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Effects of fish oil and safflower oil emulsions on diet-inducedhepatic steatosis in rats receiving total parenteral nutrition
Clinical Nutrition (1996) 15:80-83 © Pearson Professional Ltd 1996
Effects of fish oil and safflower oil emulsions on dietinduced hepatic steatosis in rats receiving total parenteral nutrition S. L. YEH 1, W. J. CHEN 2 and P. C. HUANG a 1School of Nutrition and Health Science, Taipei Medical College, Departments of 2Surgery and 3Biochemistry, College of Medicine, National Taiwan University, 7 Chung-Shan S Road, Taipei, Taiwan 100, Republic of China (Reprint requests and correspondence to VV. J. C.) ABSTRACT--This study was conducted to investigate the effects of fish oil and safflower oil emulsions in total parenteral nutrition (TPN) solutions on diet-induced hepatic steatosis. Rats were divided into a control group (C, n = 6) and four experimental groups (A, B, S, F, n = 11~14). The control group was fed a chow diet whereas the experimental groups received a high fat (15%, w/w) diet containing 0.1% (w/w) cholesterol. Group A received the high fat diet for 4 weeks, and was killed at the end of the fourth week to ensure that hepatic steatosis had occured. Groups S and group F received TPN with safflower oil or fish oil emulsions, respectively, for 1 week following experimental diet feeding for 4 weeks. Group B was fed a limited amount of the high fat diet, without cholesterol, for 1 week following 4 weeks of experimental diet in order to maintain the same body weight and cholesterol intake as the TPN groups. Diet-induced hepatic steatosis was observed in the experimental groups. Fat deposition was reversed when the total caloric and cholesterol intake was reduced. Fish oil infusion ameliorated the severity of hepatic steatosis, whereas safflower oil had no effect on liver fat deposition. These results suggest that TPN with fish oil emulsions may be beneficial to patients with diet-induced hepatic steatosis.
lesterol. The composition of the high fat diet fed to each group is shown in Table 1. After 4 weeks, rats in group A were killed to ensure the occurrence of diet-induced hepatic steatosis. The rats in groups S and F were fitted with internal jugular catheters and were infused with TPN solutions for 7 days. TPN provided 250 kcal/kg body weight/ day with 30% energy as fat. The energy density of TPN solution was 1 kcal/ml and the calorie/nitrogen ratio was 146 (kcal/g nitrogen). The basal TPN solutions were
Introduction
Total parenteral nutrition (TPN) is widely used for the treatment of nutritional depletion. Hepatic dysfunction is a well-described complication of TPN especially during hypercaloric infusion, and hepatic steatosis is the earliest and most frequently reported histological change (1). Many studies have focused on the attenuation of the development of hepatic steatosis (2-6). Previous work in this laboratory demonstrated that, unlike olive oil and safflower oil emulsions, TPN with a fat emulsion prepared with fish oil did not induce hepatic steatosis in normal rats (7). These results were similar to those found by Rustan et al (8), who reported that liver triglycerides were significantly lower in rats receiving a high fish oil diet compared with those fed a sunflower oil diet. Since diet-induced hepatic steatosis is not uncommon in the Taiwan population, this study was designed to evaluate the effect of fish oil and safflower oil emulsion on diet-induced hepatic steatosis in TPN rats.
Table 1
Composition of the experimental diet
Ingredients Fat (p/S)* Corn starch Protein (casein) Cholesterol Salt mixture t Vitamin mixture* Methyl cellulose Choline chloride DL-methionine
Materials and methods
% (w/w) 15 (1) 57 20 0.1 3.5 1 3 0.095 0.3
* P/S: polyunsaturated/saturated fatty acid t Salt mixture contains the following (mg/g): calcium phosphate dibasic 500 mg, sodium chloride 74 mg, potassium sulphate 52 mg, potassium citrate monohydrate 220 rag, magnesium oxide 24 mg, manganese carbonate 3.5 mg, ferric citrate 6 mg, zinc carbonate 1.6 mg, cupric carbonate 0.3 mg, potassium iodate 0.01 mg, sodium selenite 0.01 mg, chromium potassium sulphate 0.55 mg. Vitamin mix contains the following (mg/g): thiamine hydrochloride 0.6 mg, riboflavin 0,6 mg, pyridoxine hydrochloride 0.7 rag, nicotinic acid 3 mg, calcium pantothenate 1.6 mg, D-biotin 0.02 rag, cyanocobalamin 0.001 mg, retinyl palmitate 1.6 mg, DL-ot-tocopherol acetate 20 mg, cholecalciferol 0.25 mg, menaquinone 0.005 mg.
Animal grouping and feeding schedule Male Long-Evans rats weighing 70-100 g were used in this study. Rats were divided into a control group (C, n = 6) and four experimental groups (A, B, S, F, n = 11~14). The control group was fed with a chow diet (Purina No. 5001) whereas the experimental groups were fed with a high fat (15%, w/w) semipurified diet containing 0.1% (w/w) cho80
CLINICAL NUTRITION
isonitrogenous and identical in nutrient distribution, except for the composition of the fat emulsion (Table 2). The fat emulsion for group S was prepared with safflower oil (Taiwan Sugar Co, R.O.C.), and that of group F with fish oil (TAMA Biochemical Co, Japan). Oil emulsions were prepared with soy lecithin according to the method of Thompson et al (9). The fat emulsions contained 10 g of oil and 1.2 g of soy lecithin per 100 ml as previously described (7). The fatty acid patterns of the two fat emulsions are shown in Table 3. Rats in group B were also fed with the high fat cholesterol diet for 4 weeks. Because there is no cholesterol in TPN solutions, these rats were given the same high fat diet, but without cholesterol, for the following 7 days. Food intake was also reduced to adjust the body weight to values comparable to those of the TPN groups. The feeding schedules are shown in Figure 1. The aminals were maintained in a room at 21°C with a 12 h light/dark cycle. Infusion was started 24 h after cannulation for the TPN rats. Two ml per hour were administered on the first day. Full strength TPN was given thereafter, and continued for 7 days. Infusion speed was controlled by a Terufusion pump
Table 2 Formulationof the TPN solution(ml/1) Ingredients Glucose 50% MoriaminSN 10%* Fat emulsion20% NaC1 3% KC17% K3PO4 8.7% Ca-gluconate 10% MgSO4 10% ZnSO4 0.6% Multivitat VitaminC 5% H20 Cholinechloride (g)
260 450 173 54 7 7 7 3.5 1.5 1.5 1.5 34 1
* From ChinesePharmaceuticalsCo. Taiwan.Per 100 ml containsLeu 1250 mg, Ile 560 mg, Lys acetate 1240 mg, Met 350 mg, Phe 935 mg, Thr 650 mg, Trp 130 mg, Va1450 mg, Ala 620 rag, Arg 790 nag, Asp 380 mg, Cys 100 rag, Glu 650 mg, His 600 mg, Pro 330 mg, Set 220 mg, Tyr 35 mg, Aminoaceticacid (Gly) 1570 mg. t Yu-LiangPharmaceuticalsCo. Taiwan.Per ml containsascorbic acid 100 mg, vitaminA 2000 IU, ergocalcifero1200IU, thiamineHC1 l0 mg, riboflavin2 mg, niacinamide20 rag, pyridoxineHC1 3 rag, D-panthenol 5 mg, dl-ct-tocopherylacetate 1 mg.
Table 3 Fattyacid profiles of the lipid emulsions* Fatty a c i d 8:0 10:0 14:0 16:0 16:1 n-7 18:0 18:1 n-9 18:2 n-6 18:3 n-3 20:4 n-6 20:5 n-3 22:6 n-3
Saffloweroil
7.0 3.6 17.4 71.3 0.6
* Expressed as relativepercent.
Fish oil
7.8 15.3 9.8 1.9 11.7 4.6 1.6 2.6 31.2 13.1
1 Group
I--I
2
3
[
4
I
81
5 (week)
1
I
b+++++
Fig. 1 Feedingschedule of the all groups. . . . . . . chow diet, high fat diet with 0.1% cholesterol, === high fat diet withoutcholesterol, ++++TPN with fat emulsionmade of fish oil, ** ** * TPN with fat emulsion made of safflower oil.
(Model STC-503, Terumo Co, Japan). All animals were allowed to drink water freely during the experimental period.
Measurements and analytical procedures Animals in the control group and group A were killed after 4 weeks. The TPN rats and rats in group B were killed at the end of the fifth experimental week. After fasting overnight, bleeding time was measured in the control and TPN groups. Rats were anesthetized and the tips of their tails (5 ram) were cut off with a razor blade. The blood was allowed to flow into saline at 37°C, and the time for blood flow to stop was measured (10). Each animal was exsanguinated by drawing arterial blood from the aorta. The entire liver was rapidly excised and stored at - 7 0 ° C until assay. Blood was collected into tubes containing EDTA-Na 2 and immediately centrifuged. Plasma was analyzed for non-esterified fatty acids (NEFA), triglycerides (TG) and very low-density lipoprotein (VLDL)-TG. VLDL was obtained by ultracentrifugation at a density of 1.006. Plasma TG, VLDL-TG, and NEFA were determined by colorimetric methods after enzymatic reaction with peroxidase (Randox Co, Ireland). Liver lipid was extracted with a chloroformmethanol 2:1 mixture according to Folch et al (11). Total lipid was gravimetrically measured after drying in an evaporator to constant weight (12). Triglycefides were determined by the method of Soloni (13). Cholesterol was measured according to the method of Carlson and Goldfard (14).
Statistics Data are expressed as mean + SD. D u n c a n ' s test following analysis of variance (ANOVA) was used to compare different groups. P values less than 0.05 were considered statistically significant.
Results
Body weights There were no differences in body weight between any of the groups after feeding with rat chow or the experimental
82 EFFECT OF FAT EMULSIONS ON DIET INDUCED HEPATIC STEATOSIS IN TPN
diet for 4 weeks. No difference in body weight gain was seen between the two TPN groups during infusion for 1 week (S:1.9 + 4.6 g vs F:4.3 + 6.2 g, P > 0.05).
Liver lipid content Hepatic steatosis was observed in group A, mainly due to TG accumulation. Group B had lower total liver lipids than groups A and S. Group S had a significantly higher liver lipid content than the other TPN groups except group A. Rats in group F had lower total liver lipids than those in groups A and S but were not different from the control group and group B. The pattern of liver TG and cholesterol levels among the groups was similar to those of the total liver lipids (Table 4).
Plasma lipid concentration Groups A and F exhibited higher plasma TG levels than the control group. No differences in plasma TG, VLDL-TG and NEFA were seen among the four experimental groups (Table 5).
Bleeding time There was no difference in bleeding time between the control group and the two TPN groups. The bleeding times were 3.8 + 0.4, 3.4 + 0.6, and 3.2 _+0.8 rain for the control, safflower oil and fish oil groups, respectively.
Table 4 Group
Hepatic lipid content of the control and the experimental groups TL
TG
Chol
mg/g wet tissue C (n = 6) A (n = 11) B (n = 12) S (n = 14) F (n = 13)
64.0 116.9 92.5 109.3 79.6
_+ 7.2 a + 26.0 b _+ 21.2 ° _+ 28.5 b _+ 16.2 ac
18.5 65.3 43.4 56.2 29.0
_+ 8.8 a + 15.9 b _+ 18.1 ° -+ 25.0 b _+ 13.5 ac
4.0 10.9 7.9 8.9 7.8
_+ 0.7 a + 2.9 b _+ 2,1 c _+ 1.6 c _+ 1.9 c
TL: total lipids Values in a column with different superscripts indicate significant differences (P < 0.05).
Table 5 Plasma triglyceride (TG), very low density lipoprotein (VLDL)TG and non-esterified fatty acid (NEFA) in the control and the experimental groups Group
TG
VLDL/TG
NEFA mmol/L
0.16_+0.06 0.33_+0.16 0.34+0.15 0.33-+0.17 0.43_+0.15
0.398_+0.132 0.338_+0.085 0.331+0.161 0.432-+0.147 0.357_+0.147
mmol/L C(n=6) A ( n = 11) B ( n = 12) S ( n = 14) F ( n = 13)
0.30_+0.06 0.54_+0.32* 0.50+0.22 0.50-+0.21 0.61-+0.21"
*: significantly different from C group (P < 0.05).
Discussion
This study was designed to investigate the effects of TPN containing emulsions with different fatty acid composition on diet-induced hepatic steatosis in rats. According to a dietary survey in Taiwan 1986-1988 (15), carbohydrate and fat intake provided 49.8% and 35.6%, respectively, of the total energy. The polyunsaturated fatty acid/saturated fatty acid (P/S) ratio was 1.35. Since hepatic steatosis is not uncommon in the people of Taiwan, an experimental diet was designed with a nutrient distribution and P/S ratio close to the Chinese diet in Taiwan. This diet was used to induce hepatic steatosis in rats. Marked hepatic steatosis was observed after rats were fed with a high fat diet ad libitum for 4 weeks. Total lipid content of the livers of rats in the experimental groups was 80% higher than those of the control group. Since liver fat accumulation can be induced by TPN providing excess energy, 250 kcal/kg body weight per day was administered during the infusion period. This level of energy intake was assumed to be adequate, without causing hepatic steatosis in TPN rats (5, 16). However, the body weights of TPN rats were only marginally maintained. Cholesterol was not present in TPN solutions, so the effect of withdrawing cholesterol on hepatic steatosis should be examined. Rats in group B received a high fat diet without cholesterol for 7 days after 4 weeks of the high fat cholesterol diet. The results demonstrated that group B had significantly lower total liver lipids than group A. This suggests that hepatic steatosis induced by diet can be reversed by reducing total energy and cholesterol intake. Most commercially available fat emulsions used in TPN are prepared with soybean oil. In order to investigate the different metabolic effects of n-3 and n-6 fatty acids on hepatic lipids, safflower oil was used as fat source in this study. This is because safflower oil has a higher linoleic acid (C18:2, n-6) content than soybean oil (71.3% vs 52.4%). Infusion of a safflower oil emulsion had no effect on hepatic steatosis; the total hepatic lipid content was similar before and after TPN administration. However, the severity of hepatic steatosis was ameliorated by fish oil infusion. Total liver lipids were significantly lower in group F compared to group S and group A. Liver lipids in group F were not different from the control group. A similar result was also reported by Rustan et al (6) in an oral feeding study. Hepatic steatosis can result from an imbalance in the rate of entry, synthesis and removal of fat from the liver. This imbalance may result from alterations in 1. the rates of hepatic synthesis of fatty acids and TG 2. secretion of TG or other compounds containing the fatty acyl moiety via plasma or bile 3. uptake of TG and fatty acid 4. TG hydrolysis and fatty acid oxidation. Our study found that the plasma NEFA and VLDL-TG levels were not different between the experimental groups. Although we did not measure VLDL secretion directly, it appears that hepatic lipid uptake and VLDL secretion were
CLINICAL NUTRITION 83
not the causes of the differences of hepatic lipid content. Rustan et al (6) and Halminski et al (17) reported that fish oil feeding led to greater peroxisomal fatty acid oxidation and suppression of liver TG synthesis compared with c0-6 fatty acid rich oils. Willumsen et al (18) and Christensen et al (19) reported that the increment in peroxisomal fatty acid oxidation was attributable to the effects of EPA and DHA. It has also been reported that glucagon-stimulated adenylate cyclase activity is enhanced in the livers of fish-oil fed rats (20), and lipolysis in the liver may occur in the presence of increased cyclic AMP. Although TPN differs from oral feeding in the route of administration and the composition of nutrients, EPA and DHA in fish oil providing the fat sources in TPN may still play important roles in preventing hepatic TG accumulation. Previous work in this laboratory showed that lipid peroxidation products did not accumulate in liver tissue after administration of fish oil emulsion (7). In order to examine the possible adverse effect of fish oil, bleeding time was measured in the control and TPN groups, but no differences were found. This finding might be explained by an inadequate administration period or because the antiaggregatory effect of fish oil was not potent enough to prolong bleeding time. Nevertheless, no clinical evidence of bleeding has been observed in any fish oil feeding studies (21). The plasma TG was not different among the experimental groups, whereas the rats in group A and group F had higher plasma TG concentrations than the control group due to their high intake of fat. In the present study, fish oil infusion did not lower plasma TG compared with the safflower oil infusion group. This result is different from dietary fish oil feeding studies (22, 23), which showed that fish oil feeding reduced VLDL secretion and consequently resulted in a lower plasma TG concentration. It has been reported that an enzyme involved in TG synthesis, acyl-CoA diacylglycerol acyltransferase, is inhibited in rat hepatocytes when EPA is administered (24). We did not analyze this enzyme activity, and the reason for differences in plasma TG concentrations between oral feeding and TPN infusion is unclear. In summary, the results of this study showed that fish oil infusion ameliorated the severity of diet-induced hepatic steatosis. The precise mechanism by which fish oil mediates these effects is still unresolved. Increased flux of fatty acids through the peroxisomal oxidation pathway may play a role. It is suggested that TPN with a fat emulsion prepared with fish oil may be beneficial to patients with diet-induced hepatic steatosis.
Acknowledgments This study was supported by research grant NSC-84-2331-B002-263 from the National Science Council, R. O. C.. The authors wish to thank Ms Lihjinan Yu for her technical assistance.
Submission date: 3 October 1995 Accepted: 4 December 1995
References 1. Hall R I, Grant J P, Ross L H et al. Pathogenesis of hepatic steatosis in the parenterally fed rat. J Clin Invest 1984; 74:1658-68 2. Buzby G P, Mullen J L, Stein T P, Rosato E F. Manipulation of TPN caloric substrate and fatty infiltration of the liver. J Surg Res 1981; 31:46-54 3. Sax H C, Bower R H. Hepatic complications of total parenteral nutrition. JPEN 1988; 12:615-18 4. Li S, Nussbanm M S, McFadden D W, Dayal R, Fischer J E. Reversal of hepatic steatosis in rats by addition of glucagon to total parenteral nutrition (TPN). J Surg Res 1989; 46:557-66 5. Keim N L. Nutritional effectors of hepatic steatosis induced by parenteral nutrition in the rat. JPEN 1987; 11:18-22 6. Grant J P, Snyder P J. Use of L-glutamine in total parenteral nutrition. J Surg Res 1988; 44:506-13 7. Chen W J, Yeh S L, Huang P C. Effects of fat emulsions with different fatty acid composition on plasma and hepatic lipids in rats receiving total parenteral nutrition. Clin Nntr 1996; 15:24-8 8. Rustan A C, Christiansen E N, Drevon C A. Serum lipids, hepatic glycerolipid metabolism and peroxisomal fatty acid oxidation in rats fed n-3 and n-6 fatty acids. Biochem J 1992; 283:333-9 9. Thompson S W, Jones L D, Ferrell J F et al. Testing of fat emulsion for toxicity. HI. Toxicity studies with new fat emulsions and emulsion components. A m J Clin Nutr 1965; 16:43-60 10. Tschopp T B, Zucker M B. Hereditary defect in platelet function in rats. Blood 1972; 40(2): 217-26 11. Folch J M, Sloane S G H. A simple method for the isolation and purification of total lipid from animal tissue. J Biol Chem 1957; 226:497-509 12. Association of Official Analytical Chemist. Official methods of analysis. 14th ed. Washington, DC 1984; 17:276 13. Soloni F G. Simplified manual micromethod for determination of serum triglycerides. Clin Chem 1971; 17:529-34 14. Abell L L, Levy B B, Brodie B B, Kendall F E. A simplified method for the estimation of total cholesterol in serum and demonstration of its specificity. J Biol Chem 1951; 195:357~67 15. Lee N Y, Chu Y C, Chang C P, Shieh M J, and Kao M D. Dietary survey in Taiwan area, 1986-1988. J Chinese Nutr Soci 1991; 16:39-60 16. Yeh S L, Chen W J, Huang P C. Effect of L-glutamine on hepatic lipids at different energy levels in rat receiving total parenteral nutrition. JPEN 1994; 18:40-4 17. Halminski M A, Marsh J B, Harrison E H. Differential effects offish oil, safflower oil and palm oil on fatty acid oxidation and glycerolipid synthesis in rat liver. J Nun" 1991; 121:1554-61 18. Willumsen N, Hexeberg S, Skorve J, Lundquist M, Berge R K. Docosahexaenoic acid shows no triglyceride-lowering effects but increases the peroxisomal fatty acid oxidation in liver of rats. J Lipid Res 1993; 34:13-22 t9. Christensen E, Hagve T A, Christopherwen B O. Mitochondrial and peroxisomal oxidation of arachidonic and eicosapentaenoic acid studied in isolated liver cells. Biochim Biophys Acta 1986; 879:313-21 20. Lee C R, Hamm M W. Effect of dietary fat and cholesterol supplements on glucagon receptor binding and adenylate cyclase activity of rat liver plasma membrane. J Nutr 1989; 119:539-46 21. Simopoulos A P. Summary of the NATO advanced research workshop on dietary 0)3 and 0)6 fatty acids biological effects and nutritional essentiality. J Nutr 1989; 119:521-8 22. Nestel P J. Effects of n-3 fatty acid on lipid metabolism. Annu Rev Nutr 1990; 10:149-67 23. Groot P H E, Scheek L M, Dubeluar M L e t al. Effects of diets supplemented with lard fat or mackerel oil on plasma lipoprotein lipid concentrations and lipoprotein lipase activities in domestic swine. Atherosclerosis 1989; 7 7 : 1 - 6 24. Rustan A C, Nossen J O, Christiansen E N, and Drevon C A. Eicosapentaenoic acid reduces hepatic synthesis and secretion of triacylglycerol by decreasing the activity of acyl-coenzyme A: 1,2diacylglycerol acyltransferase. J Lipid Res 1988; 29:1417-26
Effects of fish oil and safflower oil emulsions on dietinduced hepatic steatosis in rats receiving total parenteral nutrition S. L. YEH 1, W. J. CHEN 2 and P. C. HUANG a 1School of Nutrition and Health Science, Taipei Medical College, Departments of 2Surgery and 3Biochemistry, College of Medicine, National Taiwan University, 7 Chung-Shan S Road, Taipei, Taiwan 100, Republic of China (Reprint requests and correspondence to VV. J. C.) ABSTRACT--This study was conducted to investigate the effects of fish oil and safflower oil emulsions in total parenteral nutrition (TPN) solutions on diet-induced hepatic steatosis. Rats were divided into a control group (C, n = 6) and four experimental groups (A, B, S, F, n = 11~14). The control group was fed a chow diet whereas the experimental groups received a high fat (15%, w/w) diet containing 0.1% (w/w) cholesterol. Group A received the high fat diet for 4 weeks, and was killed at the end of the fourth week to ensure that hepatic steatosis had occured. Groups S and group F received TPN with safflower oil or fish oil emulsions, respectively, for 1 week following experimental diet feeding for 4 weeks. Group B was fed a limited amount of the high fat diet, without cholesterol, for 1 week following 4 weeks of experimental diet in order to maintain the same body weight and cholesterol intake as the TPN groups. Diet-induced hepatic steatosis was observed in the experimental groups. Fat deposition was reversed when the total caloric and cholesterol intake was reduced. Fish oil infusion ameliorated the severity of hepatic steatosis, whereas safflower oil had no effect on liver fat deposition. These results suggest that TPN with fish oil emulsions may be beneficial to patients with diet-induced hepatic steatosis.
lesterol. The composition of the high fat diet fed to each group is shown in Table 1. After 4 weeks, rats in group A were killed to ensure the occurrence of diet-induced hepatic steatosis. The rats in groups S and F were fitted with internal jugular catheters and were infused with TPN solutions for 7 days. TPN provided 250 kcal/kg body weight/ day with 30% energy as fat. The energy density of TPN solution was 1 kcal/ml and the calorie/nitrogen ratio was 146 (kcal/g nitrogen). The basal TPN solutions were
Introduction
Total parenteral nutrition (TPN) is widely used for the treatment of nutritional depletion. Hepatic dysfunction is a well-described complication of TPN especially during hypercaloric infusion, and hepatic steatosis is the earliest and most frequently reported histological change (1). Many studies have focused on the attenuation of the development of hepatic steatosis (2-6). Previous work in this laboratory demonstrated that, unlike olive oil and safflower oil emulsions, TPN with a fat emulsion prepared with fish oil did not induce hepatic steatosis in normal rats (7). These results were similar to those found by Rustan et al (8), who reported that liver triglycerides were significantly lower in rats receiving a high fish oil diet compared with those fed a sunflower oil diet. Since diet-induced hepatic steatosis is not uncommon in the Taiwan population, this study was designed to evaluate the effect of fish oil and safflower oil emulsion on diet-induced hepatic steatosis in TPN rats.
Table 1
Composition of the experimental diet
Ingredients Fat (p/S)* Corn starch Protein (casein) Cholesterol Salt mixture t Vitamin mixture* Methyl cellulose Choline chloride DL-methionine
Materials and methods
% (w/w) 15 (1) 57 20 0.1 3.5 1 3 0.095 0.3
* P/S: polyunsaturated/saturated fatty acid t Salt mixture contains the following (mg/g): calcium phosphate dibasic 500 mg, sodium chloride 74 mg, potassium sulphate 52 mg, potassium citrate monohydrate 220 rag, magnesium oxide 24 mg, manganese carbonate 3.5 mg, ferric citrate 6 mg, zinc carbonate 1.6 mg, cupric carbonate 0.3 mg, potassium iodate 0.01 mg, sodium selenite 0.01 mg, chromium potassium sulphate 0.55 mg. Vitamin mix contains the following (mg/g): thiamine hydrochloride 0.6 mg, riboflavin 0,6 mg, pyridoxine hydrochloride 0.7 rag, nicotinic acid 3 mg, calcium pantothenate 1.6 mg, D-biotin 0.02 rag, cyanocobalamin 0.001 mg, retinyl palmitate 1.6 mg, DL-ot-tocopherol acetate 20 mg, cholecalciferol 0.25 mg, menaquinone 0.005 mg.
Animal grouping and feeding schedule Male Long-Evans rats weighing 70-100 g were used in this study. Rats were divided into a control group (C, n = 6) and four experimental groups (A, B, S, F, n = 11~14). The control group was fed with a chow diet (Purina No. 5001) whereas the experimental groups were fed with a high fat (15%, w/w) semipurified diet containing 0.1% (w/w) cho80
CLINICAL NUTRITION
isonitrogenous and identical in nutrient distribution, except for the composition of the fat emulsion (Table 2). The fat emulsion for group S was prepared with safflower oil (Taiwan Sugar Co, R.O.C.), and that of group F with fish oil (TAMA Biochemical Co, Japan). Oil emulsions were prepared with soy lecithin according to the method of Thompson et al (9). The fat emulsions contained 10 g of oil and 1.2 g of soy lecithin per 100 ml as previously described (7). The fatty acid patterns of the two fat emulsions are shown in Table 3. Rats in group B were also fed with the high fat cholesterol diet for 4 weeks. Because there is no cholesterol in TPN solutions, these rats were given the same high fat diet, but without cholesterol, for the following 7 days. Food intake was also reduced to adjust the body weight to values comparable to those of the TPN groups. The feeding schedules are shown in Figure 1. The aminals were maintained in a room at 21°C with a 12 h light/dark cycle. Infusion was started 24 h after cannulation for the TPN rats. Two ml per hour were administered on the first day. Full strength TPN was given thereafter, and continued for 7 days. Infusion speed was controlled by a Terufusion pump
Table 2 Formulationof the TPN solution(ml/1) Ingredients Glucose 50% MoriaminSN 10%* Fat emulsion20% NaC1 3% KC17% K3PO4 8.7% Ca-gluconate 10% MgSO4 10% ZnSO4 0.6% Multivitat VitaminC 5% H20 Cholinechloride (g)
260 450 173 54 7 7 7 3.5 1.5 1.5 1.5 34 1
* From ChinesePharmaceuticalsCo. Taiwan.Per 100 ml containsLeu 1250 mg, Ile 560 mg, Lys acetate 1240 mg, Met 350 mg, Phe 935 mg, Thr 650 mg, Trp 130 mg, Va1450 mg, Ala 620 rag, Arg 790 nag, Asp 380 mg, Cys 100 rag, Glu 650 mg, His 600 mg, Pro 330 mg, Set 220 mg, Tyr 35 mg, Aminoaceticacid (Gly) 1570 mg. t Yu-LiangPharmaceuticalsCo. Taiwan.Per ml containsascorbic acid 100 mg, vitaminA 2000 IU, ergocalcifero1200IU, thiamineHC1 l0 mg, riboflavin2 mg, niacinamide20 rag, pyridoxineHC1 3 rag, D-panthenol 5 mg, dl-ct-tocopherylacetate 1 mg.
Table 3 Fattyacid profiles of the lipid emulsions* Fatty a c i d 8:0 10:0 14:0 16:0 16:1 n-7 18:0 18:1 n-9 18:2 n-6 18:3 n-3 20:4 n-6 20:5 n-3 22:6 n-3
Saffloweroil
7.0 3.6 17.4 71.3 0.6
* Expressed as relativepercent.
Fish oil
7.8 15.3 9.8 1.9 11.7 4.6 1.6 2.6 31.2 13.1
1 Group
I--I
2
3
[
4
I
81
5 (week)
1
I
b+++++
Fig. 1 Feedingschedule of the all groups. . . . . . . chow diet, high fat diet with 0.1% cholesterol, === high fat diet withoutcholesterol, ++++TPN with fat emulsionmade of fish oil, ** ** * TPN with fat emulsion made of safflower oil.
(Model STC-503, Terumo Co, Japan). All animals were allowed to drink water freely during the experimental period.
Measurements and analytical procedures Animals in the control group and group A were killed after 4 weeks. The TPN rats and rats in group B were killed at the end of the fifth experimental week. After fasting overnight, bleeding time was measured in the control and TPN groups. Rats were anesthetized and the tips of their tails (5 ram) were cut off with a razor blade. The blood was allowed to flow into saline at 37°C, and the time for blood flow to stop was measured (10). Each animal was exsanguinated by drawing arterial blood from the aorta. The entire liver was rapidly excised and stored at - 7 0 ° C until assay. Blood was collected into tubes containing EDTA-Na 2 and immediately centrifuged. Plasma was analyzed for non-esterified fatty acids (NEFA), triglycerides (TG) and very low-density lipoprotein (VLDL)-TG. VLDL was obtained by ultracentrifugation at a density of 1.006. Plasma TG, VLDL-TG, and NEFA were determined by colorimetric methods after enzymatic reaction with peroxidase (Randox Co, Ireland). Liver lipid was extracted with a chloroformmethanol 2:1 mixture according to Folch et al (11). Total lipid was gravimetrically measured after drying in an evaporator to constant weight (12). Triglycefides were determined by the method of Soloni (13). Cholesterol was measured according to the method of Carlson and Goldfard (14).
Statistics Data are expressed as mean + SD. D u n c a n ' s test following analysis of variance (ANOVA) was used to compare different groups. P values less than 0.05 were considered statistically significant.
Results
Body weights There were no differences in body weight between any of the groups after feeding with rat chow or the experimental
82 EFFECT OF FAT EMULSIONS ON DIET INDUCED HEPATIC STEATOSIS IN TPN
diet for 4 weeks. No difference in body weight gain was seen between the two TPN groups during infusion for 1 week (S:1.9 + 4.6 g vs F:4.3 + 6.2 g, P > 0.05).
Liver lipid content Hepatic steatosis was observed in group A, mainly due to TG accumulation. Group B had lower total liver lipids than groups A and S. Group S had a significantly higher liver lipid content than the other TPN groups except group A. Rats in group F had lower total liver lipids than those in groups A and S but were not different from the control group and group B. The pattern of liver TG and cholesterol levels among the groups was similar to those of the total liver lipids (Table 4).
Plasma lipid concentration Groups A and F exhibited higher plasma TG levels than the control group. No differences in plasma TG, VLDL-TG and NEFA were seen among the four experimental groups (Table 5).
Bleeding time There was no difference in bleeding time between the control group and the two TPN groups. The bleeding times were 3.8 + 0.4, 3.4 + 0.6, and 3.2 _+0.8 rain for the control, safflower oil and fish oil groups, respectively.
Table 4 Group
Hepatic lipid content of the control and the experimental groups TL
TG
Chol
mg/g wet tissue C (n = 6) A (n = 11) B (n = 12) S (n = 14) F (n = 13)
64.0 116.9 92.5 109.3 79.6
_+ 7.2 a + 26.0 b _+ 21.2 ° _+ 28.5 b _+ 16.2 ac
18.5 65.3 43.4 56.2 29.0
_+ 8.8 a + 15.9 b _+ 18.1 ° -+ 25.0 b _+ 13.5 ac
4.0 10.9 7.9 8.9 7.8
_+ 0.7 a + 2.9 b _+ 2,1 c _+ 1.6 c _+ 1.9 c
TL: total lipids Values in a column with different superscripts indicate significant differences (P < 0.05).
Table 5 Plasma triglyceride (TG), very low density lipoprotein (VLDL)TG and non-esterified fatty acid (NEFA) in the control and the experimental groups Group
TG
VLDL/TG
NEFA mmol/L
0.16_+0.06 0.33_+0.16 0.34+0.15 0.33-+0.17 0.43_+0.15
0.398_+0.132 0.338_+0.085 0.331+0.161 0.432-+0.147 0.357_+0.147
mmol/L C(n=6) A ( n = 11) B ( n = 12) S ( n = 14) F ( n = 13)
0.30_+0.06 0.54_+0.32* 0.50+0.22 0.50-+0.21 0.61-+0.21"
*: significantly different from C group (P < 0.05).
Discussion
This study was designed to investigate the effects of TPN containing emulsions with different fatty acid composition on diet-induced hepatic steatosis in rats. According to a dietary survey in Taiwan 1986-1988 (15), carbohydrate and fat intake provided 49.8% and 35.6%, respectively, of the total energy. The polyunsaturated fatty acid/saturated fatty acid (P/S) ratio was 1.35. Since hepatic steatosis is not uncommon in the people of Taiwan, an experimental diet was designed with a nutrient distribution and P/S ratio close to the Chinese diet in Taiwan. This diet was used to induce hepatic steatosis in rats. Marked hepatic steatosis was observed after rats were fed with a high fat diet ad libitum for 4 weeks. Total lipid content of the livers of rats in the experimental groups was 80% higher than those of the control group. Since liver fat accumulation can be induced by TPN providing excess energy, 250 kcal/kg body weight per day was administered during the infusion period. This level of energy intake was assumed to be adequate, without causing hepatic steatosis in TPN rats (5, 16). However, the body weights of TPN rats were only marginally maintained. Cholesterol was not present in TPN solutions, so the effect of withdrawing cholesterol on hepatic steatosis should be examined. Rats in group B received a high fat diet without cholesterol for 7 days after 4 weeks of the high fat cholesterol diet. The results demonstrated that group B had significantly lower total liver lipids than group A. This suggests that hepatic steatosis induced by diet can be reversed by reducing total energy and cholesterol intake. Most commercially available fat emulsions used in TPN are prepared with soybean oil. In order to investigate the different metabolic effects of n-3 and n-6 fatty acids on hepatic lipids, safflower oil was used as fat source in this study. This is because safflower oil has a higher linoleic acid (C18:2, n-6) content than soybean oil (71.3% vs 52.4%). Infusion of a safflower oil emulsion had no effect on hepatic steatosis; the total hepatic lipid content was similar before and after TPN administration. However, the severity of hepatic steatosis was ameliorated by fish oil infusion. Total liver lipids were significantly lower in group F compared to group S and group A. Liver lipids in group F were not different from the control group. A similar result was also reported by Rustan et al (6) in an oral feeding study. Hepatic steatosis can result from an imbalance in the rate of entry, synthesis and removal of fat from the liver. This imbalance may result from alterations in 1. the rates of hepatic synthesis of fatty acids and TG 2. secretion of TG or other compounds containing the fatty acyl moiety via plasma or bile 3. uptake of TG and fatty acid 4. TG hydrolysis and fatty acid oxidation. Our study found that the plasma NEFA and VLDL-TG levels were not different between the experimental groups. Although we did not measure VLDL secretion directly, it appears that hepatic lipid uptake and VLDL secretion were
CLINICAL NUTRITION 83
not the causes of the differences of hepatic lipid content. Rustan et al (6) and Halminski et al (17) reported that fish oil feeding led to greater peroxisomal fatty acid oxidation and suppression of liver TG synthesis compared with c0-6 fatty acid rich oils. Willumsen et al (18) and Christensen et al (19) reported that the increment in peroxisomal fatty acid oxidation was attributable to the effects of EPA and DHA. It has also been reported that glucagon-stimulated adenylate cyclase activity is enhanced in the livers of fish-oil fed rats (20), and lipolysis in the liver may occur in the presence of increased cyclic AMP. Although TPN differs from oral feeding in the route of administration and the composition of nutrients, EPA and DHA in fish oil providing the fat sources in TPN may still play important roles in preventing hepatic TG accumulation. Previous work in this laboratory showed that lipid peroxidation products did not accumulate in liver tissue after administration of fish oil emulsion (7). In order to examine the possible adverse effect of fish oil, bleeding time was measured in the control and TPN groups, but no differences were found. This finding might be explained by an inadequate administration period or because the antiaggregatory effect of fish oil was not potent enough to prolong bleeding time. Nevertheless, no clinical evidence of bleeding has been observed in any fish oil feeding studies (21). The plasma TG was not different among the experimental groups, whereas the rats in group A and group F had higher plasma TG concentrations than the control group due to their high intake of fat. In the present study, fish oil infusion did not lower plasma TG compared with the safflower oil infusion group. This result is different from dietary fish oil feeding studies (22, 23), which showed that fish oil feeding reduced VLDL secretion and consequently resulted in a lower plasma TG concentration. It has been reported that an enzyme involved in TG synthesis, acyl-CoA diacylglycerol acyltransferase, is inhibited in rat hepatocytes when EPA is administered (24). We did not analyze this enzyme activity, and the reason for differences in plasma TG concentrations between oral feeding and TPN infusion is unclear. In summary, the results of this study showed that fish oil infusion ameliorated the severity of diet-induced hepatic steatosis. The precise mechanism by which fish oil mediates these effects is still unresolved. Increased flux of fatty acids through the peroxisomal oxidation pathway may play a role. It is suggested that TPN with a fat emulsion prepared with fish oil may be beneficial to patients with diet-induced hepatic steatosis.
Acknowledgments This study was supported by research grant NSC-84-2331-B002-263 from the National Science Council, R. O. C.. The authors wish to thank Ms Lihjinan Yu for her technical assistance.
Submission date: 3 October 1995 Accepted: 4 December 1995
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