Effects of a fat emulsion containing medium chain fatty acids and long chain fatty acids on protein and energy metabolism in partially hepatectomized rats

Clinical Nutrition (1995) 14:23-28 Pearson Professional Ltd 1995

Effects of a fat emulsion containing medium chain fatty acids and long chain fatty acids on protein and energy metabolism in partially hepatectomized rats I. K. MAGNUSSON BORG,* L. GORAN SANDBERG,* A. K. WENNBERG,* L. LINDMARK t and L. G. EKMAN* *Pharmacia Hospital Care AB, S- 112 87 Stockholm, Sweden, tLipidco, Ejdervggen 8, S-239 41 Falsterbo, Sweden, ~Pharmacia GmbH, D-8520 Erlangen, Germany (Reprints requests to IKMB) ABSTRACT--Protein and energy metabolism were examined in 34 partially hepatectomized

rats (70%), receiving total parenteral nutrition (TPN). The TPN contained either long chain triglycerides (LCT) or triglycerides comprising both medium- (MCFA) and long chain fatty acids (LCFA) on the same carbon skeleton (MLT, medium- and long chain triglyceride). The rats were divided into 4 groups with and without glucose (G) supplementation: LCT+G, LCT-G, MLT+G, MLT-G. 3 days after surgery protein synthetic rate in skeletal muscle, as evaluated from in vitro incorporation of 14C-phenylalanine into muscle protein, was significantly higher in rats receiving MLT if compared to rats receiving LCT (p < 0.05). Rats receiving MLT lost significantly less weight during the study period when compared to the LCT group (p < 0.005). Increased leucine oxidation was observed in rats receiving TPN without glucose regardless of the type of fat emulsion used (p < 0.05). In conclusion, when given to partially hepatectomized rats TPN containing both MCFA and LCFA exerts a stimulatory effect on muscle protein synthesis and preserves body weight better than an emulsion containing LCT only.

animals. These effects may be caused by the rapid increase in plasma levels of free medium chain fatty acids (MCFA), C:8 and C:10, during MCT infusion (13). High levels of MCT have also been reported to delay the utilization of LCT (15). To improve the safety and efficacy of MCTcontaining fat emulsions and to circumvent their disadvantages a structured triglyceride emulsion, containing both LCFA and MCFA bound to the same carbon skeleton, has been synthesized (MLT). This molecule has been used by others and found to be safe in both animals (16-18) and in humans (19). It has also shown promising results regarding the nitrogen balance (16, 17), albumin concentration (•6, 17) and liver nonsecretory proteins (18): The aim of this study was to further evaluate the efficacy of a MCFA and LCFA containing fat emulsion compared to a pure LCT emulsion, with respect to muscle protein synthesis, leucine oxidation and energy expenditure. The effect of glucose supplementation on the utilization of the lipid substrates was also to be determined.

Introduction

Fat emulsions provide critically ill patients with both energy and essential fatty acids. Furthermore, partial replacement of glucose by fat emulsions as the source of non-protein calories minimizes CO 2 production and thereby reduces the metabolic load on the respiratory system (1). Compared to glucose, intravenous administration of fat has shown equal (2, 3) or superior (4) effects on nitrogen economy in surgical patients receiving parenteral nutrition. Long-chain triglycerides (LCT) have, however, been reported to be metabolized slowly, particularly by low-birth-weight infants suffering from sepsis and by intensive care patients (5-7). Medium-chain triglycerides (MCT), C:8 and C: 10, have therefore been suggested as an alternative energy source (8), since they are more rapidly utilized and are not stored in adipose tissue to any appreciable extent (9-11). Side effects including metabolic acidosis (9, 11-13) and increased energy expenditure (14) have been reported after administration of MCT to 23

24

FAT EMULSION CONTAINING MCFA AND LCFA

Material and methods

Animals Male Sprague-Dawley rats (Alab, Stockholm, Sweden) weighing approximately 160 g at arrival, were kept in separate cages in a controlled environment with 21°C _+ 2°C, 40-60% relative humidity and 12 h light-dark cycle. During a period of 5-7 days rats were adapted to wear a leather harness, designed to protect the central venous catheter, tunnelized to the back of the rat. Rats were given a standard chow diet (R3, Astra Ewos AB, Srdert~ilje, Sweden) and water ad libitum during the adaptation period. The models and methods used in the study were approved by the Animal Ethical Committee in Stockholm.

Intravenous solutions 2 different fat emulsions were used, Intralipid 20% (LCT) and a 20% fat emulsion containing both MCFA and LCFA bound the same triglyceride (MLT). The MLT emulsion contained purified interesterified triglycerides from MCTs, C:8 and C:10, and LCTs derived from soy bean oil at a ratio of 40:60 weight %. The C8:C10 ratio was approximately 2.4. The structured triglycerides were synthesized by mixing MCT and LCT oils and heating the mixture in the presence of a catalyst. During the process the triglyceride fatty acids are interesterified in a random manner. The final MLT emulsion thereby contained more than 75% of chemically mixed triglycerides with both LCFA and MCFA on the same carbon skeleton. Egg phospholipids were used to prepare the MLT emulsion. TPN was given isocalorically and isonitrogenously at a dose of 200 kCal, 0.9 g N & 300 ml/kg body weight (bw)/d. In the groups receiving MLT 18 g of triglycerides/kg bw/d were administered. In the LCT groups isocaloric amounts of Intralipid 20% were given. In the groups MLT+G and LCT+G (Glucose 50%, Pharmacia, Halden, Norway) 20% of nonprotein energy was administered as glucose. The amino acid solution used in all groups was Vamin 18 (Pharmacia, Stockholm, Sweden). Vitamins, minerals and trace elements were added according to the requirement of the rat (20). The infusion rate was controlled by infusion pumps (Imed 965 Micro) modified for small animal use.

polyethylene-silicone catheter was inserted into the superior vena cave superior, via the jugular vein (20). Simultaneously, the rats were partially (70%) hepatectomized according to Higgins et al (21). The animal (n = 34) were divided into 4 groups, each receiving different nutritional treatment, LCT+G (n = 10), LCT-G (n = 7), MLT+G (n = 10) and MLTG (n = 7), respectively, to be able to distinguish between the effects of the fat emulsions and glucose as well as to detect if an interaction between fat and glucose occur. Total parenteral nutrition (TPN) was started 3 h after surgery and was then increased stepwise from 25% day 0-1, 50% day 1-2 to 100% of the dose day 2-3 to avoid overloading the liver remnant with fat. On day 2 measurements of RQ, energy expenditure and L-[1-14C]-leucine oxidation were performed using equipment for indirect calorimetry. The estimations were made during continuous TPN infusion. On day 3, protein synthesis was determined in vitro in the intact skeletal muscle extensor digitorum longus (EDL).

Indirect calorimetry On day 2 the animals were transferred to the indirect calorimetry unit for determination of energy expenditure and RQ. Total O2 consumption and COz production were measured for 21 h (22). Energy expenditure and non-protein RQ were calculated according to standard equations (23-25).

Leucine oxidation L-[1-14C]-leucine (NEN, England) was added to the TPN mixture and infused for 3 h (0.33 ~tCi/h) on day 2. Total expired J4CO2 was collected quantitatively in fractions by entrapment in 50 ml 2 M KOH, every 30 min for 3 h of infusion, every 120 min for the next 10 h and during the remaining 8 h every 240 min (26). Absorption test have shown that approximately 99% of the CO2 was trapped in the KOH solution (22). Duplicate samples were counted in a liquid scintillation counter (LKB Wallac 1217 rackbeta, Wallac Oy, Finland). The percentage leucine oxidized was calculated as the ratio between cumulative expired 14CO2 and total ~4C-leucine infused.

Protein synthesis in intact muscle Experimental design After the adaptation period (Day 0) the animals were anaesthetized (Hypnorm Vet, Janssen Pharmaceutica, Beerse, Belgium, i.m. 0.5 ml/kg body weight) and a

After induction of anaesthesia the intact extensor digitorum longus muscle (EDL) was carefully dissected from one leg. The muscle was immediately weighed and incubated for 1 h in McCoy's cell

CHNICALNUTRITION 25 culture m e d i u m (Flow Laboratories, Irvine, Scotland), containing Hepes and U-4C-phenylalanine (0.45 ~tCi/ 3 ml). The phenylalanine concentration in the incubation medium was 0.1 mM. Protein synthesis was determined as the rate of incorporation of a4C-phenylalanine into muscle proteins. The method used the principles described by Fulks et al (27) and Li et al (28), using ~4C-tyrosine. The procedure has been described in detail elsewhere (29). The contralateral EDL muscle was used as control for background values of 14C, remaining from the previous determination of leucine oxidation. This muscle was treated in an identical way except for the incubation procedure, The muscle preparations were washed in 10% trichloroacetic acid (TCA) and lipids were extracted by addition of different solvents. The muscle specimens were then freeze-dried and the fat free dry weight determined. The muscles were dissolved in Soluene 350 and incorporation of 14C-phenylalanine into muscle proteins estimated. Protein synthesis was calculated as nmol Phe/g fat free dry weight (fdw) and hour and corrected for background values. The specific activity in the medium was used as an approximation for intracellular specific activity as described elsewhere (30). The incorporation of U-~4C-phenylalanine into muscle protein was linear for at least 3 h. Data are presented as mean + standard error of the mean (SEM). Two factors crossed analysis of variance (General Linear Model) was performed at the 5% level. This makes it possible to distinguish between the effects of the two fat emulsions, glucose as well as the possible interaction between the fat and glucose energy substrates.

Results

0

-5 .-. ~ -10

~

- 15

o

-20

-25

-30 MLT+G

MLT-G

LCT+G

LCT-G

Fig. 1. Changein total body weight (%), during the whole study period. Values are expressed as mean values + SEM. Asterisks indicate a significant difference compared to the groups receiving LCT, *p < 0.05. operation. After surgery all rats lost on the average between 39 and 46 g in total, or 19-25% in body weight (Fig. 1). During the whole study period rats receiving M L T + G lost 39 + 2.3 g (19.2 + 1.3%) and rats receiving M L T - G lost 40 + 4.5 g (20.3 + 2.4%). This was significantly less than rats receiving L C T who lost 46 + 1.4 g (23.9 + 0.5%) in the group receiving L C T + G and 44 + 3.4 g (25.2 + 1.6%) in the L C T - G group, p < 0 . 0 0 5 (Fig. 1). Glucose supplementation did not influence the results and no interaction between fat and glucose was observed.

Body weight

Energy expenditure

There was no difference in body weight, 191 + 1.5 g, between rats in the 4 different groups on the day of

There was no difference in energy expenditure between the 4 groups on day 2 (Table). The RQ values

Table Energy expenditure, RQ, leucine oxidation and muscle protein synthesis measured during the study period. Values are expressed as mean values + SEM

Energy expenditure (Kcal/kg bw/day) RQ Whole body leucine oxidation (% of 14C-Leucineinfused) Muscle protein synthesis (nmol Phe/g fdw/h)

MLT+G

MLT-G

LCT+G

LCT-G

235 + 6.5

242 + 10.8

235 + 5.6

233 + 9.4

0.74 + 0.02 41.8 + 2.1t

0.69 + 0.02 45.5 + 1.8

0.72 +_0.01 41.0 + 2.9

0.69 + 0.02 50.6 + 3.1

63.9 + 5.3"

57.0 + 5.8"

48.6 + 2.8

50.8 _+4.1

* Significantly different from the groups receiving LCT, p < 0.05. Significantly different from the groups receiving TPN without glucose supplementation,p < 0.05.

26

FAT EMULSION CONTAINING MCFA AND LCFA

were 0.74 + 0.02 and 0.72 + 0.01 in the groups receiving MLT+G and LCT+G, respectively. In the group receiving MLT-G the RQ was 0.69 + 0.02 and 0.69 + 0.02 in the LCT-G group. Slightly higher RQ values were observed in the groups receiving glucose, but this trend was not statistically significant (Table). RQ values of approximately 0.7 indicate that mainly fat was oxidized. Leucine oxidation The leucine oxidation was significantly lower in the groups receiving TPN with glucose, MLT+G and LCT+G, if compared to the groups receiving TPN without glucose, MLT-G and LCT-G, p < 0.05 (Table). There was however, no difference in leucine oxidation between the two types of fat emulsions used. No interaction between fat and glucose was observed. Protein synthesis in skeletal muscle The incorporation of 14C-phenylalanine into muscle protein was significantly higher in rats receiving MLT (Table, Fig. 2). Protein synthetic rate was 63.9 _+ 5.3 and 57.0 + 5.8 nmol/g fdw/h in the groups receiving MLT+G and MLT-G, respectively. In the groups receiving LCT the synthetic rate was 48.6 _+ 2.8 nmol/g fdw/h in the LCT+G group and 50.8 _+ 4.1 nmol/g

70 60

50

!

--l-

e~0

.s= 30

10 0 MLT+G

MLT-G

LCT+G

LCT-G

Fig. 2. Muscleprotein syntheticrate measuredin vitro on day 3. Values are expressedas mean values+ SEM. Asterisks indicate a significant differencecomparedto the groupsreceivingLCT, *p < 0.05.

fdw/h in the group receiving LCT-G (Table, Fig. 2). No effect of glucose and no interaction between fat and glucose were observed.

Discussion In the present study partially hepatectomized rats receiving TPN containing both MCFA and LCFA show both an increased muscle protein synthetic rate and an improved preservation of body weight compared to rats receiving LCT only. Why would a fat emulsion containing MCFA be expected to stimulate muscle protein synthesis more than a fat emulsion containing LCT only? In the literature only a limited number of factors have been reported to stimulate protein synthesis or retard protein breakdown. Ketone bodies have however, been shown to increase protein synthesis when administered both in humans (31) and in dogs (32). MCFAs are known to be ketogenic (12, 13), in contrast to LCFAs. An increased formation of ketone bodies, although not documented in the present study, after infusion of the MCFA containing fat emulsion, may have contributed to the stimulatory effect on muscle protein synthesis in the present study. This would be in accordance with the observation that the fractional muscle protein synthesis is increased by 10% during a 13-OH butyrate infusion (31). This finding was confirmed by Umpleby et al (32) who demonstrated an increased leucine incorporation into muscle protein during infusion of ~3-OH butyrate in postabsorptive dogs. These investigations thus demonstrate that small physiological increases in blood concentrations of ~-OH butyrate can spare protein, probably by reducing the drain of amino acids for gluconeogenesis and thereby decrease amino acid oxidation. MCFAs are partly transported across the mithochondrial membrane without involvement of the carnitine-acylcarnitine translocase system and are more rapidly oxidized than LCFAs (10, 11). Due to the rapid oxidation and the limited capacity to store MCFA in adipose tissue, high levels of acetyl-CoA are generated (9). To handle the elevated levels of acetyl-CoA the demand for oxaloacetate derived from glucose will be increased. Therefore, the need for glucose during infusion of a fat emulsion containing MCFA may be initially higher than during infusion of a fat emulsion containing pure LCFA. It has also been demonstrated that plasma glucose concentrations are decreased during MCT infusions (12). Acetyl-CoA formed from fatty acid oxidation enters the Krebs cycle if fat and carbohydrate degradation are appropriately balanced. The entry of acetyl-CoA depends on the availability of oxaloacetate

CLINICALNUTRITION 27 for the formation of citrate. However, if fat utilization is predominant, acetyl-CoA follows another pathway, producing ketone bodies in the liver, the reason being that the concentration of oxaloacetate is lowered if carbohydrate is not available or properly utilized. During starvation oxaloacetate is used to form glucose and is thus unavailable for condensation with acetylC o A (33). Under such conditions the Krebs cycle is inhibited and the acetyl-CoA is instead directed towards formation o f acetoacetate and ~-OH-butyrate. To meet the requirements of glucose the oxidation o f amino acids is increased. The rats in the present study lost weight and were in a negative energy balance and it was demonstrated that leucine oxidation was significantly increased in the two groups receiving TPN without glucose supplementation, M L T - G and LCT-G. There was however no difference between the two fat emulsions, M L T and LCT with respect to leucine oxidation. The increased skeletal muscle protein synthesis in the M L T group could thus not be attributed to a compensatory mechanism but rather to a more efficient utilization o f the energy supplied. M C T / L C T mixtures have been reported to have higher elimination rates than the M L T emulsion (34) and the M L T emulsion used in the present study was eliminated more rapidly than a LCT emulsion (35). Due to the high elimination rate during infusion of a M C T / L C T mixture the release of M C F A into the blood stream is high (13). High levels of M C F A have been reported to cause toxic effects in dogs (13) and should therefore be avoided. Further, infusion of a M C T / L C T mixture has been reported to increase energy expenditure (17), which in turn may exert a negative influence on respiratory function in critically ill patients (1). In the present study no difference in energy expenditure was o b s e r v e d between rats receiving M L T and LCT emulsions. Thus, administration of a M L T emulsion may constitute a safer way to administer the M C F A s if compared to a M C T / L C T mixture due to the differences in kinetics between the two fat emulsions. The effects observed on muscle protein synthesis and body weight after administration of the M L T emulsion may probably be related to the M C F A content per se. The question remains however if the same result would have been achieved with a M C T / LCT mixture. Other investigators have compared a M C T / L C T mixture with a structured triglyceride containing both M C F A and LCFA. Both M o k et al (17) and Maiz et al (16) demonstrated significantly higher weight gain, nitrogen balance and albumin concentrations in rats receiving M L T emulsion if compared to both a M C T / LCT mixture and a L C T emulsion in burned rats.

However, others have reported conflicting results in which rats with burn injury showed no difference in muscle protein synthetic rate when receiving either LCT, M C T / L C T mixture or M L T (36). The discrepancy in results between these studies may possible be explained by different methods for evaluation o f protein kinetics. In addition, there were differences in energy intake and amount o f energy supplied from lipids between the studies. Different compositions o f the M L T emulsions were also used, from 56% M C F A / 4 4 % L C F A in the study by M o k et al (17) to 36% M C F A / 6 4 % L C F A in the study perforg)ed by Gollaher et al (36). Thus, while a positive influence of M C T / L C T mixture on protein synthesis remains to be convincingly demonstrated, the present results show that administration of an M L T emulsion is a accompanied by a significant increase in skeletal muscle protein synthetic rate. The reason for choosing partial hepatectomy as a study model was to induce a catabolic state that reproducibly causes negative nitrogen balance in the rats. It has been shown that 70% partial hepatectomy and the simultaneous insertion of an i.v. catheter, causes a negative nitrogen balance for 3 days in rats (37). In conclusion, in partially hepatectomized rats intravenous administration of fat emulsions containing structured triglycerides with both M C F A and L C F A exerts a stimulatory effect on muscle protein synthesis and preserves b o d y weight better than emulsions containing LCT only.

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Submission date: 13 May 1995; Accepted after revision: l0 October 1994

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