Nutrition Research, Vol. 14, No. 12, pp. 1831-1839, 1994 Copyright �9 1994 Elsevier Science Ltd

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EXPERIMENTAL SHORT-BOWEL SYNDROME: FREE AMINO ACID VERSUS INTACT PROTEIN IN NUTRITIONAL SUPPORT*

Antonio Carlos lglesias*, M.D., Pedro Eder Portari*, M.D., Sergio Zucoloto#, M.D., Helio Vannucchi#, M.D.

* Faculty of Medicine of University of Rio de Janeiro and # Faculty of Medicine oflh'beir~o Preto, University of S~o Panlo, Brazil.

ABSTRACT

The objective of the present study was to evaluate the role of a diet consisting of a free amino acid mixture in the metabolic and nutritional response of rats after resection of g0% of the small intestine. Forty Wistar rats were divided into four groups of 10 animals respectively receiving a free amino acid diet (AR and AS groups) and an intact protein-casein diet (CR and CS groups). Ten animals in each group were submitted to 80% jejtmo-ileal enterectomy (AR and CR), and the remaining animals were sham operated. Nitrogen balance (Nil), diet digestibility, cumulative weight variation (CWV), urinary creatinine excretion, and serum albumin levels were determined. A significant difference in NB (I 3 < 0.05) was observed between the AR and CR groups during theimmediate postoperative period (0.10 + 0.01 gN/period vs. -0.11 + 0.04 gN/period). The digestibility of the free amino acid diets also differed significantly (p < 0.05) when compared to the casein diet during the same period of time (79.2 + 1.1% vs. 46.6 + 2.2%). At the end of the experiment, CWV was -5.1% for Group AR and -7.2% for Group CR (p > 0.05) and serum albumin also showed no significant difference between groups. During the fourth week after surgery, 24-hour urinary ereatinine excretion was significantly greater for AK animals (5.95 + 2.00 rag/24 hours) than for CK animals (4.10 + 1.04 mg/24 hours). We conclude that the free amino acid diet showed a better nutritional performance during the immediate post-entereetomy period, suggesting the possibility of an early use of these diets after extensive intestinal resection.

KEY WORDS: Elemental Diet, Amino Acids, Intestinal Resection, Short- Bowel Syndrome, Nutritional Support, Nitrogen Balance.

INTRODUCTION

The short-bowel syndrome (SBS) observed after extensive resection of thesmall intestine (SI) is characterized by diarrhea, malabsorption, a negative nitrogen balance (NB), and weight loss resulting fTom the significant decrease in absorptive area of the SI (1,2).

Patients with a normal gastrointestinal tract can be fed intact protein orally, but alternative formulas for nitrogen supply are needed for patients with impaired gastrointestinal function (3-12). The selection of a nitrogen source should be based on its nutritional value, with preference given to protein sources of lfigh nutritional value (13). The protein source, the methods used for hydrolysis, the composition of the amino acid mixture, and the addition of carbohydrates and lipids to the diet may affect both intestinal absorption and nutritional value of the diet (7, 14-16).

The objective of this study was to determine the nutritional efficacy of diets having the same nitrogen and calorie contents and differing only in the molecular form of the proteins, administered to rats after 80% resection of the small bowel. The two forms of dietary protein were a free amino acid mixture and intact protein (casein).

1. Supported by CNPq grant n 40.1543-g7.0 Address correspondence and reprint requests to: Dr. Hello Vannucchi, Faculty of Medicine of RibeirSo Preto, University of Sac Paulo, 14049-900 Ribeitfio Preto, SP, Brazil.

1831

1832 A.C. IGLESIAS et al.

MATERIAL AND METHODS

Animals and diets The animals used in this study were treated according to the international rules for

biomedical research on animals established by the WHO. Forty male Wistar rats weighing 217.6 3.6 9 were divided into four groups of 10 animals each denoted CK, CS, AR and AS

according to type of surgery and diet, as described below. The diets were isonitrogen and isocaloric and the nitrogen source was a mixture of free L-amino acids (17) (Ajinomoto, Japan) (Diet A) or intact casein (Diet C) (Tables 1 and 2).

TABLE 1 Diet Composition

Diet Groups (g/100g diet) Diet A Diet C

Amino Acid Mixture 13.33 Casein 12.50 Starch 71.47 72.30 Corn Oil 10.00 10.00 Salt Mixture 4,00 4.00 Vitamin Mixture 1.00 1.00 Choline 0.20 0.20 Protein 10.75 10.75 Enerl~ Level(kcal) 420 420

TABLE 2 Composition of the Amino Acid Mixture (g/100 g diet)

Amino Acid g%

Arginine 0.60 Asparagine 0.40 Glutamic Acid 4.00 Histidine 0.30 Isoleucine 0.50 Leucine 0.75 Lysine 0.70 Methionine 0.40 Cystine 0.20 Phenylalanine 0.40 Tyrosine 0.40 Proline 0.40 Threonine 0.50 Tryptophan 0.15 Valine 0.60 Glycine 2.19 Serine 0.20 Alanine 0.64

AA DIET IN INTESTINAL RESECTION 1833

Twenty animals were anesthetized with ether and submitted to 80% jejunoileal resection, with preservation of 10% of the proximal jejunum and 10% of the distal ileum (Groups CK and AK). The remaining animals were submitted only to section and anastomosis in the midpoint of the small bowel (Groups CS and AS). The animals were kept in individual metabolic cages at a mean temperature of 25~ on a 12-hour light/12-hour dark cycle. Experimental design

The experiment was divided into five study periods, each lasting seven days. The first period was for adaptation and all animals received Diet C. On the first day of the second period (Day 0), the animals were operated and had fi'ee access to glucophysiologic solution. From the third day to the end of the experiment, Groups CR and CS had free access to Diet C and Groups AR and AS had free access to Diet A. Food ingestion was measured daily throughout the experiment. Urine and faces were collected for three days during each period for the determination of excreted nitrogen and the calculation of nitrogen balance and diet digestibility. Twenty-four-hour urine was collected during each study period for creatinine determination (lg). The animals were weighed at seven-day intervals starting on the first day of the experiment. Cumulative weight variation (CWV) was determined using the weight on the day of surgery as tile basal value and subtracting from it the remaining weights obtained in the subsequent determinations, with a total of four CWV values.

Nitrogen balance (mg/pool) was calculated by the difference between ingested and excreted nitrogen (urine + fecal nitrogen). Nitrogen was determined by the Kjeldahl method, Nitrogen digestibility (D) was calculated as D = Ni - Nf/100, where Ni is ingested nitrogen and Nf fecal nitrogen. The animals were sacrificed on the 30th postoperative day and blood was obtained by cardiac puncture for albumin measurement (Labtest Diagnosis System - Cat 19).

Statistical analyses Data are reported as mean + SD. Differences between groups were analyzed by

two-way ANOVA and by Tukey's multiple comparison test. P < 0.05 was considered significant.

RESULTS

The diets were well tolerated by tile animals although among the enterectomized animals diarrhea lasted longer (clinical observation) in the CR than in the AR group (17 vs. 12 days).

CWV data are presented in Figure I. CWV values were negative for Groups CR and AlL with values of-28.20 9.47 9 obtained for CR and of-35.20 - 8.43 9 for AR (p < 0.05). Tile remaining results showed no significant differences. The animals reached the end of the exl~eriment with a weight deficit of-7.2% for Group CR and of-5.1% for Group AK (13 > 0.05). Groups CS and AS presented a positive CWV throughout the study period. CS values were higher than AS values and this difference was significant (p < 0.05) on the occasion of the third and fourth evaluations.

Table 3 summarizes the results of diet ingestion and digestibility. No differences among groups were observed during the first period, whereas during the second period CS and AS animals ingested more food than CR and AK animals (p < 0.05). During the second period, CR animals ingested more food than AR animals (34.0+ 6.4 9 vs. 24.5+ 5.8 9) (13 < 0.05). The digestibility observed in Group CR (46.4 • 2.2%) was lower than that observed in Group AK (79.2 • 1. I%) (p < 0.05). From the third to the fifth period, ingestion continued to be greater in groups CS and AS but Group CS started to ingest more food than group AS (p < 0.05). Digestibility did not differ significantly among CS and AS groups. The differences observed for these parameters between Groups CR and AR were not significant during the last three periods. Twenty-four-hour urinary creatinine excretion was similar for all groups from the first to the third period. During the fourth period, Group AK presented a significant increase in creatinine excretion when compared to Group CR (5.95 2.00 mg/24 hours vs. 4.10 _ 1.04 mg/24 hours; p < 0.05). A difference continued to be present during the fifth period but without statistical

1834 A.C. IGLESIAS et al.

significance. The nitrogen balance data are presented in Figure 2. During the first period all

groups had a positive NB, with no difference amongst themselves (p 0.05). From the second to the filth period, Groups CS and AS presented a positive NB which was higher than that of Groups CR and AR (p < 0.05). No difference in NB was observed between CS and AS animals during the second period, but the NB of Group CR, in addition to being negative (-0.11 _ 0.04 gN/pool) was lower than that of Group AR (+0.10 - 0.01 gN/pool) (p < 0.05). From the third to the filth period, Groups CR and AR maintained a positive NB and did not differ l~om each other, whereas Group CS started to have higher NB values than Group AS (p ' 0.05).

Albumin values were 3.4 - 0.2 9% for Group CS, 3.3 - 0.2 9% for group AS, 2.9 - 0.3 9% for Group CR, and 2.8 - 0.4 9% for group AlL The values obtained for the non- resected groups did not differ from each other, nor did those obtained for the reseeted groups, but the first two values were higher than the remaining ones (p < 0.05).

132.

88

~, 4 4

~. O-

E -22- o

--44-

Groups

�9 Resected + amino acids {AR) D Resected + casein (CR) �9 Control + amino acids (AS) . . r .

o Control + casein (CS) J,

0 7 t4 ',1'1 28 't

Surgery DAY

,WCR vs AR (p(O,05) ,ll,~l, CS vs AS (p(O,05)

FIG. 1 - Cumulative animals body weight variation after enterectomy, expressed as mean + SD.

* C R v s AR (p < 0.05) ** CS vs AS (p < 0.05)

TABLE 3 Diet Consumption (g) and Digestibility (%)

Period 1 2 3 4 DC D DC D DC D DC D

Group

5 DC D

AR 46.0 83.5 24.5* 79.2** 39.9 81.9 39.0 78.9 40.0 74.0 +4.8 +1.2 +5.8 +1.1 +4.7 +7.7 +4.2 +5.2 +8.2 +3.6

CR 43.3 80.9 34.0 46.4 36.0 78.2 37.4 72.6 37.9 72.2 +3.3 +4.3 +6.4 +2.2 +4.4 +5.4 +9.7 +7.7 +6.9 +3.8

AS 42.5 85.4 60.0 93.4 46.3 91.6 43.2 93.1 43.9 91.6 +2.8 +2.8 +4.4 +1.3 +7.9 +1.9 +9.6 +1.0 +4.3 +2.0

CS 41.9 80.8 61.3 92.7 62.7 # 91.7 5-3.4 # 93.2 ~ . 8 # 91.0 +1.9 +4.5 +7.8 +1.3 +9.9 +1.0 +5.3 +1.2 +6.0 +1.8

DC = Diet consumption; D = Digestibility Mean + SD *AR < CR (p < 0.05); **AR > CR (p < 0.05); #CS > AS (p > 0.05)

AA DIET IN INTESTINAL RESECTION 1835

A

ta.

0 . 9 -

0 , 6 -

0 , 3 - _o

• 0 - o

z-0.08- -0.16-

Groups �9 Reeected + omino ocids (AR)

- IIResected + cosein (CR) ,IPR" [ ] Control + omino ocids (AS)

+ eosein (CS)

1 2 3 4 5 Study Periods

~1" AR vsCR (p<O,05) "ll-I, CS vs AS (p(O,05)

F I G . 2 - Nitrogen balance values obtained in the five different study periods, expressed as mean + SD. * ARvs CR(p <0.05) ** CS vs AS (p < 0.05)

DISCUSSION

Elemental diets are used to provide nutritional support, especially under conditions of severely impaired gastrointestinal function, as is the case for SBS (3-6, 19). Positive results with these diets have been reported in terms of weight gain and positive NB. However, the use of the protein source as inta.ct protein, partial protein hydrolysate or amino acid mixture has been questioned in terms of the nutritional value of each preparation (20). This is definitely due to the utilization of different experimental variables sucl~ as quantity and nature of the protein source, of the carbohydrates and lipids used in the diets, calorie intake, ingestion route, age, pathologic condition, and animal species studied (7, 13, 14, 19, 21-26).

In the present study diarrhea was present (clinical observation) for a longer period of time in animals fed the diet containing intact protein than in the animals fed the amino acid diet. According the literature disappearance of diarrhea seemed to be related to development of adaptative hyperplasia in the intestinal remnant (19). The changes in bile acid metabolism after extensive resections may be partly responsible for steatorrhea. One of the probable mechanisms involved is bile acid binding to undigested protein, so that the elemental diet would have a better effect than intact protein (27).

The differences observed in diet consumption may have been due to the lower palatability of the amino acid diet compared to protein, with a consequent lower food intake by

1836 A.C. IGLESIAS et al.

tile rats (! 5). Another possible explanation for tile lower ingestion of the amino acid diet may be its lfigh osmolarity, which occurs wben the amino acid concentration is 20 to 30% but not when the concentration of the amino acid mixture or protein is 5 to 10% (28). However, in other experiments as well as in the present one a lower diet consumption was observed at concentrations of the amino acid mixture of the order of 10% (19,29).

The mean body weight of tile four different groups was similar on the day of surgery, witb a significant difference between enterectomized and non- enterectomized groups occurring thereafter. CWV analysis revealed that the weight loss during the first four days niter enterectomy was significantly greater for Group AlL probably due to its lower food ingestion. The second measurement demonstrated a different behavior when compared to the previous with Group AR starting to gain weight and Groups CR continuing to present a weight loss. During tiffs phase, NB as well as diet digestibility were significantly higher in AR animals. During the third and fourth evaluations both groups presented CWV values indicating a certain degree of weight recovery although the mean values for Group CR were lower than those for Group AR. These differences may possibly reflect a better nutritional utilization of the free amino acid diet.

Urinary creatinine is considered to be a parameter representative of body muscle mass (30). In the present experiment, no differences were observed among the four groups during the first and second periods: From the third period on, CR animals started to excrete less

creatinine, with the difference reaching statistical significance in relation to AR animals during the fourth period. This fact may reflect a greater loss of muscle protein atter surgery in Group C1L as shown by the incomplete recovery of lean body mass of these animals due to deficient nitrogen incorporation.

NB is suggested to be a more appropriate parameter than weight evolution for the evaluation of the ex'tent of protein catabolism (21). The most common error in NB evaluation occurs when the nitrogen losses of all possible source of eliminations are not considered, so that highly positive nitrogen balances do not reflect the real extent of protein incorporation by the organism. In the present study, NB included only the urinary and fecal losses, but the results could be used to compare the four experimental groups. The reduction of the absorptive surface resulting from intestinal resection had a negative effect on/fiB. Among enterectomized animals, the free amino acid diet promoted better nitrogen retention during the immediate postoperative period. Althougb diet consumption was lower in AR animals, NB, and the digestibility of the amino acid diet were significantly greater than for the intact protein diet during the immediate postoperative period, with a consequent lower urinary and fecal nitrogen loss in AR animals during this phase. From the third period on, NB values were equivalent for the two groups although NB tended to increase in Group CR and to decrease in Group AR, in which this trend was due to a greater urinary nitrogen loss (31-33).

The determination of serum protein levels may reflect the visceral protein status. The concentration of these proteins may be affected by the reduction in the rate of synthesis associated with malnutrition and/or increased catabolism. Analysis of the serum albumin values at the end of the experiment showed significantly lower values for enterectomized animals, reflecting a certain degree of malnutrition.

The weight gain of rats niter extensive Sl resection is influenced by the site and extent of resection (19). Studies comparing the nutritional effect of chemically defined diets with that of laboratory rat chow atter intestinal resection (25, 26) showed that these diet maintained the nutritional status but not as well as done by the laboratory chow, However, these diets differed significantly in nitrogen amount and also with respect to the remaining components. In the present study the two experimental diets used were the same in terms of the above dietary factors, a fact that could facilitate their comparison. Under experimental conditions similar to the present ones, it was observed that after 65% resection of the distal SI the greatest weight gain was obtained with a diet whose protein source was an amino acid mixture and casein, tbllowed by casein and by the amino acid mixture admini~ered alone. However, weight gains were directly related to food intakes (19). The same was observed using an experimental model based on malnourished rats (28, 34). Itoh et al. (35) reported that an amino acid mixture simulating casein and intact casein are nutritionally equivalent. In our

AA DIET IN INTESTINAL RESECTION 1837

experiment the difference in weight gain observed between CS and AS animals was probably due to the greater ingestion observed in group CS.

Yamamoto et al. (28), in a study of three different molecular forms of the protein source offered to normal, protein-deficient and hepatectomized rats, observed that growth and NB were similar for the three different diets, except that urinary ammonium excretion was greater in the group receiving the amino acid diet. Among gastreetomized rats, intact protein showed the worst nutritional performance up to the 10th postoperative day, as also observed in the present study.

In summary, the present study showed that the amino acid mixture had a better nutritional performance during the immediate post-enterectomy phase, an important fact not only from a nutritional viewpoint but also in terms of the stimulation of intestinal adaptation. We caution against direct extrapolation of these data to humans. However, the data do indicate that in enterectomized patients the formulas selected for early nutritional support should contain an amino acid mixture as nitrogen source. Further studies are needed to evaluate this option.

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1838 A.C. IGLESIAS et al.

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Accepted for publication July 13, 1994.