Authors:James Vasta, Bernard Fried, and Joseph Sherma
HPTLC analysis has been used to compare neutral lipid profiles in the urine of humans and mice. Neutral lipids and ubiquinone were extracted from urine with chloroform-methanol, 2:1, and determined on silica gel plates with 19 lanes and a concentration zone that were developed with petroleum ether-diethyl ether-glacial acetic acid, 80:20:1. Separated zones were detected with phosphomolybdic acid reagent and quantified by visible mode slit-scanning densitometry at 610 nm. Specific detection reagents were used to confirm the identity of particular lipid classes. The studies confirmed the presence of free sterols, free fatty acids, and triacylglycerols in both human and mouse urine. Methyl esters were found in mouse but not human urine. Hydrocarbons and ubiquinone were present in both human and mouse urine, but were not quantified. Similarities in the urinary neutral lipid profiles of humans and mice suggest that mice may serve as effective models for studies of the effects of infectious and metabolic diseases in humans.
Authors:Jessica Counihan, Kathryn Hueglin, Crystal Wagner, Stephen Gadomski, Peter Zani, Bernard Fried, and Joseph Sherma
Diapause in insects typically results in metabolic adjustments that may include the levels of feeding and activity, metabolic rate, and the accumulation and utilization of reserves. The objective of this study was to characterize and quantify the various neutral lipid classes in the pitcher-plant mosquito (Wyeomyia smithii) that are associated with photoperiod-induced diapause. We used high-performance thin-layer chromatography (HPTLC) with 10 cm × 20 cm silica gel plates, petroleum ether-diethyl ether-glacial acetic acid 80:20:1 as the mobile phase, and 5% ethanolic phosphomolybdic acid solution as detection reagent to determine the percentage of replicate larvae samples (consisting of 16–31 larvae) that contained free sterols (FS), free fatty acids (FFA), triacylglycerols (TG), methyl esters (ME), and steryl esters (SE). Irrespective of the growth treatment (shortdays or long-days), the greatest concentrations of neutral lipids in all samples examined were TG. The effects of day length were such that during long-day (diapause terminating) conditions larvae contained less TG, more FS, and more FFA than larvae exposed to short-day (diapause inducing) conditions. Furthermore, the effects of body mass affected our results such that larger larvae contained more TG when in diapause, but less TG when not in diapause. Both ME and SE were only found in trace amounts in the short-day mosquitoes and not at all in long-day group. Our results are consistent with previous research that suggests TGs are important storage lipids during insect diapause.
Authors:Jamie Jarusiewicz, Bernard Fried, and Joseph Sherma
Neutral lipids and phospholpids in the apple snail
have been determined in the whole body, digestive gland-gonad complex (DGG), viscera, head-foot, shell, operculum, plasma, and hemocytes by high-performance thin-layer chromatography on silica gel plates. Plates were developed with petroleum ether-diethyl ether-glacial acetic acid, 80 + 20 + 1 (
), as mobile phase and sprayed with 5% phosphomolybdic acid to detect neutral lipids. Plates were developed with chloroform-methanol-water, 65 + 25 + 4 (
), and sprayed with 10% cupric sulfate to detect phospholipids. Triacylglycerols, free sterols, free fatty acids, steryl esters, phosphatidylcholine, and phosphatidylethanolamine were the major lipid fractions detected in the whole body, DGG, viscera, and head-foot. The major lipid fractions in the shell and operculum were free sterols and free fatty acids. The plasma and hemocytes contained free fatty acids as the major lipid fraction. The presence of these lipids suggests they are important to the metabolism and structure of the snail.
Neutral lipids were measured by scanning densitometry after postchromatographic derivatization. Neutral lipids are often derivatized with phosphomolybdic acid reagent, which reacts to form highly visible, blue zones. Uniform application of this reagent to HPTLC plates is critical if quantification of neutral lipid bands is desired. In this paper we compare the quantitative and qualitative results observed by use of three methods of application of derivatization reagent — manual spraying, manual dipping, and use of the Derivapress. Results showed that dipping is the best method of reagent application for achieving quantitative data, and spraying is the best method for qualitative purposes. The Derivapress proved to be a simple and economical device for application of phosphomolybdic acid reagent to HPTLC plates. It enabled qualitative and quantitative analysis of neutral lipids.
Authors:Ciara O’sullivan, Nevena Popovic, Bernard Fried, and Joseph Sherma
The purpose of this study was to identify and quantify various neutral and polar lipids in certain organs of mice using high-performance thin-layer chromatography (HPTLC). Four mice infected with Schistosoma mansoni and three control mice were used for this study. At 6 weeks postinfection, the mice were necropsied, and the liver, spleen, and small intestine were removed and prepared for lipid analysis. Lipids were separated on laned, preadsorbant Analtech HPTLC-HLF 20 × 10-cm silica gel plates. Neutral lipids were separated using petroleum ether-diethyl ether-glacial acetic acid (80:20:1) mobile phase and were detected by spraying with 5% ethanolic phosphomolybdic acid detection reagent. Polar lipids were separated with chloroform-methanol-deionized water (65:25:4) mobile phase and detected using 10% cupric sulfate in 8% phosphoric acid reagent. The analyzed neutral lipids were free sterols, free fatty acids, and triacylglycerols. Using HPTLC, no significant differences were found in these lipids between the infected and uninfected mice organs. The polar lipids analyzed were phosphatidylcholine (PC) and phospatidylethanolamine (PE). There was a significantly higher PC content in the liver and small intestine of the uninfected mice compared with that of infected mice.
Authors:Alexandra Hunsberger, Bernard Fried, and Joseph Sherma
Silica gel high-performance thin-layer chromatography (HPTLC) was used to study the effects of both Schistosoma mansoni infection and high temperatures on the neutral and polar lipid content of whole bodies of Biomphalaria glabrata snails. Neutral lipids were determined using petroleum ether-diethyl ether-glacial acetic acid (80:20:1) mobile phase, phosphomolybdic acid detection reagent, densitometry at 610 nm, and polar lipids with chloroform-methanol-water (65:25:4) mobile phase, cupric sulfate-phosphoric acid reagent, and scanning at 370 nm. The high-temperature experiments were done at ambient (22–24°C), 28°C, and 34°C. Snails were maintained at these temperatures for 7 days prior to necropsy. Extracts of their bodies were then analyzed by HPTLC to determine changes that occurred in the lipid content as a function of temperature and to compare unexposed to exposed cultures at each temperature. At 4 weeks postinfection (PI), the 34°C exposed snails had significantly lower amounts of free sterols than the unexposed culture. At 4 weeks PI, the 34°C exposed snails also had significantly lower amounts of free sterols than the ambient and 28°C exposed snails. At 6 weeks PI, ambient exposed snails had significantly lower free fatty acids and significantly higher phosphatidylcholine than unexposed snails. The 28°C exposed snails had significantly lower amounts of free sterols and phosphatidylethanolamine than the unexposed snails. The 28°C exposed snails also had significantly higher amounts of free sterols, triacylglycerols, and phosphatidylcholine than the ambient snails and significantly lower amounts of free fatty acids than the ambient temperature snails. The ambient exposed snails had significantly lower amounts of free sterols than the 28°C and 34°C snails. The 34°C exposed snails had significantly lower amounts of triacylglycerols than the ambient temperature and 28°C exposed snails. At 8 weeks PI, the 28°C exposed snails had significantly higher amounts of phosphatidylcholine than the unexposed snails. These findings suggest that high temperature and S. mansoni infection had individual and combined deleterious effects on the lipid metabolism of the snails.
Authors:Sharon Bandstra, Bernard Fried, and Joseph Sherma
This review examines the use of thin-layer chromatography (TLC) for the analysis of neutral lipids and phospholipids in medically and economically important gastropod molluscs (snails). It discusses methods for isolating lipids from snails, and the use of layers, mobile phases, and detection reagents for the TLC analysis of snail neutral lipids and phospholipids. Quantitative densitometric studies are reviewed, with particular emphasis on class separations of neutral lipids and phospholipids. The review considers significant findings on the effects of diet and larval trematode parasitism on lipids in snails as determined by TLC.
Authors:S. Abram, U. Abram, H. Spies, and R. Münze
A series of technetium complexes with different alkyl dithiocarbamates, dithiophosphates and xanthates has been prepared at tracer level (10–4 molar) starting from hydrazine-reduced pertechnetate and Tc(V) gluconate, respectively. Solution studies show the complexes to be neutral, lipid-soluble tracers. Their in vitro properties can be varied in a wide range by variation of alkyl substitution of the ligands.