SIMPLE AND SENSITIVE MULTI-SUGAR-PROBE GUT PERMEABILITY TEST BY HIGH-PERFORMANCE LIQUID-CHROMATOGRAPHY WITH FLUORESCENCE LABELING

Enteral intake of a mixture of inert, non-metabolic monosaccharide and disaccharide probes, followed by measurement of their urinary probe r atio, is a well known method to investigate gut permeability. However, most applications lack sensitivity, thus a large amount of especially the disaccharide lactulose has to be ingested. This may cause diarrho ea, which influences the outcome of the test. Recently, a new fluoresc ent label 9-fluorenylmethyl chloroformate hydrazine (FMOC-hydrazine) w as introduced, which reacts with reducing sugars to form stable and hi ghly fluorescent single peak derivatives in organic medium. We applied this reagent to develop a sensitive measurement of reducing sugar pro bes in aqueous samples (e.g. urine). The presented method has a linear response for each sugar derivative between 1 and 1250 pmol with an R( 2) ranging from 0.9997 for lactulose to 0.9999 for rhamnose. The limit of detection, calculated as a signal-to-noise ratio of three, was 0.0 5 pmol for lactulose and 0.01 pmol for rhamnose, xylose and 3-O-methyl -D-glucose, corresponding to urine concentrations of 0.11 mu mol/l for lactulose and 0.02 mu mol/l for rhamnose, xylose and 3-O-methyl-D-glu cose. Compared to other tests, the limit of detection is very low. Thi s enabled a reduction in the enteral intake of the disaccharide lactul ose from 6-10 g to 1.5 g, thereby minimizing the chance of introducing diarrhoea. The coefficient of variation was below 3% both in standard s and urine samples. After spiking the urine with the saccharides a re covery of 102% for lactulose, 101% for rhamnose, xylose and 3-O-methyl -D-glucose was found. In order to evaluate the presented method we com pared the lactulose rhamnose ratio measured in urine of healthy human volunteers and kept the ingested dose in agreement with literature val ues. Furthermore, the ratio was measured after 3, 6 and 9 h to establi sh the minimal response time required to measure correct ratios. We fo und that even after 3 h the ratio was stable at a value of 0.0133 whic h is comparable to literature values (0.008-0.052).