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Rapid small intestinal permeability assay based on riboflavin and lactulose detected by bis-boronic acid appended benzyl viologens.

Clinica chimica acta; international journal of clinical chemistry (2014-10-11)
Angel Resendez, Md Abdul Halim, Caroline M Landhage, Per M Hellström, Bakthan Singaram, Dominic-Luc Webb
ABSTRACT

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. 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. 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. 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.

MATERIALS
Product Number
Brand
Product Description

USP
Riboflavin, United States Pharmacopeia (USP) Reference Standard
Riboflavin, European Pharmacopoeia (EP) Reference Standard
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D-Mannitol, meets EP, FCC, USP testing specifications
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D-Mannitol, BioXtra, ≥98% (HPLC)
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Sodium phosphate, 96%
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Lactulose, European Pharmacopoeia (EP) Reference Standard
USP
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Supelco
D-Mannitol, ≥99.9999% (metals basis), for boron determination
Mannitol, European Pharmacopoeia (EP) Reference Standard
USP
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Supelco
Lactulose, Pharmaceutical Secondary Standard; Certified Reference Material