Effect of total parenteral nutrition, systemic and glutamine on gut mucosa in rats
sepsis,
S. YOSHIDA, M. J. LESKIW, M. D. SCHLUTER, K. T. BUSH, R. G. NAGELE, S. LANZA-JACOBY, AND T. P. STEIN Departments of Surgery and Molecular Biology, University of Medicine and Dentistry of New Jersey, School of Osteopathic Medicine, Camden, New Jersey 08103; and Thomas Jefferson University, Philadelphia, Pennsylvania 19128 Yoshida, S., M. J. Leskiw, M. D. Schluter, K. T. Bush, R. G. Nagele, S. Lanza-Jacoby, and T. P. Stein. Effect of total parenteral nutrition, systemic sepsis, and glutamine on gut mucosa in rats. Am. J. PhysioZ. 263 (Endocrinol. Metab. 26): E368-E373, 1992.-The effect of the combination of total parenteral nutrition (TPN) and systemic sepsis on mucosal morphology and protein synthesis was investigated. Rats were given a standard TPN mixture consisting of glucose (216 kcal . kg-l . day-‘), lipid (24 kcal 9kg-l day-l), and amino acids (1.5 g Nekg-l-day-l) for 5 days. On the 5th day the rats (n = 37) were randomized into four groups according to diet as follows: 1) control nonseptic on standard TPN, 2) control nonseptic on TPN with glutamine, 3) septic on standard TPN, and 4) septic with the TPN supplemented with glutamine. Twenty hours after the injection of Escherichia cob, the rats were given a 4-h constant infusion of [U-l*C]leucine to determine the mucosal fractional protein synthesis rates. The following results were obtained. 1) Histological examination showed that systemic sepsis caused tissue damage to the ileum and jejunum. 2) Glutamine supplementation attenuated these changes. 3) There were no visible changes to the colon either from glutamine supplementation or sepsis. 4) Sepsis was associated with an increase in mucosal protein synthesis and decreased muscle synthesis. 5) Addition of glutamine to the TPN mix further increased protein synthesis in the intestinal mucosa of septic rats. protein synthesis
pothesis in a septic rat model and 2) to determine whether the atrophy of the gut mucosa could be attenuated by adding a gut-specific nutrient, glutamine, to the infusate. Mucosal status was investigated by light and electron microscopy and by measuring the rate of mucosal protein synthesis. Glutamine was provided in the form of glycylglutamine (1).
l
GASTROINTESTINAL MUCOSA normally functions as an effective barrier to prevent the transmigration of intestinal bacteria and endotoxins into the underlying connective tissue and subsequent entry into the systemic circulation. Weakening of this barrier is associated with increased translocation of intestinal bacteria and endotoxins (17, 18). Increased permeability of the gut has been implicated as a critical component in the multiple-systems organ failure that often follows sepsis (17, 18). Thus it is possible that any factor that can cause weakening of the gut membrane barrier can have serious consequences to the patient. One such factor that has received much attention in recent years is total parenteral nutrition (TPN). TPN has been shown to cause atrophy of the gut. In rats, this atrophy can be attenuated by adding gut-specific fuels such as glutamine, ketone bodies, and short-chain fatty acids to the infusate (5, 10, 11, 17, 19). Another factor that could contribute to weakening of the gastrointestinal barrier are the changes in blood flow to the intestinal tract that can occur during sepsis (13). Septic patients are often fed by TPN. The combination of systemic sepsis of nongut origin and TPN-mediated atrophy of the gastrointestinal mucosa might lead to greater damage to the gut wall than TPN alone. The objectives of the present study were 1) to test this hy-
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METHODS
Female Sprague-Dawley rats (280-300 g) were prepared for TPN as previously described (21). They were given a standard TPN mixture consisting of glucose (2 16 kcal kg-l day-l), lipid (24 kcal kg-’ *day+, Nutrilipid; American McGaw, Irvine, CA), and amino acids (1.5 g N vkg-’ . day-l, Freamine III; American McGaw). On the 5th day the rats were randomized into the following four groups: 1) control nonseptic on standard TPN (diet I), 2) control nonseptic on TPN with glutamine (diet II), 3) septic on standard TPN, and 4) septic on TPN with glutamine. Diet I was a continuation of the standard TPN solution; diet II was isonitrogenous and isocaloric with standard TPN, but 15 g/l amino acids were removed and replaced with 11.1 g/l glycylglutamine (Ajinomoto, Tokyo, Japan). Sepsis was induced by the injection of lOlo Escherichia coli into the catheter (12). The controls were given an injection of an equal volume of saline. Twenty hours after the injection of saline or E. coli, the rats were given a constant infusion of [U-14C]leucine (2.0 &i/h) in full-strength diet for 4 h via the central venous catheter. At 24 h survival is >90%. At the end of the isotope infusion period the rats were killed. Small segments of the jejunum, ileum, and proximal and distal colon were removed and prepared for subsequent light and electron microscopy as described below. The mucosal layer of the remainder of the segments was scraped off and promptly frozen in liquid nitrogen. The gastrocnemius muscle was also removed. Samples were stored at -20°C until analyzed. Light and eZectron microscopy. The segments of intestine were prepared for transmission electron microscopy by fixing in 2.5% glutaraldehyde in 0.1 M sodium cacodylate buffer (pH 7.2) for 2 h at room temperature. After rinsing in cacodylate buffer they were postfixed/in 1% osmium tetroxide in 0.1 M cacodylate for 1.5 h at room temperature, dehydrated in a series of increasing concentrations of ethanol, and embedded in Epon-Araldite. Thick (1.0~pm) sections were cut with glass knives and stained with toluidine blue. Thin longitudinal sections through the gastrointestinal mucosa were cut with a diamond knife and mounted onto Formvar-coated slotted grids (1 x 2-mm single hole; Electron Microscopy Sciences, Fort Washington, PA). This system facilitated visualization of the entire section without the interference of grid bars. Sections were contrasted with uranyl acetate and lead citrate and stabilized by exposing briefly to a low-intensity electron beam before examining at higher magnification with a Zeiss EM 109 transmission electron microscope. BiochemicaZ analyses. The mucosa and muscle were thawed and homogenized in “Seraprep” (Pickering, Mountainview,
$2.00 Copyright 0 1992 the American Physiological
l
Society
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SEPSIS
AND
CA). The protein precipitate was separated from the supernatant by centrifugation at 10,000 g for 15 min and then washed three times with 10% Seraprep. The specific activity of the precipitate was determined by dissolving it in 1 N NaOH and measuring the total amount of protein present using the modified Lowry method (Sigma, St. Louis, MO) and then counting an aliquot of that solution for l*C in a beta counter (LKB, Bethesda, MD). To determine the specific activity of the tissue free amino acids, the first supernatant was lyophilized to dryness to remove any ketoisocaproate, reconstituted in water, and 1 ml of the aqueous layer was then removed and counted for r*C using a beta counter. The free leucine concentration was determined on a O.Ol-ml aliquot of the aqueous layer using a Waters highperformance liquid chromatography with postcolumn derivitization. Methods of calculation. Mucosal fractional protein synthesis rates (k,) were calculated by using the formula of Garlick et al. (7), where Sn is the specific activity of protein-bound leucine, Sr is the specific activity of leucine in the mucosal intracellular fluid, t is the duration of the isotope infusion (in h), and Xi = 60
$=(&J(E)-(&)
(l)
Statistical analysis of the data was done by analysis of variance, using a statistical package supplied by Hewlett-Packard (Palo Alto, CA). RESULTS
Histology. There were no visible histological changes with the colon, either from the addition of glutamine or with sepsis. However, there were very marked changes in the jejunum and ileum with sepsis. Figure 1 shows the ultrastructure of intestinal epithelial cells in the jejunal crypts of Lieberkuhn of rats exposed to TPN alone
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for 5 days. Comparison with Fig. 2 shows that sepsis selectively damaged the cells situated at the base of the crypts. This region contains primarily stem cells, which undergo mitotic activity and provide a source of cells for the crypts and villus. In normal orally fed rats, the cells reach the tip of the villus within 3 days. The most conspicuous morphological indicator of damage to these cells was the presence of abnormally swollen and distorted mitochondria throughout the cytoplasm (Fig. 2). Some of the cells appeared to be in the early stages of necrosis. Surprisingly, the cell junctions, particularly the desmosomes of the terminal bar, were less conspicuous than those in rats exposed to TPN alone for 5 days (Fig. 1). This finding may represent the first stages of weakening of the structural integrity of the gut wall. Crypt cells situated close to the lumen of the gut and epithelial cells lining the surfaces of the villi were apparently normal and comparable with controls. Similarly, cells in the surrounding lamina propria, muscularis mucosa, muscularis externa, and serosa appeared to be unaffected by sepsis, at least during the 24-h treatment period used in this study. Inclusion of glutamine was found to alleviate the sepsis-induced damage to the cells at the base of the ileal crypts described above (Fig. 3). Few cells in the crypts of glutamine-supplemented rats showed signs of mitochondrial damage or necrosis, and junctional complexes at their apical corners were prominent and comparable with those in rats exposed to TPN alone (Fig. 1). Protein synthesis. The isotope kinetics measure the ratio Sn/Sr, which can be converted into estimates of the fractional synthesis rates by Eq. 1. Systemic sepsis was associated with an increase in Sn/Sr when compared against the control rats (group I) for the jejunum,
Fig. 1. Low-magnification electron micrograph (X6,000) showing intestinal epithelial cells in jejunal crypts of Lieberkuhn rats exposed to total pare&era1 nutrition (TPN) for 2 days. L, lumen;MV, microvilli.
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Fig. 2. Low-magnification micrograph (~2,000) of intestinal epithelial cells in jejunal crypts of rata subjected to combination of TPN and sepsis. Cells at base of crypts show signs of degeneration, highlighted by presence of swollen mitochondria (arrows) containing few cristae. L, lumen.
proximal colon, and distal colon but not ileum. Sn/Sr was increased for all four gut segments in the glutamine-supplemented septic rats when compared against the control nonseptic rats (group 1, Table 1). For ileum, proximal and distal colon Sn/Sr was higher in the glutamine-supplemented rats (group 110 than in the unsupplemented septic rats (group IV). Glutamine did not affect the muscle protein fractional synthesis rate in control rats. In contrast to the mucosa where increased protein synthesis was found, sepsis
caused decreased skeletal muscle protein synthesis (group III vs. group 0. This decrease was partially attenuated by addition of glutamine to the diet (group III vs. group IV, P = 0.06; Table 1). DISCUSSION
Correlation of the histological data with isotope kinetics. This study compared isotope kinetics with histological observations. Although gross interpretation of histological findings is not problematic, interpretation of whole
Fig. 3. Electron micrograph (~2,500) of intestinal epithelium of rats exposed to TPN and sepsis, with glutamine as dietary supplement. Structural features of cells were comparable with controls. Few cells in intestinal crypts of glutamine-supplemented rats showed signs of mitochondrial damage or necrosis. L, lumen; MV, microvilli.
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SEPSIS
Table 1. Summary
AND
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of isotope kinetic data Control
Control
12 Yejunum SH SI ks
10
3,412+220 21,863+2,241 118t9
Ileum SB
+ Gln
4,225*407 20,480k 1,876 151k14
2,916*166
3,395+267
Sepsis
Sepsis + Gln
13
14
4,167k415 13,154&1,344” 247t34*
4,059+405*
4,167_+444 13,230+990§ 224+22§
3,996+308
15,992?863 132kll
22,029*3,087-b 124t16
22,152+3,071* 141t17
14,425* 1,297$§ 207+20$$
k
1,649290 30,952+2,583 43t5
1,846+128 30,43824,701 49k6
2,252+209 28,786+ 1,808 55t3*
1,950+182 22,153+2,330 65+5$§
SI ks
40,144+3,415 39&3
3 1,846*2,278 55+4-F
2,468*56 35,559+2,228 51&4*
2,563+287 29,398+4,223 68+7$§
27lt22 26,723+4,509 8.ltl.O
151t26 24,804+3,308 4.lt0.8*
22,946+ 1,395 6.2t0.4
SI
ks
Proximal colon SH SI
Distal colon SB
2,109+170
Muscle sI3
2,514+284
249t 19 22,446+ 1,703 8.1t0.9
SI
ks
197t13
Data are means t SE. Sn and Sr, specific activities of mucosal protein-bound leucine and leucine in tissue free amino acids. k,, fractional synthesis rate in %/day. Statistics by analysis of variance. * P < 0.05, control vs. sepsis. t P < 0.05, control vs. control + Gln. $ P < 0.05, sepsis vs. sepsis + Gln. Q P c 0.05, control + Gln vs. sepsis + Gln.
body isotope kinetics can be. Determination of the rate of protein synthesis requires the measurement of the amount of isotope incorporated into protein (S,), the isotopic enrichment of the precursor pool (S,), and the time course. The continuous-infusion method is a single-point assay and assumes that this point is determined at plateau. This enables the estimation of the mean specific activity of the tissue free amino acid pools. Figure 4 shows that reasonable plateaus were obtained at 4 h in the ileum and proximal colon of control (group I) and septic rats (group 111). Others have also concluded that a 4-h infusion of [ 14C] leucine is adequate for protein synthesis determinations in stressed rats (15, 22). The crucial assumption of the method is that the specific activity of leucine in the tissue free amino acids reflect the specific activity of the actual amino acid precursor pool for protein synthesis. The method has been extensively used over the last 20 years and has not been
T e 1
I 1
I 1
I I
1 1
1
2
5
4
TIME
I
9
I I
I
5
6
(h)
Fig. 4. Change of mucosal tissue free amino acids with infusion time. Each point is mean of data from 3 rats. Data are means t SE. 0, control ileum (group I); 0, septic ileum (group III); A, control proximal colon (group I); and A, septic proximal colon (group III).
unduly criticized. The values for the control rats are comparable with those reported previously by Garlick et al. (6) using the pool-flooding method. For example, Garlick reported fractional synthesis rates for jejunal mucosa of 119 t 9 %/day, we found rates of 118 t 9 %/day. The complicating factor in this study is the histological evidence for mucosal tissue damage in the jejunum and ileum of the septic rats in group 111. If there are dead cells present they will contribute unlabeled amino acids to the measured aggregate tissue free amino acids. If the cells are injured, it is possible that the precursor pool may no longer be in equilibration with the overall tissue free amino acid pool. In either case, the result would be a dilution of the measured tissue free amino acid pool. For jejunum, a significant (40%, P c 0.01) decrease in S1 was found. Although the ileal S1 data do not show a decrease in Sr, the histology shows extensive mucosal injury. Therefore, for these two tissues in the septic rats (group M), it is questionable whether S& reflects protein synthesis or tissue damage. These reservations do not apply to the proximal colon and distal colon for groups III and IV or the four gut segments in the glutamine-supplemented sepsis group (group IV), since histological examination showed no evidence of tissue damage. Further support for interpreting differences in SB/SI in terms of changes in mucosal protein synthesis comes from a comparison of group I (control) against group IV (septic + glutamine). Sr was reduced compared with group I (P < 0.02, paired t test), and SB was consistently higher (P < 0.02, paired t test) in the glutamine-treated septic rats (group IV), i.e., despite a decrease in precursor pool specific activity, the specific activity of protein-bound leucine was increased. Effect of sepsis. Electron microscopy showed that systemic sepsis results in considerable damage to the structural integrity of the wall of the small intestine only 24 h after the induction of sepsis. The morphological damage to the small intestine occurred even though the
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gut was not the primary site of sepsis. Further experimentation is required to determine why systemic sepsis has such a marked effect on the small intestine. Alteration of blood flow to the gut is one possibility (13). Irrespective of why systemic sepsis induces tissue damage to the gut, a likely consequence is enhanced translocation of bacteria. Should this occur, it would increase the likelihood for the overwhelming of the host’s defences by adding gut-originated bacteria to those introduced via the catheter, thereby further aggravating the situation. With the colon, no obvious histological damage was observed with sepsis, but there was a marked increase in the fractional synthesis rates of the colonic mucosa. The increase in protein synthesis is most likely due to increased activity by immunoglobulin A (IgA) secretory cells (S-IgA, see Ref. 5). In sepsis there is enhancement of mucosal immunity associated with increased S-IgA activity (5). About 25% of the mucosal cells are immune cells (3) Effect of glutamine. Addition of glutamine to the TPN mix was clearly beneficial. Protein synthesis rates were much higher in the glutamine-supplemented group (group IV) when compared with the control glutamine-supplemented rats (group II). Glutamine attenuated the histological damage to the jejunum and ileum caused by the sepsis. Glutamine also increased the fractional protein synthesis rate of the colonic mucosa when compared against the non-glutamine-supplemented septic rats (group I vs. group 111). Colonic mucosal protein synthesis was significantly greater in the glutamine-supplemented group (group Iv) than in the unsupplemented group (group III). Thus sepsis was associated with an increase in mucosal protein synthesis, and the increase is greatest if supplemental glutamine is supplied. Souba et al. (18) and Ardawi et al. (3) reported decreased glutamine usage and utilization by the gut after sepsis and endotoxemia. Nevertheless, glutamine-supplemented TPN protects against bacterial translocation from the gut (2, 17, 19). To explain this paradox of net decreased uptake yet apparent beneficial effects from supplemental glutamine, Ardawi et al. (3) and Ardawi and Newsholme (4) postulated that glutamine utilization by other enterocytes is decreased to allow for an increased rate of glutamine utilization by the immune cells. Our findings of improved protein synthesis with glutamine and decreased tissue damage are consistent with their hypothesis. Because -25% of the mucosal cells are immune cells, which have a critical need for glutamine, it is not surprising that these cells benefit from supplemental glutamine (3). Role of muscle. The increase in gut mucosal protein synthesis occurred at the expense of skeletal muscle. In the present study, as in other studies of stress, sepsis caused a decrease in muscle protein synthesis, and this decrease could be attenuated with glutamine (8,9,14,20). Muscle protein synthesis was greater in the septic rats given glutamine compared with septic rats not given glutamine (P = 0.06), but the improvement in protein synthesis did not attain the value found for nonseptic rats on the same diet (Table 1). We have previously shown that, in this septic rat model, endogenous glutamine
THE
GUT
production is increased by ~20% from 1,959 to 2,331 prnol kg-l h-l (23), so th e amount of dietary glutamine given, - 500 pmol . kg- l h-l corresponds to about an additional 25 % increase in glutamine availability. In summary, our studies show that 1) systemic sepsis and TPN lead to more rapid degeneration of the mucosa of the small intestine than TPN alone, 2) inclusion of glutamine in the TPN mix attenuates the histological changes caused by sepsis, and 3) systemic sepsis is associated with increased mucosal protein synthesis and decreased muscle protein synthesis, and the addition of glutamine increases protein synthesis further in the intestinal mucosa of septic rats. l
l
This work was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grant DK-35612. Address for reprint requests: T. P. Stein, Dept. of Surgery, UMDNJ-SOM, 401 Haddon Ave., Camden, NJ 08103. Received 19 April 1991; accepted in final form 23 March 1992. REFERENCES
1. Abumrad,
N. N., E. L. Morse,
H. Lochs,
P. E. Williams,
and
Possible sources of glutamine for parenteral nutrition: impact on glutamine metabolism. Am. J. Physiol. 257 (Endocrinol. Metczb. 20): E228-E234, 1989. 2. Alverdy, J. C. Effects of glutamine-supplemented diets on immunology of the gut. J. Parenter. Enteral Nutr. 14, Suppl.: 109S113s, 1990. S. A. Adibi.
3. Ardawi, M. Newsholme.
S., Y. S. Jamal,
A. A. Ashy,
H. Nasr,
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Glucose and glutamine metabolism in the small intestine of septic rats. J. Lab. Clin. Med. 115: 660-668, 1990. 4. Ardawi, M. S., and E. A. Newsholme. Glutamine, the immune system, and the intestine (Editorial). J. Lab. Clin. Med. 115: 654655, 1990. 5. Burke, D.
J.,
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Glutamine-supplemented immune function. Arch.
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improves gut
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A rapid and convenient technique for measuring the rate of protein synthesis in tissues by injection of 3H-phenylalanine. Biochem. J. 12:
6. Garlick,
P. J., M.
719-723,198O.
7. Garlick, P. J., D. J. Millward, and W. P. T. James. The diurnal response of muscle and liver protein synthesis in vivo in meal fed rats. Biochem. J. 136: 935-945, 1973. 8. Hammarqvist, E. Vinnars.
F., J. Wernerman,
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Alanyl-glutamine counteracts the depletion of free glutamine and the postoperative decline in protein synthesis is skeletal muscle. Ann. Surg. 212: 637-644, 1990.
9. Karner, and J.
10.
J., E. Roth, G. Ollenschlager, P. Furst, A. Simmel, Karner. Glutamine containing dipeptides as infusion substrates in the septic state. Surgery 106: 893-900, 1989. Koruda, M. J., R. H. Rolandelli, D. 2. Bliss, J. Hastings, J. L. Rombeau, and R. G. Settle. Parenteral nutrition supple-
mented with short-chain fatty acids: effect on the small-bowel mucosa in normal rats. Am. J. Clin. Nutr. 51: 685-689, 1990. 11. Kripke,
S. A., A. D. Fox, J. M. L. Rombeau, and R. G. Settle.
Berman,
J. A. DePaula,
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Inhibition of TPN associated colonic atrophy with B-hydroxybutyrate. Surg. Forum XxX1X: 48-50,1988.
12. Lang, C. H., C. J. Bagby, J. L. Ferguson, and J. J. Spitzer. Cardiac output and redistribution of organ blood flow in hypermetabolic sepsis. Am. J. Physiol. 246 (Regulatory Integrative Camp. Physiol. 15): R331-R337, 1984. 13. Lanza-Jacoby, S., and A. Tabares. Triglyceride kinetics, tissue lipoprotein lipase, and liver lipogenesis in septic rats. Am. J. Physiol. 258 (Endocrinol. Metab. 21): E678-E685, 1990. 14. Millward, D. J., M. M. Jepson, and A. Omer. Muscle glutamine concentration and protein turnover in vivo in malnutrition and endotoxemia. Metab. Clin. Exp. 38: SuppZ. 1: 6-13, 1989.
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AND
15. Mok, K. T., A. Maiz, A. Yamazaki, J. Sobrado, V. K. Babayan, L. L. Moldawer, B. R. Bistrian, and G. L. Blackburn. Structured medium and long chain triglyceride emulsions are superior to physical mixtures in sparing body protein in the burned rat. Metab. Clin. Exp. 33: 910-915, 1984. 16. Rennie, M. J. Muscle protein turnover and the wasting due to injury and disease. Br. Med. Bull. 41: 257-264, 1985. 17. Smith, R. J., and D. W. Wilmore. Glutamine nutrition and requirements. J. Parenter. Enteral Nutr. 14, Suppl.: 94S-99S, 1990. 18. Souba, W. W., K. Hershowitz, V. S. Klimberg, R. M. Salloum, D. A. Plumley, T. C. Flynn, and E. M. Copeland. The effects of sepsis and endotoxemia on gut glutamine metabolism. Ann. Surg. 211: 543-549, 1990. 19. Souba, W. W., K. Herskowitz, R. M. Salloum, M. K. Chen, and T. R. Austgen. Gut glutamine metabolism. J. Parenter.
Enteral Nutr. 14, Suppl. 4: 45S-5OS, 1990.
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20. Stehle, P., J. Zander, N. Mertes, S. Albers, C. Puchstein, P. Lawin, and P. Furst. Effect of parenteral glutamine peptide supplements on muscle glutamine loss and nitrogen balance after major surgery. Lancet. 1: 231-233, 1989. 21. Stein, T. P., R. C. Fried, M. J. Leskiw, M. D. Schluter, and G. P. Buzby. The effect of calorie source (glucose, LCT, MCT) on protein metabolism in septic rats. Am. J. Physiol. 250 (Endocrinol. Metab. 13): E312-E318, 1986. 22 . Teo, T. C., S. J. DeMichele, K. Selleck, V. K. Babayan, G. L. Blackburn, and B. R. Bistrian. Administration of structured lipid composed of MCT and fish oil reduces net protein catabolism in enterally fed burned rats. Ann. Surg. 210: 100-106, 1989. S., S. Lanza-Jacoby, and T. P. Stein. Leucine and 23. Yoshida, glutamine metabolism in septic rats. Biochem. J. 276: 405-409,
1991.
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sepsis,
S. YOSHIDA, M. J. LESKIW, M. D. SCHLUTER, K. T. BUSH, R. G. NAGELE, S. LANZA-JACOBY, AND T. P. STEIN Departments of Surgery and Molecular Biology, University of Medicine and Dentistry of New Jersey, School of Osteopathic Medicine, Camden, New Jersey 08103; and Thomas Jefferson University, Philadelphia, Pennsylvania 19128 Yoshida, S., M. J. Leskiw, M. D. Schluter, K. T. Bush, R. G. Nagele, S. Lanza-Jacoby, and T. P. Stein. Effect of total parenteral nutrition, systemic sepsis, and glutamine on gut mucosa in rats. Am. J. PhysioZ. 263 (Endocrinol. Metab. 26): E368-E373, 1992.-The effect of the combination of total parenteral nutrition (TPN) and systemic sepsis on mucosal morphology and protein synthesis was investigated. Rats were given a standard TPN mixture consisting of glucose (216 kcal . kg-l . day-‘), lipid (24 kcal 9kg-l day-l), and amino acids (1.5 g Nekg-l-day-l) for 5 days. On the 5th day the rats (n = 37) were randomized into four groups according to diet as follows: 1) control nonseptic on standard TPN, 2) control nonseptic on TPN with glutamine, 3) septic on standard TPN, and 4) septic with the TPN supplemented with glutamine. Twenty hours after the injection of Escherichia cob, the rats were given a 4-h constant infusion of [U-l*C]leucine to determine the mucosal fractional protein synthesis rates. The following results were obtained. 1) Histological examination showed that systemic sepsis caused tissue damage to the ileum and jejunum. 2) Glutamine supplementation attenuated these changes. 3) There were no visible changes to the colon either from glutamine supplementation or sepsis. 4) Sepsis was associated with an increase in mucosal protein synthesis and decreased muscle synthesis. 5) Addition of glutamine to the TPN mix further increased protein synthesis in the intestinal mucosa of septic rats. protein synthesis
pothesis in a septic rat model and 2) to determine whether the atrophy of the gut mucosa could be attenuated by adding a gut-specific nutrient, glutamine, to the infusate. Mucosal status was investigated by light and electron microscopy and by measuring the rate of mucosal protein synthesis. Glutamine was provided in the form of glycylglutamine (1).
l
GASTROINTESTINAL MUCOSA normally functions as an effective barrier to prevent the transmigration of intestinal bacteria and endotoxins into the underlying connective tissue and subsequent entry into the systemic circulation. Weakening of this barrier is associated with increased translocation of intestinal bacteria and endotoxins (17, 18). Increased permeability of the gut has been implicated as a critical component in the multiple-systems organ failure that often follows sepsis (17, 18). Thus it is possible that any factor that can cause weakening of the gut membrane barrier can have serious consequences to the patient. One such factor that has received much attention in recent years is total parenteral nutrition (TPN). TPN has been shown to cause atrophy of the gut. In rats, this atrophy can be attenuated by adding gut-specific fuels such as glutamine, ketone bodies, and short-chain fatty acids to the infusate (5, 10, 11, 17, 19). Another factor that could contribute to weakening of the gastrointestinal barrier are the changes in blood flow to the intestinal tract that can occur during sepsis (13). Septic patients are often fed by TPN. The combination of systemic sepsis of nongut origin and TPN-mediated atrophy of the gastrointestinal mucosa might lead to greater damage to the gut wall than TPN alone. The objectives of the present study were 1) to test this hy-
THE
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0193-1849/92
METHODS
Female Sprague-Dawley rats (280-300 g) were prepared for TPN as previously described (21). They were given a standard TPN mixture consisting of glucose (2 16 kcal kg-l day-l), lipid (24 kcal kg-’ *day+, Nutrilipid; American McGaw, Irvine, CA), and amino acids (1.5 g N vkg-’ . day-l, Freamine III; American McGaw). On the 5th day the rats were randomized into the following four groups: 1) control nonseptic on standard TPN (diet I), 2) control nonseptic on TPN with glutamine (diet II), 3) septic on standard TPN, and 4) septic on TPN with glutamine. Diet I was a continuation of the standard TPN solution; diet II was isonitrogenous and isocaloric with standard TPN, but 15 g/l amino acids were removed and replaced with 11.1 g/l glycylglutamine (Ajinomoto, Tokyo, Japan). Sepsis was induced by the injection of lOlo Escherichia coli into the catheter (12). The controls were given an injection of an equal volume of saline. Twenty hours after the injection of saline or E. coli, the rats were given a constant infusion of [U-14C]leucine (2.0 &i/h) in full-strength diet for 4 h via the central venous catheter. At 24 h survival is >90%. At the end of the isotope infusion period the rats were killed. Small segments of the jejunum, ileum, and proximal and distal colon were removed and prepared for subsequent light and electron microscopy as described below. The mucosal layer of the remainder of the segments was scraped off and promptly frozen in liquid nitrogen. The gastrocnemius muscle was also removed. Samples were stored at -20°C until analyzed. Light and eZectron microscopy. The segments of intestine were prepared for transmission electron microscopy by fixing in 2.5% glutaraldehyde in 0.1 M sodium cacodylate buffer (pH 7.2) for 2 h at room temperature. After rinsing in cacodylate buffer they were postfixed/in 1% osmium tetroxide in 0.1 M cacodylate for 1.5 h at room temperature, dehydrated in a series of increasing concentrations of ethanol, and embedded in Epon-Araldite. Thick (1.0~pm) sections were cut with glass knives and stained with toluidine blue. Thin longitudinal sections through the gastrointestinal mucosa were cut with a diamond knife and mounted onto Formvar-coated slotted grids (1 x 2-mm single hole; Electron Microscopy Sciences, Fort Washington, PA). This system facilitated visualization of the entire section without the interference of grid bars. Sections were contrasted with uranyl acetate and lead citrate and stabilized by exposing briefly to a low-intensity electron beam before examining at higher magnification with a Zeiss EM 109 transmission electron microscope. BiochemicaZ analyses. The mucosa and muscle were thawed and homogenized in “Seraprep” (Pickering, Mountainview,
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l
Society
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SEPSIS
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CA). The protein precipitate was separated from the supernatant by centrifugation at 10,000 g for 15 min and then washed three times with 10% Seraprep. The specific activity of the precipitate was determined by dissolving it in 1 N NaOH and measuring the total amount of protein present using the modified Lowry method (Sigma, St. Louis, MO) and then counting an aliquot of that solution for l*C in a beta counter (LKB, Bethesda, MD). To determine the specific activity of the tissue free amino acids, the first supernatant was lyophilized to dryness to remove any ketoisocaproate, reconstituted in water, and 1 ml of the aqueous layer was then removed and counted for r*C using a beta counter. The free leucine concentration was determined on a O.Ol-ml aliquot of the aqueous layer using a Waters highperformance liquid chromatography with postcolumn derivitization. Methods of calculation. Mucosal fractional protein synthesis rates (k,) were calculated by using the formula of Garlick et al. (7), where Sn is the specific activity of protein-bound leucine, Sr is the specific activity of leucine in the mucosal intracellular fluid, t is the duration of the isotope infusion (in h), and Xi = 60
$=(&J(E)-(&)
(l)
Statistical analysis of the data was done by analysis of variance, using a statistical package supplied by Hewlett-Packard (Palo Alto, CA). RESULTS
Histology. There were no visible histological changes with the colon, either from the addition of glutamine or with sepsis. However, there were very marked changes in the jejunum and ileum with sepsis. Figure 1 shows the ultrastructure of intestinal epithelial cells in the jejunal crypts of Lieberkuhn of rats exposed to TPN alone
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for 5 days. Comparison with Fig. 2 shows that sepsis selectively damaged the cells situated at the base of the crypts. This region contains primarily stem cells, which undergo mitotic activity and provide a source of cells for the crypts and villus. In normal orally fed rats, the cells reach the tip of the villus within 3 days. The most conspicuous morphological indicator of damage to these cells was the presence of abnormally swollen and distorted mitochondria throughout the cytoplasm (Fig. 2). Some of the cells appeared to be in the early stages of necrosis. Surprisingly, the cell junctions, particularly the desmosomes of the terminal bar, were less conspicuous than those in rats exposed to TPN alone for 5 days (Fig. 1). This finding may represent the first stages of weakening of the structural integrity of the gut wall. Crypt cells situated close to the lumen of the gut and epithelial cells lining the surfaces of the villi were apparently normal and comparable with controls. Similarly, cells in the surrounding lamina propria, muscularis mucosa, muscularis externa, and serosa appeared to be unaffected by sepsis, at least during the 24-h treatment period used in this study. Inclusion of glutamine was found to alleviate the sepsis-induced damage to the cells at the base of the ileal crypts described above (Fig. 3). Few cells in the crypts of glutamine-supplemented rats showed signs of mitochondrial damage or necrosis, and junctional complexes at their apical corners were prominent and comparable with those in rats exposed to TPN alone (Fig. 1). Protein synthesis. The isotope kinetics measure the ratio Sn/Sr, which can be converted into estimates of the fractional synthesis rates by Eq. 1. Systemic sepsis was associated with an increase in Sn/Sr when compared against the control rats (group I) for the jejunum,
Fig. 1. Low-magnification electron micrograph (X6,000) showing intestinal epithelial cells in jejunal crypts of Lieberkuhn rats exposed to total pare&era1 nutrition (TPN) for 2 days. L, lumen;MV, microvilli.
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Fig. 2. Low-magnification micrograph (~2,000) of intestinal epithelial cells in jejunal crypts of rata subjected to combination of TPN and sepsis. Cells at base of crypts show signs of degeneration, highlighted by presence of swollen mitochondria (arrows) containing few cristae. L, lumen.
proximal colon, and distal colon but not ileum. Sn/Sr was increased for all four gut segments in the glutamine-supplemented septic rats when compared against the control nonseptic rats (group 1, Table 1). For ileum, proximal and distal colon Sn/Sr was higher in the glutamine-supplemented rats (group 110 than in the unsupplemented septic rats (group IV). Glutamine did not affect the muscle protein fractional synthesis rate in control rats. In contrast to the mucosa where increased protein synthesis was found, sepsis
caused decreased skeletal muscle protein synthesis (group III vs. group 0. This decrease was partially attenuated by addition of glutamine to the diet (group III vs. group IV, P = 0.06; Table 1). DISCUSSION
Correlation of the histological data with isotope kinetics. This study compared isotope kinetics with histological observations. Although gross interpretation of histological findings is not problematic, interpretation of whole
Fig. 3. Electron micrograph (~2,500) of intestinal epithelium of rats exposed to TPN and sepsis, with glutamine as dietary supplement. Structural features of cells were comparable with controls. Few cells in intestinal crypts of glutamine-supplemented rats showed signs of mitochondrial damage or necrosis. L, lumen; MV, microvilli.
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Table 1. Summary
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of isotope kinetic data Control
Control
12 Yejunum SH SI ks
10
3,412+220 21,863+2,241 118t9
Ileum SB
+ Gln
4,225*407 20,480k 1,876 151k14
2,916*166
3,395+267
Sepsis
Sepsis + Gln
13
14
4,167k415 13,154&1,344” 247t34*
4,059+405*
4,167_+444 13,230+990§ 224+22§
3,996+308
15,992?863 132kll
22,029*3,087-b 124t16
22,152+3,071* 141t17
14,425* 1,297$§ 207+20$$
k
1,649290 30,952+2,583 43t5
1,846+128 30,43824,701 49k6
2,252+209 28,786+ 1,808 55t3*
1,950+182 22,153+2,330 65+5$§
SI ks
40,144+3,415 39&3
3 1,846*2,278 55+4-F
2,468*56 35,559+2,228 51&4*
2,563+287 29,398+4,223 68+7$§
27lt22 26,723+4,509 8.ltl.O
151t26 24,804+3,308 4.lt0.8*
22,946+ 1,395 6.2t0.4
SI
ks
Proximal colon SH SI
Distal colon SB
2,109+170
Muscle sI3
2,514+284
249t 19 22,446+ 1,703 8.1t0.9
SI
ks
197t13
Data are means t SE. Sn and Sr, specific activities of mucosal protein-bound leucine and leucine in tissue free amino acids. k,, fractional synthesis rate in %/day. Statistics by analysis of variance. * P < 0.05, control vs. sepsis. t P < 0.05, control vs. control + Gln. $ P < 0.05, sepsis vs. sepsis + Gln. Q P c 0.05, control + Gln vs. sepsis + Gln.
body isotope kinetics can be. Determination of the rate of protein synthesis requires the measurement of the amount of isotope incorporated into protein (S,), the isotopic enrichment of the precursor pool (S,), and the time course. The continuous-infusion method is a single-point assay and assumes that this point is determined at plateau. This enables the estimation of the mean specific activity of the tissue free amino acid pools. Figure 4 shows that reasonable plateaus were obtained at 4 h in the ileum and proximal colon of control (group I) and septic rats (group 111). Others have also concluded that a 4-h infusion of [ 14C] leucine is adequate for protein synthesis determinations in stressed rats (15, 22). The crucial assumption of the method is that the specific activity of leucine in the tissue free amino acids reflect the specific activity of the actual amino acid precursor pool for protein synthesis. The method has been extensively used over the last 20 years and has not been
T e 1
I 1
I 1
I I
1 1
1
2
5
4
TIME
I
9
I I
I
5
6
(h)
Fig. 4. Change of mucosal tissue free amino acids with infusion time. Each point is mean of data from 3 rats. Data are means t SE. 0, control ileum (group I); 0, septic ileum (group III); A, control proximal colon (group I); and A, septic proximal colon (group III).
unduly criticized. The values for the control rats are comparable with those reported previously by Garlick et al. (6) using the pool-flooding method. For example, Garlick reported fractional synthesis rates for jejunal mucosa of 119 t 9 %/day, we found rates of 118 t 9 %/day. The complicating factor in this study is the histological evidence for mucosal tissue damage in the jejunum and ileum of the septic rats in group 111. If there are dead cells present they will contribute unlabeled amino acids to the measured aggregate tissue free amino acids. If the cells are injured, it is possible that the precursor pool may no longer be in equilibration with the overall tissue free amino acid pool. In either case, the result would be a dilution of the measured tissue free amino acid pool. For jejunum, a significant (40%, P c 0.01) decrease in S1 was found. Although the ileal S1 data do not show a decrease in Sr, the histology shows extensive mucosal injury. Therefore, for these two tissues in the septic rats (group M), it is questionable whether S& reflects protein synthesis or tissue damage. These reservations do not apply to the proximal colon and distal colon for groups III and IV or the four gut segments in the glutamine-supplemented sepsis group (group IV), since histological examination showed no evidence of tissue damage. Further support for interpreting differences in SB/SI in terms of changes in mucosal protein synthesis comes from a comparison of group I (control) against group IV (septic + glutamine). Sr was reduced compared with group I (P < 0.02, paired t test), and SB was consistently higher (P < 0.02, paired t test) in the glutamine-treated septic rats (group IV), i.e., despite a decrease in precursor pool specific activity, the specific activity of protein-bound leucine was increased. Effect of sepsis. Electron microscopy showed that systemic sepsis results in considerable damage to the structural integrity of the wall of the small intestine only 24 h after the induction of sepsis. The morphological damage to the small intestine occurred even though the
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gut was not the primary site of sepsis. Further experimentation is required to determine why systemic sepsis has such a marked effect on the small intestine. Alteration of blood flow to the gut is one possibility (13). Irrespective of why systemic sepsis induces tissue damage to the gut, a likely consequence is enhanced translocation of bacteria. Should this occur, it would increase the likelihood for the overwhelming of the host’s defences by adding gut-originated bacteria to those introduced via the catheter, thereby further aggravating the situation. With the colon, no obvious histological damage was observed with sepsis, but there was a marked increase in the fractional synthesis rates of the colonic mucosa. The increase in protein synthesis is most likely due to increased activity by immunoglobulin A (IgA) secretory cells (S-IgA, see Ref. 5). In sepsis there is enhancement of mucosal immunity associated with increased S-IgA activity (5). About 25% of the mucosal cells are immune cells (3) Effect of glutamine. Addition of glutamine to the TPN mix was clearly beneficial. Protein synthesis rates were much higher in the glutamine-supplemented group (group IV) when compared with the control glutamine-supplemented rats (group II). Glutamine attenuated the histological damage to the jejunum and ileum caused by the sepsis. Glutamine also increased the fractional protein synthesis rate of the colonic mucosa when compared against the non-glutamine-supplemented septic rats (group I vs. group 111). Colonic mucosal protein synthesis was significantly greater in the glutamine-supplemented group (group Iv) than in the unsupplemented group (group III). Thus sepsis was associated with an increase in mucosal protein synthesis, and the increase is greatest if supplemental glutamine is supplied. Souba et al. (18) and Ardawi et al. (3) reported decreased glutamine usage and utilization by the gut after sepsis and endotoxemia. Nevertheless, glutamine-supplemented TPN protects against bacterial translocation from the gut (2, 17, 19). To explain this paradox of net decreased uptake yet apparent beneficial effects from supplemental glutamine, Ardawi et al. (3) and Ardawi and Newsholme (4) postulated that glutamine utilization by other enterocytes is decreased to allow for an increased rate of glutamine utilization by the immune cells. Our findings of improved protein synthesis with glutamine and decreased tissue damage are consistent with their hypothesis. Because -25% of the mucosal cells are immune cells, which have a critical need for glutamine, it is not surprising that these cells benefit from supplemental glutamine (3). Role of muscle. The increase in gut mucosal protein synthesis occurred at the expense of skeletal muscle. In the present study, as in other studies of stress, sepsis caused a decrease in muscle protein synthesis, and this decrease could be attenuated with glutamine (8,9,14,20). Muscle protein synthesis was greater in the septic rats given glutamine compared with septic rats not given glutamine (P = 0.06), but the improvement in protein synthesis did not attain the value found for nonseptic rats on the same diet (Table 1). We have previously shown that, in this septic rat model, endogenous glutamine
THE
GUT
production is increased by ~20% from 1,959 to 2,331 prnol kg-l h-l (23), so th e amount of dietary glutamine given, - 500 pmol . kg- l h-l corresponds to about an additional 25 % increase in glutamine availability. In summary, our studies show that 1) systemic sepsis and TPN lead to more rapid degeneration of the mucosa of the small intestine than TPN alone, 2) inclusion of glutamine in the TPN mix attenuates the histological changes caused by sepsis, and 3) systemic sepsis is associated with increased mucosal protein synthesis and decreased muscle protein synthesis, and the addition of glutamine increases protein synthesis further in the intestinal mucosa of septic rats. l
l
This work was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grant DK-35612. Address for reprint requests: T. P. Stein, Dept. of Surgery, UMDNJ-SOM, 401 Haddon Ave., Camden, NJ 08103. Received 19 April 1991; accepted in final form 23 March 1992. REFERENCES
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