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Endotoxin has long been known to be an important virulence factor for Gram negative bacteria. It is chemically classified as lipopolysaccharide (LPS) and it is one of the major constituents of the outer membrane of Gram negative bacteria. This molecule is also known to be responsible for many injurious effects of Gram negative bacterial infections and thus is clinically important.1
The pathogenesis of ankylosing spondylitis (AS) is still unknown. A microbial aetiology has been suggested, as increased faecal carriage of Klebsiella spp, as well as increased antibody values, particularily against the LPS part of K pneumoniaehave been reported in AS patients.2 3 Furthermore, Wagener et al 4 have reported that the plasma endotoxin concentrations were increased in approximately 30% of the AS patients and that a significant correlation was found between the increased endotoxin values and increased C reactive protein (CRP) concentrations. Therefore, it seems that LPS plays an important part in the pathogenesis of AS.
In this study we have examined the plasma of 28 hospital AS patients (eight females, 20 males; mean age 43 years (range 26-62)); mean erythrocyte sedimentation rate 22 mm/1st h (range 5-64) and the mean CRP value 12 mg/l (range 0-37)) from the Rheumatism Foundation Hospital, Heinola, Finland for the presence of endotoxin. The mean (SD) duration of the disease was 14.5 (9.0) years: less than five years for three patients, 5-10 years for nine patients, and more than 10 years for 16 patients.
Blood samples were taken by the Vacutainer blood collection system (Becton & Dickinson Diagnostic Instrument System, Towson, MD, USA) into Vacutainer tubes containing sodium citrate and kept in melting ice until separation of plasma. The samples were centrifuged immediately at 400 g for 10 minutes at +4°C, and plasma carefully removed and stored at −70°C until assayed. All equipment was free of endotoxin.
The concentration of endotoxin in plasma was determined by a chromogenic Limulus assay using Coatest kit (Chromogenix AB, Mölndal, Sweden) with a microtitre modification.5 6 The detection limit was 5 pg/ml; among normal controls, endotoxin values do not exceed this concentration.
None of the AS patients had plasma endotoxin concentrations exceeding the level of 5 pg/ml. This is surprising, as increased gut permeability has been found in AS patients7 and consequently gut microbes might easily pass through the mucosa to enter the circulation. The increased serum antibody concentrations against LPS of certain enterobacteria in AS patients support this scenario.3Endotoxaemia may be rapidly transitory, however. Endotoxin could leave the circulation at sites of inflammation, for example, the joints of these patients, as in another HLA B27 associated disease, reactive arthritis.8 9 Consequently, it may be that endotoxin is found in circulation only at certain stages of the disease; for instance, Wagener et al 4 showed a clear correlation between increased endotoxin and CRP values in AS patients. Endotoxin may also be bound to the specific endotoxin binding protein10 and is therefore not detected by the Limulus test. However, the same assay was used by Wagener et al,4 who found increased plasma endotoxin concentrations in one third of the AS patients and in up to half of the patients with sacroiliitis and peripheral arthritis or rheumatoid arthritis. In their study the blood sampling and handling was not reported. The Limulus assay is known to be extremely sensitive to contaminants.
In conclusion, in this study we could not confirm the reported findings on increased plasma endotoxin concentrations in patients with AS. However, this does not exclude the concept that endotoxin or LPS may play an important part in the pathogenesis of AS.
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