Differences in microbial metabolites in urine headspace of subjects with Immune Thrombocytopenia (ITP) detected by volatile organic compound (VOC) analysis and metabolomics.

ITP is an organ-specific autoimmune disorder characterised by a low platelet count whose cause is uncertain. A possible factor is food intolerance, although much of the information linking this with ITP is anecdotal. The role of food intolerance in ITP was studied by replacing a normal diet with an elemental diet (E028), but this did not increase platelet counts. Clear differences, however, were apparent between the volatile organic compounds (VOCs) in the urine headspace of patients with ITP and those present in healthy volunteers, which leads to speculation that abnormal metabolic activity of the intestinal microbiome may be a factor causing ITP. However, further work is needed to confirm this. There were also differences between the VOCs of patients on a normal diet and those on the elemental diet, and in this case, the VOCs involved are very likely to be of bacterial origin, as their production is affected by dietary manipulation. Many of these VOCs are known to be toxic.

Enteral feeding reduces metabolic activity of the intestinal microbiome in Crohn's disease: an observational study.

BACKGROUND/OBJECTIVES: Enteral feeding will induce remission in as many as 80-90% of compliant patients with active Crohn's disease (CD), but its method of action remains uncertain. This study was designed to examine its effects on the colonic microbiome. METHODS/SUBJECTS: Healthy volunteers and patients with CD followed a regimen confined to enteral feeds alone for 1 or 2 weeks, respectively. Chemicals excreted on breath or in faeces were characterised at the start and at the end of the feeding period by gas chromatography/mass spectrometry. RESULTS: One week of feeding in healthy volunteers caused significant changes in stool colour and deterioration in breath odour, together with increased excretion of phenol and indoles on the breath. Feeding for 2 weeks in patients with CD produced significant improvements in symptoms and a decrease in the concentration of C-reactive protein. The faecal concentrations of microbial products, including short-chain fatty acids (SCFAs), and potentially toxic substances, including 1-propanol, 1-butanol and the methyl and ethyl esters of SCFAs, showed significant falls. CONCLUSIONS: A significant change occurs in the production of microbial metabolites after enteral feeding in both healthy volunteers and patients with CD. Many of those detected in CD are toxic and may feasibly lead to the immunological attack on the gut microbiota, which is characteristic of inflammatory bowel disease. The reduction in the production of such metabolites after enteral feeding may be the reason for its effectiveness in CD.

Characterisation of the faecal metabolome and microbiome of Thoroughbred racehorses.

REASONS FOR PERFORMING STUDY: The intestinal bacterial community of the horse is a key determinant of intestinal and whole body health. Understanding the bacterial community structure and function is an important foundation for studies of intestinal health and disease. OBJECTIVES: To describe the faecal bacterial community and volatile organic compounds (VOCs) of the faecal metabolome of healthy Thoroughbred racehorses and to characterise responses to dietary supplementation with amylase-rich malt extract. STUDY DESIGN: Intervention study. METHODS: Faecal samples were collected noninvasively before and 6 weeks after supplementation in 8 privately owned Thoroughbred racehorses in active race training. Faecal metabolome was characterised using thermal desorption-gas chromatography-mass spectrometry (TD-GC-MS), with spectral analysis performed using AMDIS and compared against the NIST database. Taxonomic description of the faecal microbiota was achieved using error-corrected 454 pyrosequencing data from 16S rRNA gene amplicons. RESULTS: The faecal metabolome of our study population was dominated by organic acids, alcohols and ketones. We identified 81 different VOCs only 28 of which were present in >50% of samples indicating functional diversity. Faecal VOC profiles differed between first and second sampling point, some VOCs being significantly reduced post supplementation, consistent with a marked response to dietary amylase-rich malt extract. Faecal microbiota was characterised as highly diverse; samples demonstrated verifiable diversity in the range 1200-3000 operational taxonomic units (OTUs) per individual. The methods used also describe high levels of infrequent, low abundance OTUs. Faecal microbial community structure was found to be different following dietary supplementation. Differences in several low abundance bacterial taxa were detected and also some evidence of interhorse variation in response. CONCLUSIONS: The volatile faecal metabolome of Thoroughbred racehorses is dominated by organic acids, alcohols and ketones; this study demonstrates that dietary supplementation with amylase-rich malt extract may significantly alter the profile of VOCs. The faecal microbiome is highly diverse, dominated by Firmicutes and Bacteroidetes. Small but significant changes in microbial community structure were detected following dietary supplementation. This study describes the faecal metabolome and microbiome of healthy Thoroughbred racehorses against which future studies of disease and dietary intervention can be benchmarked.

Xenobiotic sulphation and its variability during inflammation: a factor in adverse drug reactions?

The interactions between disease processes and the metabolism of therapeutic drugs have not been systematically investigated. Inflammation, with the presence of pro-inflammatory cytokines, affects Phase 1 metabolism, particularly the activity of the CYP isoforms. Inflammatory factors also alter the activity of some Phase 2 enzymes, particularly the sulphotransferases (SULT isoforms) responsible for drug sulphonation and the enzyme pathway involved in the supply of sulphate for this reaction. Being ill may, therefore, in itself make drug metabolism unpredictable.

Do horses suffer from irritable bowel syndrome?

Irritable bowel syndrome (IBS) in man is not a single entity but has several causes. One of the most common forms has similarities with colic and laminitis in horses. Undigested food residues may pass from the small intestine into the colon where bacterial fermentation produces chemicals that lead to disease. In horses the consequences may be disastrous, but in healthy humans such malabsorption may not be harmful. After events such as bacterial gastroenteritis or antibiotic treatment, an imbalance of the colonic microflora with overgrowth of facultative anaerobes may arise, leading to malfermentation and IBS. It is not known whether such subtle changes may likewise be present in the microflora of horses who are susceptible to colic and laminitis. Metabolomic studies of urine and faeces may provide a suitable way forward to identify such changes in the horse's gut and thus help to identify more accurately those at risk and to provide opportunities for the development of improved treatment.

Effect of Lactobacillus plantarum 299v on colonic fermentation and symptoms of irritable bowel syndrome.

A number of recent clinical trials have promoted the use of probiotic bacteria as a treatment for irritable bowel syndrome (IBS). The recent demonstration of abnormal colonic fermentation in some patients with this condition provides an opportunity for the objective assessment of the therapeutic value of these bacteria. This study was designed to investigate the effects of Lactobacillus plantarum 299V on colonic fermentation. We conducted a double-blind, placebo-controlled, cross-over, four-week trial of Lactobacillus plantarum 299V in 12 previously untreated patients with IBS. Symptoms were assessed daily by a validated composite score and fermentation by 24-hr indirect calorimetry in a 1.4-m3 canopy followed by breath hydrogen determination for 3 hr after 20 ml of lactulose. On placebo, the median symptom score was 8.5 [6.25-11.25 interquartile range (IQR)], the median maximum rate of gas production was 0.55 ml/min (0.4-1.1 IQR), and the median hydrogen production was 189.7 ml/24 hr (118.3-291.1 IQR). On Lactobacillus plantarum 299V the median symptom score was 8 (6.75-13.5 IQR), the median maximum rate of gas production 0.92 ml/min (0.45-1.5 IQR), and the median hydrogen production 208.2 ml/24 hr (146-350.9 IQR). There was no significant difference. Breath hydrogen excretion after lactulose was reduced by the probiotic (median at 120 min, 6 ppm; placebo, 17 ppm; P = 0.019). In conclusion, Lactobacillus plantarum 299V in this study did not appear to alter colonic fermentation or improve symptoms in patients with the irritable bowel syndrome.

A review of the role of the gut microflora in irritable bowel syndrome and the effects of probiotics.

Irritable bowel syndrome (IBS) is a multi-factorial gastrointestinal condition affecting 8-22 % of the population with a higher prevalence in women and accounting for 20-50 % of referrals to gastroenterology clinics. It is characterised by abdominal pain, excessive flatus, variable bowel habit and abdominal bloating for which there is no evidence of detectable organic disease. Suggested aetiologies include gut motility and psychological disorders, psychophysiological phenomena and colonic malfermentation. The faecal microflora in IBS has been shown to be abnormal with higher numbers of facultative organisms and low numbers of lactobacilli and bifidobacteria. Although there is no evidence of food allergy in IBS, food intolerance has been identified and exclusion diets are beneficial to many IBS patients. Food intolerance may be due to abnormal fermentation of food residues in the colon, as a result of disruption of the normal flora. The role of probiotics in IBS has not been clearly defined. Some studies have shown improvements in pain and flatulence in response to probiotic administration, whilst others have shown no symptomatic improvement. It is possible that the future role of probiotics in IBS will lie in prevention, rather than cure.

A double-blind, randomized, placebo-controlled trial of essential fatty acid supplementation in the maintenance of remission of ulcerative colitis.

BACKGROUND: Essential fatty acid supplementation has been found to ameliorate certain chronic inflammatory diseases. This effect is thought to be mediated through the modulation of eicosanoid synthesis. Pro-inflammatory eicosanoids have been implicated in ulcerative colitis. AIM: To investigate the possible therapeutic benefit of essential fatty acids in quiescent ulcerative colitis to reduce the frequency of disease relapse. METHODS: A randomized, double-blind, placebo-controlled study was performed with a treatment duration of 12 months. Patients with quiescent disease received either trial medication (gamma-linolenic acid, 1.6 g, eicosapentaenoic acid, 270 mg, and docosahexaenoic acid, 45 mg, per day) or placebo (sunflower oil, 500 mg/day). The primary end-point was disease activity, assessed by a previously validated clinical index, sigmoidoscopic appearance and histology. RESULTS: Sixty-three patients were randomized, 31 to receive essential fatty acid treatment and 32 to receive placebo. Disease relapse rates were similar at 12 months (placebo, 38%; essential fatty acids, 55%), as were changes in sigmoidoscopic grade from baseline. CONCLUSIONS: The supplementation of the diet with this combination of essential fatty acids does not prolong the period of disease remission of ulcerative colitis.

A randomised controlled trial of high versus low long chain triglyceride whole protein feed in active Crohn's disease.

BACKGROUND: Polymeric feeds have shown variable efficacy in active Crohn's disease (CD) with remission rates from 36% to 82%. Meta-analyses of elemental, peptide, and whole protein feeds have shown a strong negative correlation between remission rate in CD and the long chain triglyceride (LCT) content of the feed. We performed a randomised controlled double blind trial in patients with active CD comparing two single whole protein feeds with LCT supplying 5% or 30% of the total energy. METHODS: Fifty four patients with active CD (Crohn's disease activity index (CDAI) >200, serum C reactive protein (CRP) 10 mg/l) were randomised to a high or low LCT feed for three weeks. The total amount of energy supplied by fat was identical in the two feeds. Remission was defined as a CDAI < or =150 and response as a fall in CDAI of > or =70 or a CRP <10 mg/l. RESULTS: Overall remission rate by intention to treat was 26% for the low LCT feed and 33% for the high LCT feed (p=0.38). Response was achieved in 33% with the low LCT and in 52% with the high LCT feed (p=0.27). CRP <10 mg/l was achieved in 30% in the low LCT and 33% in the high LCT group (p=0.99). Thirty nine per cent (21/54) of patients withdrew before three weeks because of inability to tolerate the diet. Excluding patients unable to tolerate the diet, remission rates were 46% for low LCT and 45% for high LCT (p=0.99). DISCUSSION: This trial has shown no difference in the effect of low and high LCT whole protein feeds in active CD. The previously reported correlation between LCT content of diet and response in active CD is unlikely to be due to LCT itself and may be due to some other component of high LCT feeds.

Colonoscopic hydrostatic balloon dilatation of Crohn's strictures.

Recurrent strictures cause considerable morbidity among many patients with Crohn's disease. We present a retrospective review of the outcome of patients who underwent endoscopic hydrostatic balloon dilatation of Crohn's strictures in our hospital over the past 7 years. All patients were under observation at Addenbrooke's Hospital, Cambridge. An initial assessment of all strictures was made radiologically, and any patient with active disease was treated before consideration of endoscopic dilatation. Dilatation was carried out with Microvasive Rigiflex balloons. Outcome after dilatation was assessed by review at subsequent clinic visits. Sixteen of 22 patients had resolution of their obstructive symptoms after dilatation, although one third of patients required more than two dilatations over the follow-up period. Six of 22 patients had persisting symptoms after endoscopic treatment, requiring surgery. There were no complications noted after any of the 71 dilatations that were performed. We conclude that hydrostatic balloon dilatation is a safe and effective alternative to surgery in the management of Crohn's strictures within the reach of the colonoscope.

Irritable bowel syndrome: is the search for lactose intolerance justified?

OBJECTIVES: To determine if confirmation of hypolactasia offers any benefit to the dietary treatment of patients with irritable bowel syndrome (IBS). METHODS: One hundred and twenty-two consecutive IBS patients (37 male, 85 female) were given lactose hydrogen breath tests (LHBT). Those with positive LHBT followed a low lactose diet for 3 weeks. Those improving on the diet were given double-blind, placebo-controlled challenges (DBPCC) with 5 g, 10 g and 15 g of lactose and a placebo, to confirm lactose intolerance. Those who did not respond to the low lactose diet followed either an exclusion or low fibre diet. Symptoms scores were kept prior to the LHBT, 8 h post-LHBT and daily whilst following any dietary change. Patients with negative LHBT returned to clinic and subsequent dietary interventions were recorded. RESULTS: LHBT was positive in 33/122 (27%) IBS patients. Syrr otom scores prior to LHBT were not significantly different between the two groups, but after LHBT the symptoms in the positive group were significantly worse. Twenty-three patients followed a low-lactose diet of which only nine (39%) improved. Six who did not improve followed an exclusion diet, three improved and all were intolerant of milk. Three tried a low fibre diet with two improving. DBPCC were inconclusive. In the negative LHBT group 35 agreed to try a diet and 24 improved (69%). Eight were intolerant of cow's milk. CONCLUSIONS: Use of a low lactose diet was disappointing in IBS patients with lactose malabsorption. Food intolerance was demonstrated in IBS patients with positive or negative LHBT and milk was identified as a problem in both groups. DBPCC were inconclusive. There appears to be little advantage in trying to separate patients who malabsorb lactose from others with IBS.

The physiological expression of inducible nitric oxide synthase (iNOS) in the human colon.

BACKGROUND: Inducible nitric oxide synthase (iNOS) is expressed in the colonic epithelium in both inflammatory bowel disease and colorectal cancer. Nitric oxide (NO), the product of this enzyme, has been implicated in the pathogenesis of both conditions. However, there are conflicting data on whether iNOS is expressed in the normal, uninflamed human colon. AIMS: To evaluate the expression of iNOS in histologically normal, non-inflamed human colonic mucosa. PATIENTS/METHODS: Reverse transcription polymerase chain reaction (RT-PCR), immunoblotting, and immunohistochemistry were used to investigate the expression of iNOS in 17 histologically normal specimens obtained at colectomy performed for colorectal neoplasia. In addition, 16 endoscopic mucosal biopsies, taken from normal individuals, were also evaluated. Eleven surgical specimens and 16 endoscopic biopsies from patients with refractory ulcerative colitis were used as inflammatory controls. RESULTS: All types of specimens expressed iNOS mRNA. Immunoblotting revealed a protein of approximately 130 kDa consistent with iNOS in mucosal extracts of 77% of normal individuals, and 85% of diseased controls. Immunolabelling localised this protein to the surface epithelium in most of the normal specimens and also to the crypt epithelium and inflammatory cells in the diseased controls. CONCLUSIONS: These findings provide evidence that iNOS is often expressed in the surface epithelium of non-inflamed human colon, suggesting that it is induced by local luminal factors, such as bacterial lipopolysaccharide (endotoxin). The resultant NO produced at this site might act as an oxidative barrier, reducing bacterial translocation and providing a means of defence against pathogenic microorganisms.

Neuronal COX-2 expression in human myenteric plexus in active inflammatory bowel disease.

BACKGROUND: Inflammatory bowel disease (IBD) is associated with changes in colonic motility which may contribute to the pain and diarrhoea associated with exacerbations of this disease. These changes may be mediated by prostaglandins which are increased in this condition. Increased expression of the inducible isoform of cyclo-oxygenase (COX-2) has been found in active IBD although its cellular distribution remains uncertain. AIMS: To evaluate the cellular distribution of COX-2 in active IBD. PATIENTS AND METHODS: Using reverse transcription-polymerase chain reaction, in situ hybridisation, and immunohistochemistry, COX-2 expression was evaluated in 12 colectomy specimens from patients with active ulcerative colitis (UC), and six specimens from patients with Crohn's colitis that had failed medical therapy. Histologically normal colon was obtained from 12 patients having resection for colorectal neoplasia and evaluated as above, acting as control specimens. RESULTS: All specimens expressed COX-2 mRNA, with some 6-8-fold increase in inflamed tissues on densitometric analysis (both UC and Crohn's) compared with controls. In situ hybridisation localised this mRNA to myenteric neural cells, surrounding smooth muscle cells, and inflammatory cells of the lamina propria in the IBD specimens, with some weaker labelling seen in the epithelium. No COX-2 labelling was seen in normal tissues. Immunohistochemistry confirmed these sites of COX-2 expression in all inflamed specimens, with absence of immunoreactivity in control tissues. CONCLUSIONS: These findings provide the first evidence of COX-2 expression in neural cells of the myenteric plexus in active IBD which, via increased prostaglandin synthesis at this site, may contribute to the dysmotilty seen in this condition.

Treatments for Crohn's disease that minimise steroid doses are associated with a reduced risk of osteoporosis.

Crohn's disease is associated with an increased prevalence of osteoporosis. Corticosteroids, commonly used to control exacerbations, appear to be a major risk factor for subsequent development of osteoporosis. Exclusion diets, avoiding foods that precipitate symptoms, frequently allow control of the disease avoiding the use of corticosteroids and may thereby reduce the risk of osteoporosis. To investigate this we performed bone mineral density measurements of the proximal femur and spine in 95 patients, 31 treated predominately by corticosteroids, 33 by dietary manipulation with a low life-time corticosteroid dose and 31 by treatments other than diets but also with a low life-time corticosteroid dose. In both groups with a low life-time corticosteroid dose bone mineral density was comparable to that of age-matched normal controls, whereas bone mineral density was significantly reduced in those treated predominately by corticosteroids. We conclude that corticosteroid therapy is an independent risk factor for osteoporosis in patients with Crohn's disease and should be used as little as possible.

Evaluation of the effectiveness of a specialist nurse in the management of inflammatory bowel disease (IBD).

OBJECTIVE: To determine the effect of a specialist nurse on the management outcome of patients with inflammatory bowel disease (IBD). DESIGN: Audit of the management of a cohort of patients in the year prior to the employment of the specialist nurse and the year immediately after. SUBJECTS: 339 patients, both male and female, with either Crohn's disease or ulcerative colitis, resident in the Cambridge health district. SETTING: Addenbrooke's Hospital NHS Trust Outpatient Centre. MAIN OUTCOME MEASURE: Health status was measured by blood tests (C-reactive protein, albumin and haemoglobin) throughout the year, symptom indices, number of clinic attendances, admissions to hospital and length of stay. Quality of life was measured via a postal questionnaire. RESULTS: Hospital visits were reduced from 1377 to 853 (38% reduction) and in-patient length of stay measured in bed-days from 516 to 417 (19% reduction). The number of patients in remission increased from 63 to 69%. Patient satisfaction improved in key areas, in particular, access to information on IBD and advice on avoidance of illness and maintaining health. Of a total of 251 calls to the telephone helpline, only 19 patients were referred for a medical opinion and five patients required hospital admission. CONCLUSION: The IBD nurse specialist is a valuable and cost-effective member of the gastroenterology team.