Background: Patients with acute severe ulcerative colitis (ASUC) often do not respond to infliximab (IFX) induction therapy. In an earlier study we showed that IFX could be measured in feces of these patients, with the highest fecal IFX concentrations in the first days after the first infusion. This “fecal loss” of IFX is believed to contribute to primary non-response. The mucosa of patients with inflammatory bowel disease is characterized by overexpression of proteases (metalloproteinases (MMPs) and high levels of proteases are found in feces. Therefore fecal IFX concentrations from patients with ASUC may be underestimated due to proteolysis.
Methods: Fecal samples of 5 patients with ASUC not receiving biological therapy, were homogenized (0.2 gram/ml) in buffers containing general protease inhibitors (EDTA free, Sigma-Aldrich®), Marimastat, broad spectrum MMP inhibitor ((Sigma-Aldrich®), a combination of the two, or no protease inhibitors at all. Ten μg/ml IFX was added to the fecal supernatants, after which samples were stored at different temperatures (4°C, 22°C and 37°C) for 24 hours. IFX concentrations were measured using validated ELISA technology by Sanquin Laboratories (Amsterdam, The Netherlands).
Results: In samples without protease inhibitors stored at 37°C IFX concentrations were (median, IQR) 0.2 μg/ml (0.1–5.2). After addition of protease inhibitors, higher IFX concentrations were observed: 0.7 μg/ml (0.5–5.9) for the general protease inhibitor, 0.2 μg/ml (IQR 0.1–4.1) for Marimastat and 0.6 μg/ml (IQR 0.5–3.4) for the combination (Figure 1). In samples without protease inhibitor, the highest concentrations were measured after incubation at a temperature of 4°C (0.8 μg/ml; 0.3–7.5) compared to the lowest concentrations measured after incubation at 37°C (0.2 μg/ml; 0.1–5.2). Overall, the IFX concentrations that were measured were >10 times lower than what was “spiked” to the samples.
Conclusions: Degradation of IFX by fecal proteases is highly relevant, since <10% of IFX added to feces could be measured with an ELISA test. This indicates that fecal loss of IFX in these patients is strongly underestimated. In order to maximize measurements of fecal IFX in patients with IBD samples should be processed in the presence of regular protease inhibitors and at a low temperature (4°C), to minimize proteolytic degradation. In real life, most of the degradation however may already take place in the gut before defecation.