Epidemiologic Survey, Functional Examination And Pathophysiologic Investigation Of The Functional Constipation In Childhood

IntroductionConstipation is a common complaint in childhood; it is a series of symptoms, but not an independent disease. The incidence reported by Thowa and Heston university hospital was 3%-8% among which 90%-95% was the functional constipation. On the base of this data, there were 20 million children involved in our country with the total amount of children was up to 300 million. Constipation does harm to the physical and mental health in children, and further body development. Till now the pathophysiological ground of the functional constipation in childhood is unclear, and fortunately, drug abuse remained the main problem in treatment. The aim of this investigation was just to work on these fields so as to resolve these problems mentioned above.With attention to the importance of constipation in medical science, there were numerous surveys carried out in the epidemiology of the functional constipation in the world, especially in adulthood. Till now, there have been many epidemiologic investigations of adult constipation and the normal stooling pattern reported in Beijing, Guangzhou, Chengdu, Chongqing, Hangzhou, Tianjin, Shanghai, Xi'an, and other cities. The government has listed the constipation survey of aged people into the "ninth five-year" project (the number is 96-906-05-09) and put lots of attention in it. The normal stooling pattern could be influenced by many factors, such as the dietary habit, the psychological factor, the circumstance, the race, the culture and so on. The children are in physiological development stage and the gastrointestinal system is still immature. So there are many differences between children and adult functional constipation. The epidemiologic investigation of the functional constipation in childhood is relatively rare, especially in China. At the beginning of these unsolved problems, the study was carried out on the cluster epidemiologic investigation of the children in representative cities in the north of China. The aim was to acquire the incidence of the functional constipation in childhood and to found the criteria of diagnosis, and further to explore the normal stooling pattern as well.Functional constipation in childhood is a complicated pathophysiologic process. The exact pathogenesis is not clear till now. Colorectal motility, function of pelvic floor muscle and disorder of intestinal flora might be either involved. In the past years, there have been numerous published issues reported that colon inertia was considered to be an important factor lead to the functional constipation. Animal experiments have demonstrated that endogenous cannabinoids could inhibit the gastrointestinal motility, mainly by the receptor CB1 distributed in the terminal nerve. Fatty acid amide hydrolase (FAAH) is a novel membrane enzyme could catalyze the hydrolysis of endogenous cannabinoids in vivo. Thus the mount of endogenous cannabinoids could theoretically be controlled by the activity and the amount of FAAH expression both in vivo and in vitro to achieve the purpose of gastrointestinal motility regulation. Till now not any reports have been published reflecting whether FAAH was expressed or acted in human gastrointestinal tract. Another aim of this study was to try hard to observe the expression of FAAH in human colon by means of the immunohistochemistry, the Western blotting and the quantitative PCR technique both on the protein and the mRNA level, and to explore its roles in the development of slow transit constipation in childhood. In addition, with the rapidly development of molecular ecology, the molecular biological technique was widely used in identification and classification of the microorganism, more and more intestinal flora have been isolated and identified, the function of these floras in gastrointestinal tract was also gradually confirmed that beyond the traditional digestion and absorption. More and further functions such as nutrition and immunity and motility charge have been explored. As revealed that gastrointestinal motility could be regulated by metabolic product of intestinal flora, especially the short-chain fatty acid, which can influence the human gastrointestinal motility through many different mechanisms. So the DNA finger prints of the normal and abnormal intestinal flora was established by PCR-SSCP and BOX-PCR method, and clone sequencing based on BOX-PCR was carried out to observe the variation of the proportion of the intestinal flora in normal and functional constipation children. Meanwhile, the gas chromatographic technique was also combined used so that the amount and proportion of the short chain fatty in the normal and functional constipation children could also be observed. From above, it is promising that the role of the intestinal flora and its metabolic product-short chain fatty acid in the pathogenesis of the functional constipation in childhood could be approached.Materials and Methods1,EpidemiologyA screening program for FC in childhood was carried out in 5 northern cities (Beijing, Tianjin, Shenyang, Changchun, and Harbin) of China according to symptoms using Rome II criteria. Random clustered sampling of the inhabitants was carried out under stratification of city, zone and school or school nursery. Sample size of each area was in proportion to the population of the area. The range of age was 2-14. All subjects studied were requested to fill in a questionnaire distributed by the teacher who was trained prior to survey. A preliminary program was firstly performed before the screening program, after that the formal screening program was performed, according to the result of the screening, a small number of patients who fulfilled the criteria were further selected to undergo detail clinical examination in the hospital including laboratory examination, colonic transit time, defecography or/and barium enema, electromyologram and anorectal manometry to exclude organic disease of the colon. Prevalence of FC of the population was then adjusted by the rate of correct diagnosis during detailed study. Potential risk factors and the relative symptoms were explored by comparing frequencies between FC group and non-FC group using X~2 and logistic analysis so that the symptomatic criteria for diagnosis could be approached.2,Physical examinationAll the subjects involved in this study were divided into 2 groups. One group was control, and 33 healthy volunteers (21 males and 12 females) aged 2-13 years (mean 5 years) were involved in this group. The other was constipation group, in which 25 patients (15 males and 10 females) aged 3-14 years (mean 7 years) referred to constipation according to the Rome II criteria were involved. In this study the simplified method of radio opaque markers was used to determine the total gastrointestinal transit time and segmental colonic transit time of the normal and constipated children, and simultaneously X ray defecography and anorectal manometry were also combined used to explore the etiology and classification of the functional constipation in childhood.3,Expression of FAAH in human colon and its mode of action inthe slow-transit constipation in children.The investigate objects were divided into functional constipation (FC) group and control group. 16 children were involved in the control group including 10 boys and 6 girls, with a mean age of 7.5 years, and 22 children in the FC group, including 15 boys and 7 girls, the mean age was 7 years. All of the subjects met the standard of STC. All of the colon samples were classified into ascending colon, descending colon and sigmoid colon basing on the position. Among the total, there were 5 ascending colon, 3 descending colon and 8 sigmoid colon samples in control group and 4 ascending colon samples, 18 descending colon samples and 22 sigmoid colon samples in constipation group obtained. Immunohistochemistry, Western blotting and Real Time PCR were performed to detect the expression of FAAH both in human colon and in FC patients. By these means, the localization and quantitative expression of FAAH and its roles in normal colon were approached. Furthermore, the immonofluorescence double-staining was used to observe the location and distribution of FAAH and CB1 receptors. Actions of FAAH on colonic function in physiological condition were further to explored. Meanwhile, quantitive detection of the activity of FAAH in human colon was done by high pressure liquid chromatography and its function in pathogenesis in children functional constipation was approached.4,Foundation of the DNA finger-print of the intestinal flora infunctional constipation in children.Investigation objects were divided into control group and functional constipation (FC) group. A Rome II criterion in childhood was used to establish the FC group. There were 15 children in FC group, including 9 boys and 6 girls, with a mean age of 6.5-year-old. There were 35 children in control group, including 24 boys and 11 girls, with a mean age of 6-year-old. Stool samples of FC group and control group were analyzed. We extracted stool bacterial genome DNA utilizing stool bacterial genome DNA extracting kit and carried out amplification of BOX AIR (primer of BOX genome repetitive sequence) and bacterial 16S rDNA V3 region primer. Then, electrophoresis and image analysis establishing on stool bacterial DNA finger print of 16S rDNA and BOX genome's repetitive sequence was carried out. The significance of PCR-SSCP and BOX-PCR on stool bacteria abstraction effect and stool bacterial flora identification was approached. Amplification products were clone-sequencing and the homology of arrays was compared utilizing BLAST instrument in GenBank database. We initially definited the genus and classification of bacteria and approached the changing tendency of intestinal flora in children FC and the significance of intestinal flora on constipation genesis.5,Variations of the short chain fatty acid in functionalconstipation in children and their probable roles of action.Quantitive detection of short-chain fatty acid content in children of control group and FC group was carried out combining gas chromatographic technique and analysis of short-chain fatty acid's changing tendency in children FC was done. Advanced, the significances of intestinal microecology bacteria and its metabolic product-short-chain fatty acid in morbility of FC in childhood were approached.Results1,EpidemiologyIn this investigation, 20,000 questionnaires were released, including 6,000 pieces in Beijing, 4000 pieces in Tianjin, 4500 pieces in Shenyang, 3000 pieces in Harbin and 2500 pieces in Changchun. Among the total, 19638 pieces were regained, including Beijing's 5452 pieces, Tianjin's 3788 pieces, Shenyang's 4066 pieces, Harbin's 2713 pieces and Changchun's 2267 pieces. The recovery rate was 98.19%. Among these, there were 19286 effective questionnaires, with an effective power of 96.43%. 913 children met children Rome II diagnostic criteria, accounting 4.73% of investigated children. The morbility rate of children FC had area and age variability. Seeing from area, Beijing had the highest morbidity of 5.02% and Changchun had the lowest of approximately 4.27 % (P<0.001). Seeing from age, infantile children (2-3 years old) hadthe highest morbidity and school age children (7-11 years old) had the lowest(p<0.001). There were no obvious differences in sex distribution. Stepwise logistic regression demonstrated that artificial feeding, adding assistant-food too early, low dietary fiber, dietary bias, coprophobia, depression, anxiety, overweight, heredity and high economic income were risk factors of FC.Constipation related symptoms were compared one by one using single factor chi-square analysis. The result revealed: the 22 symptoms (defecating less than 3 times per week, dry hard bolus-shape stool, difficult defecation, abdominal pain, nausea and vomiting and so on) could exist in control group, but FC group had a higher frequency. The statistic difference was significant. Multiple factor stepwise logistic regression analysis showed defecating less than 3 times per week, dry hard bolus-shape feces, difficult defecation, defecation needing assist of drug and so on had a higher incidence rate in FC and they had much significance on diagnosis of FC. The absence of other symptoms had no influence on diagnosis of FC. International common used Rome II criteria was established as the golden criteria to evaluate the sensitivity, specificity and diagnostic accurate rate of symptomatic standard diagnostic test in FC children. The result showed: children have 2 items above had the highest diagnostic accurate rate but medium sensitivity and specificity.2,Physical examinationIn the constipated children,the TGITT, LCTT and RSTT were significantly longer than those in controls, while the RCTT had no significant difference. Radiological presentation of the colonic transit time could be present as three forms: all the colonic segment were abnormal, only the outlet area was abnormal and/or both of them; X ray defecography demonstrated that the puborectal muscle syndrome was noted in 21 children(21/96, 21.88%), the spastic external sphincter was in 9(9/96, 9.38%), the sastic pelvic floor syndrome was in 4(4/96, 4.17%), and the rectal prolapse or intussusception was in 8(8/96, 8.33%), the perineal floor hernia was in 10(10/96, 10.42%), and the rectocele and the perineal descent syndrome were both noted in 3(3/96, 3.13%) respectively. Each syndrome has its special radiological presentations on the X ray defecography film, which could be easily distinguished from each other. The MaxP(S) of in FC group was higher and the VSI was lower than those of the control, while V had no difference.3,Expression of FAAH in human colon and its action in theslow-transit constipation in children.Immunohistochemistry, Western blotting, Real Time PCR were utilized in analysis of FAAH expression in colon wall of FC group and control group. The result showed: FAAH expressed at both protein and mRNA level in normal colon wall, regardless of ascending colon, descending colon or sigmoid colon. There was no significant deviation in different segment. Immunostaining showed FAAH mainly localized in intermuscular neuron of bowel wall, absorptive cell and intestinal gland of mucosa. In normal colon, there was a tendency that FAAH's expression location in intermuscular neurons corresponded to CBI receptor. It was found combining immunofluorescence double staining and confocal microscopy. But such alteration was not detected in absorptive cells or intestinal gland. In colon of FC children, the expression of FAAH significantly decreased. Combining high pressure liquid chromatography technique, we found that the hydrolytic activity of FAAH also significantly decreased. The difference had statistical significance.4,Foundation of the DNA finger-print of the intestinal flora infunctional constipation in children.All samples of control group and FC group were analyzed more than 3 times utilizing PCR-SSCP and BOX-PCR finger print. We got stable and repeatable pictures. There were many electrophoresis strips in pictures of objects in control group and FC group. From the location of strips, there were consistent strips and individual specific strips exist between control group and FC group. The brightness of the strips were different. Comparing with PCR-SSCP pictures, BOX-PCR pictures had more strips with distinct margins. The result of sequencing revealed: there were identical and distinct bacterial strains exist in FC group and control group. In FC group, there were obviously more strain types with significant statistic difference. Combining the strip locations of BOX-PCR finger print, predominant bacteria of control group were mainly Bacteroides, Escherichia, Bifidobacterium and Acidobacterium; predominant bacteria of FC group were mainly Bacteroides fragilis, Escherichia, Eubacterium and Clostridia.5,Variations of the short chain fatty acid in functional constipationin children and their probable roles of action.The results of gas chromatographic analysis revealed: short-chain fatty acid content in per gram dry stool decreased in children troubled by FC. Statistic difference was significant. But water content of FC group decreased with notably elevated short-chain fatty acid concentration, which was obviously higher than control group, and the short-chain fatty acids were disproportioned. Conclusions(1) FC is a common disorder in childhood in the North of China which deserves greater care. Higher prevalence is noted in age 2-3. The feeding patterns, earlier feed adding, dietary bias, fiber intake, and fear of defecation, heredity, exposure to anxiety and depression, and income might be the most important risk factors. Therefore the popular knowledge shoule be announced to the public so as to prevent FC from happening.(2) The symptomatic criterion for FC diagnosis in children in our country is:â‘ defecation frequency is less than 3 times per week;â‘¡dry, hard, bolus-shape stool is noted in most defecation;â‘¢hard defecation is noted in most defecation;â‘£defecation assisted by drugs or fingers. Those owing 2 of the above 4 and the symptoms last at least 2 months could be committed a diagnosis of FC.(3) FC in childhood could be classified into several subtypes as follow:â‘ Slow-transit constipation: disorder of the transit function was found in any of the colonic segment.â‘¡Outlet-obstruction: dynamic abnormality was only found in the anorectal region, in other words, the outlet area.â‘¢Mixed type: both the colonic transit and the outlet dynamic disorder exist. Combined use of colonic transit time, X-ray defecography, and anorectal manomatry has important significance on etiological diagnosis and classification of FC in children.(4) Expression of FAAH on protein and nucleic acid levels was both found in the normal colon in human. It mainly expresses in the myenteric plexus in colon, absorptive cells and glands on the surface of intestinal mucosa. In the myenteric plexus, FAAH have a tendency of comparative distribution with CB1 receptor.(5) In the slow transit constipation, the expression and activity of FAAH decrease. FAAH may take part in pathogenesis of the slow transit constipation in children.(6) The category of the intestinal flora increase in FC in children. The resident bacteria are similar to the normal children, but the predominant bacteria are different and disproportion exists among the intestinal flora.(7) Short-chain fatty acid content decrease with error ratio in stools of FC children. Pathogenesis of FC may relate to excessive absorption of short-chain fatty chain in bowel and increasing of local concentration of the short-chain fatty acid.