Table Of ContentPage 254 The Veliger, Vol. 45, No. 3
by means ofCellulofine GCL-300 sfgel filtration column Boer. H. H. & J. Witteveen. 1980. Ultrastructural localization
chromatography. Subsequently, each fraction containing of carbonic anhydrase in tissue involved in shell formation
enzyme activity was subjected to SDS-PAGE. This anal- and ionic regulation in the pond snail Lymnaea stagnalis.
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protein exhibiting enzyme activity. This molecular mass mals. Marine Biology Letters 1:3-13.
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To determine the amino terminal sequence, PVDF by thiophene-2-sulfonamide and sulfanilamide. Journal of
membrane transferred a 60 kDa protein was subjected to Biological Chemistry 158:567.
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N66 was cloned from Pinctada maxima. RT-PCR analysis ofaragonite orcalcite polymorphism by mollusk shell mac-
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Henry, R. P. 1996. Multiple roles ofcarbonic anhydrase in cel-
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sequence, which is in agreement with N16-3 except that andcarbonate contentduring mineralizationoftheratenam-
yraesshiidtuae e5t8ali.s,N2,00P0e)a.rlin also belongs to this family (Mi- KiESEe,l.MBio&chAe.miBc.aHeatstBiinogp.hy1s9i4c0a.AFcatcator1s28a9f:f2e2c6ti-n2g30t.he activity
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THE VELIGER
© CMS, Inc., 2002
The Veliger45(3):256-258 (July 2, 2002)
Thin Layer Chromatographic Analysis of Lutein and ^-carotene in
Biomphalaria glabrata Maintained on a High Fat Diet
YONGHYUN KIM', BERNARD FRIED-^ and JOSEPH SHERMA'
Department of Chemistry' and Department of Biology-, Lafayette College, Easton, Pennsylvania 18042, USA
Abstract. Thin layerchromatographic analysis was used to determine lutein and (3-carotene in Biomphalariaglabrata
snails maintained on a high fat diet of hen's egg yolk. The mean values of lutein in snails on a Romaine lettuce diet
were approximately X3 and X2.5 at 14 and 20 days postculture, compared to those of snails maintained on the yolk
diet. Likewise, the mean values of (i-carotene for snails on the lettuce diet were approximately Xl.5 and X6.6 at 14
and 20 days, respectively, compared to those from the snails on the yolk diet. The only significant differences in values
(Student's t-test, P < 0.05) was at day 20 at which time the mean percent of (S-carotene in the snails on the high fat
diet was significantly reduced compared to snails on the lettuce diet. The concentration oflutein in the lettuce was about
X3.5 that in the egg yolk. The concentration of ^-carotene in the lettuce was X8 that in the egg yolk. In general, the
concentration ofthese lipophilic pigments in B. glabrata reflected the content of lutein and ^-carotene in the lettuce and
egg yolk diets.
INTRODUCTION For TLC analysis, the whole-body of five individual
snails (n = 5) was prepared for both Y-L and L diets at
Numerous studies have reported the use of a hen's egg
14 and 20 days after the cultures were started. To do this,
yolk diet to observe nutrition in uninfected Biomphalaria the shell ofeach snail was gently crushed with ahammer,
gtloasbormaetsaa(nSady,ec1h8i1n6o)staonmdessna(isleseirnefveicetewdswiintFhrliaerdva&l sSchheirs-- awnhdolet-hebodsnyaiwlasbohdoymowgaesnizreedmoivned2 mwiLthoffoarcceeptso.neEiancha
ma, 1990, 1993). These studies have observed mainly the glass homogenizer. The pellet was washed twice with ac-
esfnfaeilcsts(oFrfietdhe&egSgheyromlak,di1e9t90o,n1t9h9e3)l,ipaildthcoountgehnta orfecetnhet tehteonesup(e1r0n0ata|xnLt)., Tahned tchoembwiansehdingssupewrenraetacntomwbaisnedevawpiot-h
study by Kim et al. (2001) has examined the effects of rated to dryness under nitrogen and then reconstituted
this diet on the carbohydrate content ofthe snail. Because with 200 |jlL or 300 (jlL of heptane, as necessary for the
effects of the diet on lipophilic pigments, i.e., lutein and scan areas of at least one sample zone to be bracketed
P-carotene, are not available, this study examined these within the scan areas of the standard zones in the TLC
pigments in snails maintained on hen's egg yolk. analysis. Single samples (n = 1) of the hen's egg yolk
MATERIALS and METHODS (200 mg) and the Romaine lettuce (200 mg) were extract-
ed in acetone and prepared for TLC analysis as described
Twenty juvenile Biomphalaria glabrata snails, about 7 for the snail bodies.
mm in shell diameter, were obtained from Dr. FredLewis, The standards used for TLC analysis were lutein and
Schistosomiasis Laboratory, Biomedical Research Insti- p-carotene (Sigma, St. Louis, Missouri). The solid stan-
tute (Rockville, Maryland, USA). Groups of 10 snails dards were weighed on an analytical balance and diluted
were maintained at 23—24°C in aerated glass containers with dichloromethane to prepare standard solutions of
each containing 800 mL of artificial spring water (ASW) 0.0100 (xg [jlL ' forboth lutein and P-carotene. TLC anal-
prepared as described by Ulmer (1970). One culture of yses were performed on Merck (EM Science, Gibbstown,
10 snails was fed ad libitum on boiled Romaine leaf let- New Jersey) 10 cm X 20 cm chemically bonded C-18
tuce (L diet). The other culture was fed the hen's egg silica gel plates with concentrating zone (RP-I8F254S, Art.
yolk diet ad libitum, supplemented with 500 mg of Ro- 15498). Plates were prewashed by developmentto the top
maine lettuce once a week (Y-L diet), as described in with dichloromethane-methanol (1:1) and dried in air in
Beers et al. (1995). Food and water were changed twice a fumehood. The standards (4.00, 8.00, 12.0. and 16.0 |jlL
weekly in all cultures. for each standard) and 1.00-8.00 |xL of the reconstituted
samples were applied in separate lanes in the concentrat-
3Authorto whomcorrespondence shouldbe addressed: Tele- ing zone by means of a 10-|Ji.L Drummond (Broomall,
phone: 610-330-5463, Fax: 610-330-5705, e-mail: friedb® Pennsylvania) digital microdispenser in a dark room with
lafayette.edu minimum lighting. The applied solutions were dried in
