title: Rapid small intestinal permeability assay based on riboflavin and lactulose detected by bis-boronic acid appended benzyl viologens.
abstract: 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 5 g mannitol or 10 g lactulose. Urine was collected for 6 hrs. 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 6 hrs 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 364 μM. 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 4 years.
  
  
  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.
authors.affiliation: Department of Chemistry and Biochemistry, University of California at Santa Cruz, Santa Cruz, CA 95064, United States.
authors.forename: Angel
authors.initials: A
authors.lastname: Resendez
authors.affiliation: Department of Medical Sciences, Gastroenterology and Hepatology Unit, Uppsala University, 751 85, Uppsala, Sweden.
authors.forename: Md
authors.initials: M
authors.lastname: Abdul Halim
authors.affiliation: Department of Medical Sciences, Gastroenterology and Hepatology Unit, Uppsala University, 751 85, Uppsala, Sweden.
authors.forename: Caroline M
authors.initials: CM
authors.lastname: Landhage
authors.affiliation: Department of Medical Sciences, Gastroenterology and Hepatology Unit, Uppsala University, 751 85, Uppsala, Sweden.
authors.forename: Per M
authors.initials: PM
authors.lastname: Hellström
authors.affiliation: Department of Chemistry and Biochemistry, University of California at Santa Cruz, Santa Cruz, CA 95064, United States.
authors.forename: Bakthan
authors.initials: B
authors.lastname: Singaram
authors.affiliation: Department of Chemistry and Biochemistry, University of California at Santa Cruz, Santa Cruz, CA 95064, United States; Department of Medical Sciences, Gastroenterology and Hepatology Unit, Uppsala University, 751 85, Uppsala, Sweden. Electronic address: dlw_insulin@hotmail.com.
authors.forename: Dominic-Luc
authors.initials: DL
authors.lastname: Webb
mesh_terms: D000328:Adult; D001594:Benzyl Viologen / Q000737:chemistry*; D001897:Boronic Acids / Q000737:chemistry*; D005260:Female; D006801:Humans; D007421:Intestine, Small / Q000378:metabolism*; D007792:Lactulose / Q000652:urine*; D008297:Male; D008875:Middle Aged; D010539:Permeability; D012256:Riboflavin / Q000652:urine*; D055815:Young Adult
chemical_list: D001897:Boronic Acids; D001594:Benzyl Viologen; D007792:Lactulose; D012256:Riboflavin
keywords: Gastroenterology; Intestinal permeability; Lactulose; Mannitol; Organoborane; Riboflavin