Fast sampling, rapid filtration apparatus: Principal characteristics and validation from studies ofd-glucose transport in human jejunal brush-border membrane vesicles

Abstract

Kinetic data in (brush-border) membrane vesicles which rely on the validity of the initial rate assumption for their interpretation and depend on tracer flux studies using the rapid filtration technique for their experimental measurement have been limited to some extent by the absence of techniques that would allow for real-time data analysis. In this paper, we report on our successful design of a fast sampling, rapid filtration apparatus (FSRFA) which seems to fill up this technical gap since showing the following characteristics: (i) rapid injection (5 msec) and mixing (less than 100 msec) of small amounts of vesicles (10-40 microliters) with an incubation medium (0.2-1.0 ml); (ii) fast (20 to 80 msec depending on the sample volume) and multiple (up to 18 samples at a maximal rate of 4 sec) sampling of the uptake mixture followed by rapid quenching in the stop solution (approximately 5 msec) according to a predetermined time schedule (any time combination from 0.25 to 9999 sec); and (iii) fast, automated, and sampling-synchronized filtration and washings of the quenched uptake medium (only 15-20 sec are necessary for the first filtration followed by two washings and extra filtrations). As demonstrated using adult human jejunal brush-border membrane vesicles and Na(+)-D-glucose cotransport as models, the FSRFA accurately reproduces the manual aspects of the rapid filtration technique while allowing for very precise initial rate determinations. Moreover, the FSRFA has also been designed to provide as much versatility as possible and, in its present version, allows for a very precise control of the incubation temperature and also permits a few efflux protocols to be performed. Finally, its modular design, which separates the fast sampling unit from the rapid filtration device, should help in extending its use to fields other than transport measurement.