Comparison of an enzymatic assay with liquid chromatography-pulsed amperometric detection for the determination of lactulose and mannitol in urine of healthy subjects and patients with active celiac disease

Abstract

Integrity of mucosal barrier function may be altered by different gastrointestinal (GI) diseases including celiac disease (CD) [1]. A reliable indicator of small intestine mucosal damage is provided by the ratio of urinary recovery of the disaccharide lactulose (Lac) over that of the sugar alcohol mannitol (Man) following ingestion of standardized amounts of each [2].Several methods have been proposed for the quantitative determination of Lac and Man, such as thin-layer chromatography, liquid chromatography with refractive index and liquid chromatography-tandem mass spectrometry [3]. Along with these procedures, a high performance anion-exchange chromatography (HPAEC) coupled with pulsed amperometric detection (PAD) has been set up [4]. This technique allows a fast and direct quantification of non-derived carbohydrates at picomole levels. Its implementation in routine clinical laboratory testing is, however, limited by expertise of laboratory personnel and high equipment cost. Cheaper colorimetric/enzymatic procedures have also been developed [5]. They are simple and rapid, but do not evaluate the composition of saccharides and are often subjected to many interferences. However, the use of such kits would greatly facilitate Lac and Man evaluation especially in clinical settings. To investigate the potentiality of ESAT in comparison to HPAEC-PAD as a simple and rapid tool for large scale IP detection, the aim of the present study was to compare the urinary recovery of Lac and Man determined by HPAEC-PAD and a commercially available ESAT kit in 30 healthy controls and 20 CD patients before and after 1 year on gluten-free diet (GFD). The study was consistent with the Declaration of Helsinki and the probands gave written informed consent.After an overnight fast, a pretest urine sample was collected to check for the possible presence of endogenous sugars. Then, subjects drank a sugar test solution containing 5 g of Lac, 2 g of Man and 40 g of sucrose as an osmotic filler in 100 mL water. Urine was collected for the subsequent 5 h into a flask containing chlorhexidine, centrifuged, and the supernatant stored at -20°C until analysis. For each sugar, the 5-h urinary recovery was expressed as the percentage of the ingested dose (%Lac and %Man), and the %LMR was calculated.Following a previously published HPAEC-PAD method [6,7], urine samples were centrifuged at 7000 × g and appropriately diluted and filtered before injection (25 µL). Urinary sugar concentrations were calculated from the calibration curve by peak-area analysis. HPAEC-PAD was performed by a Thermo Scientific model Dionex ICS-5000 (Sunnyvale, CA, USA) with a pulsed amperometric Brought to you by | University of Georgia Libraries Authenticated Download Date | 5/27/15 11:34 AM

Synbiotics Alter Fecal Microbiomes, But Not Liver Fat or Fibrosis, in a Randomized Trial of Patients With Nonalcoholic Fatty Liver Disease

“…Since the INSYTE study was not designed with a healthy control group, we compared the INSYTE baseline lactulose/mannitol ratio with that for healthy subjects described in the literature. Notably, the mean and median values of lactulose/mannitol ratio were ~10-fold greater than those reported for healthy subjects in the literature [33][34][35][36] , supporting the presence of altered intestinal permeability. That said, there were no significant changes in intestinal permeability between baseline and end-of-study measurements for the lactulose mannitol ratio in either the synbiotic or placebo groups.…”

Noninvasive biomarkers of gut barrier function identify two subtypes of patients suffering from diarrhoea predominant-IBS: a case-control study

“…Patients with a La/Ma ratio lower than 0.035 were considered as D-IBS(−); patients with a value equal to or higher than 0.035 were considered D-IBS(+). This cut-off value (mean + 2SD) derived from our previous study performed on a large group of healthy subjects [ 26 ].…”

Adipose Tissue-Derived Biomarkers of Intestinal Barrier Functions for the Characterization of Diarrhoea-Predominant IBS

“…All the participants (both D-IBS and HC) were subjected to all the scheduled analyses. The D-IBS patients were categorised as having normal [D-IBS(−)] or increased s-IP [D-IBS(+)], by using the sugar absorption tests [23].…”

“…Urine samples were stored at −80°C until analysis. The detection and measurement of the three sugar probes, Su, La, and Ma, in urine were performed by chromatographic analysis as described previously by our group [23]. Briefly, high-performance anion exchange chromatography coupled with pulsed amperometric detection was performed on a Dionex Model ICS-5000 with a gold working electrode and a 25 μ l peek sample loop (Dionex Corp., Sunnyvale, California, USA).…”

“…The percentages of ingested Su (%Su) together with those of La (%La) and Ma (%Ma) in urine were evaluated, and the La/Ma ratio was calculated for each sample. Based on data from controls in our laboratory, La/Ma ≥ 0.035 is indicative of increased intestinal permeability [23].…”