G Sigthorsson

A simple method for assessing intestinal inflammation in Crohn's disease

BACKGROUND AND AIMS Assessing the presence and degree of intestinal inflammation objectively, simply, and reliably is a significant problem in gastroenterology. We assessed faecal excretion of calprotectin, a stable neutrophil specific marker, as an index of intestinal inflammation and its potential use as a screening test to discriminate between patients with Crohns disease and those with irritable bowel syndrome. METHODS The validity of faecal calprotectin as a marker of intestinal inflammation was assessed in 22 patients with Crohns disease (35 studies) by comparing faecal excretions and concentrations using four day faecal excretion of111indium white cells. A cross sectional study assessed the sensitivity of faecal calprotectin concentration for the detection of established Crohns disease (n=116). A prospective study assessed the value of faecal calprotectin in discriminating between patients with Crohns disease and irritable bowel syndrome in 220 patients referred to a gastroenterology clinic. RESULTS Four day faecal excretion of 111indium (median 8.7%; 95% confidence interval (CI) 7–17%; normal <1.0%) correlated significantly (p<0.0001) with daily (median ranged from 39 to 47 mg; normal <3 mg; r=0.76–0.82) and four day faecal calprotectin excretion (median 101 mg; 95% CI 45–168 mg; normal <11 mg; r=0.80) and single stool calprotectin concentrations (median 118 mg/l; 95% CI 36–175 mg/l; normal <10 mg/l; r=0.70) in patients with Crohns disease. The cross sectional study showed a sensitivity of 96% for calprotectin in discriminating between normal subjects (2 mg/l; 95% CI 2–3 mg/l) and those with Crohns disease (91 mg/l; 95% CI 59–105 mg/l). With a cut off point of 30 mg/l faecal calprotectin has 100% sensitivity and 97% specificity in discriminating between active Crohns disease and irritable bowel syndrome. CONCLUSION The calprotectin method may be a useful adjuvant for discriminating between patients with Crohns disease and irritable bowel syndrome.

Mitochondrial damage: a possible mechanism of the “topical” phase of NSAID induced injury to the rat intestine

Background—The “topical” effect of non-steroidal anti-inflammatory drugs (NSAIDs) seems to be an important cause of NSAID induced gastrointestinal damage. Aim—To examine the possible mechanism of the “topical” phase of damage in the small intestine. Methods—Electron microscopy and subcellular organelle marker enzyme studies were done in rat small intestine after oral administration of indomethacin (doses varied between 5 and 30 mg/kg). The effect of conventional and non-acidic NSAIDs on rat liver mitochondrial respiration was measured in vitro in a Clarke-type oxygen electrode. Results—The subcellular organelle marker enzymes showed mitochondrial and brush border involvement within an hour of indomethacin administration. Electron microscopy showed dose dependent mitochondrial changes following indomethacin administration consistent with uncoupling of oxidative phosphorylation (or inhibition of electron transport) which were indistinguishable from those seen with the uncoupler dinitrophenol. Parenteral indomethacin caused similar changes, but not in rats with ligated bile ducts. A range of NSAIDs, but not paracetamol or non-acidic NSAIDs which have a favourable gastrointestinal tolerability profile, uncoupled oxidative phosphorylation in vitro at micromolar concentrations and inhibited respiration at higher concentrations. In vivo studies with nabumetone and aspirin further suggested that uncoupling or inhibition of electron transport underlies the “topical” phase of NSAID induced damage. Conclusion—Collectively, these studies suggest that NSAID induced changes in mitochondrial energy production may be an important component of the “topical” phase of damage induction.

High prevalence of NSAID enteropathy as shown by a simple faecal test

BACKGROUND The diagnosis of non-steroidal anti-inflammatory drug (NSAID) induced enteropathy is difficult, requiring enteroscopy or the use of four day faecal excretion of 111In labelled white cells. AIMS To assess faecal calprotectin (a non-degraded neutrophil cytosolic protein) as a method for diagnosing NSAID enteropathy. METHODS Single stool faecal calprotectin concentrations were compared with the four day faecal excretion of 111In labelled white cells in 47 patients taking NSAIDs. The prevalence and severity of NSAID enteropathy was assessed using this method in 312 patients (192 with rheumatoid arthritis, 65 with osteoarthritis, 55 with other conditions) taking 18 different NSAIDs. RESULTS The four day faecal excretion of 111In white cells correlated significantly with faecal calprotectin concentrations. In the group of 312 patients on NSAIDs faecal calprotectin concentrations were significantly higher than in controls, the prevalence of NSAID enteropathy being 44%. The prevalence and severity of NSAID enteropathy was independent of the particular type or dose of NSAID being taken or other patient variables. CONCLUSIONS Assay of faecal calprotectin provides a simple practical method for diagnosing NSAID enteropathy in man. Forty four per cent of patients receiving these drugs had NSAID induced enteropathy when assessed by this technique; 20% of these had comparable levels of inflammation to that previously reported in patients with inflammatory bowel disease.

Uncoupling of intestinal mitochondrial oxidative phosphorylation and inhibition of cyclooxygenase are required for the development of NSAID-enteropathy in the rat

The pathogenesis of NSAID‐induced gastrointestinal damage is believed to involve a nonprostaglandin dependent effect as well as prostaglandin dependent effects. One suggestion is that the nonprostaglandin mechanism involves uncoupling of mitochondrial oxidative phosphorylation.

Subclinical intestinal inflammation: an inherited abnormality in Crohn’s disease relatives?

BACKGROUND & AIMS One approach to unraveling the genetics of complex inherited disease, such as Crohns disease, is to search for subclinical disease markers among unaffected family members. We assessed the possible presence, prevalence, and inheritance pattern of subclinical intestinal inflammation in apparently healthy relatives of patients with Crohns disease. METHODS A total of 49 patients with Crohns disease, 16 spouses, and 151 (58%) of 260 available first-degree relatives underwent a test for intestinal inflammation (fecal calprotectin concentration). The mode of inheritance was assessed from 36 index patients (by variance component analysis) when more than 50% of relatives were studied. RESULTS Fecal calprotectin concentrations in patients with Crohns disease (47 mg/L; confidence interval [CI], 27-95 mg/L) and relatives (11 mg/L; CI, 9-14 mg/L) differed significantly (P < 0.0001) from controls (4 mg/L; CI, 3-5 mg/L), whereas that of the spouses did not (4 mg/L; CI, 3-6 mg/L; P > 0.5). Fecal calprotectin concentration was increased in 49% of all relatives studied. The increased fecal calprotectin concentration among the relatives of the 36 index patients had an inheritance pattern that was most consistent with an additive inheritance pattern. CONCLUSIONS There is a high prevalence of subclinical intestinal inflammation in first-degree relatives of patients with Crohns disease that conforms best to an additive inheritance pattern. The genetic basis for this abnormality may represent a risk factor for Crohns disease.

COX-2 inhibition with rofecoxib does not increase intestinal permeability in healthy subjects: a double blind crossover study comparing rofecoxib with placebo and indomethacin

BACKGROUND Acute and chronic use of non-steroidal anti-inflammatory drugs can increase intestinal permeability. Rofecoxib, which selectively inhibits cyclooxygenase 2 (COX-2), is a novel anti-inflammatory drug with the potential to produce minimal gastrointestinal toxic effects while retaining clinical efficacy. AIMS To assess the potential for rofecoxib to affect the intestine adversely, in comparison with placebo and indomethacin. SUBJECTS Thirty nine healthy subjects (aged 24–30 years). METHOD We performed a four period crossover trial to assess intestinal permeability before and after seven days of treatment. Permeability was measured by the urinary ratio of chromium-51 labelled ethylene diamine tetraacetate (51CrEDTA)/l-rhamnose (five hour collection). RESULTS Indomethacin 50 mg three times daily produced greater increases in intestinal permeability compared with placebo or rofecoxib (25 or 50 mg) (p⩽0.001); rofecoxib was not significantly different from placebo. Mean day 7 to baseline ratios (95% confidence intervals) for51CrEDTA/l-rhamnose were 0.97 (0.82, 1.16), 0.80 (0.68, 0.95), 0.98 (0.82, 1.17), and 1.53 (1.27, 1.85) for placebo, rofecoxib 25 mg, rofecoxib 50 mg, and indomethacin groups, respectively. Rofecoxib was generally well tolerated. CONCLUSION In this study, treatment for one week with indomethacin 50 mg three times daily significantly increased intestinal permeability compared with placebo, while treatment with rofecoxib 25 mg or 50 mg daily did not. The absence of a significant effect of rofecoxib on intestinal permeability at doses at least twice those recommended to treat osteoarthritis was consistent with other studies that have demonstrated little or no injury to the gastrointestinal mucosa associated with rofecoxib therapy.

Selective inhibition of COX-2 in humans is associated with less gastrointestinal injury: a comparison of nimesulide and naproxen

BACKGROUND Selective inhibitors of cyclooxygenase (COX)-2 may provoke less gastric damage and platelet inhibition than conventional non-steroidal anti-inflammatory drugs. AIMS We compared the biochemical and gastrointestinal effects of nimesulide, a potent and selective COX-2 inhibitor, with naproxen which exhibits no selectivity. SUBJECTS Thirty six healthy volunteers were randomised to nimesulide 100 mg or naproxen 500 mg twice daily for two weeks in a double blind, crossover study with a washout between treatments. METHODS Gastrointestinal side effects were assessed by endoscopy, and by estimation of small intestinal absorption-permeability and inflammation. Comparisons were made between variables at the end of each treatment phase. RESULTS Nimesulide caused significantly less gastric injury using the modified Lanza score (p<0.001) as well as reduced duodenum injury (p=0.039). Nimesulide had lower visual analogue scores (VAS) for haemorrhage and erosive lesions in the stomach (p<0.001) and for mucosal injection in the duodenum (p=0.039). Naproxen increased excretion of calprotectin, a marker of intestinal inflammation (5.5 (1.2) to 12.1 (2.1) mg/l) while nimesulide had no effect (treatment difference p=0.03). Naproxen abolished platelet aggregation to arachidonic acid and suppressed serum thromboxane B2 (TXB2) by 98%, indices of COX-1 activity. In contrast, nimesulide had no significant effect on platelet aggregation, although it reduced serum TXB2 by 29%. Production of prostaglandin E2 and prostacyclin by gastric biopsies, also COX-1 dependent, was inhibited by naproxen, but not by nimesulide. COX-2 activity, determined as endotoxin induced prostaglandin E2 formation in plasma, was markedly suppressed by both treatments. INTERPRETATION Nimesulide has preferential selectivity for COX-2 over COX-1 in vivo at full therapeutic doses and induces less gastrointestinal damage than that seen with naproxen in the short term.

Enantiomers of flurbiprofen can distinguish key pathophysiological steps of NSAID enteropathy in the rat

Background—Non-steroidal anti-inflammatory drugs (NSAIDs) cause gastrointestinal damage by a non-prostaglandin (PG) dependent “topical” action and by inhibiting cyclooxygenase. Aims—To discriminate between these two effects by studying some key pathophysiological steps in NSAID enteropathy following administration of (R)- and (S)-flurbiprofen, the racemic mixture, and an uncoupler, dinitrophenol. Methods—The effects of dinitrophenol, racemic, (R)-, and (S)-flurbiprofen on mitochondria were assessed in vitro and on key pathophysiological features of small intestinal damage in vivo (ultrastructure by electron microscopy, mucosal prostanoid concentrations, intestinal permeability, inflammation, and ulcer count) in rats. Results—All the drugs uncoupled mitochondrial oxidative phosphorylation in vitro, caused mitochondrial damage in vivo, and increased intestinal permeability. Dinitrophenol and (R)-flurbiprofen caused no significant decreases in mucosal prostanoid concentrations (apart from a decrease in thromboxane (TX) B2 concentrations following (R)-flurbiprofen) while racemic and (S)- flurbiprofen reduced mucosal prostanoids significantly (PGE, TXB2, and 6-keto-PGF1α concentrations by 73–95%). Intestinal inflammation was significantly greater following administration of (S)-flurbiprofen and racemate than with dinitrophenol and (R)-flurbiprofen. No small intestinal ulcers were found following dinitrophenol or (R)-flurbiprofen while both racemic and (S)-flurbiprofen caused numerous ulcers. Conclusions—Dinitrophenol and (R)-flurbiprofen show similarities in their actions to uncouple mitochondrial oxidative phosphorylation in vitro, alter mitochondrial morphology in vivo, increase intestinal permeability, and cause mild inflammation without ulcers. Concurrent severe decreases in mucosal prostanoids seem to be the driving force for the development of severe inflammation and ulcers.

Intestinal tolerability of nitroxybutyl-flurbiprofen in rats.

BACKGROUND: Nitric oxide derivatives of non-steroidal anti-inflammatory drugs (NSAIDs) are thought to be much less ulcerogenic than their parent compounds. AIM: To compare the effect and potency of flurbiprofen and nitroxybutyl-flurbiprofen to uncouple mitochondrial oxidative phosphorylation (an early pathogenic event in NSAID enteropathy), increase intestinal permeability (transitional stage), and cause macroscopic small intestinal damage. METHODS: In vitro uncoupling potency was assessed using isolated coupled rat liver mitochondria and in vivo by electron microscopy of rat small intestinal mucosa (two hours after the drugs). A dose-response study with flurbiprofen (single doses of 5, 10, 20, and 40 mg/kg) and equimolar doses of nitroxybutyl-flurbiprofen was performed; assessing their effect on intestinal permeability (at 18-20 hours), with 51Cr EDTA, and the number of pointed (< 5 mm) and longitudinal (> 5 mm) small intestinal ulcers at 24 hours. RESULTS: Flurbiprofen, but not nitroxybutyl-flurbiprofen, stimulated coupled respiration in vitro. Both drugs, however, uncoupled in vivo; in the case of nitroxybutyl-flurbiprofen possibly because hydrolysis of its ester bond released free flurbiprofen. Intestinal permeability was uniformly and equally increased with both drugs compared with controls. The number of small intestinal ulcers, pointed and longitudinal, was significantly reduced with nitroxybutyl-flurbiprofen apart from the number of longitudinal ulcers with the highest dose. CONCLUSIONS: These studies show that nitroxybutyl-flurbiprofen is associated with significantly less macroscopic damage in the small intestine than flurbiprofen but was associated with mitochondrial damage in vivo and caused similar increases in permeability of the small intestine, suggesting that its beneficial effect is on the later pathogenic stages of the damage.

The prevalence and severity of intestinal disaccharidase deficiency in human immunodeficiency virus-infected subjects

BACKGROUND Gastrointestinal symptoms are distressing features of human immunodeficiency virus (HIV) infection, and management is often empirical, including withdrawal of dietary lactose. We assessed the prevalence and severity of intestinal disaccharidase deficiency in vitro and in vivo. METHODS Fifty-four HIV-seropositive patients (19 HIV well +/- mild diarrhoea, 7 acquired immunodeficiency syndrome (AIDS) well, and 28 AIDS with diarrhoea) were studied with a combined non-invasive absorption-permeability-disaccharidase test that enables quantitative assessment of the rate of intestinal hydrolysis of lactose, sucrose, and palatinose. Thirty patients had jejunal biopsy specimens suitable for histomorphometric assessment, and 36 had in vitro disaccharidase activity measurement. RESULTS Patients with HIV (with mild diarrhoea) and AIDS (with and without severe diarrhoea) had frequent but mild histomorphometric changes in jejunal specimens. This was associated with frequent (21%-100%) and often severe in vitro jejunal disaccharidase deficiency. In vivo hydrolysis of lactose, sucrose, and palatinose was impaired in 25%-75% of patients, apart from HIV well patients, who were normal. The prevalence of the in vivo lactase and sucrase deficiency was significantly (P < 0.006) lower than in vitro and severe in about 30%. CONCLUSIONS Intestinal disaccharidase deficiency is common both in vitro and in vivo in HIV-seropositive patients but sufficiently severe to consider lactose withdrawal only in about a quarter of the patients with AIDS and diarrhoea.