Altered lipid metabolism in livers of rainbow trout fed cyclopropenoid fatty acids

EXPERIMENTAL

AND

MOLECULAR

Altered

23,

PATHOLOGY

Lipid

Metabolism

Department

of Food

D.

(19%)

in Livers

Fed Cyclopropenoid B. J. STRUTHERS,~

181-187

Fatty

J, LEE,

AND

of Rainbow Acids ‘, ’

R. 0.

SINNHUBER

Science and Technology, Oregon Cowallis, Oregon 97331 Received

November

Trout

State

University,

18, 1974

The incorporation of “C-labeled fatty acids isotopic phosphorus into liver lipids of rainbow trout (Salmo gairdneri) that had received either 200 or 300 ppm of cyclopropenoid fatty acids (CPFA) in the diet for 14 days was studied. The amount of [?‘]04 incorporated into liver phospholipids was lower in fish fed CPFA than in controls, with the greatest difference being in an unknown phospholipid. High levels of free [“Cloleic acid were found in livers from CPFA-fed fish after perfusion with this fatty acid, while none was detected in the controls. CPFA feeding lowered the incorporation of oleic acid into lipids of microsomal and mitochondrial fractions, but increased it in the 105,OOOg supematant phospholipids. The type of dietary protein fed was shown to influence CPFA effects.

Cyclopropenoid fatty acids (CPFA) inhibit fatty acid desaturation in several species, including chicken (Allen et al., 1967; Johnson et al., 1969)) rat (Raju and Reiser, 1967), pig and cow (Phelps et al., 1965), and trout (Roehm, et al., 1970). Decreased hatchability of eggs and decreased egg production have been associated with the ingestion of CPFA (Schneider et al., 1961; Shenstone and Vickery, 1959). Deleterious effects have been reported in rats (Miller et al., 1969), including increased pre- and postnatal mortality and numerous organ defects in newborns from females fed l-2% Sterculia foetida oil (0.5-l% CPFA). Sinnhuber ( Sinnhuber et al., 1968) demonstrated that CPFA were cocarcinogenic with aflatoxin Br in rainbow trout and that they reduced fish growth, These experiments were undertaken to determine if CPFA alter lipid metabolism in trout liver. MATERIALS

AND METHODS

Experimental animals and diets. Rainbow trout (Salmo gairdneri) for these experiments were spawned and reared at the Food Science Toxicology and Nutrition Laboratory. Fish used in any one experiment were of similar back1 Technical Paper No. 3926, Oregon Agricultural Experiment Station. 2 Supported in part by USPHS Grants No. ES 00263, ES 00550 and FD 00010. 3 Present address: Central Research, Ralston Purina Company, Checkerboard Square, St. Louis, MO 63188. The described work is taken in part from a Ph.D. thesis submitted by B. J. Struthers in the Department of Food Science and Technology, Oregon State University.

181 Copyright All rights

1975 by Academic Press, oP reproduction in any form

1%

STRUTHERS,

LEE

AND

SINNHUBER

COMPOSITION OF EX~IGMMC~~NT.\LL)II'TS (PERCENT) Ingredients’

Casein FPCY Gelatin I>extrin Salmon

49.5 X.7 1.5.6

oil

LL dhd(Jse

CM0 Mineral Choline Vitamin Methyl

mixd chloride mixe stercldatef

(70%)

10.0 7.7 I ..i 4.0 1.0 2.0

49.3 8.7 1 .i.6 10.0 7.7 1.5 4.0 1.0 2.0 0.02

49.5 x.7 1 T,.(i 10.0 7.7 1.5 4.0 1.0 2.0 0.03

49.5 8.7 15.6 10.0 9.2 1 .?j 2.3 1.0 2.0 -

49.5 8.7 15.6 10.0 9.2 1.5 2.5 1.0 2.0 0.02

49.3 x.7 15.6 10.0 9.2 I ..i 2.-i 1.0 2.0 0.03

a All ingredients from Nutrit.ional Biochemicals Corp., Cleveland, OH, unless noted ot.herwise. * Fish protein concentrate, a defat,ted refined ptot,ein from Pacific hake. c Carboxymethyl cellldose, Hercules Powder Co., Los Angeles, CA. d Bernhart-Tomarelli mineral mix [(1966) J. iliz,lr. 89, 4031. e Vitamin mix described by Lee et (~1. [(1967) J. Xutr. 92, 931. Vitamin E at 660 IU/kgm and vitamin A and I) at, 25,000 and 4000 IU/kgm, respect,i\-ely. f Methyl esters of Stcr~lin jot~idu oil was added at the expense of salmon oil (diets CS, C,, FZ and F, contained slight,ly less than 10% salmon oil).

(age and diet). Fish were transferred to experimental tanks prior to beginning the CPFA regimen to allow them to acclimate to their new surroundings. Groups were fed semipurified diets (Table I) twice daily, with the amount fed based on the consumption of fish fed methy stercuIate (MS). (Over 90% of CPFA fed was sterculic acid, with the remainder malvalate.) Test diets in experiments 14 contained 200 ppm of CPFA and in experiment 5, 300 ppm. Assays were conducted after the fish were on the diets 14 days. Liver size ranged between 0.8 and 1.2% of body weight, which is normal for these fish. The methyl ester of sterculic acid was prepared by transesterificntion of Sterculia foetida seed oil with the sodium methoxide method of Luddy (Luddy et al., 1960) and the cyclopropenoid was concentrated to 60-70% CPFA by vacuum distillation. In the initial injections [33P]04 (New England Nuclear) was used because of its longer tt and lower energy as compared with szP. Later, 31’Pwas used because of its availability and lower cost. The isotopes were injected intraperitoneally into four lOO-gm, six 50-gm, and four 250-gm fish/diet. The isotope was in 0.1 M acetic acid-sodium acetate buffer, pH 4.8. After 10 minutes the animals were killed by a blow on the head, the livers removed, weighed and perfused with 4°C fish Ringer’s solution (Helms and Stoff, 1958), which has a pH of 7.2 rather than 7.4, to remove blood, and frozen immediately in liquid N2 until extracted. AI1 radioactive compounds used in this study were quantitated by liquid scintillation counting ( Nuclear Chicago Model 6673 spectrometer). In Experiment 2, 250-g fish were killed as above, livers quickly excised and hand-perfused with fish Ringer’s solution to remove blood. A polyethylene

ground

LIPID

METABOLISM

IN RAINBOW

TROUT

183

catheter attached to a glass syringe was inserted into the hepatic vein aird the liver immediately perfused with 25°C Ringer’s solution containing t3”P]04. Perfusion was accomplished with a two-chambered infusion pump (Harvard Instrument Company) at a rate of 0.6 ml/minute for 10 minutes. Following perfusion, the liver surface was rinsed with cold Ringer’s solution and immediately frozen in liquid N,. Results were compared with those obtained by the in vivo method. [l-l%] oleic acid and [ l-l*C] palmitic acid ( Amersham-Searle), ( 55 &i/pmol) were dispersed in fish Ringer’s solution and perfused through livers from 65150-g fish in Experiment 3. Specific activity of the perfusate was either 1.0 or 1.25 &i/ml. Livers were perfused for 10 minutes with the radioactive solution and then 5 minutes with Ringer’s saline. The liver was immediately rinsed and frozen in liquid nitrogen until lipids were extracted. In Experiment 4, livers were perfused with [l%]oleic acid as described above and the incorporation into the mitochondria, microsomes and supernatant fraction determined. Lipids were extracted from livers and subcellular fractions by Palmer’s modification (Palmer, 1971) of the method of Bligh and Dyer ( 1959) in order to obtain highly polar phospholipids. Initial separation of lipid fractions was done by column chromatography using Silicar CC-7 ( Mallinckrodt, St. Louis, MO). Neutral lipids were eluted from the column with hexane and chloroform and phospholipids with a gradient of methanol in chloroform. The most polar phospholipids were eluted with 0.2% glacial acetic acid in methanol. Plates were prepared by the method of Skipsi (Skipsi et al., 1964) and phospholipids (PL) were identified by the elution order off the column, R;s in two solvent systems on thin layer plates and by reactions with spray reagents (Dittmer and Lester, 1964; Johnstone, 1971; Randerath, 1966; Skidmore and Entenman, 1962). The phosphate assay of Bartlett (Bartlett, 1959) was used to quantitate PL. Neutral lipids were quantitated gravimetrically or by the charring method of Blank (Blank et al., 1964). Livers were homogenized in two or three volumes of 0.05 M phosphate buffer, pH 7.2. Nuclei and debris were removed by centrifugation at 600g for 10 minutes, mitochondria were collected at 20,OOOgfor 20 minutes, washed once, and resedimented. Microsomes were sedimented at 105,OOOgin a Beckman L2 refrigerated ultracentrifuge at 4°C. Each fraction was immediately extracted by the method of Palmer ( 1971). The fatty acid composition of subcellular fractions was determined by transesterifying the lipids by the method of Luddy (Luddy et al., 1960) and identifying the methyl esters on an Aerograph Model 600-B gas chromatograph equipped with a hydrogen flame detector A lo-foot, I/&inch o.d. aluminum column packed with 15% DEGS on 545A Celite support (Anakron, Analabs, Inc., North Haven CT) was used with a column temperature of 185°C and a flow of 20 ml of nitrogen/minute. Unreported observations at our Food Toxicology and Nutrition Laboratory indicated that fish fed fish protein concentrate (FPC) were not as severely effected by CPFA as those fed casein. In Experiment 5, 250-g fish which had been reared on casein (diet C) or FPC (diet F) were fed 300 ppm of MS (diets Ct and F3). After 14 days the livers were perfused with [W]oleic acid as described above and the radioactivity of subcellular fraction lipids determined.

154

STRUTHERS,

LEE

AND

TABLE Tw:

SINNHUBER II

IN(WRPOIL\~~IUN 01,’ 1, \BP:~,KI) PO4 \NJ) F‘ I T T Y A(*~ns INTO (I’~(,L(JPI(uPI’:.uc)II)-~~D TROUT (DIET Cz COMPARED WITH DIET C WITH INCORPORATION IN CONTROLS = 100) Lipid

fraction

Percent Expt la 1’04 (inj.)

incorporation

Expt 2b PO1 (perf.)

Expt p4C]palmiticc

Neutral f Cardiolipin Phosphatidyl Phosphatidyl Phosphatidyl Phosphatidyl Total lipid

Se ethanolamine serine choline inositol

4.6 20.6 15.3 15.8 0.2 4.9 6.S

0 0.2 22.6 :30.9 00.0 400.0 4.0

64.4 14.8 OF.4 42.0 70.9 17.8 X.7

3 [W]oleicd 635 0.2 22.3 02.1 16.0 16.4 19.5

a Average of three injections, pooled four and six fish/diet/injecGon; 0.223 or 0.286 mCi of [a3P]04 or [32P]0a/100-gm fish injected 10 minut,es before death. *Livers, from four fish/diet perfused 10 minutes with fish Ringer’s solution containing 10 fiCi[32P]04/ml. c Four livers/diet were perfused with Kinger’s solution ront,aining 65,000 dpm of [l-Wlpalmitic acid/ml. dAverage of two separate perfusions, four livers/diet each, with 1 pCi and 1.25 @C of [l-W] oleic acid/ml. ‘An unknown phospholipid designated X. Values obtained from POa studies are those for X alone. Those from fatty acid incorporation include label in both neutral lipid and S.

RESULTS Feeding CPFA caused a marked drop in the incorporation of PO, into liver lipids in both injection and perfusion studies (Table II). Although there were some wide differences, the perfusion experiment did show that the liver per se, was affected. Total incorporation was down markedly as was the amount of labeling of the unknown phospholipid X. The amount of a particular phospholipid present had no clearcut effect on label incorporation. Phosphatidyl ethanolamine, phosphatidyl serine and phosphatidyl choline were the major PL present. The results of Experiment 3 (Table II) indicate that the incorporation of %-labeled fatty acids into phospholipids was also inhibited by CPFA feeding. The neutral lipid fraction, together with the unknown phospholipid (Neutral + X), contained a larger amount of activity in CPFA-fed fish when oleic acid was pelfused. This finding Ied to experiment 4 in which neutral lipids from subcellular fractions were separated. High levels of free oleic acid were found in a11 the subcellular fractions in CPFA-fed fish as compared to undetectable levels in the controls (Table III), The unknown X from fish fed CPFA had the lowest W-labeled fatty acid incorporation of all three subcellular fractions. The glycerides of the soluble fraction had a higher IeveI of *% in the CPFA-fed fish than in the controls. Fatty acid analysis showed that there was an increase in the amount of stearate in al1 the fractions from CPFA-fed fish (7 to 13%), but total saturated fatty acid levels were similar to those in the controls.

LIPID

METABOLISM

IN RAINBOW TABLE

EXPERIMENT

Subcell&r

20,OOOg 105,OOOg

pellet

105,OOOg

supernatant,

four pooled phospholipid

ACID LABEL LIPIDS 0~ LIVERS

Diet

pellet

a Values from b X, unknown respectively.

III

4 RESULTS: DISTRIBUTION OF [1J4C]O~~~c UNKNOWN PHOSPH~LIPID X AND IN NEUTRAL SUBCELLULAR FRACTIONS FROM PERFUSED

fraction

Lipid

C cz C CZ C cz livers/diet. ; oleate,

185

TROUT

fraction*

X

Oleate

60.12 4.44 43.84 5.16 14.46 1.64

0 32.18 0 26.28 0 17.23

Presented BS dpm/mgm free oleic acid ; MG, DG,

IN AN

MG,

DG,

TG

75.41 15.50 66.44 25.08 6.79 16.92

of lipid X 1Om3. TG, mono-, di-, and triglycerides,

Dietary protein did cause some differences in the response of trout to CPFA (Table IV). Incorporation of 14C into mitochondrial (20,OOOg pellet) neutral lipids was lowered in fish on both diets by CPFA, especially in those fed FPC. Labeling in neutral lipids from the 105,OOOgpellet was much higher in caseinfed than FPC-fed control fish. Feeding CPFA appeared to reduce 14C incorporation into microsomal lipids more in casein-fed fish than in those fed FPC. Phospholipids from the 105,OOOgsupernatant fraction contained higher levels of label when CPFA was fed, regardless of diet, but the cyclopropenoids reduced incorporation in the neutral lipids when casein was fed. Labeling with [33P]0, produced activity in an apparently neutral lipid fraction (X) that eluted just prior to cardiolipin from a silicic acid column. On tic plates, X exhibited migration patterns different from known phospholipids. Preparative tic caused degradation, but column chromatography resulted in the isolation of intact X. This lipid isolate gave one spot in two tic solvent systems, and both NMR and ir spectra were obtained. The spectral data are not incomTABLE INFLUENCX;

OF BOTH

PHOTNN

OF [~-W;OLEIC VALUEShRE

0 Values b CPFA,

IV DIET

AND

CPFA

ON THE

INCORPORATION

ACID INTO LIPIDS OF PERFUSED TROUT LIVERS DPM/MGM LIPID ORDPM/10-7 MOLE OF PoGa

Diet*

Casein Casein + CPFA FPC FPC + CPFA

SOURCE

106,000g pellet

20,000g pellet

105,000g supernatant

PL

NL

PL

NL

PL

NL

2,692 2,663 910 843

17,850 13,626 13,698 7,888

4,995 1,758 4,581 958

73,514 3,474 24,230 6,694

806 2,389 775 13,510

6,667 676 12,296 14,896

obtained from four pooled livers/diet. Perfusate 300 ppm, fed for 14 days prior to experiment,

contained 1.25 &irl-“C]oleic Diets C, C, F, and Fs.

acid/ml.

186 patible with characteristic amine, were

STRUTHERS,

LEE

AND

SINNHUBER

a cardiolipinlike structure. The main features of both spectra were of many lipids and no polar functional groups, such as ethanolseen in the NMR. DISCUSSION

The effect of CPFA on lipid metabolism in the trout does not appear to be limited to the well-known inhibition of acyl desaturase. However, it is possible that the abnormal metabolism seen here is a secondary effect of altered fatty acid composition. This physiological result of increased ratios of saturated to unsaturated fatty acids is not well understood, particularly in fish. Turnover rates of lipids were not specifically studied but if the incorporation of l”*C] oleic acid can be used as an indicator it would appear to be slowed considerably by CPFA. Fat content usually increased with CPFA feeding although incorporation of label was markedly reduced. In some cases increased lipid content of the livers contributed to the lower specific activity of lipid from CPFA-fed trout, but in no case did it account for all the difference. This study indicates some marked dietary effects on lipid metabolism and the response to CPFA. Some of the differences in the response of trout to CPFA in Experiment 5 as compared to Experiments 2-4 may have been due to the larger fish used. Larger fish eat a smaller percentage of their body weight per day and we have observed a definite size/response relationship when CPFA is fed. Compounds similar to the unidentified X have been reported by other workers. 0~01s and Marinetti (1972) have recently reported a minor unknown phospholipid from rat liver, “GPX” which becomes very highly labeled in a short period of time (15 minutes). GPX is not identical to X, but in four two-dimensional chromatographic systems the phosphate-containing breakdown product of X has R, values very similar to those reported for GPX. 0~01s and Marinetti postulated that GPX is an intermediate in the synthesis of phosphatidyl ethanolamine, since this also becomes rapidly labeled in their system. The most rapidly labeled lipid in the trout liver system other than X is cardiolipin, and it is possible that X is a cardiolipin precursor. Malevski (Malevski et al., 1974) f ound that trout fed CPFA did not incorporate label from acetate into protein or from amino acids into lipids as rapidly as did controls. He concluded this could be the result of abnormal mitochondria. This study agrees with the theory that CPFA damages mitochondrial function. The high level of free oleic acid found in the subcellular fractions of fish fed CPFA and the reduced incorporation could have been due to faulty energy metabolism. Preliminary tests of oxidative phosphorylation and mitochondrial oxidation indicate ATP formation is reduced in CPFA-fed trout (unpublished results). ACKNOWLEDGMENTS The authors are indebted to Mary E. Ambrose of the Southeast sity of Maryland, College Park, MD 20740, for the fish protein Putnam, T. Will and R. A. Foster, Jr., for feeding and caring experiments.

Utilization Center, Univerconcentrate and to G. B. for the fish used in these

REFERENCES ALLEN, E., JOHNSON, A. P., PEARSON, J. A., and cyclopropene

fatty

acids

of the desaturation

SHENSTONE, F. S. (1967).

of stearic

acid

in hen

liver.

Lipids

Inhibition by 2, 419-432.

LIPID BARTLEIT,

G. R.

( 1959).

METABOLISM

Phosphorus

assay

IN RAINBOW in

column

187

TROUT

chromatography.

J. Biol.

Chem.

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466466. BLANK, M. L., SCHMIT, J. A., and Pmvxx-r, 0. S. (1964). Quantitative analysis of lipids by thin-layer chromatography. J. Amer. Oil Chem. Sot. 41, 371-380. BLIGH, E. G., and DYER, W. J. ( 1959). A rapid method of total lipid extraction and puri&ation. Can. J. Biochem. Physiol. 37, 911-917. DIITMER, J. C., and LESTER, R. L. ( 1964). A simple, specific spray for the detection of phospholipids. J. Lipid Res. 5, 126-127. HOLMS, W. M., and STOFF, G. H. ( 1958). Studies of the respiration rates of excretory tissue in the cutthroat trout (Salmo clarki ckzrki). Physiol. Zool. 33, 9-14. JOHNSON, A. R., FOGERTY, A. C., PEARSON, J. A., SHENSTONE, F. S., and BERNSTEN, A. M. (1969). Fatty acid desaturase systems of hen liver and their inhibition by cyclopropene fatty acids. Lipids 4, 265-269. JOHNSTONE, P. V. ( 1971). “Basic Lipid Methodology.” Special Publication 19, College of Agriculture, University of Illinois, Urbana-Champaign, IL. LUDDY, F. E., BARFORD, R. A., and RIEMENSCHNEIDER, R. W. ( 1960). Direct conversion of lipid components to their fatty acid methyl esters. J. Amer. Oil Chem. Sot. 40, 716-717. MALEVSKI, Y., MONTGOMERY, M. W., and SINNWBER, R. 0. ( 1974). Liver fat and protein metabolism in rainbow trout (Salmo gairdneri) fed cyclopropenoid fatty acids. J. Fish. Res. Bd. Can. 31, 1093-1100. MILLER, A. M., SHEEHAN, E. T., and VAVICH, M. G. ( 1969). Prenatal and postnatal mortality of offspring of cyclopropenoid fatty acid-fed rats. Proc. Sot. Elcp. Biol. Med. 131, 61-66. OZOLS, R. F., and MARINE=, G. V. (1972). E vi ‘d ence for a new phospholipid intermediate in rat liver subcellular systems. Biochem. Biophys. Acta 280, 451-460. PALMER, F. B. ST. C. (1971). The extraction of acidic phospholipids in organic solvent mixtures containing water. Biochim. Biophys. Acta 231, 134-144. PHELPS, R. A., SHENSTONE, F., KEMMEFLER, A. R., and EVANS, R. (1965). A review of cyclopropenoid compounds: biological effects of some derivatives. PO&T. Sci. 44, 358-394. RAJU, P. K., and REISER, R. ( 1967). Inhibition of fatty acyl desaturase by cyclopropene fatty acids. J. Biol. Chem. 242, 379-384. RANDERATH, K. ( 1966). “Thin Layer Chromatography.” Academic Press, New York. ROEHM, J. N., LEE, D. J., POLITYCKA, S. D., and SINNHUBER, R. 0. (1970). The effect of dietary sterculic acid on the hepatic lipids of rainbow trout. Lipids 5, 80-84. SCHNEIDER, D. L., VAVICK, M. G., KWLNICK, A. A., and KEMMERER, A. R. (1961). Effect of Stesculia foetida oil on mortality of the chick embryo. PO&T. Sci. 40, 1644-1648. SHENSTONE, F. S., and VICKERY, J. R. (1959). Substances in plants of the order Malvales causing pink whites in stored eggs. Poultr. Sci. 38, 1055-1070. SINNHUBER, R. O., LEE, D. J., WALES, J. H., and AYRES, J. L. (1968). Dietary factors and hepatoma in rainbow trout (Salma gairdneri). II. Cocarcinogenesis by cyclopropenoid fatty acids and the effect of gossypol and altered lipids on aflatoxin-induced liver cancer. J. Nat. Cancer Inst. 41, 1293-1301. SKIDMORE, W. C., and ENTENMAN, C. ( 1962). Two dimensional chromatography of rat liver phosphatides. J. Lipid Res. 3, 471475. SKIPSI, V. P., PETERSON, R. F., and BARCLAY, M. ( 1964). Quantitative analysis of phospholipids by thin layer chromatography. Biochem. J. 90, 374-378.