Stool Specimens of Children with In ammatory Bowel Disease

#e aim of the study was to analyze the di!erences in the activity of β-glucuronidase and β-glucosidase in stool specimens of children with Inammatory Bowel Diseases (IBD) and healthy subjects. #e disease activity was determined according to the PCDAI scale (Crohn disease) and Truelove-Witts scale (Ulcerative colitis). Enzyme activity was determined by spectrophotometry. #ere was a correlation between the level of β glucosidase activity in stool and patient’s age in the group of healthy controls, but not in the IBD group. β-glucosidase activity in IBD and healthy subjects stool specimens did not di!er significantly. #e activity of β-glucuronidase in children with IBD was two times lower than in the healthy group and was correlated with age in children with IBD, but not in the group of healthy ones. K e y w o r d s: faecal enzymes, β-glucuronidase, β-glucosidase, inammatory bowel disease * Corresponding author: P. Szachta, Institute of Microecology, 10 Sielska, 60-129 Poznań, Poland; phone: +48 602441399; fax: +48 618626335; e-mail: pszachta@instytut-mikroekologii.pl Mroczyńska M. et al. 3 320 uid (Walaszek, 1993). #e increased concentration of enzyme of microbial origin is characteristic for colorectal cancer (De Moreno de leBlanc and Perdigon, 2005). Increased levels of β-glucuronidase in stool seem to be physiological for men, especially aged 25 to 60 years. What is interesting, elevated enzyme level doesn’t occur in healthy non-pregnant women (Kim and Jin, 2001). #e activity of this enzyme is stimulated by contact with toxic substances, tobacco smoke or carcinogens. On the other hand, low-calorie and vegetarian diet is a useful tool to reduce the level of β-glucuronidase in the stool (Reddy et al., 1974; Mykkänen et al.,1997). A diet rich in red meat and protein increases the amount of the enzyme, because of stimulating microbial producers growth. It should be emphasized, that antibiotic treatment leads to increased level of the enzyme in the colon (Żółtaszek et al., 2008). #is observation indicates the important role microbiota play in regulating β-glucuronidase levels in the colon. β-glucosidase, the second analyzed enzyme is produced the most intensively by Bacteroides uniformis, Clostridium paraputrificum, clostridioformes Clostridium, Enterococcus faecalis. #ere are no studies evaluating β-glucosidase activity in health and disease. #e enzyme’s main role is hydrolysis of glycosides to aglycones and sugar components. Glycosides aren’t hydrolyzed in the higher part of gastrointestinal tract part, but are hydrolyzed in the colon by bacterial β-glycosidases. As a result, potentially toxic substances (f.ex. quercetin), may be formed in the large intestine. It is thought that production of harmful substances could be higher among people eating a lot of vegetables, cause of their richness in glycosides. #e activity of β-glucuronidase and β-glucosidase in stool specimens of children with IBD has not yet been studied. Because of procarcinogenic and proinammatory activity of enzymes and the fact that the activity of the enzymes is higher in stool specimens of patients with cancer, we attempt to assess enzyme activity in the stool of children with organic gastrointestinal disease. 68 children with IBD hospitalised in the Department of Paediatric Gastroenterology and Metabolic Diseases participated in the study. #e activity of the disease was evaluated for each patient. Diagnosis of the disease was made according to the generally accepted scheme of diagnostic proceeding, including: medical history (anamnesis), physical (subjective) examination, and laboratory tests, endoscopic examination, radiological imaging and histopathological examination. In CD patients, the Pediatric Crohn’s Disease Activity Index (PCDAI) was determined (0–10 points – no activity; 11–25 points – mild state; 26–50 points – moderate state; > 51 points – severe state). In CU patients activity of the disease was evaluated using Truelove-Witts index (0–5 points – mild state; 5–8 points – moderate state; 9–12 points – severe state). CI is regarded as a indeterminate colitis, which can proceed on full-blown IBD. #e state of disease activity wasn’t assessed in CI manifestation. #e control group consisted of 38 healthy children. #e study obtained approval from the Ethical Committee at the “Karol Marcinkowski” Medical University in Poznań. A sample of stool was collected from each child to evaluate β-glucuronidase and β-glucosidase activity. Prior to collecting faeces samples an informed consent for participation in the analysis was obtained (from patient or his/her guardian, for children under the age of 16 years). For determination of enzyme activity, stool specimens were suspended in phosphate bu!er, subjected to homogenization, and sonication with appropriately selected parameters of the device. #e enzymatic activity of β-glucuronidase (β-glucosidase) was determined by spectrophotometry, specifying the amount of phenolphthalein (p-nitrofenol) released during the hydrolysis of 10 mM phenolphthaleinβ-D-glucopyranoside (20 mM p-nitrophenol-β-Dglucosidase). #e samples were centrifuged aXer sonication. #en the collected supernatant (containing the enzyme) was transferred to test tubes and 1 ml of 0.02 M phosphate bu!er pH = 7, and 0.1 ml of phenolphthalein-β-D-glucopyranoside (p-nitrophenolβ-D-glucopyranoside) was added to it. #e samples prepared in such a way were incubated in a water bath for 15 min at 37°C (for 60 min at 37°C), aXer that the reaction was stopped by adding glycine bu!er (sodium hydroxide). #e contents of the tubes were mixed thoroughly and the absorbance level was measured at wavelength λ = 540 nm, (λ = 450 nm) with regard to similarly prepared and heated control sample, which contained water instead of the substrate. A unit of activity was adopted as such amount of phenolphthalein (for β-glucuronidase) and p-nitrophenol (for β-glucosidase) expressed in mM released during the reaction throughout 1 h per 1 mg of protein. #e total concentration of protein in bacterial cells was determined by the Lowry method. All statistical analyses were performed using Statistica 7.0. #e activity of β-glucuronidase and β-glucosidase in stool samples was compared between study and control groups. Di!erences in the levels of enzymes, depending on the age of the children were also analyzed. Correlations between β-glucuronidase and β-glucosidase activity and clinical disease manifestation (CU, CD, CI), activity state and phase of treatment were evaluated. To determine the reason for different enzyme activities between the study and control group, the disparity in number of bacteria producing β-glucuronidase and β-glucosidase was checked. Statistical significance for the confidence interval 0.05 was determined using One-Way ANOVA test. Short communication 3 321 #e characteristics of study and control group, in view of average age and number of children in each group, is presented in table I. #e study group was divided into three subgroups, according to disease clinical manifestation (CD, CU, CI) disease activity state. #e range of β-glucuronidase activity in stool specimens of children with IBD and healthy ones is presented in table II and for β-glucosidase in table III. β-glucuronidase activity was statistically significantly di!erent in stool samples of children with IBD and healthy children. Enzyme mean value in children with IBD stool specimens was twice lower than in the control group, but wasn’t statistically di!erent in di!erent IBD clinical manifestations (CD, CU, CI). It is worth noting that the enzyme activity was highly di!erentiated in stool specimens of children with CI. β-glucuronidase activity in the study group and didn’t depend on age, but in control group did. In stool specimens of healthy children enzyme activity correlated with age. #e results are presented in figure 1 (A, B, C, D). #ere were no statistically significant di!erences in β-glucosidase activity in stool specimens of children with IBD and healthy ones. In stool specimens of children with CI enzyme activity was highly di!erentiated and also higher than in the stool of healthy children. β-glucosidase activity in stool samples wasn’t statistically significantly di!erent in CU and CD. β-glucuronidase activity in control group didn’t depend on age, but in IBD group correlated with age. #ese correlations are presented in figure 2. β-glucuronidase and β-glucosidase activity range in IBD clinical manifestations (CU, CD, CI) is presented in table IV. Data are presented before and aXer treatment. We found no statistically significant di!erences in enzyme activity in stool specimens before and aXer treatment, regardless of type of treatment (antibiotics, steroids or antibiotics and steroids together) #is is the first study evaluating β-glucuronidase and β-glucosidase activity in stool specimens of children with IBD. #e control group consisted of healthy children, without any organic changes in the gastrointestinal tract. β-glucuronidase has a rather negative e!ect on human health. #e enzyme disrupts glucuronidation, which is very important element of the Total patient’s number 38 25 26 17 Disease activity state – No Mild Moderate Severe Mild Moderate Severe activity state state state state state state – Patients number in group – 10 2 8 3 18 4 4 – Age 1–18 6–18 1.5–18 3–17.5 Mean age 6.63 14.1 13.8 11.6 Table I Study and control group characteristic