Simple and sensitive multi-sugar-probe gut permeability test by high-performance liquid chromatography with fluorescence labelling.

Enteral intake of a mixture of inert, non-metabolic monosaccharide and disaccharide probes, followed by measurement of their urinary probe ratio, 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 diarrhoea, which influences the outcome of the test. Recently, a new fluorescent label 9-fluorenylethyl chloroformate hydrazine (FMOC-hydrazine) was introduced, which reacts with reducing sugars to form stable and highly fluorescent single peak derivatives in organic medium. We applied this reagent to develop a sensitive measurement of reducing sugar probes in aqueous samples (e.g., urine). The presented method has a linear response for each sugar derivative between 1 and 1250 pmol with an R2 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 0.05 pmol for lactulose and 0.01 pmol for rhamnose, xylose and 3-O-methyl-D-glucose, corresponding to urine concentrations of 0.11 micromol/l for lactulose and 0.02 micromol/l for rhamnose, xylose and 3-O-methyl-D-glucose. Compared to other tests, the limit of detection is very low. This enabled a reduction in the enteral intake of the disaccharide lactulose from 6-10 g to 1.5 g, thereby minimizing the chance of introducing diarrhoea. The coefficient of variation was below 3% both in standards and urine samples. After spiking the urine with the saccharides a recovery of 102% for lactulose, 101% for rhamnose, xylose and 3-O-methyl-D-glucose was found. In order to evaluate the presented method we compared the lactulose rhamnose ratio measured in urine of healthy human volunteers and kept the ingested dose in agreement with literature values. Furthermore, the ratio was measured after 3, 6 and 9 h to establish the minimal response time required to measure correct ratios. We found that even after 3 h the ratio was stable at a value of 0.0133 which is comparable to literature values (0.008-0.052).

Comparison of enteral feeding and total parenteral nutrition after liver transplantation.

Total parenteral nutrition is used for nutritional support in patients undergoing orthotopic liver transplantation but is associated with complications. We compared the efficacy and tolerability of early enteral feeding with total parenteral nutrition after liver transplantation. 24 patients were studied: 14 received enteral feeding and 10 total parenteral nutrition. A double-lumen enteral tube was used to deliver the feed directly into the jejunum with the second lumen of the tube being used for gastric aspiration. Enteral feeding was started post-operatively within 18 h, was well-tolerated, and of comparable efficacy to total parenteral nutrition. The median number of days for patients to start eating (4) and to achieve 70% of estimated requirements orally (5) did not differ significantly between the two groups. Mid-arm circumference, triceps skinfold thickness, and biceps skinfold thickness were, by comparison with pre-operative values, maintained on the tenth postoperative day in both groups. Early postoperative absorptive capacity, as assessed by a combined carbohydrate test, was reduced significantly in both groups but insufficiently to be of nutritional concern. Intestinal mucosal integrity, as assessed by an intestinal permeability test, was maintained throughout. We conclude that the practical aspects of enteral feeding after liver transplantation are surmountable and that enteral feeding is as effective at maintaining nutritional status as total parenteral nutrition, and has potential benefits in terms of reduced complications and costs.

A novel HPLC method for the simultaneous quantification of monosaccharides and disaccharides used in tests of intestinal function and permeability.

The quantification of monosaccharides and disaccharides used as probes in intestinal function and permeability tests can be technically demanding, detracting from the value of this approach to the indirect assessment of intestinal damage. In this study, a procedure is described for the simultaneous quantification of rhamnose, lactulose, 3-O-methyl-D-glucose and xylose in urine by HPLC using an anion exchange column with pulsed amperometric detection. This method is relatively fast and simple to perform, requiring no pre-treatment of urine samples or post-column derivatization. Accuracy and precision of determinations are illustrated by analytical recoveries (mean percentage +/- S.D., CV., n = 30) for multiple batch analyses of a diluted urine sample containing 20 mg/l of rhamnose (100 +/- 6.8, 6.2%), lactulose (100 +/- 6.1, 5.5%), 3-O-methyl-D-glucose (98 +/- 5.9, 5.5%) and xylose (104 +/- 7.1, 6.5%). Linearity of standard curves indicated that the lower limit for accurate quantification was 0.1 mg/l for all four sugars. Urinary recoveries following oral administration of these sugars to control dogs were determined as a baseline for the investigation of intestinal damage in this species and comparison of chromatograms illustrated enhanced permeability in dogs with gluten-sensitive enteropathy.

Intestinal permeability and function in patients infected with human immunodeficiency virus. A comparison with coeliac disease.

The relationships among intestinal permeability, advancing human immunodeficiency virus (HIV) infection, and the presence of diarrhoea or weight loss were investigated in 51 HIV patients and 20 healthy controls. Ten patients with untreated coeliac disease were also investigated for comparison. Fasting subjects drank an isosmolar test solution containing D-xylose, lactulose (LL), L-rhamnose (R) and 3-O-methyl-D-glucose. Urine was collected for 5 h, test sugar content being subsequently measured by thin-layer chromatography for the dosing sugars. Intestinal permeability (LL/R excretion ratio) and recovery of D-xylose and 3-O-methyl-D-glucose in urine were abnormal in patients with HIV disease, and especially those with diarrhoea, as they were in coeliac disease. Patients with coeliac disease and HIV disease, especially when diarrhoea and/or weight loss were present, had significantly reduced 5-h excretion of L-rhamnose, D-xylose, and 3-O-methyl-D-glucose. These data indicate that abnormal permeability and reduced intestinal absorption capacity are common in HIV patients, occur at all stages of HIV disease, especially in the presence of diarrhoea, and, with the exception of lactulose permeation, are relatively similar to the alterations seen in coeliac disease.

Cardiopulmonary bypass impairs small intestinal transport and increases gut permeability.

Gastrointestinal damage occurs in 0.6% to 2% of patients after cardiopulmonary bypass (CPB), and carries a mortality of 12% to 67%. The incidence of subclinical gastrointestinal damage may be much greater. We examined the effects of nonpulsatile, hypothermic CPB on intestinal absorption and permeability in 41 patients. Bowel mucosal saccharide transport and permeation were evaluated using 100 mL of an oral solution containing 3-O-methyl-D-glucose (0.2 g), D-xylose (0.5 g), L-rhamnose (1.0 g), and lactulose (5.0 g) to assess active carrier-mediated, passive carrier-mediated, transcellular, and paracellular transport, respectively, with a 5-hour urine analysis. Patients were studied before, immediately after, and 5 days after CPB. Immediately after CPB there was a decrease in urinary excretion of 3-O-methyl-D-glucose (from 34% +/- 2.2% to 5.2% +/- 0.7%; p < 0.0001), D-xylose (from 25.4% +/- 1.4% to 4.1% +/- 0.8%; p < 0.0001), and L-rhamnose (from 8.3% +/- 0.6% to 2.6% +/- 0.4%; p < 0.0001). The permeation of 3-O-methyl-D-glucose and D-xylose returned to normal levels 5 days after CPB, but that of L-rhamnose remained significantly below pre-CPB values at 6.6% +/- 0.5% (p = 0.004). However, the permeation of lactulose increased after CPB (from 0.35% +/- 0.04% to 0.59% +/- 0.1%; p = 0.018), and the lactulose/L-rhamnose gut permeability ratio increased markedly (from 0.045 +/- 0.04 to 0.36 +/- 0.08; normal = 0.06 to 0.08; p = 0.004). Patients who had a CPB time of 100 minutes or more had a greater increase in gut permeability (p = 0.049).(ABSTRACT TRUNCATED AT 250 WORDS)

Simultaneous quantification of mannitol, 3-O-methyl glucose, and lactulose in urine by HPLC with pulsed electrochemical detection, for use in studies of intestinal permeability.

The percentage of an oral dose of mannitol, 3-O-methyl glucose, and lactulose excreted in urine is used in noninvasive investigation of active and passive intestinal mucosal transport. We developed a high-pressure liquid-chromatographic method involving anion exchange and pulsed electrochemical detection that allows the simultaneous determination of all three sugar probes in urine. Sample preparation is simple: diluting, mixing with internal standard (melibiose), and desalting. With use of a Dionex 250 x 40 mm Carbopac PA-1 column and elution with an isocratic mixture of 120 mmol/L NaOH and 0.5 mmol/L zinc acetate, all sugars were resolved within 10 min. The standard curve of the method is linear to the following concentrations: mannitol 125 mg/L, 3-O-methyl glucose 300 mg/L, and lactulose 40 mg/L. The minimal detectable concentration of lactulose is 0.4 mg/L. Analytical recovery of the sugars is between 89.0% and 99.5%. The precision of estimation (CV) ranges from 1.76% to 5.6% overall. Reference intervals were established from results for 28 healthy children. The method is adaptable for the study of carbohydrates at low concentrations in other biological fluids.

Misoprostol reduces indomethacin-induced changes in human small intestinal permeability.

This study examined whether indomethacin-induced increases in small intestinal permeability in man are prevented by concomitant administration of a prostaglandin analog (misoprostol). Twelve male volunteers were tested as baseline, following misoprostol alone (200 micrograms, at -16, -12, -8.5, -4, -1.5, and +4 hr); following indomethacin alone (75 mg, at -8; 50 mg, -1 hr); and following coadministration of misoprostol and indomethacin as specified above. A 100-ml test solution containing 3-O-methyl glucose (0.2 g), D-xylose (0.5 g), L-rhamnose (1.0 g), and [51Cr]EDTA (100 microCi) was ingested at 8 AM, and a 5-hr collection made for marker analysis to assess active and passive carrier-mediated transport and trans- and intercellular permeation, respectively. Indomethacin increased the permeation of [51Cr]EDTA selectively, and this increase was significantly reduced by the coadministration of misoprostol. These changes were mirrored by changes in [51Cr]EDTA-L-rhamnose urine excretion ratios, which indicates that paracellular permeability was specifically altered. This study supports the suggestion that NSAIDs alter intestinal permeability by a mechanism involving reduced prostaglandin synthesis and indicates that coadministration of misoprostol with NSAIDs may reduce the frequency and severity of NSAID-induced small intestinal inflammation.