Measuring intestinal fluid transport in vitro: Gravimetric method versus non-absorbable marker
Comparative Biochemistry and Physiology Part A: Molecular & Integrative Physiology
Reason for embargo
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.
We are grateful to Ian and Tony McClure, the local fishermen of Flookburgh, Cumbria (U.K.) for collecting the flounder used in this study, and to Jan Shears for assistance with fish husbandry at Exeter (U.K.). We thank Ray Hurley and Debbie Fretz in Miami (U.S.A.) for supplying the toadfish. This work was supported by the Biotechnology and Biological Sciences Research Council (BBSRC) grants BBS/S/A/2004/11078 and BB/F009364/1 to R.W.W., and National Science Foundation (NSF) grants IAB0743903 and 1146695 to M.G.
This is the author accepted manuscript. The final version is available from Elsevier via the DOI in this record.
Vol. 194, pp. 27 - 36