J. Dutta

Synthesis of diene prostaglandins in freshwater fish.

Biosynthesis of PGD2, PGE2 and PGF2α in four species of freshwter fish,Tilapia mossambica, Cyprinus carpio, Heteropneustes fossilis andClarius batrichus was studied. Both arachidonic acid and PGH2 were used as substrates. When PGH2 replaced arachidonic acid in the enzymic reaction, there was a 3- to 4-fold increase in PGE2 synthesis, but no such increase in the synthesis of PGF2α and PGD2 was observed.

Enzymatic reactions on thin-layer chromatographic plates : I. Lipolysis of triglycerides and separation of products on a single plate

Abstract A novel method for the lipase deacylation of triglycerides is described in which 0.1–0.2 ml of the enzyme solution in 1 M tris buffer (pH 8.2) containing 2–4 mg of protein is applied as a band on a 0.5-mm thick silica gel thin-layer chromatographic plate. Over this band, 1–4 mg of triglyceride in n-hexane is applied evenly and the plate is incubated at 40° for 1–2 min. The reaction is stopped by exposure to hydrogen-chloride vapour and the products are removed from the reaction zone by three consecutive developments in diethyl ether up to 2 cm from the line of application. These are resolved by re-developing the plate in n-hexane—diethyl ether—acetic acid (80:20:1.5) up to 14 cm. The sn-2-monoglyceride and sn-1,2(2,3)-diglyceride bands located by iodine vapour are extracted and the fatty acid compositions evaluated by gas-liquid chromatography for the determination of fatty acid distribution in the glyceride molecule. The method, when applied to groundnut oil, goat depot fat, body fat of shol fish (Channa striatus) and yeast (Rhodotorula glutinis) fat, produced representative sn-1,2(2,3)-diglycerides which can be used for the stereospecific analysis of triglyceride.

Comparison of the effects of dietary fish oils with different n-3 polyunsaturated fatty acid compositions on plasma and liver lipids in rats.

The effects of dietary fish oils with different n−3 polyunsaturated fatty acid compositions on plasma lipid profiles in rats have been studied. Forty-eight male rats, previously maintained on a cholesterol-free diet for 15 days, were fed for 60 days with diets supplemented with 10% fat of either marine hilsa fish (Hilsa ilisa, family clupeidae) or fresh-water chital fish (Notopterus chitala, family notopteridae). The diets had similar levels of total saturated (35–41%), monounsaturated (43–47%) and n−3 polyunsaturated (9–10%) fatty acids. Cholesterol contents of the diets were adjusted to 0.85%; γ-linolenic acid (3.3%) in chital oil and eicosapentaenoic acid (4.9%) in hilsa oil diets were the major n−3 contributors. The percentage of eicosapentaenoic acid in the chital oil diet was 0.57 times that of the hilsa oil diet, but the eicosapentaenoic (EPA) to arachidonic acid (AA) ratio in the latter (4.08) was 3.2 times that of the former (1.27). Sixty days of hilsa oil diet feeding decreased the levels of cholesterol (53.3±2.9 to 50.0±1.1 mg/dL), triacylglycerol (75.7±3.8 to 64.3±2.6 mg/dL) and phospholipid (55.8±1.5 to 51.7±3.1 mg/dL) in rat plasma. Similar treatment with chital oil diet elevated the plasma cholesterol level (53.3±2.9 to 62.3±7.6 mg/dL) while triacylglycerol and phospholipid contents remained unaltered. Both the dietary treatments decreased the levels of linoleic and arachidonic acids in liver but only under the hilsa oil diet did the eicosapentaenoic acid percentage increase markedly (0.8±0.06% to 5.5±0.06%) at the expense of arachidonic acid. This study strongly suggests that the hypolipidemic effect depends on the composition of the n−3 polyunsaturated fatty acids rather than on the total n−3 polyunsaturated fatty acid content of the dietary fish oil.

The role of hydrophobic substances in leaves in adaptation of plants to periodic submersion by tidal water in a mangrove ecosystem

SUMMARY (1) Three species of plants, Acanthus ilicifolius, Avicennia officinalis and Bruguiera gymnorhiza, grow abundantly in the tidal mangrove area of the Sunderbans, India. (2) The leaves from plants not normally submerged and from those periodically submerged have been analysed for their hydrophobic components (lipid, wax ester and hydrocarbons). The weights of hydrocarbons and wax esters per unit leaf area of the submerged plants were about three times greater than those of emergent plants; this apparently makes them tolerant to flooding. (3) In addition, the area, thickness, fresh weight to dry weight quotient, loss in weight on drying, and chlorophyll content of leaves were all higher in the periodically submerged plants. The great increases in leaf area and chlorophyll content may help in overcoming the limitations imposed on photosynthesis due to periodic submergence.

Preferential oxidation of linolenic acid compared to linoleic acid in the liver of catfish (Heteropneustes fossilis and Clarias batrachus)

The fate of [1-14C] linoleic acid and [1-14C] linolenic acid in the liver slices and also in the liver tissues of live carnivorous catfish,Heteropneustes fossilis andClarias batrachus, was studied. Incorporation of the fatty acids into different lipid classes in the live fish differed greatly from the tissue slices, indicating certain physiological control operative in vivo. The extent of desaturation and chain elongation of linoleic and linolenic acids into long-chain polyunsaturated fatty acids was low. Linolenic acid was oxidized (thus labeling the saturated fatty acid with liberated14C-acetyl-CoA) in preference to linoleic acid, and this oxidation also seemed to be under physiological control since both of the fatty acids were poorly oxidized in the tissue slices and in the killed fish. These fish can therefore recognize the difference in the acyl chain structures of linoleate and linolenate. The higher oxidation of liolenic acid and poor capacity for its conversion to longer chain, highly unsaturated derivatives indicates a higher demand for the dietary supply of these essential fatty acids in these two species.

Lipid contaminants in commercial lipases.

During lipolysis of wax esters on thin layer chromatography (TLC) plates (1), abnormally high errors for stearic (18:0), palmitic (16:0) and oleic (18: t ) acids were found in the hydrolysates. These anomalous results prompted us to examine the source of the contaminations, which was found to be the lipase used. We have analyzed 2 commercial lipase samples that were used for the lipolysis of wax esters and developed a procedure for the preparation of commercial lipase free of fatty acid (FA) contaminants. Pancreatic lipase from 300 mg each from Sigma Chemical Co. (St. Louis, MO, Type II, crude from porcine pancreas) and from Calbiochem (Los Angeles, CA, B grade from porcine pancreas) were mixed vigorously with 15 ml of redistilled diethyl ether and centrifuged. The supernatnats were carefully removed with a syringe and saved. The extract ion procedure with diethyl ether was repeated 6 times for each lipase sample. The pooled etherial extracts were dried over anhydrous sodium sulphate and the solvent was evaporated and weighed. The lipases were then washed 6 times with 15 ml protions of acetone. The acetone extracts were also pooled and the solvent was evaporated and weighed. The etherial extracts were spotted on preparative TLC plates that were developed using a solvent system of light petroleum ether (40 C60 C) diethyl ether/acetic acid (80:20:1.5, v/v). The bands were visualized by putting the plates in an iodine chamber. The bands were identified by comparing the Rf values with those of known standards. Finally, the various bands were scraped off the plates and the compounds were extracted using diethyl ether, the solvent was evaporated and then weighed. The free fatty acid (FFA) bands from the 2 lipase samples were methylated using diazomethane (2). To each of the methyl ester samples, 50 /~g of methyl pentadecanoate (15:0) were added and analyzed by gas liquid chromatography (GLC) using a 10% DEGS column. From the chromatograms, peak areas were determined and each of the components was identified and quantified. Beeswax and Avicennia officinalis leafwax were hydrolyzed with extracted and unextracted lipases on TLC plates, using 1 mg lipase and 1 mg wax esters, according to the method des-

Enzymatic reactions on thin-layer chromatographic plates : II. Phospholipase A2 hydrolysis of phosphatidylcholine and separation of the products on a single plate

A procedure for the phospholipase A2 hydrolysis of phosphatidylcholine on a thin-layer chromatographic plate and subsequent separation of the products on the same plate is described. A 0.2-0.8-mg amount of Russells viper venom (phospholipase A2) in 0.2 ml of 0.005 M calcium chloride solution was applied on a 0.5-mm silica gel G plate as a band over which 2-5 mg of egg phosphatidylcholine in 0.2 ml of diethyl ether containing 5% of methanol was evenly applied. After the reaction had proceeded for 15-20 min in a diethyl ether-saturated chamber at 25 degrees, the plate was developed with chloroform-methanol-water (65:25:4). The bands were identified and their contents extracted. The extent of hydrolysis under different reaction conditions was evaluated from the amount of lysophosphatidylcholine formed. Approximately 74.6% (maximum) conversion was obtained within 15 min at 25 degrees using a substrate to enzyme ratio of 4:1. The acyl group distributions in the 1- and 2-positions of hen egg phosphatidylcholine obtained from the gas-liquid chromatographic analysis of the methyl ester corresponding to the lyso and free fatty acid band agreed with those obtained by the method of Wells and Hanahan. The method is also applicable to phosphatidylethanolamine.

Preparation ofcis,cis,cis-5,8,11-eicosatrienoic acid from arachidonic acid.

Arachidonic acid was reduced by hydrazine to yield isomeric eicosatrienoic acids with other products. Methylcis,cis,cis-5,8,11-eicosatrienoate was isolated from the products by silver ion chromatography and preparative gas liquid chromatography in 8% yield. The structure was confirmed by spectral studies and oxidative degradation.

Use of argentation column chromatography in the identification of fish oil fatty acids by GLC: Application to cod liver oil

The fatty acid methyl esters from cod liver oil have been fractionated into seven fractions, depending on degree of unsaturation, by a silver nitratetreated silicic acid column. The fractions have been analyzed by gas liquid chromatography and the components identified by usual techniques.

Preparation of fatty acid methyl esters from triglycerides on thin layer plates.

A simple and convenient procedure for the preparation of methyl esters from triglycerides with potassium methoxide (KOMe) as catalyst on thin layer chromatoplates has been described. The triglyceride was separated and the reaction was carried out on a thin layer chromatoplate without extraction of triglyceride. A final extraction with CS2 was sufficient for injection of the product into a gaschromatograph. The method has been tested with coconut, peanut, and watermelon seed oils. The method is applicable to 10–20 mg of oil sample and is rapid.