choline level required by the rats to maintain normal liver lipid concentrations

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Quarterly Journal of Experimental Physiology (1973) 58, 275-284 FATTY ACID COMPOSITION OF TISSUE LIPIDS IN CHOLINE DEFICIENT RATS. By J. S. CEAHL* and C. C. KRATZING.

1 Quarterly Journal of Experimental Physiology (1973) 58, FATTY ACID COMPOSITION OF TISSUE LIPIDS IN CHOLINE DEFICIENT RATS. By J. S. CEAHL* and C. C. KRATZING. Department of Physiology, University of Queensland, St Lucia, Queensland, 467, Australia. (Received for publication 18th December 1972) (Revised version 26th March 1973) Rats fed a choline deficient diet for three weeks had up to 38% of their liver weight in lipids, compared with 6.5% in choline supplemented controls. This large increase in liver lipids was due to an accumulation of triglycerides, whose fatty acid composition was not altered in choline deficiency although the plasma triglyceride concentration of stearic acid was increased and that of oleic acid was decreased. In choline-deficient rats, the concentration of arachidonic acid was decreased in liver phospholipids while that of docosapolyenoic acids was increased. There were more widespread changes in the cholesterol ester fatty acids of liver and plasma in choline deficient rats. It is proposed that selective impairment of transport of lipid species, depending on fatty acid composition, occurs in choline deficiency. In rats fed a choline deficient diet the concentration of liver lipid is increased, while that of plasma is decreased [Lombardi, Pani and Schlunk, 1968]. This increase in liver lipids is due mainly to triglycerides, which are normally removed from the liver as components of very low density lipoproteins (VLDL). In choline deficiency, the plasma VLDL concentration is also reduced [Lombardi et al., 1968; Olsen et al., 1958], and it has been suggested that the fatty liver of choline deficiency is due to a failure of the liver to secrete VLDL. It remains to be established if this is the primary cause. The environmental temperature at which rats are kept is a factor which determines liver lipid accumulation in choline deficiency [Chahl and Kratzing, 1966a and b]: with increasing environmental temperature the fatty liver gets more severe. Chahl and Kratzing [1966b] have also shown that the dietary choline level required by the rats to maintain normal liver lipid concentrations increases with increasing environmental temperature, over the range -35, but these effects were not significant if a low protein, choline deficient diet was fed [Chahl and Kratzing, 1966a]. In rats maintained at low temperatures, the proportion of unsaturated fatty acids in adipose tissue was higher than that of rats maintained at higher temperatures [Mefferd, Nyman and Webster, 1958; Young and Cook, 1955]. Liver lipid concentration in choline deficient rats was reduced when oleic acid was included in the diet [Benton, Harper and Elvehjem, 1956]. These observations suggest that in the rat the role of choline may be to transfer saturated fatty acids out of the liver, and it is likely that if dietary choline were to affect fatty acid metabolism, changes would be apparent in the fatty acid profiles of liver, plasma and adipose tissue lipids. Experiments were designed to see what change occurted in the proportion * Present address: Department of Human Biology, Faculty of Medicine, University of Papua and New Guinea, Post Office Box 5623, Boroko, Papua, New Guinea. 275

2 276 Chahl and Kratzing of saturated and unsaturated fatty acids in the tissue lipids of rats fed a choline deficient diet. The rats were made to live at 33 because of the greater effect of choline deficiency on liver lipid accumulation at higher temperatures [Chahl and Kratzing, 1966b]. It was thought that variations of the fatty acid composition of lipids from rats at low temperatures might be difficult to interpret in the absence of simultaneous increase in liver lipid concentration. METHODS Rats. The rats were male white albinos originating from the Wistar strain, and inbred for more than twenty years at the Department of Physiology, University of Queensland, Brisbane. They were 4-6 weeks old at the start of the experiment and weighed between 7-1 g. Two groups of six rats each were kept in individual cages at an environmental temperature of 33+2 for 3 weeks and fed either the choline-supplemented or the choline-deficient diet, diet B of Chahl and Kratzing [1966b]. The dietary fatty acid composition was myristic 2.45%, myristoleic -81%, palmitic 23-11%, palmitoleic 3-68%, stearic 17.21%, oleic 42-78%, linoleic 15.4% and arachidic 1-96%. Food and water were provided ad libitum. At the end of the experiment, under ether anaesthesia samples of blood were collected from the posterior vena cava into a heparinized syringe, and liver and samples of peri-renal adipose tissue also collected. Liver plasma, and adipose tissue lipids, were extracted by the method of Wheeldon and Collins [1957]. Lipid analyses. Total liver lipids were determined gravimetrically. Separation of lipids. Liver and plasma lipids were separated into their major classes by thin layer chromatographic techniques. Glass plates (2 x 2 cm) were coated with a -3 mm layer of Silica Gel G (Merck) using the apparatus and methods of Chahl and Kratzing [197]. Samples of lipid solutions were applied to the adsorbent surface by means of the multisample applicator of Chahl and Kratzing [1969]. To separate the major lipid classes, plates were developed in petroleum ether (6-8 ): diethyl-ether : acetic acid, 8 :2: 1 by volume. Solvents used were of distilled grade. Antioxidants were not used in the thin layer separation of lipids. In order to separate liver phospholipids, lipid solutions were applied onto the plates and immediately placed in a light-proof tank continuously flushed with a stream of dry N2 to evaporate the solvents. The plates were developed in chloroform : methanol : water, 65 : 25 : 4 by volume, and the separated lipid components made visible, either by exposure to iodine vapour, or by spraying with.5% Rhodamine B in 95% ethanol and examining for fluorescence in ultraviolet light. The Rhodamine B stain was used for lipid samples separated for gas chromatography. Major lipids were identified by reference to standard lipids (Hormel Institute TLC Model Mixture 2A). Phospholipid classes were identified by paper chromatography of the glyceryl phosphoryl bases [Dawson, 1958]. Quantitative analysis. Liver triglycerides were determined by the method of Amenta [1964]. Quantitative recovery of tripalmitin (Sigma, 99%) and corn oil triglycerides was obtained with this method [Chahl and Kratzing, 197]. Total liver phospholipids were determined using the method of Rouser, Siakotos and Fleischer [1966]. Liver lecithin was determined after thin layer separation of phospholipids [Chahl, 1971]. Plasma free fatty acids were determined by the method of Dole [1956]. Liver cholesterol and cholesterol esters were taken to be the difference between total liver lipids and the sum of triglycerides and phospholipids. Gas chromatography of the methyl esters of fatty acids. After separation of lipids by TLC, lipid species containing the esterified and free fatty acids were analyzed for their

3 Fatty Acids in Choline Deficient Rats 277 component fatty acids. The fatty acids were converted to their methyl esters [Stoffel, Chu and Ahrens, 1959] and analyzed using a Perkin-Elmer Gas Chromatograph (Model 81) equipped with dual columns and a flame ionization detector. Stainless steel columns, 12 ft long, i.d in., were packed with 6-8 chromosorb W. acid washed (John Mansville, U.S.A.), and coated with 15% diethylene glycol succinate. Nitrogen (dry and oxygen free) was used as the carrier gas with the inlet pressure at 5 p.s.i. and a flow rate of 3 ml. per min at 21 C. The injector temperature was at 2 C, the oven at 19 C and the detector at 26 C. Fatty acid methyl ester peaks were identified by reference to NIH type mixtures KA and KC (National Institute of Health, Bethesda, Maryland) and GLC reference mixtures 1, 4-6 and methyl arachidonate (Hormel Institute, University of Minnesota). The proportion of esters in any given sample was determined from the product of height of peak and the retention time of the sample [Carroll, 1961]. Quantitative results of standard mixtures agreed with the stated composition data to within 5% for major components (i.e. those more than 1% of total mixture) and to within 1% for the minor components (i.e. those less than 1% of the total mixture). Results were statistically analyzed using Student's t-test. RESULTS Rats fed the choline deficient diet had a greater amount of liver lipid than those offered choline in the diet (Table I) which was due to changes in neutral lipids (Table II). The mean value of liver triglycerides was 23-4 mg/g of lipid-free liver in the choline supplemented group and 5-2 mg/g in the choline-deficient group. The mean liver cholesterol and cholesterol ester content in the choline supplemented group was 12 mg/g of lipid-free liver, and 9 mg/g in the cholinedeficient group. There was no change in the total liver phospholipid content with choline deficiency but the proportion of lecithin in phospholipids decreased from to 38.37%. Table III shows the distribution of fatty acids in liver lipids. Despite a large increase in the total triglyceride content in choline deficiency, the relative fatty acid composition of the triglyceride fraction was similar to that in the choline supplemented group. Two other changes occurred in the choline deficient group: firstly, the arachidonic acid content of phospholipid fatty acids was decreased and that of docosapolyenoic acids was increased; and secondly, the proportion of stearic acid in cholesterol esters was decreased, whilst that of linoleic acid was increased. The fatty acid composition of plasma and adipose tissue lipids is shown in Table IV. Stearic acid concentration of plasma phospholipids was lowered in choline deficiency. In plasma triglycerides, the concentration of stearic acid increased and that of oleic acid decreased in rats fed the choline-free diet. The plasma concentrations of cholesterol esters containing palmitic, oleic and linoleic acids decreased, those of myristoleic and palmitoleic acid increased, and those of arachidonic acid remained unchanged, in choline deficiency. There were no significant changes in plasma or adipose tissue fatty acids when rats were given the choline-free diet. Tissue fatty acid profiles appeared to be similar to those of dietary lipids and were not affected by the presence of choline in the diet. An exception was linoleic acid, found at a higher concentration in tissue lipids than in the diet.

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6 28 Chahl and Kratzing ) CD ) H -H "- -H-"'-H 1 Co CZ CO 11 cli -A< -H 4- ) )a Cw _-14 L _ l o E o co 1 ) t- 1 M t 11 CD tc C 1) cq -H cs -H-"-H--H ei-h ei-h--h -f c:xe:eo cq wqoc w CO e4 1 _O14 _ )1 co _ -C-) Q 4 -H -4 H +-H-H1-H -HC -H4 -f -H c~ q~~~~ ~ ~~~~~ 5d+ d O Ca ) m C E- t- 1 t1 O * * - CO 1 H1H -f A es cq m cc 11 COC9 CB 11 ' -H -4 > CiO C:_ I~~ -H 4CqI4 tg~-d C gl -4 t co o t-o~ c o411 CO X CO COC CO 1 CO 1 C Ca C) < CO 1 CO CO CO CO b CO ) ) O ) CO X~~ E- X X _C CO - CO L CO s4 _ C1 _ A - _HiHC O+~+e+L A *E~)w. CIO, = to Ci = ) -H1" -H ~~~~~~ ) ) ) * CD Co *-.* _e 6 h1 G ) OC: H CA CO -H*.- O~~~~ C -H i - Ce > c6c4)64 ; -4 CD 1 Co 1 CD 1 co 1 C~~~1 " ~ ~ 4a + C o > 4 H C).- *_s# *H4, $ Hq, Ci *- ; CC) ~ O o D ~~~~~~a iv V V-- * +-+?=$ '

Fatty Acids in Choline Deficient Rats 281 DIsCUSSION Triglycerides and cholesterol esters The present study confirms observations [Ridout et al., 1954; Tinoco et al.

7 Fatty Acids in Choline Deficient Rats 281 DIsCUSSION Triglycerides and cholesterol esters The present study confirms observations [Ridout et al., 1954; Tinoco et al., 1965], that fatty liver in choline deficient rats is due to increased deposition of triglycerides in the liver. Liver cholesterol esters are also increased when choline free diets are fed [Tinoco et al., 1965; Wells and Hogan, 1968] and it is likely that the large increase in cholesterol and cholesterol ester fraction noted in the present study was due to increase in the cholesterol ester species. No change in the free cholesterol fraction has been reported [Tinoco et al., 1965]. Triglyceride fatty acids There was a close similarity in the fatty acid composition of liver triglycerides in the choline-deficient and choline-supplemented groups, despite an almost twenty-fold increase in the triglyceride content of the liver in the choline deficient group (Tables 1-4). This does not support the hypothesis that fatty liver in choline deficiency results from a lack of transfer of saturated fatty acids out of the liver. There was no significant change in the triglyceride fatty acid composition of adipose tissue but there was a small, but significant, change in the triglyceride fatty acids of plasma, where in the choline deficient group the stearic acid concentration was less than the control group. Not much significance can be attached to these observations, since in choline deficiency liver triglyceride fatty acids have not altered even though a large amount of triglycerides had accumulated in the liver (Tables I, II and III). The fatty acid profiles of liver, plasma and adipose tissue triglycerides bear a close similarity to the composition of dietary fatty acids (Tables III and IV) [Chalvardjian, 1966; Glende and Cornatzer, 1965; Miller and Ellis, 1965]. Phospholipid fatty acids Beare-Rogers [1969] has reported an inverse relationship between the concentration of arachidonic acid in liver phospholipids and liver lipid content. In the present study the arachidonic acid concentration of liver phospholipids was decreased in choline deficient rats (Table III). This finding is also in agreement with the observations by Chalvardjian [1966] and Tinoco et al. [1965]. The relationship between arachidonic acid concentration of liver phospholipids and liver triglyceride content has not been satisfactorily explained, since Chalvardjian [1966] observed that when 3% fat was included in the diet of choline deficient rats, the liver lipid content increased despite an increase in the concentration of arachidonic acid in liver phospholipids. It is also difficult to reconcile the relationship between arachidonic acid levels and fat accumulation in the liver with the finding that, although the arachidonic acid concentration in total phospholipids was decreased, the proportion present in liver lecithin was not altered in choline deficient rats [Beare-Rogers, 1969]. Yamamoto, Sano and Isozaki [1969] report that the proportion of arachidonate in liver lecithins of choline deficient rats is increased. These conflicting reports from different laboratories do not support the notion that arachidonic acid

8 282 Chahl and Kratzing content of lecithins has any direct effect on liver lipid accumulation. It is more likely that liver lipid accumulation is related to total lecithin concentration of the liver and it has been consistently observed that liver lecithin levels are lowered in choline deficiency [Beare-Rogers, 1969; Blumenstein, 1968]. This was also confirmed in the present study (Table II). The very large increase in the docosapolyenoic acids (Table III) is probably the result of failure to methylate phosphatidyl ethanolamine for its conversion to phosphatidyl choline. It is well established that the availability of methyl groups for lecithin synthesis is severely restricted in choline deficiency [Snyder and Cornatzer, 1958]. There has been extensive speculation as to the role of lecithin species derived from phosphatidyl ethanolamine in the transport of triglycerides out of the liver, but no definite evidence that they are involved [Beare-Rogers, 1969; Blumenstein, 1968; Tinoco, Shannon and Lyman, 1964]. Liver lecithin contains very little docosapolyenoic acids and these are not changed in choline deficiency [Beare-Rogers, 1969]. In part, the much larger concentration of docosapolyenoic acids in liver phospholipids found in our experiment (24% in our study compared with 6% in Beare-Rogers [1969]) may be due to the more severe fatty liver that is produced under our experimental conditions, 38% liver weight as lipid as compared with 1% in the experiments of Beare-Rogers [1969]. Cholesterol ester fatty acids Changes in cholesterol ester fatty acids in our experiments were different from those previously reported. The data of Miller and Ellis [1965] suggests that in liver cholesterol esters the fatty acid composition is not affected by choline deficiency. The composition of oleic acid and linoleic acid reflects more closely the dietary fatty acid composition. This also appears to be the case in the data of Chalvardjian [1966] but only when 3% of the diet contained lipids. In our experiments the stearic acid concentration was decreased (P <.1) and that of oleic and linoleic fatty acids was increased. Changes in plasma cholesterol esters in choline deficiency were also different from those reported previously by Chalvardjian [1966] and Tinoco et at. [1964]. No significant changes were observed by Tinoco et al. [1964] but their data was obtained from starved rats. No strict comparison can be made between our data and the data of Chalvardjian [1966], because of the differences in the fat content of the diet. In choline deficiency we noticed that the changes were widespread and very marked (Table IV). The concentrations of myristoleic and palmitoleic were increased while that of palmitic, oleic and linoleic acids were decreased. It is likely that these differences in composition of the cholesterol esters observed in our experiments, are the result of a decreased transport of these particular fatty acid cholesterol esters out of the liver in choline deficiency. This might be due to the reduction in concentration of the very low density plasma lipoproteins which has been reported to occur in choline deficiency [Lombardi et al., 1968; Olson et al., 1958], and which are the major vehicle for the transport of cholesterol esters [Gidez, Roheim and Eder, 1967]. The selective

Fatty Acids in Choline Deficient Rats impairment of the transport of other lipid species differing in fatty acid composition is also likely, but it remains to be established whether change in a

9 Fatty Acids in Choline Deficient Rats impairment of the transport of other lipid species differing in fatty acid composition is also likely, but it remains to be established whether change in a particular lipid species is the primary failure in choline deficiency which leads to a fatty liver. ACKNOWLEDGMENTS This work was supported by funds from the Australian Research Grants Committee. REFERENCES AMENTA, J. S. (1964). A rapid chemical method for quantification of lipids by thin layer chromatography. Journal of Lipid Research, 5, BEARE-ROGERS, J. L. (1969). Liver phospholipids of rats deprived of dietary choline. Canadian Journal of Biochemistry, 47, BENTON, D. A., HARPER, A. E. and ELVEHJEM, C. A. (1956). The effects of different dietary fats on liver fat deposition. Journal of Biological Chemistry, 218, BLUMENSTEIN, J. (1968). Studies in phospholipid metabolism 2. Incorporation of metabolic precursors of phospholipids in choline deficiency. Canadian Journal of Physiology and Pharmacology, 46, CARROLL, K. K. (1961). Quantitative estimation of peak areas in gas-liquid chromatography. Nature (London), 191, CHAHL, J. S. (1971). Quantitative determination of phospholipids. Experientia, 27, CHAHL, J. S. and K.RATZING, C. C. (1966a). Environmental temperature and choline requirements in rats. I. Choline deficiency in rats at various temperatures. Journal of Lipid Research, 7, CHAHL, J. S. and KRATZING, C. C. (1966b). Environmental temperature and choline requirements in rats. II. Choline and methionine requirements for lipotropic activity. Journal of Lipid Research, 7, CHAHL, J. S. and KRATZING, C. C. (197). Simple apparatus for preparative and quantitative thin layer chromatography. Laboratory Practice, 19, CHAHL, J. S. and KRATZING, C. C. (1969). A multisample applicator for quantitative thin layer chromatographic analysis. Clinica Chimica Acta, 26, CHALVARDJIAN, A. (1966). Mode of action of choline. I. Fatty acid composition of liver, serum and adipose tissue of choline deficient rats. Canadian Journal of Biochemistry, 44, DAWSON, R. M. C. (1958). A hydrolytic procedure for the identification and estimation of individual phospholipids in biological samples. Biochemical Journal, 75, DOLE, V. P. (1956). A relation between non-esterified fatty acids in plasma and the metabolism of glucose. Journal of Clinical Investigation, 35, GIDEZ, L. I., ROHEIM, P. S. and EDER, H. A. (1967). Turnover of cholesteryl esters of plasma lipoproteins in the rat. Journal of Lipid Research, 8, GLENDE, E. A. and CORNATZER, W. E. (1965). Fatty acid composition of rat liver lipids during choline deficiency. Journal of Nutrition, 86, LOMBARDI, B., PANI, P. and SCHLUNK, P. F. (1968). Choline deficiency fatty liver: Impaired release of hepatic triglycerides. Journal of Lipid Research, 9, MEFFERD, R. B. Jr., NYMAN, M. S. and WEBSTER, W. W. (1958). Whole body lipid metabolism of rats after chronic exposure to adverse environments. American Journal of Physiology, 195, MILLER, G. J. and ELLIs, W. W. (1965). Effects of dietary lipid and diethyl-stilbestrol upon liver fatty acids of choline-deficient rats. Journal of Nutrition, 86, OLSON, R. E., VESTER, J. W., GURSEY, D., DAVIS, N. and LONGMAN, D. (1958). The effect of low protein diets upon serum cholesterol in man. American Journal of Clinical Nutrition, 6, RIDOUT, J. H., LUCAS, C. C., PATTERSON, J. M. and BEST, C. H. (1954). Changes in chemical composition during the development of 'cholesterol fatty livers'. Biochemical Journal, 58, ROUSER, G., SIAROTOS, A. N. and FLEISCHER, S. (1966). Quantitative analysis of phospholipids by thin-layer chromatography and phosphorus analysis of spots. Lipids, 1, I 1.

10 284 Chahl and Kratzing SNYDER, F. and CORNATZER, W. E. (1958). Effect of choline on the incorporation of 35S-1-methionine into liver protein of rats with fatty livers. Biochimica Biophysica Acta, 27, STOFFEL, W., CHU, F. and ARRENS, E. H. JR. (1959). Analysis of long chain fatty acids by gas liquid chromatography. Micro-method for preparation of methyl esters. Analytical Chemistry, 31, TINOCO, J., SHANNON, A. and LYMAN, R. L. (1964). Serum lipids in choline deficient male and female rats. Journal of Lipid Research, 5, TINOCO, J., SHANNON, A., MILJAMICH, P., BABCOCK, R. and LYMAN, R. L. (1965). Liver lipids of choline deficient rats. Biochemical Journal, 94, WELLS, I. C. and HOGAN, J. M. (1968). Effect of dietary deficiencies of lipotropic factors on plasma cholesterol esterification and tissue cholesterol in rats. Journal of Nutrition, 95, WHEELDON, L. W. and COLLINS, F. D. (1957). Studies on phospholipids. 1. The determination of amino nitrogen in unhydrolysed phospholipids. Biochemical Journal, 66, YAMAMOTO, A., SANO, M. and IsozANi, M. (1969). Studies on phospholipid metabolism in choline deficient fatty liver. Journal of Biochemistry (Tokyo), 65, YOUNG, D. R. and COOK, S. F. (1955). Body lipids in small mammals following prolonged exposure to high and low temperatures. American Journal of Physiology, 181,

Turnover of Individual Cholesterol Esters in Human Liver and Plasma*

Journal of Clinical Investigation Vol. 45, No. 7, 1966 Turnover of Individual Cholesterol Esters in Human Liver and * P. J. NESTEL t AND E. A. COUZENS (From the University of Melbourne Department of Medicine,