Correlation of lactulose-to-mannitol ratios in plasma and urine for intestinal permeability assessment in pigs.

OBJECTIVE: The lactulose-to-mannitol ratio test is a test to assess the disorders associated with gut permeability. The test requires an oral administration of the mixture of lactulose and mannitol and urine collection. The urinary ratio of lactulose to mannitol is an indicator of intestinal permeability. Due to the complexity of urine collection in animal studies, plasma exposure ratios of lactulose to mannitol compared to their urinary concentration ratios were evaluated following an oral administration of the sugar mixture in pigs. ANIMALS: 10 pigs were orally dosed with a solution of lactulose and mannitol mixture. PROCEDURES: Plasma samples were collected at predose, 10 and 30 minutes and 2, 4, and 6 hours postdosing, and cumulated urinary samples were collected at 6 hours for liquid chromatography-mass spectrometry analysis. The ratios of pharmacokinetic parameters of lactulose to mannitol and the plasma sugar ratios at a single time point or the mean values of several time points were compared to their urinary sugar ratios. RESULTS: The results revealed that the lactulose-to-mannitol ratios of AUC0-6h, AUCextrap, and Cmax were correlated to the urinary sugar ratios, and the plasma sugar ratios of a single time point at 2, 4, or 6 hours and the mean values of those time points were also appropriate to replace their urinary ratios in pigs. CLINICAL RELEVANCE: Following an oral administration of lactulose and mannitol mixture, blood collection, and assay can be an option for assessing intestinal permeability, especially in animal studies.

Accelerated Caco-2 cell permeability model for drug discovery.

INTRODUCTION: By culturing Caco-2 cells according to a new and optimized protocol, it has been possible to accelerate the cell culture process in such a way that the cells can be used for experiments after only 6 days. The accelerated Caco-2 model has been compared to the traditional model (requiring 21-25 days of culture) in terms of tightness of the junctions, ability to rank chemical compounds for apparent permeability, active efflux and to discriminate P-gp substrates. METHODS AND RESULTS: In the new protocol, Caco-2 cells were cultured with the classical Caco-2 medium supplemented with puromycin. The initial cell seeding density was increased two times compared to the traditional procedure and the presence of a low concentration of puromycin in the culture medium reduced the Caco-2 permeability of mannitol. Bi-directional studies were performed with known P-gp substrates (rhodamine 123, digoxin and saquinavir) and with a total of 20 marketed drugs covering a wide range of physicochemical characteristics and therapeutic indications. Strong correlations were obtained between the apparent permeability in absorptive (Papp A→B) or secretory (Papp B→A) of the drugs in the accelerated model and in the traditional models and comparable efflux ratios were observed in the two studied models. DISCUSSION: The new protocol reduces costs for screening and leads to higher throughput compared to traditional Caco-2 cell models. This accelerated model provides short time-feedback to the drug design during the early stage of drug discovery.

Assessment of intestinal permeability using sugar probes: influence of urinary volume.

BACKGROUND: As the accuracy of the "Sugar Test" is currently debated, this study was conducted to focus on how urine volumes may impact the test results. METHODS: Fifty-five subjects, 23 healthy and 32 with Irritable Bowel Syndrome (IBS), were enrolled. Lactulose and D-mannitol dissolved in water were administered to all the participating subjects; the urine excreted was collected and the total urine volume was measured. The urine samples were analyzed by High Performance Liquid Chromatography (HPLC). The results were expressed as percentage of urine recovery of lactulose and D-mannitol and lactulose/D-mannitol ratio (LMR). RESULTS: All subjects were divided into two groups: subjects with urine volume < 500 mL and subjects with urine volume > or = 500 mL. Urine analysis showed that the mean LMR was significantly lower in subjects with urine volume > or = 500 mL than in subjects with urine volume < 500 mL (0.02 +/- 0.02 vs 0.04 +/- 0.04; p < 0.05). A significant increase in D-mannitol recovery was found to be associated with greater urine volumes (p < 0.001). CONCLUSIONS: The urine volume may influence urinary excretion of sugar probes. Intake of liquids should therefore be carefully monitored before and during the test and the volume of urine produced over the period of collection should be precisely measured.

Role of 99mTc-mannitol and 99mTc-PEG in the assessment of paracellular integrity of cell monolayers.

AIM: To assess the role of 99mTc-mannitol and 99mTc-polyethylene glycol 4000 in the evaluation of paracellular integrity of Caco-2 and Madine-Darby canine kidney (MDCK) cell monolayers, and confirm it in the presence of absorption promoters. METHODS: Radiolabelling of mannitol and polyethylene glycol was performed by a simple reduction method. Transepithelial electrical resistance values were measured to gain information regarding the integrity of tight junctions of Caco-2 and MDCK cell monolayers. Permeabilities of 99mTc-mannitol/99mTc-polyethylene glycol across cell monolayers were studied in the absence and presence of absorption promoters, namely dimethyl-beta-cyclodextrin, chitosan hydrochloride and sodium lauryl sulfate, and during recovery studies to assess paracellular integrity. RESULTS: Values for the apparent permeability coefficient (Papp) of Tc-mannitol were found to be 0.286 x 10 cm x s(-1) and 0.507 x 10 cm x s(-1) in Caco-2 and MDCK cell monolayers, respectively, whereas corresponding values for 99mTc-polyethylene glycol were 0.046 x 10 cm x s(-1) and 0.065 x 10 cm x s(-1). The insignificant Papp values of the marker molecules demonstrated the paracellular integrity of the cell monolayers. Significant increases in the Papp values in the presence of absorption promoters and their combinations due to opening of paracellular pathways and a return of Papp values to almost baseline values during recovery studies confirm the role of these marker molecules in the assessment of paracellular integrity of cell monolayers. CONCLUSION: 99mTc-labelled marker molecules can be attractive, useful and viable alternatives to the conventionally used markers in the assessment of paracellular integrity because of the absence of tissue-damaging corpuscular radiation and the ease of production of radiochemically pure and stable molecules at a reasonable cost.

Experimental assessment of chemotherapy-induced early intestinal damage in colon cancer the lactulose-mannitol permeability test.

AIMS AND BACKGROUND: Although chemotherapy plays an important role in the management and cure of cancer, it has undesiderable side effects mostly affecting the bone marrow and gastrointestinal tract, which greatly limit patient compliance and treatment efficacy. METHODS: The lactulose-mannitol test was used to assess intestinal mucosa damage 48 hours after the end of the first adjuvant chemotherapy cycle with 5-fluorouracil (5-FU) and levamisole in 12 patients with colon cancer. Fifteen age- and sex-matched subjects were studied as controls. The excreted amount of lactulose and mannitol was expressed as the percentage of the administered doses recovered in the urine as well as their ratio. RESULTS: The percent urinary recovery of lactulose was significantly (P < 0.001) higher in colon cancer patients (1.1 +/- 0.5%) than in the control group (0.3 +/- 0.03%), whereas the mannitol recovery was only slightly reduced in the former. As a result, the lactulose/mannitol excretion ratio was significantly (P < 0.001) higher in colon cancer patients (0.07 +/- 0.03) than in the control group (0.01 +/- 0.01). CONCLUSIONS: As assessed by the lactulose-mannitol test, the combined chemotherapy regimen with 5-FU and levamisole affects mainly the barrier function of the intestinal mucosa rather than its absorption capacity. The toxic effect seems to be attributable to the 5-FU molecule rather than to levamisole. The lactulose-mannitol test is a simple, safe and reliable tool to evaluate chemotherapy-induced early damage to the intestinal epithelium, in particular when new kinds of substances are being administered. Its use in clinical practice seems appropriate to establish the correct timing of drug administration, thereby enhancing treatment efficacy and improving patient compliance.

Assessment of hypolactasia and site-specific intestinal permeability by differential sugar absorption of raffinose, lactose, sucrose and mannitol.

The sugar absorption test is a non-invasive test for investigating intestinal permeability by simultaneous measurement of four probe sugars. In this study, we evaluated the utility of raffinose, lactose, sucrose and mannitol as probe sugars and calculated their urinary recovery as a percentage of ingested dose (mol/mol) and the recovery ratios of raffinose/mannitol, lactose/ raffinose and sucrose/raffinose. The reference ranges for these ratios, established from 39 healthy volunteers, are 0.005-0.015, 0.13-0.63 and 0.09-0.47, respectively. This sugar absorption test was performed in three patient groups. i) In 109 patients with aspecific gastrointestinal symptoms of whom intestinal histology was studied by duodenal biopsies: the urinary raffinose/mannitol recovery ratio highly correlated with gradation of duodenal damage; the sensitivity and specificity of the raffinose/mannitol ratio for detection of intestinal damage were 93% and 91%, respectively, using a cut-off level of 0.020. ii) In 70 patients in whom intestinal lactase activity was investigated by the lactose tolerance test: the urinary lactose/raffinose recovery ratio provided high diagnostic accuracy for hypolactasia (sensitivity 81% and specificity 89% at a cut-off level of 0.70). In analogy with the lactose/raffinose ratio, we suppose that the sucrose/raffinose ratio can be used as a marker of hyposucrasia. iii) In 40 patients with localized small intestinal damage, Crohn's disease of the ileum (n = 21) and celiac disease with histologically proven duodenal damage (n = 19): the raffinose/mannitol recovery ratio was increased in 100% of patients with celiac disease and in 81% of patients with Crohn's disease; increased lactose/raffinose recovery ratio (hypolactasia) and increased sucrose/raffinose (hyposucrasia) were present in 89% and 95% of celiac patients and 19% and 0% of Crohn's disease patients, respectively. The combination of the raffinose/mannitol ratio and sucrose/raffinose ratio appears to be an indication of the distribution of intestinal damage.

Estimation of organ transport function for recirculating indicator dilution curves.

The transport function of an indicator through an organ allows the calculation of important physiological parameters, but its estimation, especially in the presence of recirculation, can be difficult. In this paper, we estimate the transport function of 3H-mannitol (an extracellular tracer of glucose) in the human leg skeletal muscle. To do so, an indicator bolus is administered into the femoral artery and its recirculating dilution curves are nonuniformly sampled in both the femoral artery and the femoral vein. A new deconvolution-based method is used to simultaneously estimate the indicator transport function and the organ plasma flow. Subsequently, the indicator mean transit time and distribution volume are calculated. The reliability of the method is assessed by Monte Carlo simulation. The ability to estimate parameters, like mean transit time and extracellular distribution volume, is critical to the study of pathophysiologic states such as diabetes, insulin resistance, and hypertension.

Assessment of intestinal permeability changes induced by nonsteroidal anti-inflammatory drugs in the rat.

Intestinal permeability was investigated as an alternative to intestinal ulceration for measuring nonsteroidal anti-inflammatory drug (NSAID) gut damage in the rat and developed as a method for routine measurement. NSAID dose-response curves produced using the two indices of damage showed that intestinal permeability is as sensitive and reproducible as ulceration, although changes could not be detected before visible ulceration occurred. Lactulose, [51Cr]-EDTA and [14C]-carboxyinulin were compared as possible in vivo markers of rat intestinal permeability. Measurement of [51Cr]-EDTA permeation was found to be the most sensitive and reproducible method. Dose-response curves produced by measuring [51Cr]-EDTA permeation were used to compare the potency of the two NSAIDs piroxicam and (S+) ibuprofen; piroxicam was found to be 10 times more potent in increasing intestinal permeability than (S+)-ibuprofen. These studies show that intestinal permeability measurement is a useful alternative to other methods of assessing NSAID adverse effect and is easily and rapidly performed.

Analysis of multiple sugar probes in urine and plasma by high-performance anion-exchange chromatography with pulsed electrochemical detection. Application in the assessment of intestinal permeability in human immunodeficiency virus infection.

Mannitol, 3-O-methylglucose and lactulose administered orally are used to investigate small intestinal absorption pathways and mucosal integrity. Current methods of analysis include thin-layer chromatography, gas chromatography (GC) and enzymatic analysis, which require separate estimation of mono- and disaccharides and for GC, prior derivatization. We describe a high-pressure anion-exchange chromatographic method coupled with pulsed electrochemical detection allowing simultaneous measurement of all three sugars and its clinical application in monitoring intestinal damage in human immunodeficiency virus (HIV) infection. Sample preparation is simple and fast. All sugars are resolved within 10 min. Mean recovery is 93.3% for all sugars and the overall relative standard deviation is 4.2%. Intestinal permeability (lactulose/mannitol ratio) rises with disease progression to AIDS, indicating mucosal damage. The greatest increase in permeability is associated with chronic diarrhoea. The method is an ideal non-invasive test to assess gut mucosal damage in HIV infection.

Differential sugar absorption for the assessment of canine intestinal permeability: the cellobiose/mannitol test in gluten-sensitive enteropathy of Irish setters.

Intestinal permeability was assessed in Irish setters with gluten-sensitive enteropathy by oral administration of an isotonic solution of cellobiose and mannitol, and measurement of their urinary excretion ratio. The cellobiose/mannitol ratio was increased in affected Irish setters fed a wheat-containing diet compared both with littermates reared on a cereal-free diet, with no evidence of jejunal damage, and clinically healthy Irish setters. The ratio fell following six weeks on a gluten-free diet to be comparable with control values, and subsequently increased after six weeks gluten challenge. The results indicate that the cellobiose/mannitol test may be useful for the detection of mucosal damage and for monitoring the response to therapy.