This article is accompanied by an editorial. Please see: Duggan C. Glutamine-based oral rehydration solutions: the magic bullet revisited?J Pediatr Gastroenterol Nutr 1998;26:533-535.
Cholera is a disease caused by the toxin of Vibrio cholerae, which causes secretory diarrhea and severe dehydration and circulatory collapse. Cholera is now endemic throughout most of Latin America and has become endemic in northeast Brazil since 1992 when 31,803 cases and 398 deaths were reported (1,2), for an incidence of 21.4 per 100,000 inhabitants and a mortality rate of 1.3%.
The dehydration resulting from cholera and other diarrheal diseases constitutes the major complication and cause of death with these illnesses(1,3). In addition, diarrheal diseases are a major contributor to malnutrition in northeast Brazil and in other tropical developing areas (4). The World Health Organization (WHO) recommended a glucose-based oral rehydration solution that is considered the primary means of treatment for diarrheal diseases including cholera, but this solution fails to reduce the volume of stool or the duration of the diarrheal illness (5). The mortality rate associated with dehydration and malnutrition can be reduced by early treatment with oral rehydration solution (ORS) with feeding, especially breast feeding(6,7).
Vibrio cholerae produces intestinal secretion of water and electrolytes primarily through its enterotoxin, cholera toxin (molecular weight 84 kDa). This toxin is composed of two subunits, A and B, which are responsible for binding to the specific monosialoganglioside (GM1) receptor (for the B subunit), and stimulation by the A subunit of adenylate cyclase by adenosine diphosphate ribosylation of the Gsα protein in the intestinal epithelium (8). The secretory diarrhea results from the stimulation of chloride secretion and the inhibition of neutral sodium chloride absorption. Critical to oral rehydration therapy is the preservation during acute cholera of sodium-coupled absorption with organic substrates such as glucose and neutral amino acids (8). It has been suggested that optimum exploitation of this phenomenon could lead to an improved ORS formulation that would not only successfully replace the deficit of salts and water in diarrhea but would also actively induce absorption and healing of the disrupted barrier function, with reduction of stool volume and duration of diarrhea(9-11).
Glutamine, the principal substrate of the enterocyte, is absorbed by a sodium-coupled contransport in the intestinal mucosa(12-15). In addition, glutamine stimulates mucosal growth and repair in rats after chemo- or radiation therapy(10,16,17). Therefore an oral rehydration solution containing glutamine would be an effective treatment for cholera and other diarrheal diseases. In this work, the effects of a glutamine-based oral rehydration solution on the absorption of sodium, chloride, potassium and water are evaluated, using the perfused rabbit ileal segment model of secretory diarrhea induced by cholera toxin.
MATERIALS AND METHODS
New Zealand White rabbits of both sexes, weighing 1.5 to 2 kg, were fasted for 18 hours with water ad libitum before each experiment. Intestinal perfusion was achieved in loops that were ligated after administration of ketamine anesthesia (35 mg/kg, intramuscularly) and xylazine (5 mg/kg intramuscularly) and a median 8 to 10-cm laparotomy for visualization of the small bowel. An approximate 30-cm segment of terminal ileum was washed with 20 ml of phosphate-buffered saline (pH 7.4). Both proximal and distal ends of the segment were ligated around multiperforated polyvinyl cannula (internal diameter 0.0892 cm, outer diameter 0.2619 cm, wall 0.8636 cm; ColeParmer; Niles, IL, U.S.A.) for perfusion studies. The test solutions were warmed to 37°C, maintained at pH 7.4, and introduced through the proximal cannula, using a pump (900 Holter Roller; Extracorporeal Medical Specialties, King of Prussia, PA, U.S.A.). Perfusion was maintained at a slow rate of 0.6 ml/min throughout the experiment. At the end of the perfusion, the animals were killed, and the dry weight (after desiccation at 90 °C for 48 hours) of the intestinal segment was used for flux calculations.
Drugs and Reagents
Phenolsulfonphthalein (PSP; 50 mg/ml) was used as a non-absorbable marker for calculation of sodium, potassium, chloride, and water net fluxes. Phenolsulfonphthalein was measured spectrophotometrically (Spectrophotometer Model B382; Micronal S.A. Aparelhos de Precisão; São Paulo, SP, Brazil), according to the method developed by Schedl and Clifton(18). Sodium and potassium concentrations in the perfusate were measured by flame photometry (Flame Photometer Model 443 and 21; Instrumentation Laboratory; Lexington, MA, U.S.A.). The colorimetric method for chloride (Labtest Bio. Diagnósticos; Belo Horizonte, Brazil) was used according to manufacturer's instructions. The osmolality of the perfusion samples was measured with a vapor pressure osmometer (Model 5100C; Wescor; Logan, Utah, U.S.A.).
Groups of five animals each were used to test the following oral rehydration solutions (at pH 7.4): (a) modified Ringer's solution (RS), (b) WHO ORS and (c) glutamine-based ORS (Table 1). In treated animals, approximately 30 ml of modified Ringer's solution containing cholera toxin at 1 μg/ml was perfused in the segments for 30 minutes before the initiation of the perfusion with oral rehydration solutions, without cholera toxin and containing PSP marker. Control-treated animals were treated identically but without cholera toxin in the first 30 minutes of perfusion. Thereafter, samples were taken every 10 minutes throughout the 180-minute study period for measurement of electrolyte and PSP concentrations. The volume of the intestinal loops for control was 21 ± 7.5 ml, not statistically different from that of the other groups. There was a small back diffusion(loss) of PSP marker in the cholera toxin (CT)-treated intestinal loops using Ringer's solution, but it was not statistically different from that of control animals (percentage of PSP recovered: RS + CT = 69 ± 16% vs. RS control= 87 ± 12%; n = 5, p > 0.05 by Student'st- test).
The calculations of net fluxes were done, using the Microsoft Excel 4.0 Version for Macintosh (Microsoft Corporation, Cupertine, CA, U.S.A.), and groups were compared using the analysis of variance. Data are presented as mean values ± standard error of the mean. Differences with p< 0.05 were considered statistically significant.
Cholera toxin was obtained from ICN Biomedicals; Aurora, OH, U.S.A.; phenolsulfonphthalein, glutamine, and glucose from Sigma Chemical; St. Louis, MO, U.S.A. salts for perfusion solutions (NaCl, KCl, CaCl2, NaHCO3 and NaH2PO4) and sodium citrate from Sigma.
Cholera toxin caused significant sodium secretion (from a control net absorptive flux of 4.29 ± 2.19 μEq/g per minute to a net secretion of -14.34 ± 2.18 μEq/g per minute; p < 0.05;Fig. 1,). In addition, changes were seen in net water transport with cholera toxin (0.04 ± 0.01 vs. -0.15 ± 0.02 ml water/g per minute; p < 0.05) compared with that of control animals with Ringer's solution without cholera toxin. Initial effects of cholera toxin were noted at 30 and 60 minutes for water and sodium, respectively.
Figures 2 and 3 show the effects of WHO and glutamine-based ORS solutions on sodium and water transport when comparing the results of modified Ringer's solution with those in loops treated with cholera toxin. The WHO ORS significantly reduced intestinal sodium secretion (from-14.34 ± 2.18 to -0.50 ± 0.48 μEq sodium/g/ minutes;p < 0.05). Similarly, water secretion was significantly reduced from -0.15 ± 0.02 to -0.01 ± 0.01 ml/g/ minute (p < 0.01). In contrast, glutaminebased ORS had a much greater effect than WHO glucose-based ORS on net sodium obsorption, reducing the net sodium secretion by 10.31 ± 1.21 μEq/g per minute (p < 0.01), and a greater effect on net water absorption (0.076 ± 0.008 ml/g per minute(p < 0.01). In addition, the effect of cholera toxin was abolished when in the presence of glutamine-based ORS (12.9 ± 1.093μEq sodium/g per minute and 0.107 ± 0.011 ml water/g per minute).
The data shown in Table 2 demonstrate the effects of cholera toxin in a modified Ringer's solution, WHO-based ORS, and glutamine-based ORS on the absorption of chloride and potassium, as well as on the osmolality and pH of the luminal solutions after cholera toxin treatment. The WHO-based ORS increased chloride absorption by 10.13 ± 1.51μEq/g per minute and the net secretory effect of cholera toxin was reduced but not abolished by WHO ORS to -0.86 ± 1.11 μEq/g per minute(p < 0.05). In contrast, cholera toxin did not induce significant chloride secretion when glutamine-based ORS was used (16.90 ± 2.05 and 9.68 ± 1.24 μEq chloride/g per minute; p > 0.05).
Both WHO ORS and glutamine-ORS significantly increased potassium absorption when compared with that with modified Ringer's solution (net WHO ORS = 3.4± 0.2 μEq/g per minute, p < 0.05; net glutamine-ORS = 2.9 ± 0.2 μEq/g per minute, p < 0.05 vs. net Ringer's = 0.4 ± 0.1 μEq/g per minute). The glutamine-based ORS, however, was more effective than WHO ORS at reducing the potassium secretion induced by cholera toxin (Table 2).
Cholera toxin also reduced the osmolality of the intestinal fluid secreted(osmolality of control group = 22.34 ± 3.340 vs. osmolality CT = -21.74± 2.270 μOsm/g per minute), an effect that was significantly reduced by either WHO or glutamine-ORS (Table 2). Although cholera toxin significantly reduced the luminal fluid pH from 7.55 ± 0.05 to 7.36 ± 0.02 (p < 0.05), pH was not altered by WHO ORS or by glutamine-based ORS (Table 2).
Histologic studies were done on slices stained by hematoxylin-eosin in perfused segments of intestinal loops from control animals, RS, WHO ORS and glutamine-ORS, with and without cholera toxin; and they did not show significant changes.
The basis for oral rehydration therapy was first introduced by Craig Wallace et al. (19) at the same time that Curran and Schultz and Zalusky observed sodiumcoupled glucose absorption by intestinal mucosa (19,20). Patients with cholera or animals treated with cholera toxin maintain intestinal mucosal absorption of substrate-coupled sodium absorption (19-21). These observations have enabled the replacement of sodium and water, using glucose-based oral rehydration solutions in patients with cholera and other diarrheal diseases (1).
Recently, an oral rehydration formula based on glutamine has been studied in vitro and in perliminary clinical studies. Rhoads et al. determined the effects of glutamine on intestinal electrolyte transport in piglet jejunum mounted in Ussing chambers (12). They concluded that glutamine promotes electroneutral salt absorption in the small intestine. In addition, they reported a small electrogenic Na+-absorptive response to L-glutamine. In stripped rabbit ileum mounted in Ussing chambers, Lima et al. have also demonstrated that the absorption of sodium in the presence of glutamine was more effective than with glucose(13-15). Moreover, this contransport was maintained in the presence of cholera toxin treatment(21). The results of the current study in a perfused intact intestinal segment further extend and confirm the superiority of glutamine in fully reversing the effects of cholera toxin on net water, sodium, and potassium transport. Because all ORS preparations used in this study had similar osmolality, it further suggests that glutamine was superior in net water, sodium, and potassium absorption. Other reports have also shown that glutamine-based oral rehydration solution is more effective than is glucose in infected pigs with rotavirus or Cryptosporidium spp(22,23). Argenzio et al. examined the effect of glutamine in experimental porcine cryptosporidiosis (22), and showed that glutamine was superior to glucose for use in oral rehydration solution. Rhoads et al. studied the transport response to mucosal L-glutamine after intestinal injury caused by porcine rotavirus. They found that glutamine and alanine were more effective than glucose to stimulate NaCl absorption in porcine rotavirus (23). Nath et al. demonstrated that intestinal sodium absorption in diarrheagenic Escherichia coli-infected rabbit can be enhanced by administering enteral glutamine(24).
Several new oral rehydration solutions have been studied with different amino acid and glucose polymers as substrates for sodium contransport. For example, oral rehydration solutions containing glucose and L-alanine appeared to be superior in treating cholera to the standard WHO-recommended glucose-based oral rehydration therapy (25). Preliminary results from Indonesia in studies of adult patients with cholera, comparing a glutamine-based solution (with 90 mmol/l each of glutamine and glucose) with the standard WHO ORS in adults with cholera, demonstrated a significant 25% reduction in the stool volume in the first 24 hours and a 30% reduction in the total stool fecal volume loss in the group treated with glutamine-based oral rehydration compared with stool volume reduction in the group treated with the WHO oral rehydration fluid (26,27).
Ribeiro et al. studied an oral rehydration solution containing L-glutamine and compared the efficacy of adding 90 mmol/l L-glutamine with 90 mmol/l L-glucose to the standard WHO oral rehydration solution to treat infants between 1 month and 1 year of age with acute noncholera diarrhea and dehydration (28). They showed that a glutamine-based ORS was well tolerated and had similar effect to the standard WHO ORS in treating dehydration in infants with noncholera diarrhea. The ORS solution containing glutamine used in this report had less glutamine and higher osmolality than did our glutamine-ORS preparation. We can not extrapolate results from animal to human, but on the basis of our results, we can at least hypothesize that an ORS preparation with lower osmolality and higher glutamine concentration will potentially work better than the standard WHO solution on net intestinal water and electrolytes absorption, even for noncholera diarrhea.
Although it is estimated that glutamine-ORS would cost more than standard WHO ORS, glutamine-ORS has a potential benefit of healing damaged intestine resulting from the action of several infectious agents and their virulent factors. This hypothesis is undergoing testing by our group in animals, as well as in patients with HIV infection and children living in a high endemic area for diarrheal diseases.
In summary, the data in this study demonstrate clearly that an oral rehydration solution based on L-glutamine is more effective in inducing the absorption of sodium and water than is a solution based on glucose in perfused intestinal segments treated with cholera toxin. In addition, a solution based on L-glutamine is superior to that with glucose in enhancing chloride and potassium absorption in intestinal loops treated with cholera toxin. We conclude that glutamine-based oral rehydration fluids deserve further clinical studies in cholera and other related diarrheal diseases to measure the experimental data shown herein.
Acknowledgment: This work was supported in part by CNPq/UFC and by the National Institutes of Health (NIAID International Collaborative Infectious Diseases Research award) grant # PO1-AI26512 (RLG and AAML).
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