ+] and Its Determinant Prion Protein Rnq1 on Yeast Lipid Profiles.

The baker's yeast Saccharomyces cerevisiae harbors multiple prions that allow for the creation of heterogeneity within otherwise clonal cell populations. However, in many cases, the consequences of prion infection are entirely unclear. Predictions of prion-induced changes in cell physiology are complicated by pleotropic effects, and detection is often limited to relatively insensitive cell growth assays that may obscure many physiological changes. We previously showed that silica gel high performance thin-layer chromatography-densitometry (HPTLC) can be used to empirically determine prion-induced changes in lipid content in yeast. Here, we conduct pair-wise quantifications of the relative levels of free sterols, free fatty acids, and triacylglycerols [petroleum ether-diethyl ether-glacial acetic acid (80:20:1, v/v/v) mobile phase and phosphomolybdic acid (PMA) detection reagent]; steryl esters, methyl esters, and squalene [hexane-petroleum ether-diethyl ether-glacial acetic acid (50:20:5:1, v/v/v/v) and PMA]; and phosphatidylethanolamine, phosphatidylcholine, and phosphatidylinositol (chloroform-diethyl ether-acetic acid (65:25:4.5, v/v/v) and cupric sulfate-phosphoric acid) in otherwise clonal prion-infected ([RNQ+]) and prion-free ([rnq-]) cells in both stationary- and logarithmic-growth phases. We detected multiple statistically significant differences between prion-infected and prion-free cells that varied by growth phase, confirming our pr evious observations that prions exert distinct influences on cell physiology between stationary- and log-phase growth. We also found significant differences between cells expressing or lacking the Rnq1 protein which forms the [RNQ+] prion, providing new clues to the as yet unresolved normal biological function of this prion-forming protein. This investigation further emphasizes the utility of HPTLC-densitometry to empirically determine the effects of prions and other presumed innocuous gene deletions on lipid content in yeast, and we expect that additional analyses will continue to resolve the physiological effects of prion infection.

Review of the Determination of the Antioxidant Activity of Foods, Food Ingredients, and Dietary Supplements by Thin Layer Chromatography-Direct Bioautography, Spectrometry, and the Dot-Blot Procedure.

This paper reviews a selection of the most important studies on antioxidants in foods (including beverages), food ingredients, and dietary supplements by effect-directed analysis using TLC with DPPH*, ABTS*+, and ß-carotene direct bioautography. Total antioxidant activity by visible mode spectrometry (colorimetry) with TLC used offline to obtain additional analytical results, mostly for identification and quantification of phenolic compounds in samples, is also discussed. Finally, dot-blot assay for total antioxidant activity, carried out on a TLC plate without mobile-phase development, is reviewed as an alternative with possible advantages compared with spectrometry.

Biennial Review of Planar Chromatography: 2015-2017.

The most important advances in planar chromatography published between November 1, 2015, and November 1, 2017, are reviewed in this paper. Included are an introduction to the current status of the field; student experiments and reviews; apparatus and techniques for sample preparation and TLC separations; detection and identification of separated zones; quantitative analysis; preparative layer chromatography; and thin-layer radiochromatography. Selected applications are given in the various sections of the review.

Biennial Review of Planar Chromatography: 2013-2015.

The most important advances in planar chromatography published between November 1, 2013 and November 1, 2015 are reviewed in this paper. Included are an introduction to the current status of the field; student experiments, books, and reviews; apparatus and techniques for sample preparation and TLC separations; detection and identification of separated zones; quantitative analysis; preparative layer chromatography; and thin layer radiochromatography. Selected applications are given in the various sections of the review.

Effects of Echinostoma caproni miracidia dose on the amino acid contents of Biomphalaria glabrata as determined by high-performance thin-layer chromatography.

The effects of 5, 20, and 40 miracidia dose exposures of Echinostoma caproni on the amino acid contents of Biomphalaria glabrata were studied using high performance thin-layer chromatography-densitometry. Amino acids were identified and quantified in whole bodies of exposed snails and in the uninfected matched controls at 2 and 4 weeks post-exposure. Using cellulose layers with the mobile phase 2-butanol-pyridine-glacial acetic acid-deionized water (39:34:10:26) and ninhydrin detection reagent [2% ninhydrin in acetone-n-butanol (1:1)], five amino acids were identified, i.e., leucine/isoleucine, valine, alanine, glycine, and ornithine, by hRF value comparison and color differentiation. Quantitatively, there was a marked elevation in the amounts of four of these five amino acids (isoleucine/leucine, valine, alanine, and ornithine) across dose levels at 4 weeks post-infection (P<0.05). Elevation of the amino acid content in the high dose snail group suggested that some changes occurred in the amino acid metabolism of the snails in that group as a function of miracidia dose.

Review of advances in the thin layer chromatography of pesticides: 2012-2014.

Publications reporting techniques and applications of thin layer chromatography (planar chromatography) for the separation, detection, qualitative, and quantitative determination, and preparative isolation of pesticides and their metabolites are reviewed for the period from November 1, 2012 to November 1, 2014. Analyses are described for a variety of sample types and pesticide classes. In addition to references on residue analysis, studies such as pesticide structure - retention relationships, identification and characterization of natural and synthesized pesticides, metabolism, bioactivity, degradation, soil mobility, and lipophilicity are covered.

Effects of Echinostoma caproni miracidia dose on the neutral and polar lipids of Biomphalaria glabrata as determined by high-performance thin-layer chromatography.

The effects of a 5 versus 25 miracidia exposure of Echinostoma caproni on the lipid composition of Biomphalaria glabrata was studied using high performance thin layer chromatography (HPTLC)-densitometry. A 50 miracidia dose was not used because such a high level of exposure caused severe snail mortality by 3 weeks post-exposure (PE). Lipids were determined in the digestive-gland gonad complex (DGG) of the exposed snails and in the uninfected matched controls at 2 and 4 weeks PE. Extraction of lipids from DGGs was carried out by the Folch method with chloroform-methanol (2:1), and extracts were analyzed on Analtech HPTLC-HLF pre-adsorbent silica gel plates with measurement of separated bands using a CAMAG Scanner 3. For neutral lipids the mobile phase was petroleum ether-diethyl ether-glacial acetic acid (80:20:1) and the detection reagent was 5% ethanolic phosphoric acid, and for polar lipids chloroform-methanol-deionized water (65:25:4) mobile phase and 10% cupric sulfate in 8% phosphoric acid detection reagent were used. No significant differences in the concentrations of free sterols, free fatty acids, triacylglycerols, phosphatidylcholine, and phosphatidylethanolamine were seen at 2 weeks PE in any of the groups. At 4 weeks PE, the free fatty acid concentration increased significantly in the snails exposed to 25 miracidia compared to that of the 5 miracidia/snail group or the controls. Elevation of the free fatty acid fraction in the high dose snail group suggested that some changes occurred in the lipid metabolism of the snails in that group as a function of miracidia dose.

Effects of Schistosoma mansoni infection on lutein and ß-carotene concentrations in Biomphalaria glabrata snails as determined by quantitative high performance reversed phase thin-layer chromatography.

High performance thin-layer chromatography was used to determine the concentration of ß-carotene and lutein in the whole body and digestive gland-gonad complex (DGG) of uninfected Biomphalaria glabrata snails and those infected with Schistosoma mansoni for 6 and 8 weeks. Pigments were extracted from the snails using acetone and separated on EMD Millipore reversed phase C-18 plates with concentration zone using petroleum ether-acetonitrile-methanol (1:1:2) mobile phase. After development, two yellow pigment zones, lutein and ß-carotene, were identified with respective Rf values of 0.55 and 0.13 and then quantified by densitometry. Statistical analysis of the weight percentages of each pigment showed a significant decrease (P < 0.05) in the concentration of ß-carotene in the DGGs of infected B. glabrata at 6 and 8 weeks post-infection compared to the uninfected snails. No significant differences were seen in the concentrations of ß-carotene in the whole body of the uninfected versus infected snail samples. Changes in the lutein concentration of the infected DGG and whole snail bodies were insignificant compared to the uninfected controls. In conclusion, larval S. mansoni infection caused a significant decrease in the ß-carotene concentration of the DGG at 6 and 8 weeks post infection.

Review of advances in the thin layer chromatography of pesticides: 2010-2012.

Techniques and applications of thin layer chromatography (planar chromatography) for the separation, detection, qualitative and quantitative determination, and preparative isolation of pesticides and their metabolites and some related pollutants are reviewed for the period from November 1, 2010 to November 1, 2012. Analyses are described for a variety of samples types and pesticide classes. In addition to references on residue analysis, studies such as pesticide structure - retention relationships, identification and characterization of natural and synthesized pesticides, metabolism, degradation, mobility, lipophilicity, and mechanism of action are covered.

Metabolic profiling of Echinostoma caproni and Schistosoma mansoni in their definitive and intermediate hosts.

This review examines metabolic profiling of Schistosoma mansoni and Echinostoma caproni in their definitive and intermediate hosts. The earlier coverage of the literature on metabolic profiling was reviewed by Wang et al. 2010, Advances in Parasitology, 73, 373-404 and covered mainly studies using proton nuclear magnetic resonance spectroscopy. The methods focused upon in our review are mainly chromatographic. In the studies reviewed, various metabolites were analyzed in hosts infected with either E. caproni or S. mansoni and compared to the uninfected controls.

Biennial review of planar chromatography: 2009-2011.

The most important advances in the planar chromatography published between November 1, 2009 and November 1, 2011 are reviewed in this paper. Included are an introduction to the current status of the field; history, student experiments, books, and reviews; theory and fundamental studies; apparatus and techniques for sample preparation and TLC separations (sample application and plate development with the mobile phase); detection and identification of separated zones (chemical and biological detection, TLC/MS, and TLC coupled with other methods); techniques and instruments for quantitative analysis; preparative layer chromatography; and thin layer radiochromatography. Selected applications are given in the various sections of the review, especially for modern HPTLC-densitometry.

Glycogen, not dehydration or lipids, limits winter survival of side-blotched lizards (Uta stansburiana).

Climate change is causing winters to become milder (less cold and shorter). Recent studies of overwintering ectotherms have suggested that warmer winters increase metabolism and decrease winter survival and subsequent fecundity. Energetic constraints (insufficient energy stores) have been hypothesized as the cause of winter mortality but have not been tested explicitly. Thus, alternative sources of mortality, such as winter dehydration, cannot be ruled out. By employing an experimental design that compared the energetics and water content of lizards that died naturally during laboratory winter with those that survived up to the same point but were then sacrificed, we attempt to distinguish among multiple possible causes of mortality. We test the hypothesis that mortality is caused by insufficient energy stores in the liver, abdominal fat bodies, tail or carcass or through excessive water loss. We found that lizards that died naturally had marginally greater mass loss, lower water content, and less liver glycogen remaining than living animals sampled at the same time. Periodically moistening air during winter reduced water loss, but this did not affect survival, calling into question dehydration as a cause of death. Rather, our results implicate energy limitations in the form of liver glycogen, but not lipids, as the primary cause of mortality in overwintering lizards. When viewed through a lens of changing climates, our results suggest that if milder winters increase the metabolic rate of overwintering ectotherms, individuals may experience greater energetic demands. Increased energy use during winter may subsequently limit individual survival and possibly even impact population persistence.

Review of advances in the thin layer chromatography of pesticides: 2008-2010.

Techniques and applications of thin layer chromatography (planar chromatography) for the separation, detection, qualitative and quantitative determination, and preparative isolation of pesticides and their metabolites and other related compounds are reviewed for the period from November 1, 2008 to November 1, 2010. Analyses are described for a variety of samples types and pesticide classes. In addition to references on residue analysis, studies such as pesticide structure-retention relationships, identification and characterization of plant pesticides and synthesized pesticides, metabolism, degradation, mobility, identification of biomarkers for detection of herbicide effects in plants, and lipophilicity are covered.

Review of HPTLC in drug analysis: 1996-2009.

Analytical methods for drug substances, formulations, and clinical samples developed and validated on HPTLC plates during the period 1996-2009 are reviewed. Procedures, materials, and instrumentation for the different steps in the HPTLC procedure and validation of results; applications to bulk drugs, formulations, stability studies, biological samples (e.g., urine and plasma), and hydrophobicity studies; and prospects for the use of HPTLC for drug analysis in the future are described.