Eur J Clin Pharmacol (1992) 43:189-192

© Springer-Verlag 1992

Bacterial acetylation of 5-aminosalicylic acid in faecal suspensions cultured under aerobic and anaerobic conditions R. A. van Hogezand 1, H. M. Kennis 2, A. van Schaik 1, J. P. Koopman 2, P. A. M. van Hees 1, and J.H.M. van Tongeren 1 1Department of Medicine, Division of Gastroenterology, St. Radboud Hospital and 2 Central Animal Laboratory, University of Nijmegen, P. O. Box 9101, 6500 Nijmegen, The Netherlands Received: September 19,1991/Accepted in revised form: March 13,1992

Summary. A f t e r oral administration of 5-aminosalicylic acid a substantial p r o p o r t i o n of the acetylated f o r m is excreted in the faeces. We have studied the role of the faecal microflora in acetylating 5-aminosalicylic acid. In faeces i n c u b a t e d u n d e r b o t h aerobic and anaerobic conditions there was acetylation of 5-aminosalicylic acid. Of the anaerobic bacteria isolated f r o m a 108 faecal dilution 44 % were able to acetylate 5-aminosalicylic acid. We conclude that the n o r m a l faecal microflora contribute to the acetylation of 5-aminosalicylic acid.

Key words: 5-Aminosalicylic acid, Faecal bacteria, Sulphasalazine; acetyl-5-aminosalicylic acid, drug m e t a b o lism

5-Aminosalicylic acid ( 5 - A S A , mesalazine), the active c o m p o n e n t of salicylazosulphapyridine (sulphasalazine, Salazopyrin ®, SASP), is used in treating i n f l a m m a t o r y bowel disease. A f t e r oral administration most of the 5 - A S A is a b s o r b e d in the proximal gut and excreted in the urine [1]. H o w e v e r , if it is given as a slow-release formulation (Asacol ®, Salofalk ®, or Pentasa ®) or as a diazo derivative (olsalazine, D i p e n t u m ®) 5 - A S A is liberated in the m o r e distal parts of the gut [2-8]. A substantial p r o p o r t i o n of the 5 - A S A in the faeces is acetylated [3-8], and the ability of the faecal bacterial flora and of the intestinal m u c o s a to acetylate 5 - A S A has b e e n r e p o r t e d [9-12]. We have shown that a c e t y l - 5 - A S A was not effective in patients with idiopathic proctitis [13] and so intraluminal acetylation inactivates 5 - A S A . In this study we have investigated the acetylation of 5 - A S A in faecal suspensions cultured u n d e r aerobic and anaerobic conditions and the acetylation by individual anaerobic bacteria.

Methods We introduced fresh faeces from five healthy subjects into an anaerobic glove box with an oxygen concentration below 5 x 10-4 kPa [14]. Faeces from each subject were diluted (1:2) in broth [15],

36 ml of each dilution was mixed thoroughly with 4 ml of 5-ASA (5000 gmol. l- 1;765 gg. ml - 1). As a control 36 ml of broth was used. Aliquots of i ml were made and two of each series were immediately analysed for (acetyl)-5-ASA [16]. Half of the remainder was incubated aerobically or anaerobically at 37 °C. After 3, 6, 12, and 24 h and 2, 3, and 7 days the aliquots were analysed for (acetyl)5-ASA. To detect the capability of faecal bacteria to acetylate 5-ASA fresh faeces from one subject were introduced into an aerobic glove box and eight dilutions with broth (10-1 to 10- 8) were made. Eighteen ml of each dilution was mixed with 2 ml of 5-ASA (5000 gmol. 1 1).Aliquotsofl mlweremadeandtwoofeachmixtureserieswere immediately analysed for (acetyl)-5-ASA, 8 were incubated aerobically, and 8 anaerobically at 37°C. After 1, 2, 4, and 8 days the aliquots were analysed. To investigate the capacity of cultured faecal bacterial dilutions, in a separate set of experiments 0.1 ml of each fresh faecal dilution with broth (10 -1 to 10*) was spread on to 6 or more agar plates and incubated anaerobically. After 7 days the growth was harvested and suspended in 8 aliquots with 18 ml of broth to a turbidity of Mac Farland number 5 and mixed with 2 ml of 5-ASA (5000 gmol.l-l). Aliquots of 1 ml were made and of each series two were immediately analyzed for (acetyl)-5-ASA. Half of the remainder were incubated aerobically or anaerobically at 37°C. After 1, 2, 4, and 8 days the aliquots were analysed. In order to determine the total amount of viable anaerobic bacteria we spread 0.1 ml of the 10 -6 to 10 -8 dilutions on to three sheep blood-agar plates and incubated than anaerobically for 7 days. Of the 10-1 to 10 5dilutions, 0.1 ml was spread on to 3 sheep blood-agar plates and 3 Levine-agar EMB plates (DIFCO) and incubated aerobically for 48 h for determination of the numbers of aerobic bacteria and Enterobacteriaceae. Finally, we derived subcultures from the anaerobically incubated agar plates (10 .8 dilution). Suspensions were made with 4.5 ml of broth mixed with 0.5 ml of 5-ASA (5000 gmol-l-1), and incubated anaerobically at 37 °C. After 2 days (acetyl)-5-ASA was measured.

Results Figure 1 shows the m e a n faecal concentrations of 5 - A S A , acetyl-5-ASA, and total 5 - A S A ( 5 - A S A + acetyl5 - A S A ) after aerobic and anaerobic incubation with 5 - A S A . T h e m e a n concentration of 5 - A S A fell after 7 days u n d e r b o t h aerobic and anaerobic conditions and a c e t y l - 5 - A S A was p r o d u c e d in b o t h cultures. T h e r e was

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no acetyl-5-ASA produced in the control incubation. Total 5-ASA fell only under aerobic conditions. Under aerobic conditions 5-ASA and total 5-ASA fell in diluted faeces. However, when diluted more than 10 .4 there was no difference in total 5-ASA between the first and the last days. Under anaerobic conditions 5-ASA concentration fell but total 5-ASA did not. In Fig.2 acetyl-5-ASA production (aerobic and anaerobic) is shown in relation to the dilution factor on days 0, 1, 2, 4, and 8. Acetyl-5-ASA production was maximal in t h e 10 .4 dilution and in the 10 _6 dilution under aerobic and anaerobic conditions respectively. At 10-8 dilution in both conditions nearly no acetyl-5-ASA was formed. In the experiment designed to study the influence of cultured faecal bacterial dilution no changes occured over 8 days (Fig.3). The number of bacteria in the 10 -I faecal dilution was about 10l°.ml 1 and that in the 10 -s dilution was about 102. ml-1. The total number of viable anaerobic bacteria was 1.7 (1.5-1.8) x 1011 per g of faeces: 1.8 (1.7-1.9) x 107 aerobic bacteria and 1.9 (1.9-1.9)x 107 Enterobacteriaceae.

Eighty subcultures were measured in the 10-s dilution from anaerobically incubated agar plates. Acetyl-5-ASA production was small (below 50 gmol. 1 1 after 2 days) in 13 cultures (16%), moderate (50-200gmol-1-1) in 6 (7.5 % ), and substantial (over 200 gmol. 1-1) in 16 (20 % ).

Discussion

Rectal and oral 5-ASA formulations are effective in the treatment of proctocolitis and Crohn's disease [5, 17, 18]. In studies with sulphasalazine or 5-ASA analogues a substantial part of the 5-ASA in the faeces is acetylated [3-8]. We have shown that 5-ASA is acetylated in diluted faecal suspensions. Under aerobic conditions, part of the 5-ASA is not only acetylated but also transformed into substances which could not be detected in our assay. This transformation is probably oxidative, for which faeces are required, because it could not be demonstrated in anaerobic cultures and in the more dilute suspensions under aerobic conditions. Production of acetyl-5-ASA was most pronounced in the 10 -4 and 10 -6 faecal dilutions cultured aerobically and anaerobically respectively. This can be explained by insufficient bacterial nutrition or by the presence of higher concentrations of inhibitory factors in the less dilute samples. In the experiment used to study the capacity of cultured faecal bacterial dilutions more or less constant ace-

191 References

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Fig.3a, b. Concentrations of acetyl-5-ASA after incubation of 5-ASA with bacterial dilutions in broth, derived from faecal dilutions, cultured under (a) aerobic and (b) anaerobic conditions at 37 °C, and determined at zero time and after 1, 2, 4, and 8 days

t y l - 5 - A S A concentrations were f o u n d in the dilutions 10 -1 to 10 -7 b o t h in the aerobic and anaerobic cultures. D u r i n g culture, all the bacteria present in the sample had the chance to grow to large numbers. This is p r o b a b l y w h y no differences were seen b e t w e e n the dilution steps f r o m 10 -~ to 10 -7. O n l y in the 10 8 dilution were low values found, p r o b a b l y because of elimination of acetylating bacteria caused by dilution. In the e x p e r i m e n t with diluted faeces (Fig. 2) the same p h e n o m e n o n was found, with one difference: in the anaerobic culture also almost no acetyl5 - A S A was p r o d u c e d in the 10- 7 dilution. It can therefore be concluded that in faecal dilutions higher t h a n 10 -6 to 10-7 only small a m o u n t of acetylating bacteria were present. This is in a c c o r d a n c e with the finding that only a small a m o u n t (20 % ) of the pure cultures f r o m a 10 -8 dilution was albe to acetylate 5 - A S A substantially. Intraluminal acetylation of 5 - A S A has b e e n described [9-12]. We s h o w e d that p r o d u c t i o n of a c e t y l - 5 - A S A , a m o n g others, is p e r f o r m e d by b o t h a n a e r o b i c and aerobic colonic bacteria. A c e t y l a t i o n in h u m a n gut wall has also b e e n postulated [19, 20]. T h e therapeutic effect of a c e t y l - 5 - A S A is controversial. A c e t y l - 5 - A S A in e n e m a s or suppositories was not effective in proctitis or proctocolitis [13, 21], but successful t r e a t m e n t with a c e t y l - 5 - A S A e n e m a s was claimed by Will o u g h b y et al. [22]. If a c e t y l - 5 - A S A is ineffective, it is possible that a substantial part of 5 - A S A offered to the colon as salicylazosulphapyridine, A s a c o l ®, Salofalk ®, Pentasa ®, D i p e n t u m ®, or as a rectal f o r m u l a t i o n of 5 - A S A could be inactivated by colonic bacteria.

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