Novel analytical approach to a multi-sugar whole gut permeability assay

Abstract

Many pathophysiological conditions are associated with increased gastrointestinal permeability, reflecting an elevated risk of endotoxaemia, inflammation, and sepsis. Permeability tests are increasingly used in clinical practice to obtain information on gastrointestinal functioning, but tests are often restricted to the small intestine, and require large oral sugar doses. Therefore, a novel multi-sugar assay was developed, allowing assessment of whole gut permeability changes in urinary and plasma samples collected at regular intervals from 10 healthy volunteers at baseline and after intake of monosaccharides (rhamnose and erythritol) and disaccharides (sucrose, lactulose, and sucralose). Samples were analyzed by isocratic cation-exchange LC-MS. Sample preparation and detection conditions were optimized. After centrifugation, chromatographic separation was achieved on an IOA-1000 column set at 30°C. Column effluent was mixed with ammonia for sugar-ammonium adduct formation. The lower limit of detection was 0.05 μmol/L for disaccharides and 0.1 μmol/L for monosaccharides. Linearity for each probe was between 1 and 1000 μmol/L (R(2): 0.9987-0.9999). Coefficients of variation were <5% in urine, and <9% in plasma. Recovery data were within the 90% to 110% range at all spiked concentrations. This highly sensitive novel LC-MS approach resulted in a significant decrease of the detection limit for all sugar probes, allowing a 5-fold reduction of the commonly used lactulose dose and the addition of sugar probes to also assess the gastroduodenal and colon permeability. In combination with its extended application in plasma, these features make the novel assay a promising tool in the assessment of site-specific changes in gastrointestinal permeability in clinical practice.