Comp. Biochem. Physiol., 1975, VoL 50B, pp. 109 to 111. Pergamon Press. Printed in Great Britain
THE BILIARY BILE ACIDS OF THE CHANNEL CATFISH, ICTALURUS PUNCTATUS, AND THE BLUE CATFISH, ICTALURUS FURCATUS* THOMAS F. KELLOGG Department of Biochemistry, Agricultural and Forestry Experiment Station, Mississippi State University, Mississippi State, Mississippi 39762, U.S.A. (Received 8 March 1974)
Abstract--1. The biliary bile acids of the channel catfish, Ictalurus punctatus, and the blue catfish, Ictalurus furcatus, have been determined. 2. In both species the biliary bile acids are composed of taurocholic acid: 83%--I. punctatus, 84% --I. furcatus ; taurochenodesoxycholic acid: 15~--I. punctatus, 16%--1. furcatus ; taurodesoxycholic acid: 2~.--I. punctatus only. 3. These bile acids are consistent with the phylogenetic placement of these species in suborder silurodei. INTRODUCTION HASLEWOOD(1967a, b) has reviewed the evolutionary aspects of bile salt metabolism and pointed out the usefulness of differences in bile salt composition in assitming phylogenetic relationships among fishes. In recent years, intensive aquaculture of the channel catfish, lctaluruspunctatus, has stimulated interest in the biochemistry of this and related species (Stickney et aL, 1971 ; Wilson, 1973). This paper reports the biliary bile acid composition of the channel catfish, L punctatus, and the blue catfish, lctalurusfurcatus. MATERIALS AND METHODS Animals The fish were reared in cages suspended in a local pond. A high quality, commercially available, floating pelleted catfish ration was fed. The fish were sacrificed after 1 year on the ration, in June, with a body weight of 300--400g. Chemicals All solvents used were glass redistiiled, analytical grade. Bile acid standards were obtained from Steraloids, Pawling, New York, and the chenodesoxycholic acid from Weddel Pharmaceuticals Ltd., London. The taurodesoxycholic acid was synthesized by the method of Lack et al. (1973) and purity confirmed by TLC. Other reagents were of analytical grade.
ANALYTICAL METHODS The preliminary extraction and purification methods of Haslewood (1967a) were followed. The gall bladders of eleven channel catfish (Ictalurus punetatus) and eleven * Mississippi Agricultural and Forestry Experiment Station Publication No. 2739.
blue catfish (Ictalurus furcatus) were removed and stored by species in a large excess of ethanol. The ethanol-bile solution was sonicated for 15 rain, heated to boiling and the solution filtered into round-bottom flasks. The flasks were taken to dryness in vaeuo at 60°C and the last traces of water removed as the benzene azeotrope. The solids were extracted with 3 x 50 ml of hexane and the hexaue (containing fats and cholesterol) discarded. The remaining solid was extracted with 3 x 50 ml of methanol and the filtered methanol extract dried in vacuo in a tared flask. The bile salts from the methanol extract weighed 2.07 g from L punctatus and 1.09 g from L furcatus. TLC on silica gel-G plates with Hofmann's System SVIII (1964), followed by sulfuric acid spray and heating, revealed compounds from both species with the mobility of taurocholate and a dihydroxytaurine conjugated bile salt with the mobility of taurochenodeoxycholate or taurodeoxycholate (both Rt, 0-23). Fifty nag of bile salt from each species was saponified in Teflon beakers containing 10 ml of 1.25 N NaOH for 2 hr at 120°C in an autoclave. The samples were cooled and acidified to pH 1 with HCI and diluted to 50 ml with water. They were extracted with 3 x 50 ml of chloroform and the pooled extracts dried in vaeuo. The free bile salts were taken to 24 ml with ethyl acetate. TLC of the nonconjugated bile acids was on silica gel-G in isooctaneethyl acetate--acetic acid (10 : 10 : 2) and detected by the anisaldehyde method of Kritchevsky et al. (1963). Mobility and color reaction revealed that each sample contained cholic and cbenodesoxycholic acids. A sample of the free bile acids was methylated by diazomethane (Schlenk & Gellerman, 1960) and analyzed by TLC on silica gel-G in acetone--benzene (3:7). This analysis also revealed methyl eholate and methyl chenodeoxycholate in the two samples. The trifluoroacetates of the bile acid methyl esters were prepared by reaction of the methyl esters with trifiuoroacetyl imidazole. These derivatives were gas chromatographed on 3~o QF-1 on Gas Chrom Q in in a silieated 4 mm i.d. 2-m glass U-tube column in a Hewlett-Packard
109
110
THOMASF. KELLOC_.,-G
7610A gas chromatograph equipped with a flame ionization detector and a Model 3370B digital electronic integrator with teletype output. Column temperature was 250°C, injectors and detector temperatures were 260 and 280°C, respectively. Carrier gas was N~ at 27 ml/min and 40 psig inlet pressure. Additional portions of eluted bands of chenodesoxycholic and cholic acids were prepared for i.r. spectroscopic examination by the micro KBr method of Garner & Packer (1968) on a Perkin-Elmer Model 337 grating i.r. spectrophotometer.
cholic acid. Comparison of the i.r. spectras of the isolated chenodesoxycholic and cholic acids with those of standards confirmed the identification of the substances. Acknowledgements--This work was supported by Biochemistry Research Fund, Mississippi State University. The author acknowledges the co-operation and assistance of Dr. R. Wilson and Mr. D. Garling in collecting the bile samples and Mr. W. Poe for the infrared analysis.
Table 1. TLC of bile acids and derivatives
Derivative
Sample
R/
Color of detected compound under visible light
Conjugated bile salts
1. punctatus 1. punctatus L furcatus L furcatus Taurocholate standard Taurochenodesoxycholate standard Glycochenodesoxycholate standard
0'14 0'23 O.14 0'23 0.13 0'22 0.59
All spots black
Free bile acids
L punctatus L punctatus L furcatus L filrcatus Cholic acid standard Desoxycholic standard Chenodesoxycholic standard Hyodeoxycholic standard
0.09 0'31 0'09 0.31 0.09 0.35 0.31 0.24
Purple Purple Purple Purple Purple Gray Purple Purple
Bile acid methyl esters
L punctatus L punctatus L furcatus L furcatus Methylcholate standard Methylchenodesoxycholate standard
0"08 0"35 0"08 0"35 0.08 0-34
All spots black
RESULTS AND DISCUSSION
REFERENCES
As can be seen from Table 1, the bile samples from each species revealed two bile acids by TLC. When chromatographed as the native conjugates, the free bile acids, and the methyl esters, the mobility was identical with that of cholic and chenodesoxycholic standards of the same derivative. Also, the mobility on Hofmann's System SVIII indicates that the biliary acids are taurine conjugates. The gas chromatographic separation, as the trifluoroacetates, revealed that the bile from L punctatus also contained 2~o desoxycholic acid. By comparison with appropriate standards, the bile acids were calculated to be of the following composition: L punctatus--15~o chenodesoxycholic acid, 83~o cholic acid and 2~o desoxycholic acid; L furcatus--16% chenodesoxycholic acid and 84~
GARNERH. R. & PACKERH. (1968) New technique for the preparation of KBr pellets from microsamples. AppL Spectrose. 22, 122-123. HASLEWOOD G. A. D. (1967a) Bile Salts. Methuen, London. HASLEWOODG. A. D. (1967b) Bile salt evolution. J. Lipid Res. 8, 535-550. HOFMANN A. F. (1964) Thin Layer Chromatography of Bile Acids and their Derivatives it) New Biochemical Separations (Edited by JAMESA. T. & MORRISL. J.), p. 269. Van Nostrand, London. KRrrCHEVSY D., MARTAKD. & ROTHBLATG. (1963) Detection of bile acids in thin layer chromatography. Analyt. Biochem. 5, 388-392. LACKL., DORRrrY F. O., JR., WALKERT. & SINGLETARY G. D. (1973) Synthesis of conjugated bile acids by means of a peptid~ coupling agent. J. Lipid Res. 14, 367-370.
Biliary bile acids of the catfish SCm~N~ H. & GrZ.LERMANJ. L. (1960) Esterification of fatty acids with diazomethane on a small scale. Analyt. Chem. 32, 1412-1414. STIcm,~Y R. R. & ANDREWS J. W. (1971) Combined effects of dietary lipids and environmental temperature on growth, metabolism and body composition of channel catfish (Ictalurus punctatus). J. of Nutrit. 101, 1703-1710.
111
WILSON R. (1973) Nitrogen metabolism in channel catfish, lctalurus punctatus--I. Tissue distribution of aspartate and alanine aminotransferases and glutarnic dehydrogenase. Comp. Biochem.Physiol. 46B, 617-624.
Key Word Index--lctalurus punctatus ; Ictalurus furcatus; channel catfish; blue catfish; bile acids; bile salts; taurocholate; taurochenodeoxycholate.
THE BILIARY BILE ACIDS OF THE CHANNEL CATFISH, ICTALURUS PUNCTATUS, AND THE BLUE CATFISH, ICTALURUS FURCATUS* THOMAS F. KELLOGG Department of Biochemistry, Agricultural and Forestry Experiment Station, Mississippi State University, Mississippi State, Mississippi 39762, U.S.A. (Received 8 March 1974)
Abstract--1. The biliary bile acids of the channel catfish, Ictalurus punctatus, and the blue catfish, Ictalurus furcatus, have been determined. 2. In both species the biliary bile acids are composed of taurocholic acid: 83%--I. punctatus, 84% --I. furcatus ; taurochenodesoxycholic acid: 15~--I. punctatus, 16%--1. furcatus ; taurodesoxycholic acid: 2~.--I. punctatus only. 3. These bile acids are consistent with the phylogenetic placement of these species in suborder silurodei. INTRODUCTION HASLEWOOD(1967a, b) has reviewed the evolutionary aspects of bile salt metabolism and pointed out the usefulness of differences in bile salt composition in assitming phylogenetic relationships among fishes. In recent years, intensive aquaculture of the channel catfish, lctaluruspunctatus, has stimulated interest in the biochemistry of this and related species (Stickney et aL, 1971 ; Wilson, 1973). This paper reports the biliary bile acid composition of the channel catfish, L punctatus, and the blue catfish, lctalurusfurcatus. MATERIALS AND METHODS Animals The fish were reared in cages suspended in a local pond. A high quality, commercially available, floating pelleted catfish ration was fed. The fish were sacrificed after 1 year on the ration, in June, with a body weight of 300--400g. Chemicals All solvents used were glass redistiiled, analytical grade. Bile acid standards were obtained from Steraloids, Pawling, New York, and the chenodesoxycholic acid from Weddel Pharmaceuticals Ltd., London. The taurodesoxycholic acid was synthesized by the method of Lack et al. (1973) and purity confirmed by TLC. Other reagents were of analytical grade.
ANALYTICAL METHODS The preliminary extraction and purification methods of Haslewood (1967a) were followed. The gall bladders of eleven channel catfish (Ictalurus punetatus) and eleven * Mississippi Agricultural and Forestry Experiment Station Publication No. 2739.
blue catfish (Ictalurus furcatus) were removed and stored by species in a large excess of ethanol. The ethanol-bile solution was sonicated for 15 rain, heated to boiling and the solution filtered into round-bottom flasks. The flasks were taken to dryness in vaeuo at 60°C and the last traces of water removed as the benzene azeotrope. The solids were extracted with 3 x 50 ml of hexane and the hexaue (containing fats and cholesterol) discarded. The remaining solid was extracted with 3 x 50 ml of methanol and the filtered methanol extract dried in vacuo in a tared flask. The bile salts from the methanol extract weighed 2.07 g from L punctatus and 1.09 g from L furcatus. TLC on silica gel-G plates with Hofmann's System SVIII (1964), followed by sulfuric acid spray and heating, revealed compounds from both species with the mobility of taurocholate and a dihydroxytaurine conjugated bile salt with the mobility of taurochenodeoxycholate or taurodeoxycholate (both Rt, 0-23). Fifty nag of bile salt from each species was saponified in Teflon beakers containing 10 ml of 1.25 N NaOH for 2 hr at 120°C in an autoclave. The samples were cooled and acidified to pH 1 with HCI and diluted to 50 ml with water. They were extracted with 3 x 50 ml of chloroform and the pooled extracts dried in vaeuo. The free bile salts were taken to 24 ml with ethyl acetate. TLC of the nonconjugated bile acids was on silica gel-G in isooctaneethyl acetate--acetic acid (10 : 10 : 2) and detected by the anisaldehyde method of Kritchevsky et al. (1963). Mobility and color reaction revealed that each sample contained cholic and cbenodesoxycholic acids. A sample of the free bile acids was methylated by diazomethane (Schlenk & Gellerman, 1960) and analyzed by TLC on silica gel-G in acetone--benzene (3:7). This analysis also revealed methyl eholate and methyl chenodeoxycholate in the two samples. The trifluoroacetates of the bile acid methyl esters were prepared by reaction of the methyl esters with trifiuoroacetyl imidazole. These derivatives were gas chromatographed on 3~o QF-1 on Gas Chrom Q in in a silieated 4 mm i.d. 2-m glass U-tube column in a Hewlett-Packard
109
110
THOMASF. KELLOC_.,-G
7610A gas chromatograph equipped with a flame ionization detector and a Model 3370B digital electronic integrator with teletype output. Column temperature was 250°C, injectors and detector temperatures were 260 and 280°C, respectively. Carrier gas was N~ at 27 ml/min and 40 psig inlet pressure. Additional portions of eluted bands of chenodesoxycholic and cholic acids were prepared for i.r. spectroscopic examination by the micro KBr method of Garner & Packer (1968) on a Perkin-Elmer Model 337 grating i.r. spectrophotometer.
cholic acid. Comparison of the i.r. spectras of the isolated chenodesoxycholic and cholic acids with those of standards confirmed the identification of the substances. Acknowledgements--This work was supported by Biochemistry Research Fund, Mississippi State University. The author acknowledges the co-operation and assistance of Dr. R. Wilson and Mr. D. Garling in collecting the bile samples and Mr. W. Poe for the infrared analysis.
Table 1. TLC of bile acids and derivatives
Derivative
Sample
R/
Color of detected compound under visible light
Conjugated bile salts
1. punctatus 1. punctatus L furcatus L furcatus Taurocholate standard Taurochenodesoxycholate standard Glycochenodesoxycholate standard
0'14 0'23 O.14 0'23 0.13 0'22 0.59
All spots black
Free bile acids
L punctatus L punctatus L furcatus L filrcatus Cholic acid standard Desoxycholic standard Chenodesoxycholic standard Hyodeoxycholic standard
0.09 0'31 0'09 0.31 0.09 0.35 0.31 0.24
Purple Purple Purple Purple Purple Gray Purple Purple
Bile acid methyl esters
L punctatus L punctatus L furcatus L furcatus Methylcholate standard Methylchenodesoxycholate standard
0"08 0"35 0"08 0"35 0.08 0-34
All spots black
RESULTS AND DISCUSSION
REFERENCES
As can be seen from Table 1, the bile samples from each species revealed two bile acids by TLC. When chromatographed as the native conjugates, the free bile acids, and the methyl esters, the mobility was identical with that of cholic and chenodesoxycholic standards of the same derivative. Also, the mobility on Hofmann's System SVIII indicates that the biliary acids are taurine conjugates. The gas chromatographic separation, as the trifluoroacetates, revealed that the bile from L punctatus also contained 2~o desoxycholic acid. By comparison with appropriate standards, the bile acids were calculated to be of the following composition: L punctatus--15~o chenodesoxycholic acid, 83~o cholic acid and 2~o desoxycholic acid; L furcatus--16% chenodesoxycholic acid and 84~
GARNERH. R. & PACKERH. (1968) New technique for the preparation of KBr pellets from microsamples. AppL Spectrose. 22, 122-123. HASLEWOOD G. A. D. (1967a) Bile Salts. Methuen, London. HASLEWOODG. A. D. (1967b) Bile salt evolution. J. Lipid Res. 8, 535-550. HOFMANN A. F. (1964) Thin Layer Chromatography of Bile Acids and their Derivatives it) New Biochemical Separations (Edited by JAMESA. T. & MORRISL. J.), p. 269. Van Nostrand, London. KRrrCHEVSY D., MARTAKD. & ROTHBLATG. (1963) Detection of bile acids in thin layer chromatography. Analyt. Biochem. 5, 388-392. LACKL., DORRrrY F. O., JR., WALKERT. & SINGLETARY G. D. (1973) Synthesis of conjugated bile acids by means of a peptid~ coupling agent. J. Lipid Res. 14, 367-370.
Biliary bile acids of the catfish SCm~N~ H. & GrZ.LERMANJ. L. (1960) Esterification of fatty acids with diazomethane on a small scale. Analyt. Chem. 32, 1412-1414. STIcm,~Y R. R. & ANDREWS J. W. (1971) Combined effects of dietary lipids and environmental temperature on growth, metabolism and body composition of channel catfish (Ictalurus punctatus). J. of Nutrit. 101, 1703-1710.
111
WILSON R. (1973) Nitrogen metabolism in channel catfish, lctalurus punctatus--I. Tissue distribution of aspartate and alanine aminotransferases and glutarnic dehydrogenase. Comp. Biochem.Physiol. 46B, 617-624.
Key Word Index--lctalurus punctatus ; Ictalurus furcatus; channel catfish; blue catfish; bile acids; bile salts; taurocholate; taurochenodeoxycholate.
