Gut 1996; 38: 531-537 531 Comparative effects of dietary nucleoside- nucleotide mixture and its components on endotoxin induced bacterial translocation and Gut: first published as 10.1136/gut.38.4.531 on 1 April 1996. Downloaded from small intestinal injury in protein deficient mice AA Adjei, K Yamauchi, Y C Chan, M Konishi, S Yamamoto Abstract nucleotides or cytidine provide a better Background-Nucleoside-nucleotide mix- response. ture has been shown to improve gut mor- (Gut 1996; 38: 531-537) phology and reduce the incidence of bacterial translocation in protein deficient Keywords: nucleosides, nucleotides, bacterial mice. translocation, protein deficiency, endotoxin. Aims-To compare the reparative effect of nucleoside-nucleotide mixture and their individual components on mainte- The gastrointestinal tract plays a primary part nance of gut integrity and bacterial trans- in the digestion and absorption of nutrients, location based on their differential but it is also essential as a barrier to enteric metabolism and utilisation. flora, preventing host invasion by micro-organ- Methods-ICR (CD-1) mice were ran- isms or bacterial endotoxins.1 2 Nevertheless, domised into eight groups of 10 animals recent data suggest that under certain clinical each and fed 20% casein diet (control), and experimental conditions, this intestinal protein free diet, or protein free diet barrier function seems to be impaired leading supplemented with 3 M cytidine, uridine, to the translocation of bacteria or endotoxin to thymidine, inosine, guanosine monophos- the mesenteric lymph nodes, spleen, liver, and phate, or nucleoside-nucleotide mixture bloodstream, initiating the septic process that for four weeks. On the fourth week, each perpetrates multiple organ failure.3 Some com- mouse was injected lipopolysaccharide mercially available parenteral (Aminosyn) and http://gut.bmj.com/ intraperitoneally (50 [ug/500 Al) and the enteral formulas (Osmolite) used for nutri- incidence of bacterial translocation, tional treatment of critically ill or immuno- caecal bacterial populations, and the ileal compromised patients fail to support gut histology, noted 48 hours later. mucosal structure or function. This occurs Results-The death rate in the control because the formulas do not contain gluta- group was 40%/o compared with 10% in the mine, fibre, or nucleic acid components, which nucleoside-nucleotide mixture and 20% are required for normal enterocyte growth or on September 25, 2021 by guest. Protected copyright. each in the individual components repair after an insult and during periods of groups, respectively. Bacterial trans- critical illness.4 location to the mesenteric lymph node There are a number of factors that partici- did occur in 100% of the surviving mice pate in the modulation of the renewal of the fed the control diet in comparison with absorptive epithelium and in the repair of the 44Gb (nucleoside-nucleotide), 50%/o (cyti- gut under abnormal conditions.5 Nitrogen dine), 75°/0 (thymidine), 750/o (uridine), containing substrates seem to be of great 63% (inosine), and 63% (guanosine importance for gut development. Dietary monophosphate). Histologically, the nucleotides have been described as improving damage to the gut was more distinct in growth in weanling rats fed a low protein the protein free diet group. Villous diet,6 and as stimulating the in vitro and in Department of Bacteriology height, crypt depth, and wall thickness in vivo growth of intestinal bifidobacteria.7 8 A A Adjei the nucleoside-nucleotide mixture group Oral supplementation of total parenteral mean (SEM) (5.01 (0.34); 0-87 (0.14); 0.33 nutrition with nucleoside-nucleotide mixture and Research Centre of Comprehensive (0.10)), were respectively, higher com- prevented the intestinal mucosal atrophic Medicine pared with the protein free diet (3.34 changes in the ileum of rats induced by total K Yamauchi (0.34); 0.61 (0.03); 0.18 (0.04)) group. In parenteral nutrition.9 Nucleoside supplemen- Y C Chan the M Konishi cytidine group, crypt depth (0.86) tation increased the rate of maturation and S Yamamoto (0.08)), and wall thickness (0.30 (0.002)) growth in the young rat, as determined by were higher. The same measurements in mass, RNA, DNA, and protein concentra- University of the the components groups tended to be tions and of brush border Ryukyus, Okinawa, activity enzymes.10 Japan higher than the protein free diet group. Nucleotide supplementation restored the bio- Caecal bacterial populations were, how- chemical indices of the small intestine at Correspondence to: Dr AA Adjei, Department of ever, similar in all groups. proximal and distal sites, and improved Bacteriology, University of Conclusions-These results suggest that intestinal changes after induction of chronic the Ryukyus, Nishihara, Okinawa 903-01, Japan. dietary nucleosides and nucleotides are diarrhoea.11-14 Dietary nucleotides are also Accepted for publication essential nutrients for intestinal repair; implicated in the maintenance of the 31 October 1995 and that a mixture of nucleosides and immune response.)5 16 Intraperitoneal or oral 532 Adjei, Yamauchi, Chan, Konishi, Yamamoto administration of nucleoside-nucleotide mix- were randomised into eight groups according ture improved gut morphology and reduced the to the dietary treatment. Each diet was started incidence of bacterial translocation in protein four weeks before the administration of endo- deficient mice.17 18 These substrates have been toxin, and was continued until the mice were on studied in normal humans under stress, and killed. On the fourth week the diet, mice Gut: first published as 10.1136/gut.38.4.531 on 1 April 1996. Downloaded from animal models." 1-4 19 Whereas much attention were intraperitoneally given lipopolysaccharide have been focused on the use of combinations (50 ,ug/500 gl) and killed by cervical disloca- of nucleosides, nucleotides or nucleic acids in tion 48 hours later, and the incidence of bacte- the improvement of gut integrity and function, rial translocation, caecal bacterial populations, one question that has not been considered con- and the damage to the intestine were investi- clusively is which components of nucleic acids, gated. nucleotides, nucleosides are effective. This question is of clinical significance because of the differential metabolism and utilisation of Endotoxin individual nucleotides and nucleosides, as Lipopolysaccharide (from Escherichia coli evidenced by some investigators.2022 To 026:B6, Wako Chemical, Tokyo, Japan) was answer this question, this study was designed to dissolved in pyrogen free saline to a final con- see the effect(s) of dietary nucleosides and centration of 50 ,ug/500 gl; 500 ,l was injected nucleotides on the incidence of endotoxin intraperitoneally into each animal used for the induced bacterial translocation, intestinal experiment. We used 50 ,ug lipopolysaccharide bacterial populations, and morphology of the because previous results showed that this dose gut in protein deficient mice and to evaluate the and timing did not cause death, yet resulted relative importance of nucleosides and in bacterial translocation in 100% of the nucleotides. We used protein deficient mice injected mice.17 18 Lipopolysaccharide was because protein deficiency is the most common used because it has been shown that protein cause of immunosuppression worldwide,23 malnutrition alone does not induce trans- and, in combination with endotoxin, results in location.24 deficiency of the gut and potentiates bacterial translocation.24 Diet The control mice were fed 20% casein diet Methods (nucleic acid free), and the remaining groups of mice were fed protein free diet, or protein Animals free diet supplemented with 3 M guanosine Specific pathogen free ICR (CD-1) mice were monophosphate (guanosine group), inosine http://gut.bmj.com/ obtained from Kyudo Breeding Laboratories (inosine group) (purines), cytidine (cytidine (Kumamoto, Japan) and used for the experi- group), uridine (uridine group), thymidine ment. Weight matched (29+±2 g) 8 week old (thymidine group) (pyrimidines), or a nucleo- female mice were kept in a constant tempera- side-nucleotide mixture (nucleosides-nucleo- ture (25±2°C) and humidity (50-700%) room tide group) per kg diet throughout the with a 12 hour light period from 0800 to investigation. The mixture (Otsuka Pharma- 2000. Animal care was in compliance with ceutical Factory, Tokushima, Japan) was on September 25, 2021 by guest. Protected copyright. applicable guidelines from the Ryukyus developed to compensate for a decrease in the University Policy on Animal Care and Use. intrinsic pools of purines and pyrimidines The mice were kept for one week before the because of the increased enzyme activity in onset of the experiment to acclimatise to our salvage under various abnormal conditions.25 laboratory conditions. During this period the It consisted of inosine (8 g/l), guanosine mice received normal non-purified diet monophosphate (12.2 g/l), cytidine (7.3 g/l), (Nihon Clear, Osaka, Japan). This standard uridine (5.5 g/l), and thymidine (1.8 g/l) at a mouse diet contains 25.5% protein and 4.3% molar ratio of 4:4:4:3:1. The diets were made fat by weight. isonitrogenous and isocaloric by adding an appropriate amount of glycine. Table I shows the composition of the diets. The nucleotide Study protocol content of the control diet and protein free After the period of acclimatisation, the mice diet was negligible as determined by high TABLE I Composition ofexperimental diets (g/kg) Protein Nucleoside- Guanosine Control free diet nucleotide Uridine Thymidine Cytidine Inosine monophosphate Casein 200 0 0 0 0 0 0 0 Glycine 9-6 9-6 0 0 0 0 0 0 Nucleic acid 0 0 6-5 5-5 5-5 5.5 6-0 9-2 Carbohydrate* 670 860-4 863-5 864-5 864-5 864-5 864 860-8 Corn oil 50 50 50 50 50 50 50 50 Mineral mixturet 50 50 50 50 50 50 50 50 Vitamin mixture* 10 10 10 10 10 10 10 10 Cellulose 20 20 20 20 20 20 20 20 *ay-corn starch:sucrose, 2:1 ratio.