title: Measuring intestinal fluid transport in vitro: Gravimetric method versus non-absorbable marker.
abstract: The gut sac is a long-standing, widely used in vitro preparation for studying solute and water transport, and calculation of these fluxes requires an accurate assessment of volume. This is commonly determined gravimetrically by measuring the change in mass over time. While convenient this likely under-estimates actual net water flux (Jv) due to tissue edema. We evaluated whether the popular in vivo volume marker [(14)C]-PEG 4000, offers a more representative measure of Jvin vitro. We directly compared these two methods in five teleost species (toadfish, flounder, rainbow trout, killifish and tilapia). Net fluid absorption by the toadfish intestine based on PEG was significantly higher, by almost 4-fold, compared to gravimetric measurements, compatible with the latter under-estimating Jv. Despite this, PEG proved inconsistent for all of the other species frequently resulting in calculation of net secretion, in contrast to absorption seen gravimetrically. Such poor parallelism could not be explained by the absorption of [(14)C]-PEG (typically <1%). We identified a number of factors impacting the effectiveness of PEG. One was adsorption to the surface of sample tubes. While it was possible to circumvent this using unlabelled PEG 4000, this had a deleterious effect on PEG-based Jv. We also found sequestration of PEG within the intestinal mucus. In conclusion, the short-comings associated with the accurate representation of Jv by gut sac preparations are not overcome by [(14)C]-PEG. The gravimetric method therefore remains the most reliable measure of Jv and we urge caution in the use of PEG as a volume marker.
authors.affiliation: Biosciences, College of Life and Environmental Sciences, University of Exeter, Exeter EX4 4QD, UK. Electronic address: jwhittamore@pathology.ufl.edu.
authors.forename: Jonathan M
authors.initials: JM
authors.lastname: Whittamore
authors.affiliation: Rosenstiel School of Marine and Atmospheric Science, University of Miami, Miami, FL 33419, USA; Department of Biology, University of West Georgia, Carrollton, GA 30118, USA.
authors.forename: Janet
authors.initials: J
authors.lastname: Genz
authors.affiliation: Rosenstiel School of Marine and Atmospheric Science, University of Miami, Miami, FL 33419, USA.
authors.forename: Martin
authors.initials: M
authors.lastname: Grosell
authors.affiliation: Biosciences, College of Life and Environmental Sciences, University of Exeter, Exeter EX4 4QD, UK.
authors.forename: Rod W
authors.initials: RW
authors.lastname: Wilson
mesh_terms: D000818:Animals; D001826:Body Fluids / Q000378:metabolism*; D005399:Fishes; D066298:In Vitro Techniques; D007413:Intestinal Mucosa / Q000378:metabolism*; D011092:Polyethylene Glycols / Q000378:metabolism
chemical_list: D011092:Polyethylene Glycols
keywords: Gut sac; PEG 4000; Polyethylene glycol; Teleost; Water