A Yazar, K Büyükafþar*, G Polat+, C Pata, #A Kanýk, EN Tiftik, +Ö Baðdatoðlu
Departments of Internal Disease, *Pharmacology, +Biochemistry, # and Biostatistics, Faculty of Medicine, Mersin University, Turkey
Corresponding author: Dr Aziz Yazar, Mersin University, Faculty of Medicine , Department of Internal Medicine, Zeytinlibahce Cad. Eski Otogar Yani , 3079 Mersin Turkey
e-mail: azizyazar@yahoo.com
SMJ 2005 50(1): 27-29
Abstract: Background and Aims: Postprandial increase of 5-hydroxytryptamine (5-HT) has been implicated in irritable bowel syndrome (IBS). There is evidence that nitric oxide (NO) may act as a mediator of 5-HT-evoked secretions in the colon. Our aim is to investigate the role of urinary 5-hydroxyindole acetic acid (5-HIAA) and plasma NO levels (with diarrhoea) in IBS patients. Methods: Nineteen (with constipation) IBS patients (group 1), 22 IBS patients (group 2) and 18 healthy controls (group 3) were included in the study. The diagnosis of IBS was made according to the Rome I Criteria. The urine was collected for determination of 5-HIAA and venous blood was collected from each subject for the measurement of plasma NO levels. Results: The levels of urinary 5-HIAA mmol/day and plasma NO mmol/l of group 1 (22,4±2,2 and 29,4±2 respectively) were significantly higher than group 3(14,2±2,3 and 21,3±2,1 respectively) (p=0,036 and p=0,019 respectively). The NO level of group 1 was also significantly higher than group 2(21,8±1,9) (p=0,021). The 5-HIAA level of group 1 was higher than group 2 (15,2±2,1) and the difference was marginally significant (p=0,055). There was no difference between group 2 and group 3 with respect to 5-HIAA and NO levels. Conclusions: The results of this preliminary study lend support to the involvement of 5-HT in some symptomatology of diarrhoea predominant IBS. Furthermore, NO may be one of the effector mediators of the 5-HT-induced symptoms in these patients.
Key words: 5-hydroxyindole acetic acid, irritable bowel syndrome, nitric oxide
Introduction
The heterogeneity of the clinical expression of irritable bowel syndrome (IBS) is well known and any part of the gastrointestinal system (GIS) may be involved.1,2 Psychoneurotic behaviour, emotional stress, specific food intolerances, and enteric infections have been implicated as causal factors in the pathogenesis of IBS.3 However, the pathophysiologic mechanism underlying the disorder is not fully understood. Recent studies have suggested that 5-hydroxytryptamine (5-HT) is involved in the pathogenesis of the IBS. 5-HT and its receptors are distributed widely throughout the body, including the gastrointestinal tract which has around 95% of the total body 5-HT source.4 5-HT in the gastrointestinal tract is located predominantly in enterochromaffin cells but also in the enteric nervous system. Almost all of the 5-HT in the blood comes from the gastrointestinal tract.5,6 The enzymes monoamine oxidase and aldehyde dehydrogenase found in the kidney and the liver convert 5-HT to 5-hydroxyindole acetic acid (5-HIAA) which is excreted in the urine.
At least seven different 5-HT receptors have been identified, and each type of 5-HT receptors may have several subtypes.7 It has been shown that 5-HT induces contractions in isolated colon preparations of the guinea-pig8 and these contractions are mediated by 5-HT2, 5-HT3, and 5-HT4 receptors.9,10 In isolated tissue preparations 5-HT also induces intestinal secretion of fluid and electrolytes.11,12 Accordingly, alosetron, a 5-HT3 receptor antagonist alleviates pain and bowel-related symptoms in diarrhoea predominant IBS patients.13 There are parellels between the features of diarrhoea predominant IBS patients and the effect of 5-HT on motility by means of diarrhoea, increased gastrointestinal transit14 and intestinal secretion.15 The mechanism responsible for the secretory effect and smooth muscle phasic contractile response to 5-HT in the colon is unclear. In recent studies it has been suggested that 5-HT receptors may be also localized on nonadrenergic, noncholinergic (NANC) inhibitory motoneurons (also called nitrergic neurons) and the secretory effect of 5-HT may be mediated by nitric oxide(NO) release from these motoneurons.11,16-18 Because 5-HT may be responsible for some symptoms of diarrhoea predominant IBS patients and NO may be a neurotransmitter in 5-HT-induced secretion of fluid and electrolytes, we aimed to determine urinary 5-HIAA and plasma NO levels in diarrhoea predominant IBS patients.
Materials and methods
This study was carried out in the department of Internal Medicine of University Hospital. Three groups were included in the study: group one, 19 patients (five male, 14 female) with diarrhoea predominant IBS; group two, 22 (three male, 19 female) with constipation predominant IBS; and group three, 18 (five male, 13 female) healthy controls. The diagnosis of IBS was made according to the international working team (Rome I) criteria.19 All the patients have had symptoms of IBS more than two years and all the following tests were normal in IBS patients: complete blood count, erythrocyte sedimentation rate, stool hemoccult test, stool culture, stool test for ova and parasites, urine analysis and rectosigmoidoscopic examination performed to patients over 40 yr. Diarrhoea predominant patients were put on two-weeks trial of lactose-free diet for lactose intolerance. Patients older than 50 years, with any abdominal surgery or abdominal organic disease were not included in this study. None of the patients were on any medication at the time of the study.
All the tests performed for IBS patients were also normal for the controls. Subjects over 50 years, having previous abdominal surgery, organic bowel disease, IBS or on any medication were not included in the control group. For quantitative analysis of 5-HIAA, 24-h urine samples were collected without preservatives. Bananas, walnuts and tomatoes were banned from the diet for 72 hours before urine collection. Every patient was given a list containing low nitrate (<100 mmol/day) and carbohydrate-rich (85% of the diet) diet. Specimens were refrigerated during collection. On receipt in the laboratory, the urine specimens were thoroughly mixed and the total volume measured and recorded. Aliquots were removed for determination. The pH of the urine was then adjusted to between two and three by addition of six M HCl. Urine samples were analyzed using reversed-phase HPLC with electrochemical detection.20
Analysis of nitrite and nitrate was made using a procedure based on the Griess reaction.21 Blood samples were obtained and centrifuged at 4000 rpm for 10 min. Serum samples were then separated and stored at –700C until used for assay. Equal volumes of serum and potassium phosphate buffer were placed in an ultrafilter tube and centrifuged at 4000 rpm for 45 min. The ultrafiltrates were used in the test. Nitrates were quantitatively converted to nitrites for analysis. Enzymatic reduction of nitrate to nitrite was carried out using coenzymes (NADPH, FAD) in the presence of nitrate reductase in step of incubation assay. N-1-(naphthyl)-ethylenediamine dihydrochloride, sulfanilamide and incubation solutions were mixed. Sodium nitrite of 1.00 mM was used as standard for determination of nitrite and potassium nitrate of 80 mM was used as standard for determination of nitrate. These mixtures were incubated for five min at room temperature in dimmed light and measured at 540 nm. (Nitric oxide colorimetric assay, 1-756-281, Roche Diagnostics GmbH, Mannheim, Germany). The measurements of urinary 5-HIAA and plasma NO levels were performed in a blind fashion.
Data were entered on computer and analysed by SPSS 9.05 program. The results are expressed as means ± standart errors. Statistical analyses were performed first of all by Kolmogorov- Simirnov test to assess the distrubition of the groups.22 After determination the distrubition of the groups One way ANOVA and Tukey Post Hoc tests were used to compare the groups. P values less than 0,05 were considered to be statistically significant.
Results
The mean of age of group one, group two and group three was 40,1 ± 1,9 yr, 37,3 ± 2,1 yr and 36,3 ± 2,1 yr respectively (p=0.42). The gender distribution of the three groups was not different (Chi-Square test, p=0,484). There was significant difference between the three groups with respect to 5-HIAA (p=0.024) (Table 1). The urinary 5-HIAA level in group one was significantly and marginally significantly higher than group three and group two respectively (p=0,036 and p=0,055 respectively). There was no difference between group two and group three with respect to 5-HIAA levels (p=0,94). There was difference between the three groups with respect to plasma NO levels (p=0,009). The level of NO in group one was significantly higher than group two and group three (p=0,021 and p=0,019 respectively) (Table I). We also investigated if there was any correlation between the levels of 5-HIAA and NO in the groups by Pearson’s correlation analysis. There was no significant correlation (group one; r=0,326, p=0,17, group two; r=-0,358, p=0,1, and group three; r=0,07, p=0,76). NO and 5-HIAA levels of the three groups were shown in Fig 1.
Discussion
Recent studies have suggested that neurotransmitters are involved in the pathogenesis of the IBS. Neurotransmitters that may have an important role in IBS include 5-HT, calcitonin gene-related peptide, acetylcholine, substance P, pituitary adenylate cyclase-activating polypeptide, NO, and vasoactive intestinal peptide.23 It has been shown that 5-HT induces fluid and electrolyte secretion in the small intestine and colon11,12,24 and stimulates phasic contractions of the colon.16 5-HT has been implicated in the secretory diarrhoea of carcinoid syndrome, which is associated with increased plasma 5- HT and urinary 5-HIAA, intestinal secretion in response to cholera toxin and intestinal hypersensitivity reactions.25-27 Tropisetron a 5-HT3 receptor antagonist attenuates in some patients the secretory diarrhoea of carcinoid syndrome, which is associated with increased plasma 5-HT and urinary 5-HIAA concentrations.28
5-HT may be involved in the physiological control of gastrointestinal motility and responsible for the diarrhoea of IBS patients. Bearcroft et al.29 found serum 5-HT levels in diarrhoea predominant IBS patients higher than the controls at 0.5, 2 and 2.5 hours of postprandial period. However, they didn’t find any difference between diarrhoea predominant IBS patients and controls with respect to preprandial and postprandial urinary 5-HIAA. In contrast to Bearcroft’s study we asked the patients to collect their urine for 24 hours. Because psychoneurotic behaviour and emotional stress have been implicated on the pathogenesis of the IBS and being in the hospital for a few hours may aggrevate the symptoms of IBS which could be resulted in the increases of plasma 5-HT levels, we let the patients go on their usual daily life, and hence we minimized the stress on our patients during urine collection. However, in our study the finding of increased level of 5-HIAA in diarrhoea predominant IBS group was in accordance of Bearcroft’s results.
5-HT induces electrolyte secretion and stimulates phasic contractions in the colon in vivo.11,16-18 The exact mechanism(s) of 5-HT induced phasic colonic contraction, electrolytes and fluid secretion and eventually diarrhoea was not identified yet. Stoner et al.11 showed a significant chloride secretion response to 2-methyl-5- HT, a 5-HT3 receptor agonist and this secretion was significantly reduced by Nw-nitro-L-arginine methyl ester (L-NAME), a NO synthase inhibitor. In the same study sodium nitroprusside (SNP), a NO donor also induced the chloride secretory response. Kuwahara et al.30 reported that addition of 5-HT to the serosal side of guinea pig of distal colon produced a concentration-dependent increases in short-circuit current caused by chloride secretion. NG-nitro-L-arginine significantly reduced the 5-HT-evoked responses and the NO donor SNP increased basal short-circuit current mainly because of chloride secretion. These results provide evidence that NO generation may be a part of the neural secretory reflex to 5-HT. In our study the NO level of diarrhoea predominant IBS group was higher than the other two groups. In several studies it has been suggested that NO, the nitrergic neurotransmitter is a partial mediator of 5-HT,11,17,30 and there may be other transmitter(s) in the neural secretory response to 5-HT. Dykhuizen et al.31 measured plasma nitrate concentrations in diarrhoea predominant IBS, inflammatory bowel disease, infective gastroenteritis and healthy controls. However, they found a significant plasma nitrate increase only in infective gastroenteritis group.
NO is a prominent mediator of NANC or nitrergic neural activity in the enteric nervous system. NO activates soluble guanylate cyclase to catalyze the conversion of guanosine 5’-triphosphate to cyclic guanosine 5’-monophosphate (cGMP) in the target cell.32 cGMP activates gated ion channels, phosphodiesterases, and protein kinase G. Cyclic nucleotides are known to be potent stimulants of active chloride secretion by direct interaction with the apical chloride channel in the crypt enterocyte.11,33 Accordingly, it has been reported that NO release causes laxative and purgative effect in response to castrol oil34 which is parellel to our study.
In the present study, the lack of any patients with other diarrhoeal disorders limits the interpretation of our results. Blocking the effects of NO might perhaps help to elucidate the effects of NO on 5-HT and its metabolites. Examination of these possibilities would strengthen our results. In the study although increased 5-HT level is the cause or the result of the diarrhoea remains unclear, other findings such that alosetron, a novel 5-HT3 receptor antagonist caused the relief of diarrhoeal symptoms in IBS patients, give support to the notion that 5-HT and/or NO are likely the reason of diarrhoea as 5-HT as well as NO are ones of the effective mediators in the electrolyte secretion and propulsive movement of the gut.4,11,33 Furthermore, several catharthic agents such as castor oil, phenolphthalein, bisacodyl, magnesium sulfate, bile salts, senna, and cascara induced NO formation that causes diarrhoea.34,35 In order to strengthen this hypothesis, a next study would be performed in which the level of 5-HT is detected in diarrhoea predominant IBS patients when they have constipation or after they are treated with antidiarrhoeal agents or serotonin antagonists.
In summary, the urinary 5-HIAA and plasma NO levels were higher in diarrhoea predominant IBS patients than in those with constipation predominant IBS group and controls. These results lend support to the involvement of 5-HT in the symptomatology of diarrhoea predominant IBS and NO may be a mediator of these symptoms.
REFERENCES
1 Kellow JE, Phillips SF. Altered small bowel motility in irritable bowel syndrome is correlated with symptoms. Gastroenterology 1987;92:1885-93.
2 Drossman DA, McKee RC, Sandler RS, et al. Psychological factors in the irritable syndrome. A multiple study of patients and non-patients with irritable bowel syndrome. Gastroenterology 1988;95:701-8.
3 Collins SM. Is the irritable gut inflamed gut? Scand J Gastroenterol 1992;27 suppl 192:102-5.
4 Kim DY, Camilleri M. Serotonin: a mediator of the brain-gut connection. Am J Gastroenterol 2000;95:2698-709.
5 Bertaccini G. Tissue 5-hydroxytryptamine and urinary 5-hydroxyindoleacetic acid after partial or total removal of the gastrointestinal tract in the rat. J Physiol (Lond) 1960;153:239-49.
6 Da Prada M, Tranzer JP, Pletscher A. Storege of 5-hydroxytryptamine in human blood platelets. Experimentia 1972;28:1328-9.
7 Barnes NM and Sharp T. A review of central 5-HT receptors and their function. Neuropharmacology 1999;38:1083-152.
8 Costa M, Furness JB. The sites of action of 5-hydroxytryptamine in nervemuscle preperations from the guinea-pig small intestine and colon. Br J Pharmacol 1979;65:237-48.
9 Sato N, Keiichirg H, Setoguchi M. The pharmacological characteristics of 5- HT4 receptors in the isolated guinea-pig ascending colon. Proceedings of the 6th annual meeting of the Japanese Pharmacological Society 1991;55:393P.
10 Briejer MR, Meulemans AL, Schuurkes JAJ. Site related distribution of 5-HTreceptors in guinea-pig colon ascendens. Eur J Physiol 1991;419:R5.
11 Stoner MC, Scherr AM, Lee JA, Wolfe LG, Kellum JM. Nitric oxide is a neurotransmitter in the chloride secretory response to serotonin in rat colon. Surgery 2000;128:240-5.
12 Rolfe NE, Levin RJ. Neural and non-neural activation of electrogenic secretion by 5-hydroxytryptamine in the rat ileum in vivo. Physiol Scand 1998;162:469- 74.
13 Camilleri M, Northcutt AR, Kong S, Dukes GE, McSorley D, Mangel AW. Efficacy and safety of alosetron in women with irritable bowel syndrome: a randomised, placebo-controlled trial. Lancet 2000;355:1035-40.
14 Vassallo M, Camilleri M, Phillips SF, Brown ML, Chapman NJ, Thomforde GM. Transit trough the proximal colon influences stool weight in the irritable bowel syndrome. Gastroenterology 1992;102:102-8.
15 Oddsson E, Rask-Madson J, Krag E. A secretory epithelium of the small intestine with increased sensitivity to bile acids in irritable bowel syndrome associated with diarrhoea. Scand J gastroenterol 1978;13:409-16.
16 Graf S, Sarna SK. 5-HT-induced colonic contractions: enteric locus of action and receptor subtypes. Am J Physiol 1997;273:G68-G74.
17 Franks CM, Hardcastle J, Hardcastle PT. Involvement of nitric oxide in the response to 5-hydroxytryptamine in the rat in-vivo. J Pharm Pharmacol 1994;46:387-9.
18 Matsuda H, Li Y, Yoshikawa M. Possible involvement of 5-HT and 5-HT2 receptors in acceleration of gastrointestinal transit by escin Ib in mice. Life Sci 2000;66:2233-8.
19 Drossman DA. Functional gastrointestinal disorders: Diagnosis and treatment. Boston, Little, Brown, 1994.
20 Rosano TG, Whitley RJ: Catecholamines and serotonin. In Burtis CA, Ashwood ER, eds. Tietz Textbook of Clinical Chemistry, 3rd ed. Saunders Company, Philadelphia, 1999:1596-7.
21 Green LC, Wagner DA, Glogowski J, Skipper PL, Wishnok JS, Tannenbaum SR. Analysis of nitrate, nitrite, and [15N]nitrate in biological fluids. Anal Biochem 1982;126:131-8.
22 Zor JH. Testing for goodness of fit. In: Zor JH, ed. Biostatistical analysis. Prentice-Hall, New Jersey 1999:478-83.
23 Horwitz BJ and Fisher RS. The irritable bowel syndrome. N Eng J Med 2001;344:1846-50.
24 Kadowaki M, Kuramoto H, Kuwahara A. Morphological relationship between seretonergic neurons and nitrergic neurons for electrolytes secretion in the submucus plexus of the guinea-pig distal colon. Brain Res 1999;831:288-91.
25 Feldman JM. Carcinoid tumours and syndrome. Semin Oncol 1987;14:237- 46.
26 Nilsson O,Cassuto J,Larsson PA, et al. 5-hydroxytryptamine and cholera secretion:a histochemical and physiological study in catsGut 1983;24:542- 48.
27 Castro GA, Harari Y, Russell D. Mediators of anaphylaxis-induced ion transport changes in small intestine. Am J Physiol 1987;253:G540-8.
28 Anderson JV, Coupe MO, Morris JA, Hodgson HJ, Bloom SR. Remission of symptoms in carcinoid syndrome with a new 5-hydroxytryptamine M receptor antagonist. Br Med J 1987;294:1129.
29 Bearcroft CP, Perrett D, Farthing MJG. Postprndial plasma 5- hydroxytryptamine in diarrhoea predominant irritable bowel syndrome: a pilot study. Gut 1998;42:42-6.
30 Kuwahara A, Kuramoto H, and Kadowaki M. 5-HT activates nitric oxidegenerating neurons to stimulate chloride secretion in guinea pig distal colon. Am J Physiol 1998;275:G829-34.
31 Dykhuizen RS, Masson J, McKnight G, et al. Plasma nitrate concentration in infective gastroenteritis and inflammatory bowel disease.Gut 1996;39:393- 95.
32 Moncada S, Palmer RMJ, Higgs EA. Nitric oxide physiology, pathophysiology and pharmacology. Pharmacol Rev 1991;43:109-42.
33 Stoner MC, Kellum JM. Both serotonin and niric-oxide donor cause chloride secretion in rat colonocytes by stimulating cGMP. Surgery 2001;130:236-41.
34 Mascolo N, Izzo AA, Autore G, et al. Nitric axide and castrol oil-induced diarrhea. J Pharmacol Exp Ther 1994;268:291-5.
35 Izzo AA, Mascolo N, Capasso F. Nitric oxide as a modulator of intestinal water and electrolyte transport. Dig Dis Sci 1998;43:1605-20.