THE VELIGER © CMS, Inc., 2003 The Veliger46(4):325-328 (October6, 2003) Thin Layer Chromatographic Analysis of Neutral Lipids and Phospholipids in Helisoma trivolvis (Colorado Strain) Maintained on a High Fat Diet JESSICA L. SCHNECK', BERNARD FRIED- \ and JOSEPH SHERMA' Department of Chemistry' and Department of Biology-, Lafayette College, Easton, Pennsylvania 18042, USA Abstract. Thin layer chromatographic analysis was used to determine neutral lipids and phospholipids in Helisoma trivolvis (Colorado strain) snails maintained on a high fat diet ofhen's egg yolk. Whole snail bodies, the digestive gland- gonad complexes (DGGs), and plasma samples were analyzed for lipids. The mean percentage values of free sterols, free fatty acids, triacylglycerols, methyl esters, steryl esters, phosphatidylcholine (PC), and phosphatidylethanolamine (PE) in snail whole bodies fed yolk were 1.9, 2.3, 9.2, 2.8, 63, 3, and 5.6 times higher, respectively, than those fed lettuce. The mean percentage values of free sterols, free fatty acids, triacylglycerols, methyl esters, steryl esters, PC, and PE in the DGGs of snails fed yolk were 3.0, 2.2, 3.0, 6.4, 58, 1.4, and 3.4 times higher, respectively, than those on the lettuce diet. Only free fatty acids and PC were detected in plasma, and the respective concentrations were 1.9 and 3.4 times higher in the plasma of the snails on the yolk diet compared to those on the lettuce diet. This research shows that H. trivolvis, in addition to Biomphalaria glabrata, is a useful pulmonate to study the effects of a high fat diet on hyperlipidemia and hyperlipemia in snails. INTRODUCTION MATERIALS and METHODS Fried et al. (1989) induced elevated levels oflipids in the Stock cultures of Helisoma trivolvis (Colorado strain) tissues and hemolymph of the medically important pla- were maintained from eggs to sexually mature adults at norbid snail Biomphalaria glabrata (Say, 1816) by feed- 23 ± 1°C in aerated glass jars each containing 10 to 20 ing snails on a high lipid diet of hen's egg yolk. Biom- snails in 800 mL of artificial spring water (ASW). The phalaria glabrata maintained on the egg yolk diet have ASW was prepared as described by Ulmer (1970). One been used to study nutrition in uninfected snails and those culture of 15 snails with shell lengths ranging from 16- infected with larval schistosomes and echinostomes (see 20 mm was maintainedadlibitum on boiledRomaine leaf reviews in Fried & Sherma, 1990, 1993). lettuce (L diet) for 20 weeks. Anotherculture of 13 snails One ofus (BF) has maintained a continuous culture of was maintained on a boiled leaflettuce diet for 16 weeks a Colorado strain of the planorbid Helisoma trivolvis and then on a boiled hen's egg yolk diet (Y diet) ad (Say, 1816) for about 15 years. This strain is refractory libitum for an additional 4 weeks. Food and water were to miracidial infection by schistosomes andechinostomes, changed three times per week in all cultures. lacks melanin, has an orange-red body, and has been used For TLC analysis, the whole bodies and digestive extensively in neurobiology (see Kater, 1974). This snail gland-gonad complexes (DGGs) of four individual snails is easy to maintain in the laboratory on a leaf-lettuce diet, (n = 4) on each of the Y and L diets were prepared. and an earlier study by Park et al. (1991) examined neu- Hemolymph from three snails was pooled to make a sam- tral lipids in an H. trivolvis strain maintained on a lettuce- ple containing 300 )jlL of blood from the L diet (n = 1): Tetramin diet. To date the only studies on the effect of likewise, hemolymph was pooled from three snails on the high fat diet on snails are those that use B. glabrata. The Y diet to prepare a 125 |jlL sample from that population purpose of this study was to show the usefulness of H. (n = 1). Shells of all snails were removed by gently trivolvis as a model to study the effects of a high lipid crushing with a hammer, and the snail bodies were re- diet on hyperlipidemia and hyperlipemia in a pulmonate moved with forceps. The DGGs were dissected free of snail. H. trivolvis is easier to maintain in simple cultures the visceral mass using forceps, and pooled hemolymph at room temperature than is B. glabrata and provides a was removed using a Pasteur pipet. useful model for experimental dietary studies. Whole bodies and DGGs were prepared by homoge- nizing each sample in 1 mL of chloroform-methanol (2: 1) in a glass homogenizer. The sample was washed twice ^Author to whom correspondence should be addressed: Tele- with 1 mL of chloroform-methanol (2:1), and the super- phone: 610-330-5463; Fax: 610-330-5705; e-mail: friedb® natant was passed through a glass wool filter and treated lafayette.edu with Folch wash (0.88% KCl, 1 mL). Pooled hemolymph Page 326 The Veliger, Vol. 46, No. 4 was centrifuged for 6 min at 8000 rpm, and the plasma as described for the neutral lipids. Plates were developed was removed and treated with 2 mL ofchloroform-meth- with chloroform-methanol-water (65:25:4) mobile phase, anol (2:1), and then passed through a glass wool filter. dried for 5 min under cool air in a fume hood, sprayed The filtrate was treated with 1 mL of Folch wash. All with a 10% cupric sulfate solution, and then heated for samples were placed in a refrigerator at 4°C for 1 hr to 10 min at 140°C to form brown bands on a white back- separate the biphasic layer. The upper hydrophilic layer ground. was discarded using a Pasteur pipet, and the lipophilic Quantitative densitometric analysis was done with a layer was transferred to a 7 mL glass vial and dried under Camag TLC Scanner II using a tungsten light source set nitrogen gas in a water bath at 40°C. Plasma samples of at 610 nm for neutral lipids and a deuterium source set the L and Y diets were each reconstituted with 75 |xL of at 370 nm for polar lipids. Other scanner settings were a chloroform-methanol (2:1). DGG and whole body sam- slit width 4, slit length 4, and scanning speed of 4 mm/ ples ofthe L diet were reconstituted with 200 (jlL ofchlo- sec. The CATS-3 software was used to create a linear roform-methanol (2:1) and the Y diet samples were re- regression calibration curve relating the weights of the constituted with 400 |jlL of this solvent. standard zones (0.400-3.20 |jLg for neutral lipids and The standard for neutral lipid analysis was Non-Polar 0.500-4.00 [xg for polar lipids). Weights ofneutral lipids Lipid Mixture-B (Matreya, Inc.. Pleasant Gap, Pennsyl- and polar lipids in sample aliquots were automatically vania), which contained 20.0% each ofcholesteryl oleate, interpolated from the curve. Ifthe areas ofmore than one methyl oleate, triolein, oleic acid, and cholesterol and a aliquot of a particular sample were bracketed within the total lipid concentration of 25.0 mg/mL. The standard calibration curve, the weight corresponding to the sample was used to represent steryl esters, methyl esters, tria- area closest to the areas ofthe middle two standards was cylglycerols, free fatty acids, and sterols, respectively, in used to calculate the weight percent of the lipid. The the samples. The standard (1.00 mL) was placed in a 25 equation used for calculation of weight percent in the mL volumetric flask and diluted with chloroform-metha- whole bodies and DGGs is: nol (2:1) to prepare a standard solution containing 0.200 |xg/[xL for each of the components. TLC analysis was % Lipid (w*R*Dilution Factor*100) performed on Whatman high performance LHPKDF sil- |jLg snail sample ica gel plates, 10 X 20 cm, containing 19 scored lanes w = |jLg interpolated from calibration curve, and a preadsorbent spotting area. Plates were precleaned R = [reconstitution volume ((jLL)]/[spotted volume by development to the top with dichloromethane-metha- (fJiL)]. nol (1:1). The standards (2.00, 4.00, 8.00, and 16.00 |jlL) and reconstituted samples (2.00, 4.00, and 8.00 ixL) were The equation used for calculation of the concentration applied to the preadsorbent zone in individual lanes with (mg/dL) of lipid in the blood samples is: a 10-|jlL Drummond (Broomall. Pennsylvania) digital mi- (w*R) crodispenser. Plates were developed with petroleum ether- mg/dL lipid (V*10) diethyl ether-glacial acetic acid (80:20:1) mobile phase when analyzing methyl ester, triacylglycerol, free fatty V = plasma volume (fxL). acid and sterol content in the samples, and with hexane- Some of the tissue samples had to be diluted or con- petroleum ether-diethyl ether-glacial acetic acid (50:25:5: centrated to obtain a bracketed area within the calibration 1) when analyzing steryl esters. All plates were devel- curve, and then an appropriate dilution factor was includ- oped for a distance of9 cm in a rectangular Camag (Wil- mington. North Carolina) TLC twin-trough chamber con- ed in the calculations. For several samples, the largest taining about 50 mL ofthe mobile phase and a saturation aliquot spotted gave a zone with a scan area below that of the lowest standard. Therefore, the exact concentra- pad (Anal Tech, Newark, Delaware). Developed plates tions of the lipids in these samples could not be deter- were blown dry in a fume hood with air for 5 min, mined. In these cases, the limit of detection was calcu- sprayed with 5% ethanolic phosphomolybdic acid (PMA) lated, and one-half of this value was used for statistical solution, and heated at 115°C until blue bands appeared calculations of Student's t-test. on a yellow background. The standard for polar lipid analysis. Polar Lipid Mix RESULTS AND DISCUSSION (Matreya, Inc.), contained 25.0% each ofcholesterol (ste- rol), phosphatidylethanolamine (PE), phosphatidylcholine The samples analyzed for neutral lipids showed zones (PC), and lysophosphatidulcholine (LPC) and a total lipid with comparable migration to the standards at R, values concentration of 25.0 mg/mL. The standard (1.00 mL) of0.19 (cholesterol), 0.23 (oleic acid), 0.53 (trolein), and was placed in a 25 mL volumetric flask and diluted with 0.67 (methyl oleate) in the petroleum ether-diethyl ether- chloroform to prepare a standard solution of0.250 |xg/|j.L glacial acetic (80:20:1) mobile phase. The R, value of for each component. TLC analysis was done on the same cholesteryl oleate sample and standard zones was 0.61 in plates and with the same sample and standard volumes the hexane-petroleum ether-diethyl ether-glacial acetic 1 J. L. Schneck et al., 2003 Page 327 Table 1 Weight percents of lipids in the whole bodies of snails maintained on the egg yolk (Y) and the lettuce (L) diets. Lipid L* Y* Free Sterols 0.0170 0.0048** 0.0330 0.0023 Free Fatty Acids 0.0168 0.0043 0.0393 0.011 Triacylglycerols 0.0221 0.011** 0.203 0.039 Methyl Esters 0.00224 0.0016^ 0.00635 0.041 Steryl Esters 0.00287 0.00070** 0.181 0.025 Phosphatidylcholine 0.0104 0.0011 0.0310 0.0088 Phosphatidylethanolamine 0.00244 0.00061 0.0136 0.0041 *Snail bodies; Mean (weight %) ± standard error; n = 4 individual snails for each sample. **Concentration significantly reduced (Student's t-test, P < 0.05) compared with snails on the Y diet. "Two data points were below the limit of quantification. Weight % values of 0.000685 and 0.000510 were used for calculations, which were one-halfthe limit ofquantification for the two samples. acid (50:20:5:1) mobile phase. Samples analyzed for po- esters, steryl esters, PC, and PE in snails fed the Y diet lar lipids showed sample and standard zones at Rf values were 3.0, 2.2, 3.0, 6.4, 58, 1.4, and 3.4 times higher, re- of 0.28 (phosphatidylcholine, PC) and 0.47 (phosphati- spectively, than those on the L diet. The amount of steryl dylethanolamine, PE). The lysophosphatidylcholine esters in the snails on the Y diet was significantly greater (LPC) in the polar lipid standard was not detected with than that on the L diet (Student's t-test, P < 0.05). the cupric sulfate reagent at the concentrations applied, Table 3 shows the lipids detected in the plasma of the and zones that might have been LPC were not seen in snails on the L and Y diets. Each sample consisted of samples. pooled plasma from three snails, and only one analysis Table 1 presents lipid percentages for whole bodies of was made foreach diet. Only free fatty acids and PC were the snails fed the L and the Y diets (n = 4 for each found in the pooled plasma samples. The values of the sample). Mean values offree sterols, free fatty acids, tria- free fatty acids and PC were 1.9 and 3.4 times higher, cylglycerols, methyl esters, steryl esters, PC, and PE in respectively, in the Y diet than the L diet snails. The snails fed on the Y diet were 1.9, 2.3, 9.2, 2.8, 63, 3.0. sample size precluded t-test analysis. and 5.6 times higher, respectively, than the whole bodies Snails maintained on the Y diet showed a gross differ- ofsnails fed theLdiet. The concentrations offree sterols, ence in appearance compared to those on the L diet. The triacylglycerols, and steryl esters in the snails fed the Y DGGs of snails fed the L diet were dark green-brown in diet were significantly higher than those fed the L diet color, while the DGGs of snails on the Y diet were yel- (Student's t-test, P < 0.05). low-white in color. The difference in the appearance of Table 2 presents percentages of lipids in the DGGs of the DGGs in snails on the Y diet was apparent at about snails fed the lettuce versus yolk diets. The mean values 1 week after the snails were placed on the hen's egg yolk for free sterols, free fatty acids, triacylglycerols, methyl diet. The appearance of the snail DGGs is an apparent Table 2 Weight percent of lipids in the DGGs of snails maintained on the egg yolk (Y) and the lettuce fed (L) diets. Lipid Y* Free Sterols 0.0198 0.0049 0.0593 0.030 Free Fatty Acids 0.0265 0.011 0.0593 0.030 Triacylglycerols 0.0692 0.012 0.207 0.103 Methyl Esters 0.00290 0.00080" 0.0185 0.00924 Steryl Esters 0.00186 0.00012'^=' 0.108 0.058 Phosphatidylcholine 0.0334 0.0035 0.0480 0.011 Phosphatidylethanolamine 0.0073 0.00089 0.0248 0.0066 * Snail bodies; Mean (weight %) ± standard error; n = 4 individual snails for each sample (unless indicated otherwise). ** Concentration significantly reduced (Student's t-test, P < 0.05) compared with snails on the Y diet. "One data point was below the limit ofquantification. A weight % value of 0.00134 was used for calculations, which was one-half the limit ofquantification. ^All ofthe four data points were below the limit ofquantification. Weight % values of0.00160, 0.00174, 0.00200, and 0.00212 were used for calculations, which were one-halfthe limit ofquantification for the four samples. — Page 328 The Veliger, Vol. 46, No. 4 Table 3 acids, and steryl esters; this finding is in accord with our results on H. trivolvis maintained on the Y and L diets. Concentration (mg/dL) of lipids in pooled plasma sam- The average triacylglycerol content in snail DGG on the ples of snails maintained on the egg yolk (Y) and the L diet, 0.0221%, was considerably less than thatreported lettuce fed (L) diets. by Park et al. (1991), which was 0.69% for snails on a Lipid L* Y=i lettuce-Tetramin diet. These differences probably reflect dietary differences in the two studies. Free Fatty Acids 6.10 In conclusion, the results of this study show that the Phosphatidylcholine 1.92 6.57 Colorado strain of H. trivolvis is a useful model to study * Snail plasma, pooled: n I with plasma pooled from 3 the effects of a high lipid diet on hyperlipidemia and hy- snails. perlipemia in a pulmonate snail. Acknowledgments. The work of J. L. Schneck was supported by a grant from the Lafayette College EXCEL Program. reflection of the different diets used in the study. The shells of snails maintained on the L diet were less sus- LITERATURE CITED ceptible to cracking than those on the Y diet. Snails on the L diet yielded more hemolymph per snail, about 100 Fried, B., S. Schafer, T. S. Lillie & J. Sherma. 1989. Dietary- (jlL per snail, than the snails on the Y diet, which yielded pionddau)c.edThheypVeerlliigpeirde3m2i:a23i0n-2B3i2o.mphalaria glabrata (Gastro- about 50 |xL per snail. Fried, B. & J. Sherma. 1990. Thin-layerchromatography oflip- All five major classes of neutral lipids and two phos- ids found in snails. Journal of Planar Chromatography pholipids (PC and PE) were found in detectable quantities Modern TLC 3:290-299. in the DGGs and whole bodies of the snails maintained Fried, B. & J. Sherma. 1993. Effects of a high fat diet on the on both diets. The plasma of H. trivolvis, however, only lipid composition of Biomphalaria glabrata (Planorbidae: showed quantifiable amounts of free fatty acids and PC. Gastropoda). Trends in Comparative Biochemistry and Physiology 1:941-958. Park et al. (1991) reported that the free fatty acids were Kater, S. B. 1974. Feeding in Helisoma trivolvis: the morpho- the major lipid in snail hemolymph of H. trivolvis (Col- logical and physiological bases of a fixed action pattern. orado strain) maintained on a leaf lettuce-Tetramin diet. American Zoologist 14:1017-1036. We also found that free fatty acid was the main lipid Park. Y.. B. Fried & J. Sherma. 1991. Densitometric thin-layer fraction in the plasma of this snail maintained on both chromatographic studies on neutral lipids in two strains of Helisoma trivolvis (Gastropoda). Comparative Biochemistry diets. and Physiology 100B:127-130. The previous study on H. trivolvis (Colorado strain) Ulmer, M. J. 1970. Notes on rearing snails in the laboratory. Pp. maintained on a lettuce-Tetramin diet (Park et al., 1991) 143-144 in A. J. Maclnnis & M. Voge (eds.). Experiments showed that triacylglyerols were at the highest concen- and Techniques in Parasitology. W. H. Freeman: San Fran- tration in snail DGG, followed by free sterols, free fatty cisco.