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Rapid small intestinal permeability assay based on riboflavin and lactulose detected by bis-boronic acid appended benzyl viologens
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Gastroenterology/Hepatology.
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Gastroenterology/Hepatology.
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Gastroenterology/Hepatology.
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2015 (English)In: Clinica Chimica Acta, ISSN 0009-8981, E-ISSN 1873-3492, Vol. 439, 115-121 p.Article in journal (Refereed) Published
Abstract [en]

BACKGROUND: Although organoboronic acids are efficient high-throughput sugar sensors, they have not been pursued for gut permeability studies. A modification of the lactulose/mannitol assay is described by which small intestinal permeability is assessed at the time of urine collection using a lactulose/riboflavin ratio.

METHODS: Volunteers ingested 50mg riboflavin and either 5g mannitol or 10g lactulose. Urine was collected for 6hrs. Riboflavin was assayed by autofluorescence. Riboflavin was removed by C18 solid phase extraction. Lactulose and mannitol were then assayed using 1,1'-bis(2-boronobenzyl)-4,4'-bipyridinium (4,4'oBBV) coupled to the fluorophore HPTS.

RESULTS: The temporal profile over 6hrs for riboflavin paralleled mannitol. Riboflavin recovery in urine was 11.1±1.9 % (mean±SEM, n=7), similar to mannitol. There was selective binding of 4,4'oBBV to lactulose, likely involving cooperativity between the fructose and galactose moieties. Lower limits of detection and quantification were 90 and 364μM. The lactulose assay was insensitive to other permeability probes (e.g., sucrose, sucralose) while tolerating glucose or lactose. This assay can be adapted to automated systems. Stability of 4,4'oBBV exceeds 4years.

CONCLUSIONS: Riboflavin measured by autofluorescence combined with lactulose measured with 4,4'oBBV represents a useful new chemistry for rapid measurement of intestinal permeability with excellent stability, cost and throughput benefits.

Place, publisher, year, edition, pages
2015. Vol. 439, 115-121 p.
Keyword [en]
Intestinal permeability, Organoborane, Gastroenterology, Lactulose, Mannitol, Riboflavin
National Category
Gastroenterology and Hepatology
URN: urn:nbn:se:uu:diva-236349DOI: 10.1016/j.cca.2014.09.031ISI: 000347499700021PubMedID: 25300228OAI: oai:DiVA.org:uu-236349DiVA: diva2:764124

De två första författarna delar första författarskapet.

Available from: 2014-11-18 Created: 2014-11-18 Last updated: 2016-05-25Bibliographically approved
In thesis
1. Gut peptides in gastrointestinal motility and mucosal permeability
Open this publication in new window or tab >>Gut peptides in gastrointestinal motility and mucosal permeability
2016 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Gut regulatory peptides, such as neuropeptides and incretins, play important roles in hunger, satiety and gastrointestinal motility, and possibly mucosal permeability. Many peptides secreted by myenteric nerves that regulate motor control are also produced in mucosal epithelial cells. Derangements in motility and mucosal permeability occur in many diseases. Current knowledge is fragmentary regarding gut peptide actions and mechanisms in motility and permeability.

This thesis aimed to 1) develop probes and methods for gut permeability testing, 2) elucidate the role of neuropeptide S (NPS) in motility and permeability, 3) characterize nitrergic muscle relaxation and 4) characterize mechanisms of glucagon-like peptide 1 (GLP-1) and the drug ROSE-010 (GLP-1 analog) in motility inhibition.

A rapid fluorescent permeability test was developed using riboflavin as a transcellular transport probe and the bisboronic acid 4,4'oBBV coupled to the fluorophore HPTS as a sensor for lactulose, a paracellular permeability probe. This yielded a lactulose:riboflavin ratio test.

NPS induced muscle relaxation and increased permeability through NO-dependent mechanisms. Organ bath studies revealed that NPS induced NO-dependent muscle relaxation that was tetrodotoxin (TTX) sensitive. In addition to the epithelium, NPS and its receptor NPSR1 localized at myenteric nerves. Circulating NPS was too low to activate NPSR1, indicating NPS uses local autocrine/paracrine mechanisms.

Nitrergic signaling inhibition by nitric oxide synthase inhibitor L-NMMA elicited premature duodenojejunal phase III contractions in migrating motility complex (MMC) in humans. L-NMMA shortened MMC cycle length, suppressed phase I and shifted motility towards phase II. Pre-treatment with atropine extended phase II, while ondansetron had no effect. Intestinal contractions were stimulated by L-NMMA, but not TTX. NOS immunoreactivity was detected in the myenteric plexus but not smooth muscle.

Food-intake increased motility of human antrum, duodenum and jejunum. GLP-1 and ROSE-010 relaxed bethanechol-induced contractions in muscle strips. Relaxation was blocked by GLP-1 receptor antagonist exendin(9-39) amide, L-NMMA, adenylate cyclase inhibitor 2´5´-dideoxyadenosine or TTX. GLP-1R and GLP-2R were expressed in myenteric neurons, but not muscle.

In conclusion, rapid chemistries for permeability were developed while physiological mechanisms of NPS, nitrergic and GLP-1 and ROSE-010 signaling were revealed. In the case of NPS, a tight synchrony between motility and permeability was found.


Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2016. 58 p.
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine, ISSN 1651-6206 ; 1233
Gut regulatory peptides, Neuropeptides, Gastrointestinal mucosal permeability, Gastrointestinal motility, GLP-1, NPS, ROSE-010
National Category
Medical and Health Sciences
urn:nbn:se:uu:diva-294390 (URN)978-91-554-9607-4 (ISBN)
Public defence
2016-06-14, Enghoffsalen, Entrance 50, Uppsala University Hospital, Uppsala, 09:00 (English)
Available from: 2016-05-24 Created: 2016-05-19 Last updated: 2016-06-15

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