Current Medical Research and Opinion (1996), 13, No. 7, 417-425

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The effect of 5-aminosalicylate and para-aminosalicylate on the synthesis of prostaglandin E2 and leukotriene B4 in isolated colonic mucosal cells

Christoph Schmidt, M.D.,
Thomas Fels, M.D.,
Bernhard Baumeister, M.D.
and
Hans Vetter, M.D.

Medical Poliklinik,
University of Bonn, Germany

Accepted: 19th December 1995

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Summary

The effect of 5-aminosalicylate (5-ASA) and para-aminosalicylate (PAS) on the synthesis of prostaglandin E2 (PGE2) and leukotriene B4 (LTB4) was studied in 24 healthy volunteers. Both drugs are successfully used in the treatment of chronic inflammatory bowel disease, but the biochemical pathway of their anti-inflammatory action is still unknown. Biopsies were taken from the descending colon and were isolated biochemically. 5-ASA, PAS and a control were added to the suspension of isolated colonic mucosal cells and incubated over 0-45 min.

Both 5-ASA and PAS did not alter the PGE2 production but, compared with PAS and the control, 5-ASA decreased the LTB4 synthesis in a dose-related fashion. As a result, the LTB4 PGE2 ratio was significantly diminished by 10-4 mol/l 5-ASA. These findings are consistent with those of other authors, indicating that 5-ASA, at least in part, modulates the colonic eicosanoid synthesis. In contrast, PAS did not influence the mucosal production of PGE2 and LTB4 and therefore must exert some other biochemical action in order to explain its therapeutic effects in the treatment of Crohn's disease or ulcerative colitis.

Key Words

Prostaglandin E 2 - Leukotriene B4 - Cell isolation - Crohn's disease - Ulcerative colitis - 5-Aminosalicylic acid - Para-aminosalicylic acid

Introduction

5-Aminosalicylic acid (5-ASA) and para-aminosalicylate (PAS or 4-aminosalicylate) are used in the treatment of Crohn's disease and ulcerative colitis. Although the clinical benefits of both drugs are well established, their pathophysiological effect is still undefined. 5-ASA is the pharmacologically active component of sulfasalazine, a drug which has been used for chronic inflammatory bowel disease for decades. Sulfasalazine consists of a sulfonamide moiety (sulfapyridine), which is thought to be responsible for most of its side effects, and of the salicylate component. Sulfasalazine and 5-ASA are known to prevent recurrences of ulcerative colitis, but are also effective in the therapy of acute disease. Para-aminosalicylate has been used in the treatment of tuberculosis. Because its chemical structure is related to 5-ASA, it was recently evaluated with success for chronic inflammatory bowel disease.

Crohn's disease and ulcerative colitis are both characterized by diarrhoea, ulceration and inflammatory infiltration of intestinal mucosa. Eicosanoid mediators including prostaglandin E2 (PGE2) and leukotriene B4 (LTB4) play an important role in this inflammatory process and their synthesis is increased in the intestinal mucosa of patients with chronic inflammatory bowel disease.12, 16 Both steroids and sulfasalazine are effective in decreasing elevated levels of PGE2 and LTB4 in these diseases

The purpose of this study is to evaluate whether 5-ASA and PAS have the same effects on colonic synthesis as steroids and sulfasalazine in decreasing the mucosal production of eicosanoids

Subjects and methods

The prostaglandin E2 and leukotriene B4 syntheses in 24 healthy volunteers (17 males and 7 females) were investigated. They were aged between 18 and 78 (mean 47), and all gave a written consent to colonoscopy (Fuji flexible endoscope). Persons on steroids, sulfasalazine, 5-ASA, PAS or non-steroidal anti-inflammatory drugs were excluded from the study.

Eight biopsies were taken by colonoscopy from the distal end of the descending colon, placed in physiological saline, transported to the laboratory, and weighed. The biopsies were similar in size and had an average weight of 5 mg. Additional specimens were obtained for histological examination to exclude any pathological findings.

To isolate mucosal cells from the biopsies, the specimens were incubated in medium A (0.5 m M NaH2PO4, 20 mM NaHCO3, 1 mM Na2HPO4, 70 mM NaCl, 5 mM KCl, 11 mM glucose, 50 mM HEPES, 2 mM EDTA and 20 g/l bovine serum albumin, pH 7.4) for 30 min, taken out, and then added to medium B (analogous to medium A, but including 1 mM MgCl2, 1 mM CaCl2, 10 g/l bovine serum albumin, 0.25 mg/ml pronase, and without EDTA) for a further 60 min. The suspension was centrifuged to sediment the isolated cells and passed through a 60 mm nylon filter to remove mucus and remaining cell clumps. Then the cells were resuspended with medium C (analogous to medium B, but with 1 g/l bovine serum albumin) and the concentration of the suspension was determined by cytometry under light microscopy. To identify dead cells, trypan blue was added, which stained less than 6% of cells in each assay. The suspension was diluted to a concentration of 500 000 cells/ml with medium C, and transferred into vials.

Concurrently, 5-aminosalicylate and para-aminosalicylate were diluted with distilled water to a concentration of 10-4 or 10-6 mol/l. Then 3 sets of vials were set up: 5-ASA, PAS and a control (distilled water). They were incubated in a water bath at 37°C for 0 (to determine the basal PGE2 concentration of the incubation medium), 10, 20, 30 or 45 min, respectively. The calcium ionophore A 23187 (dissolved in DMSO, final concentration 10 µmol/l) was added for the last 15 min to the 45 min vials. At the end of each incubation time, the suspensions were recentrifuged (12 000 U/min), and the supernatant was carefully decanted, frozen and stored for later determination of PGE2 and LTB4 by radioimmunoassay (125I PGE2 RIA and 3H LTB4 RIA by NEN/DuPont, Dreieich, Germany). Light microscopy showed no evidence of remnant cells in the supernatant.

For statistical analysis, the Wilcoxon test for pairs and the Mann-Whitney U-test for independent samples were performed.

Results

Figures 1 and 2 show the accumulation of PGE2 at 0 to 45 min with 5-ASA, PAS or the control (distilled water). 5-ASA and PAS were added in different concentrations of 10-4 mol/l (Figure 1) and 10-6 mol/l (Figure 2), respectively; each vial contained 500 000 cells per ml. At 0 min the prostaglandin E2 concentration was the same in all vials. This initial PGE2 concentration was determined by the prostaglandin accumulation in the medium before the start of the incubation. There was a marked increase of about 250% for the first 10 min, followed by a much smaller rise during the next 20 min (minute 20 and 30). The stimulation with the calcium ionophore led to another significant increase at the end of the incubation (minute 45). No difference was found in the PGE2 synthesis between 5-ASA, PAS and the control, unrelated to the concentrations of 5-ASA or PAS used.

The leukotriene B4 concentration was below the detection limit of the radioimmunoassay until stimulated by the calcium ionophore. The LTB4 accumulation after 45 min incubation is shown in Figure 3. Compared with PAS and the control, vials with 5-ASA reveal a diminished LTB4 synthesis when incubated with 10-4 mol/l 5-ASA as opposed to 10-6 mol/l. Because of this dose-related effect of 5-ASA, the LTB4/PGE2 ratio decreases significantly ( p < 0.05) when 5­ASA was used in a higher concentration (10-4 mol/l). PAS did not change this ratio.

Figure 1.
Prostaglandin E 2 concentration of the cell suspension during 45 min of incubation with 10-4 mol/l 5-ASA, PAS or control (distilled water). The suspension was stimulated by a calcium ionophore between minutes 30 and 45

Figure 2.
Prostaglandin E2 concentration of the cell suspension during 45 min of incubation with 10-6 mol/l 5-ASA, PAS or control (distilled water). The suspension was stimulated by a calcium ionophore between minutes 30 and 45

Figure 3.
Leukotriene B4 concentration of the cell suspension after 45 min of incubation. 5-ASA, PAS or distilled water was added to the suspension at different concentrations. Compared with PAS or the control, 10-4 mol/l 5-ASA markedly decreased the LTB4 synthesis

Discussion

The effect of 5-aminosalicylate (5-ASA) and para-aminosalicylate (PAS) on the synthesis of prostaglandin E2 (PGE2) and leukotriene B4 (LTB4) was studied in 24 healthy volunteers. Isolated mucosal cells were separated from colonic biopsy specimens, and the accumulation of PGE2 and LTB4 were measured after incubating the cell suspension for 0-45 min with 5-ASA or PAS. The use of this cell isolation technique has several advantages over incubated biopsies, surgically resected specimens or rectal dialysis. The eicosanoid synthesis in two biopsy specimens from the same patient is always different, even if size and site of biopsy are identical. The use of two aliquots taken from a cell suspension can reduce this intra-individual variability. This technique is therefore ideal for comparing the effects of drugs, like 5-ASA and PAS. The PGE2 and LTB4 syntheses are affected by numerous factors including other hormones, nervous stimuli and perfusion. Within a homogeneous suspension, these factors are not eliminated but controlled. However, the conditions in vitro are different from the physiological environment in vivo. Although the incubation medium was chosen to simulate this environment, results obtained by cell isolation must be critically analysed.

As shown in Figures 1 and 2, the PGE2 concentration in all suspensions rose significantly at the beginning of the incubation. Following this, there was only a moderate further increase between minutes 10 and 30. This suggested a reduced capacity of the isolated cells to produce prostaglandins, caused either by an increased mucosal catabolism or, more likely, by the shortage of substrate, as neither arachidonic acid nor any other predecessor of PGE2 were added to the medium. The stimulation with the calcium ionophore A 23187, however, led to another marked rise of PGE2 at minute 45. This drug increased the permeability of cell membranes to calcium. As the arachidonate metabolism is integrated with Ca2+ mobilization, calcium ionophores allow an energy- and carrier-independent influx of Ca2+ across impermeable membranes and thus stimulate the synthesis of eicosanoids to their maximum. The fact that consecutive measurements of PGE2 concentrations showed increasing values, even though separate vials were used for different incubation times, underlined the reliability of our measurements. Leukotriene B4 became detectable only after stimulation with the calcium ionophore between minutes 30 and 45.

The aim of this study was to find out whether 5-ASA and PAS exert their therapeutic action on patients with inflammatory bowel disease by altering the PGE2 and LTB4 synthesis of colonic mucosa. 5-ASA and PAS are chemically related to sulfasalazine which has, in addition to steroids, been used for the treatment of Crohn's disease and ulcerative colitis for decades. 5-ASA was shown to be the pharmacologically active component of sulfasalazine, whose sulfapyridine moiety is responsible for most of its side-effects. PAS was used in the treatment of tuberculosis before it was proved to be as effective as 5-ASA for chronic inflammatory bowel disease. Para-aminosalicylate is chemically characterized as 4-aminosalicylate and is thus closely related to 5-ASA.

Although steroids and sulfasalazine have been used for a long time in the therapy of Crohn's disease and ulcerative colitis, it is not known how these drugs influence the pathophysiology of these diseases. There is some evidence that the inflammatory process is, at least in part, influenced and caused by prostaglandins and leukotrienes. These eicosanoids are found to be significantly increased in active disease and both PGE2 and LTB4 are decreased by steroids or sulfasalazine.18 The rate of PGE2 synthesis correlates well with disease activity3 and is also a reliable predictor of the response to drug treatment. Patients with increased PGE2 levels show a substantial risk of relapse.4

Prostaglandin E2 acts synergistically with other mediators of inflammation,13 causes central hyperthermia and sensitizes local receptors to pain.7 PGE2 is a potent vasodilator, increases vascular permeability causing local oedema, and mediates the secretory diarrhoea observed in Crohn's disease and ulcerative colitis. Furthermore, PGE2 regulates the immune response by enhancing it at low concentrations, but suppressing it at higher concentrations.9 In addition, PGE2 promotes the cytoprotective activity in intestinal mucosa.2 Therefore prostaglandin E2 does not only contribute to negative aspects of the disease, but may also activate healing mechanisms. This may explain why isolated modulation of the PGE2 level does not necessarily improve the course of chronic inflammatory bowel disease. Inhibition of prostaglandin E2 synthesis by indomethacin, for example, does not improve but, in fact, worsens the symptoms of Crohn's disease and ulcerative colitis.8

Leukotriene B4 amplifies and modulates the inflammatory response in active Crohn's disease or ulcerative colitis. LTB4 induces neutrophil aggregation and degranulation, increases microvascular permeability and induces the release of lysosomal enzymes.6, 10

As 5-ASA and PAS are chemically related to sulfapyridine, it was suggested that they exert their pharmacological action in the same way as sulfapyridine (and steroids), which alter the eicosanoid metabolism in intestinal mucosa. The results of our study showed that there were considerable differences between 5­ASA and PAS. The leukotriene synthesis was markedly decreased in concentrations of 10-4 mol/l, whereas 10-6 mol/l did not show this effect (Figure 2). This finding underlines the dose-related effect of 5-ASA in altering the leukotriene production of colonic mucosal cells. PAS or the control (distilled water, the diluent for 5-ASA and PAS) did not alter LTB4 synthesis. The prostaglandin synthesis was not affected by 5-ASA (or PAS), as shown in Figures 1 and 2. As a result, 5-ASA decreased the LTB4/PGE2 ratio significantly ( p < 0.05). This could contribute to the therapeutic effect of 5-ASA. By shifting the LTB4/PGE2 ratio towards prostaglandin E2, the inflammatory process in inflammatory bowel disease is diminished. LTB4 is a potent mediator of inflammation, whereas PGE2, as discussed before, exerts a more ambivalent role by worsening, but also ameliorating, the inflammatory process. In addition, as only healthy subjects were examined, the influence of 5-ASA on the eicosanoid metabolism may be underestimated. We feel that the potential number of untreated patients with chronic inflammatory bowel disease would be too small for statistical evaluation. However, it is quite likely that the increased LTB4 and PGE2 synthesis in patients with active disease are much more affected by 5-ASA than in healthy subjects with normal eicosanoid levels. The alteration of mucosal eicosanoid production has been reported by other authors previously.17

No effect of para-aminosalicylate on the eicosanoid synthesis of colonic mucosa could be demonstrated in our study. There was no alteration either in PGE2 or LTB4 production in the cell suspension. Again these findings are consistent with other studies, indicating that PAS does not alter the eicosanoid metabolism.11 However, this result is confusing for two reasons. Firstly, the clinical benefit of PAS in the treatment of Crohn's disease and ulcerative colitis is well established.1, 15 Secondly, the chemical structure of PAS (= 4-ASA) is quite similar to 5-ASA. Nevertheless, our study confirms the striking fact that two chemically closely related drugs, which show the same clinical effect on inflammatory bowel disease, exert their action by different mechanisms. 5­Aminosalicylate alters the LTB4/PGE2 ratio in colonic mucosa, but the way para-aminosalicylate works remains unknown.

The enhanced PGE2 and LTB4 production in patients with active Crohn's disease or ulcerative colitis might be the consequence rather than the cause of the inflammatory process in these diseases.14 However, as LTB4 and PGE2 amplify and modulate the signs and symptoms of chronic inflammatory bowel disease, the pharmacological alteration of these mediators of inflammation contributes to the healing process in these patients. The growing understanding of how empirical drugs like steroids and sulfasalazine, as well as 5-ASA and PAS, influence the clinical course of chronic inflammatory bowel disease, will provide a better knowledge of the pathophysiology and thus of newly developed therapeutic means in the treatment of Crohn's disease and ulcerative colitis.

Acknowledgements

The authors thank Beate Schiermeyer-Dunkhase for skilled technical assistance.

References

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