|Year : 2002 | Volume
| Issue : 4 | Page : 178-182
Faecal excretion of brush border membrane enzymes in patients with clostridium difficile diarrhoea
R Katyal , C Vaishnavi , K Singh
Department of Gastroenterology, PGIMER, Chandigarh - 160 012, India
Department of Gastroenterology, PGIMER, Chandigarh - 160 012, India
PURPOSE: To look for the presence of intestinal brush border membrane (BBM) enzymes in the faecal samples of patients with Clostridium difficile association. METHODS: One hundred faecal samples were investigated for C.difficile toxin (CDT). Simultaneous assays for faecal excretion of intestinal BBM enzymes viz., disaccharidases, alkaline phosphatase (AP) and leucine aminopeptidase (LAP) were also done. RESULTS: C.difficile toxin was detected in 25 (25%) of the samples with a titre ranging from 10 to 160. No significant difference (p>0.05) was seen between the CDT positive and negative groups with any of the disaccharidases studied. However, significant increase (p<0.05) in the AP excretion was observed in the CDT positive patients compared to the CDT negative group. In contrast to this, a significant decrease (p<0.001) in the LAP enzyme excretion was observed in the latter group. CONCLUSIONS: The results of this study suggest that there is a significant disturbance in the intestinal BBM enzymes in patients with C.difficile diarrhoea.
|How to cite this article:|
Katyal R, Vaishnavi C, Singh K. Faecal excretion of brush border membrane enzymes in patients with clostridium difficile diarrhoea. Indian J Med Microbiol 2002;20:178-82
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Katyal R, Vaishnavi C, Singh K. Faecal excretion of brush border membrane enzymes in patients with clostridium difficile diarrhoea. Indian J Med Microbiol [serial online] 2002 [cited 2020 Jun 3];20:178-82. Available from: http://www.ijmm.org/text.asp?2002/20/4/178/6952
Clostridium difficile, a spore bearing anaerobic organism, is the only major pathogen commonly identified in patients with antibiotic associated diarrhoea or colitis. The frequency of isolation of C.difficile is directly correlated with the severity of disease on the basis of both anatomical and clinical parameters. The incidence of C.difficile infection rises with increasing age, particularly so in the elderly. This is partly because of increased use of antibiotics in older patients, and also due to the presence of predisposing factors such as gastrointestinal malignancy and major surgical procedures., Profuse, watery, green, foul smelling or bloody diarrhoea along with abdominal cramps are the hallmarks of pseudomembranous colitis, which is a potentially fatal complication of the disease. Inflammation is also an important feature and has been demonstrated by us in earlier studies.,
Two high molecular weight protein exotoxins, toxin A and toxin B, are produced by C.difficile. Of these, toxin A is an enterotoxin which upon injection into rabbit ileal loop elicits severe epithelial damage associated with haemorrhage and fluid secretion. It binds to cells that express the Gal a1-3 Gal ß1-4 G1c NAc trisaccharide receptor in the brush border membrane (BBM). This results in a fluid response because of the erosion and an increased permeability of the intestinal mucosa. Toxin A accounts for all or nearly all of the changes within the gastrointestinal tract.
Epithelial cells of the gastrointestinal tract undergo biochemical, ultrastructural and morphological changes during infection. Mucosal enzymes can serve as markers of intestinal damage. In an earlier study, urinary excretion of renal BBM enzymes like leucine aminopeptidase (LAP), alkaline phosphatase (AP) and maltase in a systemic disease have been reported by one of the present authors (CV). Since a similar mechanism could result in the excretion of BBM enzymes in diarrhoea, it prompted us to look for the presence of intestinal BBM enzymes in the faecal samples of patients with C.difficile association.
| ~ Materials and Methods|| |
C.difficile toxin assay
One hundred faecal samples, collected in stericol vials (Himedia, India) were received consecutively in the microbiology section of the Department of Gastroenterology with a specific request for CDT assay. These samples belonging to all age-groups formed the basis of the present investigation. Apart from other pathogens that were routinely looked for, C.difficile toxin (CDT) assay was done as described earlier. In brief, 50 ml of a 1 in 5 diluted faecal supernatant was taken on a clean glass slide to which ready-to-use C.sordelli antitoxin coated latex beads were added. The slide was gently rocked manually and looked for agglutination. A sample was considered CDT positive when agglutination occurred within 2 minutes. A known positive faecal sample obtained from a patient with antibiotic-associated diarrhoea constituted the positive control. Negative controls comprised of either or both of (i) an unreactive faecal sample from a healthy volunteer who had no antibiotic exposure for 6 weeks prior to testing and (ii) uncoated latex beads plus diluted sample. Doubling dilutions ranging from 1 in 10 to 1 in 640 were done for all the CDT positive samples and agglutination tests were repeated with them for further titration. The titre of the toxin was recorded as the highest dilution of the faecal supernatant which gave a positive agglutination reaction. The advantages of using C.sordelli coated beads for CDT assay have been described in detail earlier. During analysis, two groups viz., CDT positive group and CDT negative group were formed. The latter group served as control to the former group. Faecal excretion of intestinal enzymes were studied in both the groups.
Faecal Excretion of BBM Enzymes
The following BBM enzymes were estimated in the faecal samples. Disaccharidases viz., sucrase, lactase and maltase were estimated by the method of Dahlquist. The activity of LAP was measured according to the method of Goldberg and Ruttenberg using L-leucine-p-nitroanilide as substrate. Enzymatic hydrolysation liberated p-nitroaniline which was diazotized with sodium nitrite and converted to an azodye. The pink coloured product thus formed was measured at 540 nm spectrophotometrically. Alkaline phosphatase levels were assayed by the method of Naftalin et al using 4-methyl-p-nitrophenol phosphate as the substrate. Protein concentration in the faecal supernatant was estimated as described by Lowry et al with bovine serum albumin as the standard.
The two tailed (unpaired) Student's t-test was employed to compare the samples in the CDT positive and CDT negative groups. P<0.05 was taken as statistically significant.
| ~ Results|| |
Of the 100 patients whose faecal samples were analyzed, there were 60 males and 40 females. Seventy-two of 100 patients had diarrhoea while 97% had received antibiotics. C.difficile toxin was positive in 25 (25%) of the one hundred samples examined and all of them were on antibiotics. The CDT titre ranged from 10 to 160. Faecal lactoferrin was present in 12 of the samples that were positive for CDT (data not shown). No other pathogen was isolated from any of these samples.
The disaccharidases were estimated as mmole/min/mg protein in both CDT positive and negative samples [Figure - 1]. The mean sucrase levels excreted in the faeces in the CDT positive and negative groups were 215.98 ± 42.67 and 73.19 ± 11.52 respectively. Though increased excretion in the CDT positive group was observed, it did not reach the level of significance. The mean value of lactase excretion was 148.63 ± 31.56 in CDT positive patients as compared to 91.18 ± 14.16 in CDT negative group. Similarly the mean maltase levels were 113.43 ± 19.07 and 141.36 ± 15.62 m moles/min/mg protein in the CDT positive and negative groups respectively. However, no significant difference (p>0.05) was seen between both the CDT positive and negative groups with any of the disachharidases studied.
A significant increase (p<0.05) in the mean value of AP excretion was observed in CDT positive patients (35.26 ± 5.34) compared to CDT negative group (22.84 ± 2.18). In contrast to this, a significant decrease (p<0.001) in the LAP enzyme excretion was observed in the CDT positive group with a mean value of 17.76 ± 2.11 compared to 46.00 ± 6.86 in the CDT negative group [Figure - 1]. The excreted enzymes correlated with the toxin titres.
In the 25 CDT positive group there were 18 samples that were positive for AP and the same number of samples had lowered LAP enzymes. The sensitivity of both these enzymes was 72%, whereas the specificity for AP was 76% and for LAP was 98%.
| ~ Discussion|| |
Intrinsic host factors such as increasing age and severity of underlying diseases influence the risk of acquisition of C.difficile in the hospital. C.difficile associated diarrhoea and colitis are well reported in literature.,,
The intestinal BBM is a functional entity with a digestive and absorptive surface in membrane transport. The enzymes located on the BBM have an active role in absorption of carbohydrates and proteins in enterocytes. The carbohydrates reaching the large bowel are broken down by anaerobic microflora and absorbed as short chain fatty acids.
The proximal colon is the major site of the digestive-absorptive function of the large bowel but the distal parts of the large bowel also play a role in digestion/absorption as reported by Borkje et al. The presence of brush border enzymes in large intestinal mucosa may indicate a digestive-absorptive function similar to that in small intestine. The BBM enzyme levels in colon/rectum correspond to only 5-20% of that in upper small intestine.,
Mucosal enzymes shed in faeces can serve as markers of intestinal damage. Primary carbohydrate malabsorption arising from mucosal damage with concomitant reduction in digestive/absorptive capacity could lead to diarrhoea in C.difficile infection. Even though statistically insignificant, the mean sucrase levels excreted in the CDT positive group was apparently much higher than that obtained in the CDT negative group. This probably indicated a disturbance in carbohydrate metabolism.
Previous quantitative disaccharidase studies in mice showed that alterations in lactase activities were most pronounced in the brush border regions of the upper small intestine. In another study it was shown that intestinal lactase activity, although reduced dramatically was still sufficient to cope with the dietary lactase load. This suggested that the level of lactase activity in normal mucosa is far in excess of the minimum required to hydrolyse dietary lactose. Since in C.difficile infections, the disease pathologically involves the colon largely with only the ileum of the small bowel involved, no change in lactase activity was detected in the present study. Similarly, no change in the faecal excretion of maltase was seen in either of the groups.
The subcellular distribution of a range of marker enzymes in mucosal cells of small intestine is well established. Their activities have been amply investigated in intestinal diseases involving inflammation and mucosal damage., Alkaline phosphatase (AP) is a villus - tip cell marker. In studies by Peters et al the AP varied significantly among segments in human large bowel, with the ascending part having significantly higher activity than that of rectum. The uneven distribution of AP may be explained by the biological variation of this particular enzyme.
Alkaline phosphatase was found to be reduced in the small intestine of mice with rotavirus diarrhoea as reported by Collins et al. In the present study, a highly significant increase in excretion of AP was seen in the faecal samples of CDT positive group compared to those without C.difficile association. The damaged BBM and consequent destruction of epithelial cells may result in the shedding of intestinal BBM enzymes which are ultimately excreted in the faeces. Therefore, the estimation of these enzymes in the stool may reflect the extent of intestinal damage.
Leucine amino peptidase is another mucosal enzyme present intracellularly in all animal tissues and is secreted in the mucosa of the small intestine. This enzyme is active in the terminal phase of protein digestion. A significant reduction in LAP activity in the jejunum and ileum of rotavirus infected mice was also reported earlier. In C.difficile infection, large protein loss (hypoalbuminaemia) occurs. In an earlier study Katyal et al reported increased LAP activity in mice under the stress of malnutrition. However, in the present study reduced excretion of LAP in the faeces might suggest a decreased secretion/availability of the enzyme due to protein losses.
It is difficult to study the enzymatic activity in the human intestine as it would involve taking of multiple biopsies endoscopically which is ethically not justified. Even though many confounding variables may influence the degradation rates of these enzymes, this preliminary study may give an insight into the enzymatic disturbances in the intestine. In conclusion, faecal excretion of mucosal enzymes may reveal intestinal disturbances and can be used as an adjunct to colonoscopy but not replace it. Colonoscopy has other benefits which include direct visualization of lesions including removal of tissues for biopsy. Estimation of the enzymes on the other hand will reveal only the mucosal disturbance due to an infective process, the aetiology of which again has to be determined.
| ~ Acknowledgements|| |
We express our gratitude to the World Health Organisation, Geneva and Statens Seruminstitut, Denmark for the gift of C.sordelli antitoxin. We are thankful to Mr. DN Singh and Ms. Riti for their technical help.
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