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Effects of parenteral infusion with medium-chain triglycerides and safflower oil emulsions on hepatic lipids, plasma amino acids and inflammatory mediators in septic rats
Clinical Nutrition (2000) 19(2): 115–120 © 2000 Harcourt Publishers Ltd DOI:10.1054/clnu.1999.0088, available online at http://www.idealibrary.com on
ORIGINAL ARTICLE
Effects of parenteral infusion with medium-chain triglycerides and safflower oil emulsions on hepatic lipids, plasma amino acids and inflammatory mediators in septic rats S.-L. YEH*, C.-Y. CHAO†, M.-T. LIN†, W.-J. CHEN† *Institute of Nutrition and Health Science, Taipei Medical College, Taipei, †Department of Surgery, College of Medicine, National Taiwan University, Taipei, Taiwan, Republic of China (Correspondence to: W-JC, Department of Surgery, National Taiwan University Hospital, 7 Chung-Shan S. Road, Taipei, Taiwan 100, Republic of China)
Abstract—This study was designed to investigate the effects of preinfusion with total parenteral nutrition (TPN) using medium-chain triglycerides (MCT) versus safflower oil (SO) emulsion as fat sources on hepatic lipids, plasma amino acid profiles, and inflammatory-related mediators in septic rats. Normal rats, with internal jugular catheters, were divided into two groups and received TPN. TPN provided 300 kcal/kg/day with 40% of the non-protein energy provided as fat. All TPN solutions were isonitrogenous and identical in nutrient composition except for the fat emulsion, which was made of SO or a mixture of MCT and soybean oil (9:1) (MO). After receiving TPN for 6 days, each group of rats was further divided into control and sepsis subgroups. Sepsis was induced by cecal ligation and puncture, whereas control rats received sham operation. All rats were classified into four groups as follows: MCT control group (MOC, n = 8), MCT sepsis group (MOS, n = 8), safflower oil control group (SOC, n = 8), and safflower oil sepsis group (SOS, n = 11). The results of the study demonstrated that the MOS group had lower hepatic lipids than did the SOS group. Plasma leucine and isoleucine levels were significantly lower in the SOS than in the SOC group, but no differences in these two amino acids were observed between the MOC and MOS groups. Plasma arginine levels were significantly lower in septic groups than in those without sepsis despite whether MCT or safflower oil was infused. Plasma glutamine and alanine levels, however, did not differ between septic and non-septic groups either in the SO or MO groups. No differences in interleukin-1b, interleukin-6, tumor necrosis factor-a, and leukotriene B4 concentrations in peritoneal lavage fluid were observed between the two septic groups. These results suggest that catabolic reaction is septic rats preinfused MCT is not as obvious as those preinfused safflower oil. Compared with safflower oil, TPN with MCT administration has better effects on reducing sepsis-induced liver fat deposition. Preinfusion with MCT before sepsis, however, had no effect on inflammatory-related cytokines or leukotriene in peritoneal lavage fluid. In addition, plasma arginine appears to be a more sensitive indicator than glutamine for septic insult. © 2000 Harcourt Publishers Ltd
triglycerides (MCT) has been suggested as an alternative energy source in septic patients because of its special features (7). In vivo and in vitro studies have shown that MCTbased fat emulsions are cleared more rapidly than long-chain triglycerides (LCT) emulsions (8–11). Hamawy et al. (12) observed that after bilateral septic femur fracture, rats infused with MCTs had lower pulmonary infection and greater ability to fend off bacteria invasion. Sobrado et al. (13) also demonstrated that, in burned guinea-pigs, MCTbased fat emulsion showed an advantage over conventional emulsions, because animals with MCTs emulsion may avoid reticuloendothelial system (RES) overload, and thus clear bacteria more effectively. The decreased RES overload observed with MCT-based fat emulsions was explained by their more rapid hydrolysis by lipolytic enzymes than for LCT emulsions (7). In this study we infused MCT or
Key words: MCT; safflower oil; sepsis; lipids; cytokines; leukotriene
Introduction Sepsis is one of the major causes of death for critically ill patients. When bacterial toxins insult the body, abnormal nutrient metabolism may occur (1). Glucose intolerance is commonly found in septic patients (2, 3). For this reason, it has been suggested that exogenous fat rather than carbohydrates may be a preferred energy source. However, the experimental evidence is controversial. Some studies have reported that lipid is effectively utilized in the septic state (4, 5), whereas impaired lipid utilization in stressed and septic patients has also been reported (2, 3, 6). Medium-chain 115
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safflower oil (SO) emulsion before sepsis induction to investigate the effect of MCTs versus LCT on the septic state. In addition, plasma amino acid profiles and levels of interleukin (IL)-1b, IL-6, tumor necrosis (TNF)-a, as well as leukotriene (LT) B4 in plasma and peritoneal lavage fluid (PLF) were analysed, to evaluate the effects of the two lipid emulsions on catabolic conditions and inflammatory reaction of sepsis. Materials and methods Animals and surgical procedures Male Wistar rats (Charles River, Wilmington, MA, USA) weighing 200–250 g were used in this study. All rats were housed in temperature and humidity controlled rooms, and were allowed free access to a standard rat chow for 1 week prior to the experiment. After overnight fasting, rats were anesthetized with intraperitoneal pentobarbital (50 mg/kg), and the right internal jugular vein was cannulated with a Silastic catheter (Dow Corning, Midland, MI, USA) under sterile conditions. The catheter was tunneled subcuctaneously to the back of the neck, and exited through a coil spring, which was attached to a swivel allowing free mobility of animals inside individual metabolic cages. The animals were maintained in a 21°C room with a 12 h light/dark cycle. Infusion was started 24 h after cannulation. Two ml per h was administered on the first day. Full-strength TPN was given thereafter, and continued for a period of 6 days. Infusion speed was controlled by a Terufusion pump (Model STC-503, Terumo Co., Tokyo, Japan). All animals were allowed to drink water freely during the experimental period. TPN solution preparation and grouping Rats were assigned into two groups according to body weight to make average weights between groups as similar as possible. All basal TPN solutions were isonitrogenous and identical in nutrient composition except for the composition of the fat emulsion (Table 1). A 20% (w/v) lipid emulsion was prepared with 200 g/l oil and 12 g/l of soy lecithin. The particle size and the stability of the fat emulsion has been reported in a previous communication (14). The oils used were SO (Taiwan Sugar Co., Taipei, ROC) or a mixture of 9 parts MCT (Bristol-Myer Ltd, NY, USA) and 1 part soybean oil (Taiwan Sugar Co., Taipei, ROC); the latter soybean oil was used to prevent essential fatty acid deficiency. The SO emulsions contain 71.3% linoleic acid (C18:2, n-6); the MCT mixture contains 64.6% C8:0, 20% C10:0 and 8% linoleic acid as described previously (14). The experimental groups received TPN solutions with 40% non-protein energy provided as fat at an energy level of 300 kcal/kg body weight/d. The nutritional compositions of glucose, protein, and fat in TPN solutions were 50.3%, 16.7%, and 33% respectively. The energy density of the TPN solution was 1 kcal/ml and the calorie/nitrogen ratio was 150 (kcal/g nitrogen). The TPN solution without fat was prepared every second day in a laminar flow hood, the sterilized fat emulsions were added
Table 1 Formulation of TPN Solution (ml/l) Ingredients Glucose 50% Fat emulsion Moriamin 10%* NaCl 3% KCl 7% K3PO48.7% Ca-gluconate 10% MgSO4 10% ZnSO4 0.6% Infuvita** Distilled water Choline chloride (g)
252 190 420 35 10 10 10 4 2 7 59 1
*From Chinese Pharmaceuticals Co., Taiwan. Per deciliter contains: Leu 1250 mg, Ile 560 mg, Lys acetate 1240 mg, Met 350 mg, Phe 935 mg, Thr 650 mg, Trp 130 mg, Val 450 mg, Ala 620 mg, Arg 790 mg, Asp 380 mg, Cys 100 mg, Glu 650 mg, His 600 mg, Pro 330 mg, Ser 220 mg, Tyr 35 mg, aminoacetic acid (Gly) 1570 mg. **Yu-Liang Pharmaceuticals Co., Taoyuan, Taiwan. Per milliliter contains: Ascorbic acid 20 mg, vitamin A 660 IU, ergocalciferol 40 IU, thiamine HCl 0.6 mg, riboflavin 0.72 mg, niacinamide 8 mg, pyridoxine HCl 0.8 mg, D-panthenol 3 mg, dl-α-tocopheryl acetate 2 mg.
to the TPN solution daily just before use. The TPN solution was infused for the entire day at room temperature. After TPN administration for 6 days, each experimental group was further divided into septic and control subgroups. There were four groups classified as follows: safflower oil sepsis group (SOS, n = 11), MCT sepsis group (MOS, n = 8), safflower oil control group (SOC, n = 8), and MCT control group (MOC, n = 7). Sepsis of rats was induced by cecal ligation and puncture (CLP) according to the method of Wichterman et al. (15). Briefly, rats were lightly anesthetized with ether. The abdomen was opened through a midline incision and the cecum was punctured twice with an 18-gauge needle and was replaced into the abdomen. The abdominal wound was closed in layers. For confirmation of sepsis, blood culture was performed in half of the septic rats 22–24 h after surgery, and positive cultures were obtained in all of them. Clinical symptoms of the septic rats were similar to those described by Wichterman et al. (15). Sham operation with manipulation of the cecum but without ligation or puncture of the cecum was performed on control rats. Measurements and analytical procedures Twenty-four hours after surgery, all rats were anesthetized and sacrificed by drawing arterial blood from the aorta of the abdomen. Blood samples were collected in tubes containing heparin and centrifuged immediately. Liver lipids were extracted with a 2:1 chloroform-methanol mixture according to Folch et al. (16). Total lipids were gravimetrically measured after drying in an evaporator to constant weight (17). TG was determined by the method of Soloni (18). Cholesterol was measured according to the method of Carlson and Goldfard (19). Amino acid was analysed by standard ninhydrin technology (Beckman Instrument, model 6300, Palo Alto, CA, USA), after deproteinization of the plasma with 50% salicylic acid (20). IL-1b, IL-6 and TNF-a levels in plasma and PLF were measured using commercially
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available enzyme-linked immunosorbent assay (ELISA) in microtiter plates, with antibodies specific for rat IL-1b, IL-6, and TNF-a having been coated onto the wells of the microtiter strips provided (BioSource International, Inc., Camarillo, CA, USA). LT B4 levels in plasma and peritoneal lavage fluid were also measured by ELISA, the surface of the microtiter plates were precoated with mouse monoclonal antibody. Acetylcholinesterase covalently coupled to LTB4 was used as the enzymatic tracer (Cayman Chemical Co., Ann Arbor, MI, USA). The cytokines and the leukotriene concentrations in peritoneal lavage fluid were expressed in pg/mg protein. Protein levels were measured by Lowry et al.’s method (21). Twenty-four hour urine specimens were collected during the last 3 infusion days for determination of the nitrogen balance. Nonprotein nitrogen in urine was measured by a colorimetric method (Wako Phamaceuticals Co., Osaka, Japan).
Table 3 Hepatic lipids content of the all groups Group
TG
Chol mg/g liver
TL
SOC (n = 8) MOC (n = 8) SOS (n = 11) MOS (n = 8)
16.6 ± 4.3ab 5.7 ± 1.4c 22.0 ± 9.8a 14.7 ± 6.4b
3.8 ± 0.3b 3.8 ± 0.5b 4.7 ± 0.9a 3.4 ± 0.2b
55.2 ± 9.5b 39.1 ± 5.9c 70.2 ± 9.9a 52.3 ± 6.4b
Significance of F value* Treament effect Oil effect Treatment x oil effect
0.000 0.000 NS
NS 0.001 0.001
0.000 0.000 NS
Values are means ± SD. The groups are described in the footnote to Table 2. Values with different superscripts in the same column are significantly different from one another at P < 0.05, as determined by Duncan’s multiple range test. TG: triglyceride; Chol: cholesterol; TL: total lipids. *a 2 3 2 analysis of variance; P < 0.05 indicate significant difference; NS: non-significant. Treatment: control vs sepsis; Oil: safflower oil vs medium chain triglyceride.
Statistics Data are expressed as means ± SD. The data were analysed as a two-way experiment with testing of the interaction of diet and treatment. Specific comparison of group means was made when F values were significant for diet or treatment. Differences among groups were analysed by analysis of variance using Duncan’s test. A P value < 0.05 was considered statistically significant. Results There were no differences in initial body weights between MCT and SO infusion groups at the beginning of TPN administration. All rats gained weight after 6 days of infusion, but neither weight gain nor nitrogen retention was significantly different between the two TPN groups (Table 2). The hepatic total lipid content of septic rats was higher than in those without sepsis, regardless of whether the rats were preinfused with MCT or SO. The hepatic lipid deposition was mainly due to TG accumulation, TPN with SO produced higher liver total fat content than those with MCT both in septic and non-septic groups (Table 3). The amino acid profiles showed that arginine concentration was significantly lower in septic groups than in the corresponding groups without sepsis. Lower leucine and isoleucine concentrations were observed in the SOS group than in the SOC group, whereas no differences in plasma
Table 2 Body weight and nitrogen retention of the safflower oil (SO) and MCT (MO) infusion groups Group
Initial weight
Weight gain/6 d (g)
SO (n = 19) MO (n = 16)
215.9 ± 19.8 218.1 ± 19.5
Nitrogen retention (mg/d)
10.8 ± 4.2 9.7 ± 4.6
261.9 ± 104.1 220.1 ± 107.6
Values are means ± SD. There is no significant difference between the two groups.
concentrations of the three branch chain amino acids (BCAAs) were observed between MOC and MOS groups. No significant differences in glutamine (GLN) and alanine concentrations were seen between rats with and without sepsis, either in MCT or SO infusion groups (Table 4). Most plasma concentrations of IL-1b, IL-6, and TNF-a were undetectable in both MOS and SOS groups. No difference in plasma LTB4 levels was observed between the two septic groups (MOS 66.0 ± 23.5 vs SOS 57.4 ± 33.8 pg/ml). There were no significant differences in concentrations of IL-1b, IL-6, TNF-a, and LTB4 in PLF between the two septic groups (Table 5). Discussion In this study we used CLP as a sepsis model. This model is considered to be a simple and reproducible model of sepsis in rats (15). Since TPN was administered before CLP and the rat was not fed orally, the cecum may not have contained much fecal material. This created a chronic sublethal animal model of sepsis. Most commercialized LCT emulsions are made from soybean oil which is rich in linoleic acid, and MCTs were mixed with LCTs. In order to specify the distinctly different effects between medium-chain fatty acid and linoleic acid, SO and MCT oil were used to prepare the fat emulsions in this study. The results in this study show that body weight gain and nitrogen retention after TPN administration did not differ between the SO and MO groups. Some studies have demonstrated that TPN with MCT-based emulsion resulted in a more positive nitrogen balance than with LCT emulsions (22, 23). A study by Stein et al. (24), however, failed to disclose any difference between the two emulsions in septic rats, as was observed in this study. Whether MCT-based emulsions have a better effect on nitrogen utilization than do LCT emulsions remains controversial. Hepatic lipid contents were significantly higher in the septic groups than in the corresponding groups without
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Table 4 Plasma amino acid concentrations of all groups Group
Glutamine
Alanine
Arginine nmol/ml
Valine
Leucine
Isoleucine
SOC (n = 8) MOC (n = 8) SOS (n = 11) MOS (n = 8)
654.0 ± 151.4 483.9 ± 81.8# 519.3 ± 135.9 441.3 ± 55.3#
486.9 ± 120.4 426.3 ± 102.6 411.1 ± 139.5 473.4 ± 170.7
156.0 ± 37.5 122.1 ± 29.2 78.6 ± 32.1* 67.5 ± 30.8*
170.2 ± 37.5 154.0 ± 36.4 133.3 ± 21.0 146.8 ± 36.0
156.7 ± 29.0 129.8 ± 38.9 116.0 ± 23.4* 129.5 ± 33.5
92.1 ± 18.4 73.8 ± 16.6 61.0 ± 9.9* 77.0 ± 24.2
Significance of F value& Treatment effect Oil effect Treatment x Oil effect
0.019 0.009 NS
NS NS NS
0.000 NS ND
NS NS NS
NS NS 0.041
NS NS 0.010
Values are means ± SD. The groups are described in the footnote to Table 2. *Significantly different from corresponding group without sepsis at P < 0.05, as determined by Duncan’s multiple range test. #Significantly different from SOC group at P < 0.05, as determined by Duncan’s multiple range test. & A 2 3 2 analysis of variance; P < 0.05 indicate significant difference; NS: non-significant. Treatment: control sepsis; Oil: safflower oil vs medium chain triglyceride.
Table 5 Concentrations of interleukin (IL)-1β, IL-6, tumor necrosis factor (TNF)-α, and leukotriene (LT) B4 in peritoneal lavage fluid in septic groups Group
IL-1β
IL-6
TNF-α
LT B4
4.8 ± 6.7 3.3 ± 3.4
3.9 ± 1.4 3.6 ± 1.0
pg/mg protein SOC (n = 11) MOC (n = 8)
57.8 ± 44.1 37.1 ± 34.2
19.7 ± 15.1 11.6 ± 8.2
No significant difference was seen between the two septic groups.
sepsis. Studies by Lanza-Jacoby and Tabares (25) and Rosato et al. (26) reported that septic insult resulted in liver lipogenesis and fat deposition. In this study the groups preinfused with SO had higher liver fat than did those groups prefed MCT, suggesting that rats preinfused with MCT emulsion had lower sepsis-induced fat deposition in the liver. The lesser extent of liver fat deposition observed in MCT-infused groups may be explained by the carnitineindependent transport of medium-chain fatty acids across the mitochondrial membrane, and the rapid oxidation of medium-chain fatty acids in the liver (27). A higher rate of hydrolysis of MCTs than LCTs by lipolytic enzymes may also play a role. BCAAs are the only essential amino acids primarily oxidized by skeletal muscle (28–30). BCAAs supply energy to the muscle, and nitrogen for the glucose-alanine cycle and GLN synthesis (28–30). The results in this study showed that plasma leucine and isoleucine levels were significantly lower in the SOS group than in the SOC group. The effects might be explained by either a reduced output of leucine and isoleucine from muscle or an increase removal of these amino acids from plasma. The first possibility, however, can be excluded, because several studies have shown that oxidation rate of BCAAs in muscle is stimulated by stress and catabolic diseases (31, 32). A study by Odessey et al. (29) revealed that BCAAs stimulate de novo synthesis of alanine and GLN, thus offering energy substrate for the liver, kidney and gut. GLN is the most abundant free amino acid in the plasma and tissue pool (33). It has been reported that, following the catabolic stage, a large quantity of GLN is
extracted from the circulation by visceral organs for use as energy substrate (34), and plasma GLN falls in catabolic states (35). In this study we did not observe differences in plasma GLN and alanine levels between rats with and without sepsis. It is possible that the BCAAs were extracted from plasma to maintain the homeostasis of GLN and alanine pool in the body in the early sepsis. The finding of lower levels of leucine and isoleucine in the SOS group than in the SOC group, but no difference of these two amino acids in the two MO groups may indicate that, the removal of BCAAs from circulation is increased in septic rats preinfused SO to compensate the depletion of GLN and alanine resulting from catabolic reaction of sepsis. The catabolic reaction of septic rats preinfused MCT is not as obvious as those preinfused SO. Plasma arginine levels were significantly lower in septic groups than in those without sepsis. Arginine is the physiological substrate of nitric oxide synthetase enzymes and is used as a nitrogen donor to produce nitric oxide (NO) (36). It is known that during sepsis NO production is increased to act directly as a cytotoxic agent against microbial pathogens (37). The lower plasma arginine levels observed in septic groups than control groups in this study can be explained by the cellular uptake of arginine from plasma for NO synthesis in sepsis. Surgical injury and infection stimulate the production of a variety of endogenous mediators. TNF-a, IL-1b, and IL-6 are major mediators of the acute phase (38). TNF-a and IL-1b are primarily responsible for non-hepatic manifestations of the response of accelerated catabolism (1), and IL-6 is responsible for the hepatic component of the synthesis of acute-phase proteins (1). Although cytokines have beneficial effects in injury, exaggerated or prolonged secretion of these proteins, however, is detrimental to the host (39). Baigrie et al. (38) reported that the systemic response of IL-1b and IL-6 to surgical trauma increased with the severity of the surgical insult. Because of short half-lives of these cytokines and their rapid degradation in a variety of tissue within a few hours (38), cytokines in plasma were no detectable at the time point at which we took measurements. However, cytokine levels in PLF were measured, and no
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differences in their levels between the two septic groups were found. LTB4 is also known as a potent inflammatory mediator which enhances chemotaxis (40). No difference in plasma or PLF concentrations of LTB4 between the two septic groups was observed. These results suggest that, compared with SO emulsion, preinfusion with MCT before septic insult did not have a better effect on reducing local inflammatory-related cytokine and leukotriene production. In conclusion, the results of this study suggest that plasma arginine is a more sensitive indicator than GLN for septic insult. TPN with MCT infusion before sepsis maintain plasma branch-chained amino acid levels after sepsis, whereas catabolic reaction is obvious in rats preinfused SO. Compared with SO, TPN with MCT administration has better effects on reducing sepsis-induced liver fat deposition. However, infusion with MCT before septic insult did not have an effect on reducing the production of inflammatory mediators in the location of the injurious stimulus. Whether the systemic level of inflammatory mediators is altered by the infusion of different fat emulsions requires further investigation.
Acknowledgments This study was supported by research grant NSC 87-2314-B-038-006 from the National Science Council, Republic of China. The authors wish to thank Ms Lih-jiuan Yu for her technical assistance.
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ORIGINAL ARTICLE
Effects of parenteral infusion with medium-chain triglycerides and safflower oil emulsions on hepatic lipids, plasma amino acids and inflammatory mediators in septic rats S.-L. YEH*, C.-Y. CHAO†, M.-T. LIN†, W.-J. CHEN† *Institute of Nutrition and Health Science, Taipei Medical College, Taipei, †Department of Surgery, College of Medicine, National Taiwan University, Taipei, Taiwan, Republic of China (Correspondence to: W-JC, Department of Surgery, National Taiwan University Hospital, 7 Chung-Shan S. Road, Taipei, Taiwan 100, Republic of China)
Abstract—This study was designed to investigate the effects of preinfusion with total parenteral nutrition (TPN) using medium-chain triglycerides (MCT) versus safflower oil (SO) emulsion as fat sources on hepatic lipids, plasma amino acid profiles, and inflammatory-related mediators in septic rats. Normal rats, with internal jugular catheters, were divided into two groups and received TPN. TPN provided 300 kcal/kg/day with 40% of the non-protein energy provided as fat. All TPN solutions were isonitrogenous and identical in nutrient composition except for the fat emulsion, which was made of SO or a mixture of MCT and soybean oil (9:1) (MO). After receiving TPN for 6 days, each group of rats was further divided into control and sepsis subgroups. Sepsis was induced by cecal ligation and puncture, whereas control rats received sham operation. All rats were classified into four groups as follows: MCT control group (MOC, n = 8), MCT sepsis group (MOS, n = 8), safflower oil control group (SOC, n = 8), and safflower oil sepsis group (SOS, n = 11). The results of the study demonstrated that the MOS group had lower hepatic lipids than did the SOS group. Plasma leucine and isoleucine levels were significantly lower in the SOS than in the SOC group, but no differences in these two amino acids were observed between the MOC and MOS groups. Plasma arginine levels were significantly lower in septic groups than in those without sepsis despite whether MCT or safflower oil was infused. Plasma glutamine and alanine levels, however, did not differ between septic and non-septic groups either in the SO or MO groups. No differences in interleukin-1b, interleukin-6, tumor necrosis factor-a, and leukotriene B4 concentrations in peritoneal lavage fluid were observed between the two septic groups. These results suggest that catabolic reaction is septic rats preinfused MCT is not as obvious as those preinfused safflower oil. Compared with safflower oil, TPN with MCT administration has better effects on reducing sepsis-induced liver fat deposition. Preinfusion with MCT before sepsis, however, had no effect on inflammatory-related cytokines or leukotriene in peritoneal lavage fluid. In addition, plasma arginine appears to be a more sensitive indicator than glutamine for septic insult. © 2000 Harcourt Publishers Ltd
triglycerides (MCT) has been suggested as an alternative energy source in septic patients because of its special features (7). In vivo and in vitro studies have shown that MCTbased fat emulsions are cleared more rapidly than long-chain triglycerides (LCT) emulsions (8–11). Hamawy et al. (12) observed that after bilateral septic femur fracture, rats infused with MCTs had lower pulmonary infection and greater ability to fend off bacteria invasion. Sobrado et al. (13) also demonstrated that, in burned guinea-pigs, MCTbased fat emulsion showed an advantage over conventional emulsions, because animals with MCTs emulsion may avoid reticuloendothelial system (RES) overload, and thus clear bacteria more effectively. The decreased RES overload observed with MCT-based fat emulsions was explained by their more rapid hydrolysis by lipolytic enzymes than for LCT emulsions (7). In this study we infused MCT or
Key words: MCT; safflower oil; sepsis; lipids; cytokines; leukotriene
Introduction Sepsis is one of the major causes of death for critically ill patients. When bacterial toxins insult the body, abnormal nutrient metabolism may occur (1). Glucose intolerance is commonly found in septic patients (2, 3). For this reason, it has been suggested that exogenous fat rather than carbohydrates may be a preferred energy source. However, the experimental evidence is controversial. Some studies have reported that lipid is effectively utilized in the septic state (4, 5), whereas impaired lipid utilization in stressed and septic patients has also been reported (2, 3, 6). Medium-chain 115
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safflower oil (SO) emulsion before sepsis induction to investigate the effect of MCTs versus LCT on the septic state. In addition, plasma amino acid profiles and levels of interleukin (IL)-1b, IL-6, tumor necrosis (TNF)-a, as well as leukotriene (LT) B4 in plasma and peritoneal lavage fluid (PLF) were analysed, to evaluate the effects of the two lipid emulsions on catabolic conditions and inflammatory reaction of sepsis. Materials and methods Animals and surgical procedures Male Wistar rats (Charles River, Wilmington, MA, USA) weighing 200–250 g were used in this study. All rats were housed in temperature and humidity controlled rooms, and were allowed free access to a standard rat chow for 1 week prior to the experiment. After overnight fasting, rats were anesthetized with intraperitoneal pentobarbital (50 mg/kg), and the right internal jugular vein was cannulated with a Silastic catheter (Dow Corning, Midland, MI, USA) under sterile conditions. The catheter was tunneled subcuctaneously to the back of the neck, and exited through a coil spring, which was attached to a swivel allowing free mobility of animals inside individual metabolic cages. The animals were maintained in a 21°C room with a 12 h light/dark cycle. Infusion was started 24 h after cannulation. Two ml per h was administered on the first day. Full-strength TPN was given thereafter, and continued for a period of 6 days. Infusion speed was controlled by a Terufusion pump (Model STC-503, Terumo Co., Tokyo, Japan). All animals were allowed to drink water freely during the experimental period. TPN solution preparation and grouping Rats were assigned into two groups according to body weight to make average weights between groups as similar as possible. All basal TPN solutions were isonitrogenous and identical in nutrient composition except for the composition of the fat emulsion (Table 1). A 20% (w/v) lipid emulsion was prepared with 200 g/l oil and 12 g/l of soy lecithin. The particle size and the stability of the fat emulsion has been reported in a previous communication (14). The oils used were SO (Taiwan Sugar Co., Taipei, ROC) or a mixture of 9 parts MCT (Bristol-Myer Ltd, NY, USA) and 1 part soybean oil (Taiwan Sugar Co., Taipei, ROC); the latter soybean oil was used to prevent essential fatty acid deficiency. The SO emulsions contain 71.3% linoleic acid (C18:2, n-6); the MCT mixture contains 64.6% C8:0, 20% C10:0 and 8% linoleic acid as described previously (14). The experimental groups received TPN solutions with 40% non-protein energy provided as fat at an energy level of 300 kcal/kg body weight/d. The nutritional compositions of glucose, protein, and fat in TPN solutions were 50.3%, 16.7%, and 33% respectively. The energy density of the TPN solution was 1 kcal/ml and the calorie/nitrogen ratio was 150 (kcal/g nitrogen). The TPN solution without fat was prepared every second day in a laminar flow hood, the sterilized fat emulsions were added
Table 1 Formulation of TPN Solution (ml/l) Ingredients Glucose 50% Fat emulsion Moriamin 10%* NaCl 3% KCl 7% K3PO48.7% Ca-gluconate 10% MgSO4 10% ZnSO4 0.6% Infuvita** Distilled water Choline chloride (g)
252 190 420 35 10 10 10 4 2 7 59 1
*From Chinese Pharmaceuticals Co., Taiwan. Per deciliter contains: Leu 1250 mg, Ile 560 mg, Lys acetate 1240 mg, Met 350 mg, Phe 935 mg, Thr 650 mg, Trp 130 mg, Val 450 mg, Ala 620 mg, Arg 790 mg, Asp 380 mg, Cys 100 mg, Glu 650 mg, His 600 mg, Pro 330 mg, Ser 220 mg, Tyr 35 mg, aminoacetic acid (Gly) 1570 mg. **Yu-Liang Pharmaceuticals Co., Taoyuan, Taiwan. Per milliliter contains: Ascorbic acid 20 mg, vitamin A 660 IU, ergocalciferol 40 IU, thiamine HCl 0.6 mg, riboflavin 0.72 mg, niacinamide 8 mg, pyridoxine HCl 0.8 mg, D-panthenol 3 mg, dl-α-tocopheryl acetate 2 mg.
to the TPN solution daily just before use. The TPN solution was infused for the entire day at room temperature. After TPN administration for 6 days, each experimental group was further divided into septic and control subgroups. There were four groups classified as follows: safflower oil sepsis group (SOS, n = 11), MCT sepsis group (MOS, n = 8), safflower oil control group (SOC, n = 8), and MCT control group (MOC, n = 7). Sepsis of rats was induced by cecal ligation and puncture (CLP) according to the method of Wichterman et al. (15). Briefly, rats were lightly anesthetized with ether. The abdomen was opened through a midline incision and the cecum was punctured twice with an 18-gauge needle and was replaced into the abdomen. The abdominal wound was closed in layers. For confirmation of sepsis, blood culture was performed in half of the septic rats 22–24 h after surgery, and positive cultures were obtained in all of them. Clinical symptoms of the septic rats were similar to those described by Wichterman et al. (15). Sham operation with manipulation of the cecum but without ligation or puncture of the cecum was performed on control rats. Measurements and analytical procedures Twenty-four hours after surgery, all rats were anesthetized and sacrificed by drawing arterial blood from the aorta of the abdomen. Blood samples were collected in tubes containing heparin and centrifuged immediately. Liver lipids were extracted with a 2:1 chloroform-methanol mixture according to Folch et al. (16). Total lipids were gravimetrically measured after drying in an evaporator to constant weight (17). TG was determined by the method of Soloni (18). Cholesterol was measured according to the method of Carlson and Goldfard (19). Amino acid was analysed by standard ninhydrin technology (Beckman Instrument, model 6300, Palo Alto, CA, USA), after deproteinization of the plasma with 50% salicylic acid (20). IL-1b, IL-6 and TNF-a levels in plasma and PLF were measured using commercially
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available enzyme-linked immunosorbent assay (ELISA) in microtiter plates, with antibodies specific for rat IL-1b, IL-6, and TNF-a having been coated onto the wells of the microtiter strips provided (BioSource International, Inc., Camarillo, CA, USA). LT B4 levels in plasma and peritoneal lavage fluid were also measured by ELISA, the surface of the microtiter plates were precoated with mouse monoclonal antibody. Acetylcholinesterase covalently coupled to LTB4 was used as the enzymatic tracer (Cayman Chemical Co., Ann Arbor, MI, USA). The cytokines and the leukotriene concentrations in peritoneal lavage fluid were expressed in pg/mg protein. Protein levels were measured by Lowry et al.’s method (21). Twenty-four hour urine specimens were collected during the last 3 infusion days for determination of the nitrogen balance. Nonprotein nitrogen in urine was measured by a colorimetric method (Wako Phamaceuticals Co., Osaka, Japan).
Table 3 Hepatic lipids content of the all groups Group
TG
Chol mg/g liver
TL
SOC (n = 8) MOC (n = 8) SOS (n = 11) MOS (n = 8)
16.6 ± 4.3ab 5.7 ± 1.4c 22.0 ± 9.8a 14.7 ± 6.4b
3.8 ± 0.3b 3.8 ± 0.5b 4.7 ± 0.9a 3.4 ± 0.2b
55.2 ± 9.5b 39.1 ± 5.9c 70.2 ± 9.9a 52.3 ± 6.4b
Significance of F value* Treament effect Oil effect Treatment x oil effect
0.000 0.000 NS
NS 0.001 0.001
0.000 0.000 NS
Values are means ± SD. The groups are described in the footnote to Table 2. Values with different superscripts in the same column are significantly different from one another at P < 0.05, as determined by Duncan’s multiple range test. TG: triglyceride; Chol: cholesterol; TL: total lipids. *a 2 3 2 analysis of variance; P < 0.05 indicate significant difference; NS: non-significant. Treatment: control vs sepsis; Oil: safflower oil vs medium chain triglyceride.
Statistics Data are expressed as means ± SD. The data were analysed as a two-way experiment with testing of the interaction of diet and treatment. Specific comparison of group means was made when F values were significant for diet or treatment. Differences among groups were analysed by analysis of variance using Duncan’s test. A P value < 0.05 was considered statistically significant. Results There were no differences in initial body weights between MCT and SO infusion groups at the beginning of TPN administration. All rats gained weight after 6 days of infusion, but neither weight gain nor nitrogen retention was significantly different between the two TPN groups (Table 2). The hepatic total lipid content of septic rats was higher than in those without sepsis, regardless of whether the rats were preinfused with MCT or SO. The hepatic lipid deposition was mainly due to TG accumulation, TPN with SO produced higher liver total fat content than those with MCT both in septic and non-septic groups (Table 3). The amino acid profiles showed that arginine concentration was significantly lower in septic groups than in the corresponding groups without sepsis. Lower leucine and isoleucine concentrations were observed in the SOS group than in the SOC group, whereas no differences in plasma
Table 2 Body weight and nitrogen retention of the safflower oil (SO) and MCT (MO) infusion groups Group
Initial weight
Weight gain/6 d (g)
SO (n = 19) MO (n = 16)
215.9 ± 19.8 218.1 ± 19.5
Nitrogen retention (mg/d)
10.8 ± 4.2 9.7 ± 4.6
261.9 ± 104.1 220.1 ± 107.6
Values are means ± SD. There is no significant difference between the two groups.
concentrations of the three branch chain amino acids (BCAAs) were observed between MOC and MOS groups. No significant differences in glutamine (GLN) and alanine concentrations were seen between rats with and without sepsis, either in MCT or SO infusion groups (Table 4). Most plasma concentrations of IL-1b, IL-6, and TNF-a were undetectable in both MOS and SOS groups. No difference in plasma LTB4 levels was observed between the two septic groups (MOS 66.0 ± 23.5 vs SOS 57.4 ± 33.8 pg/ml). There were no significant differences in concentrations of IL-1b, IL-6, TNF-a, and LTB4 in PLF between the two septic groups (Table 5). Discussion In this study we used CLP as a sepsis model. This model is considered to be a simple and reproducible model of sepsis in rats (15). Since TPN was administered before CLP and the rat was not fed orally, the cecum may not have contained much fecal material. This created a chronic sublethal animal model of sepsis. Most commercialized LCT emulsions are made from soybean oil which is rich in linoleic acid, and MCTs were mixed with LCTs. In order to specify the distinctly different effects between medium-chain fatty acid and linoleic acid, SO and MCT oil were used to prepare the fat emulsions in this study. The results in this study show that body weight gain and nitrogen retention after TPN administration did not differ between the SO and MO groups. Some studies have demonstrated that TPN with MCT-based emulsion resulted in a more positive nitrogen balance than with LCT emulsions (22, 23). A study by Stein et al. (24), however, failed to disclose any difference between the two emulsions in septic rats, as was observed in this study. Whether MCT-based emulsions have a better effect on nitrogen utilization than do LCT emulsions remains controversial. Hepatic lipid contents were significantly higher in the septic groups than in the corresponding groups without
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Table 4 Plasma amino acid concentrations of all groups Group
Glutamine
Alanine
Arginine nmol/ml
Valine
Leucine
Isoleucine
SOC (n = 8) MOC (n = 8) SOS (n = 11) MOS (n = 8)
654.0 ± 151.4 483.9 ± 81.8# 519.3 ± 135.9 441.3 ± 55.3#
486.9 ± 120.4 426.3 ± 102.6 411.1 ± 139.5 473.4 ± 170.7
156.0 ± 37.5 122.1 ± 29.2 78.6 ± 32.1* 67.5 ± 30.8*
170.2 ± 37.5 154.0 ± 36.4 133.3 ± 21.0 146.8 ± 36.0
156.7 ± 29.0 129.8 ± 38.9 116.0 ± 23.4* 129.5 ± 33.5
92.1 ± 18.4 73.8 ± 16.6 61.0 ± 9.9* 77.0 ± 24.2
Significance of F value& Treatment effect Oil effect Treatment x Oil effect
0.019 0.009 NS
NS NS NS
0.000 NS ND
NS NS NS
NS NS 0.041
NS NS 0.010
Values are means ± SD. The groups are described in the footnote to Table 2. *Significantly different from corresponding group without sepsis at P < 0.05, as determined by Duncan’s multiple range test. #Significantly different from SOC group at P < 0.05, as determined by Duncan’s multiple range test. & A 2 3 2 analysis of variance; P < 0.05 indicate significant difference; NS: non-significant. Treatment: control sepsis; Oil: safflower oil vs medium chain triglyceride.
Table 5 Concentrations of interleukin (IL)-1β, IL-6, tumor necrosis factor (TNF)-α, and leukotriene (LT) B4 in peritoneal lavage fluid in septic groups Group
IL-1β
IL-6
TNF-α
LT B4
4.8 ± 6.7 3.3 ± 3.4
3.9 ± 1.4 3.6 ± 1.0
pg/mg protein SOC (n = 11) MOC (n = 8)
57.8 ± 44.1 37.1 ± 34.2
19.7 ± 15.1 11.6 ± 8.2
No significant difference was seen between the two septic groups.
sepsis. Studies by Lanza-Jacoby and Tabares (25) and Rosato et al. (26) reported that septic insult resulted in liver lipogenesis and fat deposition. In this study the groups preinfused with SO had higher liver fat than did those groups prefed MCT, suggesting that rats preinfused with MCT emulsion had lower sepsis-induced fat deposition in the liver. The lesser extent of liver fat deposition observed in MCT-infused groups may be explained by the carnitineindependent transport of medium-chain fatty acids across the mitochondrial membrane, and the rapid oxidation of medium-chain fatty acids in the liver (27). A higher rate of hydrolysis of MCTs than LCTs by lipolytic enzymes may also play a role. BCAAs are the only essential amino acids primarily oxidized by skeletal muscle (28–30). BCAAs supply energy to the muscle, and nitrogen for the glucose-alanine cycle and GLN synthesis (28–30). The results in this study showed that plasma leucine and isoleucine levels were significantly lower in the SOS group than in the SOC group. The effects might be explained by either a reduced output of leucine and isoleucine from muscle or an increase removal of these amino acids from plasma. The first possibility, however, can be excluded, because several studies have shown that oxidation rate of BCAAs in muscle is stimulated by stress and catabolic diseases (31, 32). A study by Odessey et al. (29) revealed that BCAAs stimulate de novo synthesis of alanine and GLN, thus offering energy substrate for the liver, kidney and gut. GLN is the most abundant free amino acid in the plasma and tissue pool (33). It has been reported that, following the catabolic stage, a large quantity of GLN is
extracted from the circulation by visceral organs for use as energy substrate (34), and plasma GLN falls in catabolic states (35). In this study we did not observe differences in plasma GLN and alanine levels between rats with and without sepsis. It is possible that the BCAAs were extracted from plasma to maintain the homeostasis of GLN and alanine pool in the body in the early sepsis. The finding of lower levels of leucine and isoleucine in the SOS group than in the SOC group, but no difference of these two amino acids in the two MO groups may indicate that, the removal of BCAAs from circulation is increased in septic rats preinfused SO to compensate the depletion of GLN and alanine resulting from catabolic reaction of sepsis. The catabolic reaction of septic rats preinfused MCT is not as obvious as those preinfused SO. Plasma arginine levels were significantly lower in septic groups than in those without sepsis. Arginine is the physiological substrate of nitric oxide synthetase enzymes and is used as a nitrogen donor to produce nitric oxide (NO) (36). It is known that during sepsis NO production is increased to act directly as a cytotoxic agent against microbial pathogens (37). The lower plasma arginine levels observed in septic groups than control groups in this study can be explained by the cellular uptake of arginine from plasma for NO synthesis in sepsis. Surgical injury and infection stimulate the production of a variety of endogenous mediators. TNF-a, IL-1b, and IL-6 are major mediators of the acute phase (38). TNF-a and IL-1b are primarily responsible for non-hepatic manifestations of the response of accelerated catabolism (1), and IL-6 is responsible for the hepatic component of the synthesis of acute-phase proteins (1). Although cytokines have beneficial effects in injury, exaggerated or prolonged secretion of these proteins, however, is detrimental to the host (39). Baigrie et al. (38) reported that the systemic response of IL-1b and IL-6 to surgical trauma increased with the severity of the surgical insult. Because of short half-lives of these cytokines and their rapid degradation in a variety of tissue within a few hours (38), cytokines in plasma were no detectable at the time point at which we took measurements. However, cytokine levels in PLF were measured, and no
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differences in their levels between the two septic groups were found. LTB4 is also known as a potent inflammatory mediator which enhances chemotaxis (40). No difference in plasma or PLF concentrations of LTB4 between the two septic groups was observed. These results suggest that, compared with SO emulsion, preinfusion with MCT before septic insult did not have a better effect on reducing local inflammatory-related cytokine and leukotriene production. In conclusion, the results of this study suggest that plasma arginine is a more sensitive indicator than GLN for septic insult. TPN with MCT infusion before sepsis maintain plasma branch-chained amino acid levels after sepsis, whereas catabolic reaction is obvious in rats preinfused SO. Compared with SO, TPN with MCT administration has better effects on reducing sepsis-induced liver fat deposition. However, infusion with MCT before septic insult did not have an effect on reducing the production of inflammatory mediators in the location of the injurious stimulus. Whether the systemic level of inflammatory mediators is altered by the infusion of different fat emulsions requires further investigation.
Acknowledgments This study was supported by research grant NSC 87-2314-B-038-006 from the National Science Council, Republic of China. The authors wish to thank Ms Lih-jiuan Yu for her technical assistance.
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