Metal ions bound to phospholipids after isolation by silicic acid column chromatography

South Asian nations are facing the challenge of increasing nitrogen pollution with the Indo-Gangetic Plain having some of the highest levels of atmospheric ammonia pollution globally. However, there is a lack of in-country research to evaluate the possible impact of nitrogen-related pollutants on South Asian biodiversity. In the Himalayas, there is an opportunity to utilize lichens from natural habitats to establish field-based references for better future tracking of changes in ecosystem health relevant to the wider South Asian region. In this study, we assessed the natural chemical variability of two lichens (Usnea spp. and Hypotrachyna spp.) based on thallus nitrogen and metal ion contents along with their physico-chemical and oxidative responses in two 1-km long transects from two forests of Nepal representing local gradients. Our results revealed a moderate concentration of total Kjeldalh nitrogen (0.36-0.98 % DM in Chandragiri, KTM and 0.57-2.04 % DM in Ghorepani, ACA), as well as ammonium (40.42-159.84 mg/L in Chandragiri, KTM and 80.60-280.64 mg/L in Ghorepani, ACA) and considerable amount of metal ions in both lichens, though with the highest values for lichens collected from the Ghorepani, ACA (from Western Nepal). A noteworthy background concentration of atmospheric ammonia was also observed at both sites. The highest variation in physico-chemical responses, such as electrical conductivity, chlorophyll content, chlorophyll degradation, chlorophyll fluorescence, and phenolic content was observed in the lichens from the same area, consistent with the higher levels of air pollution. Moreover, there appeared to be associated impacts on oxidative responses such as radical scavenging and catalase activities. Furthermore, the metal ions in the lichen thalli were found to originate from both anthropogenic and natural sources in Chandragiri, KTM and few of the metal ions were deposited from long-range transport mechanisms in Ghorepani, ACA, which signifies the diverse sources of pollution in the study areas. The sampling line-wise variation in thallus chemistry signifies the local pollution gradient in both sites. Further, environmental covariables (slope, elevation, crown settings, wind pattern) were observed to affect the lichen abundance and accumulation of nitrogen and metal ions. In comparison, Hypotrachyna spp. showed greater potential to accumulate pollutants and variability in physico-chemical and oxidative responses. From this study, we conclude that a range of physico-chemical and biochemical responses of the target lichens can be used as proxies for the bioindication of nitrogen and metal ion pollution to assess lichen’s health and ecological functioning. Wider studies covering large spatial extent and cellular mechanisms of lichen response are now recommended to fully understand the functional biology explaining contrasting responses between lichen species in different geographic settings of Nepal and South Asia. Keywords: Lichens; Bioindicators; Pollution; Ecosystem; Reference

Some commercial preparations of common natural conjugated bile salts contain impurities (e.g., amines, lipids, and calcium) that are likely to affect their physicochemical properties. A method was developed for purifying commercial preparations of sodium salts of glycine- and taurine-conjugated bile acids. The method consists of passage of a dilute aqueous solution of the sodium bile salt through three columns in sequence: graphitized carbon, a hydrophobic bonded octadecylsilane (C18) cartridge, and a calcium-chelating resin. The final solution was extracted with chloroform, and the purified bile salt was then isolated by freeze-drying, with a yield of 65-75%. Each bile salt purified by this method was compared with the corresponding bile salt purified by conventional adsorption chromatography on a silicic acid column, using a mixture of methanol and chloroform as eluant. Purity was assessed by visible spectra, by surface tension measurements (using the maximum bubble-pressure method and a Wilhelmy wire method), by chloroform extractability of impurities in the conjugated bile acid, by liposome solubilization, and by chemical analysis of the calcium content. Both purification methods removed colored and surface-active impurities, but the new method was always as or more effective than silicic acid column chromatography. Calcium ion, present in commercial bile salts in concentrations up to 16 mmol/mol bile salt, was removed completely by the three-column method, but not by silicic acid chromatography. The new method is thus a simple, rapid, and efficient procedure for purification of the sodium salts of glycine- and taurine-conjugated bile acids for physicochemical measurements, in which elimination of surface-active impurities and polyvalent cations is desired.

SUMMARY Normal human serum phospholipids were fractionated by silicic acid column chromatography. A large number of fractions was obtained with which it was possible to study the effect of fatty acid composition on elution rate. Phospholipids containing unsaturated fatty acids eluted from the silicic acid column more rapidly than their saturated analogues. It was found that lecithin could not be cleanly separated from sphingomyelin by any concentration of methanol or multiple-solvent elution scheme. The fatty acid compoeitions of the individual phospholipids were studied. Phosphatidyl ethanolamine was the most unsaturated phospholipid of serum; lecithin, sphingomyelin, and lysolecithin showed less unsaturation, in that order. In all phospholipids, 20 and 22 carbon unsaturated fatty acids were present. Long-chain saturated, aa well aa odd-chain saturated, fatty acids were present and were particularly prominent in the sphingomyelin fraction. In previous studies reported by this laboratory (1, 2), data on the phospholipid and phospholipid fatty acid composition of human serum and serum lipoprotein fractions were presented. Other authors (3, 4, 5) have published data from similar studies. Most of these latter studies have presented evidence for the presence of lysolecithin as well as the phosphatidyl ethanolamine, lecithin, and sphingomyelin fractions reported in the work from this laboratory. The procedure previously used here did not show the presence of lysolecithin; if any were present in the samples it would have been included in the lecithin fraction. A more extensive chromatographic fractionation of the total serum phospholipid was undertaken to improve the resolution of the individual phospholipids and attempt to establish the presence or absence of lysolecithin as well as other minor components. As a large number of fractions was taken during the chromatographic run, a large initial amount of phospholipid was necessary to permit subsequent analysis of the fatty acid moiety of the individual phospholipids. The subfractionation of the major elution peaks provided

Group separation of bile acids and salts by silicic acid column chromatography

A procedure has been developed for the separation of the glycosyl diglycerides from the phosphatides of Gram-positive bacteria on columns of silicic acid. The method utilizes mixtures of acetone in chloroform for elution of the glycosyl diglycerides, followed by increasing amounts of methanol in chloroform for elution of the phosphatides. The course of the fractionation was followed by means of phosphorus and carbohydrate determinations and by paper chromatography. The completeness of the separation of the phosphatides from the sugar-containing lipids was shown also by chromatographing a total lipid extract containing P32-labeled phosphatides.

The elution characteristics of methanol‐benzene solvent systems were determined by separating a mixture of polar and nonpolar fatty methyl esters by liquid chromatography on silicic acid columns. A series of curves were plotted showing the relationship between the elution volume of each component and methanol concentration of the stationary phase. The resulting graphs serve as a basis for predicting elution conditions for separating other polar materials. Adsorption isotherms were plotted from equilibrium studies of methanol‐benzene systems on silicic acid. Methanol concentrations of the effluents from various columns were determined by refractive index. An abrupt concentration change occurs in the methanol content of the effluent when the mobile solvent is either richer or poorer in methanol than the equilibrated solvent. Elution position of this abrupt change depends upon the concentration of methanol in both the mobile and the stationary phases. The procedure has been rigorously standardized because small variations in the amount of methanol on the column create large differences in elution volumes.

Bone is the most common site to which breast cancer cells metastasize. We found that osteoblast-like MG63 cells and human bone tissue contain the bile acid salt sodium deoxycholate (DC). MG63 cells take up and accumulate DC from the medium, suggesting that the bone-derived DC originates from serum. DC released from MG63 cells or bone tissue promotes cell survival and induces the migration of metastatic human breast cancer MDA-MB-231 cells. The bile acid receptor farnesoid X receptor (FXR) antagonist Z-guggulsterone prevents the migration of these cells and induces apoptosis. DC increases the gene expression of FXR and induces its translocation to the nucleus of MDA-MB-231 cells. Nuclear translocation of FXR is concurrent with the increase of urokinase-type plasminogen activator (uPA) and the formation of F-actin, two factors critical for the migration of breast cancer cells. Our results suggest a novel mechanism by which DC-induced increase of uPA and binding to the uPA receptor of the same breast cancer cell self-propel its migration and metastasis to the bone.

Quantitative elution of acidic and neutral glycolipids of brain and spinach leaves from silicic acid columns with acetone was demonstrated. Cerebrosides and sulfatides of brain and sulfolipid and glycosyl diglycerides of spinach leaves were eluted quantitatively with acetone while prospholipids remained on the column. The observations provide the basis for an analytical procedure employing column and quantitative thin-layer chromatography (TLC). Sephadex column chromatography is utilized for separation of lipids from nonlipids; silicic acid column chromatography for separation into neutral lipid, glycolipid and phospholipid fractions; and quantitative TLC for analysis of lipid classes of each column fraction.

The distribution of phospholipids derived from Micrococcus cerificans was determined under a variety of nutritive conditions. Cells were grown with hexadecane, heptadecane, or acetate serving as the sole carbon source. Total lipid was isolated by chloroform-methanol extraction, and the phospholipid fraction was isolated by silicic acid column chromatography. The phospholipids were characterized by silicic acid chromatography, by thin-layer chromatography, and by identification of water-soluble products resulting from acid hydrolysis of purified phospholipids. Major phospholipids characterized were phosphatidylethanolamine, phosphatidylglycerol, and cardiolipin. Minor phospholipids were phosphatidylglycerol phosphate and phosphatidylserine. Trace amounts of methylated derivatives of phosphatidylethanolamine were determined by incorporation of (14)C from (14)C-methylmethionine. These experiments demonstrated the presence of phosphatidyl-N-methylethanolamine, phosphatidyl-N,N'-dimethylethanolamine, and phosphatidylcholine in trace quantities. Pulse labeling with (14)C-serine demonstrated the direct incorporation of serine into phosphatidylserine followed by decarboxylation to phosphatidylethanolamine.

Determination of metal ions in Paris polyphylla var. Yunnanensis by ICP-OES and its influence on hemostasis

We investigated a new procedure in which silicic acid column chromatography and fluorometry are used to assay plasma vitamin A. Using tritium-labeled vitamin A acetate and vitamin A alcohol as radioactive standards, we identified two fluorescent components in plasma extracts chromatographed on silicic acid columns. One unidentified fluorescent component was eluted first with hexane or petroleum ether. Vitamin A, the second fluorescent peak, was quantitatively eluted with isopropanol. The isopropanol eluates could be immediately assayed fluorometrically without further treatment. The unidentified compound was sensitive to ultraviolet irradiation, which destroyed it slower than vitamin A is destroyed. Values correlated well for plasma vitamin A concentrations, determined by this procedure and by the Neeld and Pearson modification of the Carr-Price colorimetric method (r = 0.871). By our technique, we found 34 µg of vitamin A per 100 ml of plasma in 778 preschool children.

1. Chromatography of rat-liver lipids on a column of silicic acid or a mixture of silicic acid and Hyflo Super-Cel, with chloroform-methanol mixtures, gave monophosphoinositide-containing fractions which were invariably contaminated by the presence of nitrogen-containing phospholipids. The behaviour of the inositide was extremely sensitive to column loading and the results with different batches of silicic acid were not reproducible. 2. However, when chromatography on an alumina column was used, the solvent system chloroform-methanol-water (23:23:4, by vol.) completely eluted the neutral lipids, choline-containing phospholipids and phosphatidylethanolamine. An increase of the water content of the solvent to 14% (by vol.) then led to the elution of the monophosphoinositide component, now free from nitrogen-containing phospholipids, but still contaminated by the presence of a phospholipid, which from its properties was taken to be polyglycerophosphatide. 3. Most of the polyglycerophosphatide could be removed from a rat-liver lipid extract by silicic acid chromatography with chloroform-methanol (19:1, v/v). The other phospholipids were then eluted and applied to an alumina column, whereby a monophosphoinositide fraction of much greater purity was obtained. 4. Further purification of the monophosphoinositide was achieved by chromatography on a mixture of silicic acid and cellulose powder. The final product was virtually pure by thin-layer chromatography and gave the expected analysis for monophosphoinositide.

This study examines the solubility of humic acids as a function of solution pH and their interactions with di- and trivalent metal ions at different pH values. It has been observed that humic acids and their salts interact with divalent transition metal ions in aqueous solutions at low pH values. The results demonstrate that the amount of metal ions – specifically copper, cobalt, and nickel – bound to humic acids increases almost linearly with their initial concentration. Moreover, the amount of bound metal ions rises as the pH of the medium increases. Investigations into the interactions of iron, aluminum, and chromium ions with humic acids at varying pH levels show that, at low pH values and low metal ion concentrations, the amount of metal ions bound to humic acids is directly proportional to their concentration in the aqueous solution.

The structures of the polar lipid classes of plants and animals are presented, their nomenclature discussed, and suggestions are presented for clarification of nomenclature. The three general types of quantitative chromatographic procedures (column chromatography, thin‐layer chromatography, and combinations of column and thin‐layer chromatography) available for polar lipids are reviewed and a new quantitative two‐dimensional thin‐layer chromatographic procedure is presented. Useful quantitative procedures employing columns of cellulose, silicic acid, silicic acid mixed with silicate, magnesium silicate, and ion exchange celluloses are presented. New findings with diethylaminoethyl cellulose columns are described. New quantitative procedures employing silicic acid, magnesium silicate, or diethylaminoethyl cellulose column chromatography with quantitative thin‐layer chromatography are described.

ABSTRACTLipids were extracted from fresh egg yolk using hexane:isopropanol (3:2.v/v) and separated on a silicic acid column into triacylglycerol (TG), phospholipid (PL), cholesteryl ester and cholesterol fractions using hexane, ethyl acetate and methanol. TG and PL fractions were cholesterol‐free. Oxidative stability at 35° C of crude lipids, TG, PL and combined TG + PL was evaluated over 12 days by measuring conjugated dienes, conjugated trienes and thiobarbituric acid reactive substances. PL and TG exhibited significantly higher oxidation than the crude lipids (alpha = 0.01). Combined TG + PL was not significantly different than the crude lipids (alpha = 0.01), showing much higher stability than individual TG or PL fractions.