TY - JOUR
T1 - Measuring intestinal fluid transport in vitro
T2 - Gravimetric method versus non-absorbable marker
AU - Whittamore, Jonathan M.
AU - Genz, Janet
AU - Grosell, Martin
AU - Wilson, Rod W.
N1 - Funding Information:
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.
Publisher Copyright:
© 2016 Elsevier Inc.
PY - 2016/4/1
Y1 - 2016/4/1
N2 - 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 [14C]-PEG 4000, offers a more representative measure of Jv in 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 [14C]-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 [14C]-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.
AB - 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 [14C]-PEG 4000, offers a more representative measure of Jv in 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 [14C]-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 [14C]-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.
KW - Gut sac
KW - PEG 4000
KW - Polyethylene glycol
KW - Teleost
KW - Water
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U2 - 10.1016/j.cbpa.2016.01.004
DO - 10.1016/j.cbpa.2016.01.004
M3 - Article
C2 - 26794612
AN - SCOPUS:84955586643
SN - 1095-6433
VL - 194
SP - 27
EP - 36
JO - Comparative Biochemistry and Physiology - A Physiology
JF - Comparative Biochemistry and Physiology - A Physiology
ER -