| pA2 online © Copyright 2004 The British Pharmacological Society |
131P
GKT, University of London Winter Meeting December 2003 |
|
Comparison
of three different microdialysis membranes for the recovery of [125I]substance
P in vitro |
|
Print abstractThe undecapeptide substance P (SP) is widely accepted as an important modulator in the central nervous system, for example in the spinal cord dorsal horn (Snijdelaar et al., 2000). While SP has been the subject of much research, in common with other high molecular weight (MW) substances (Kendrick, 1990), microdialysis studies of SP release are hampered by low diffusion across commonly employed dialysis membranes. The present series of experiments were to determine the characteristics of three 'high flux' dialysis membranes with a higher MW cut-off on the in vitro recovery of 125I-SP employing a push-pull perfusion system.
Microdialysis probes of concentric design were equipped with a 1.5 mm length of either a cuprammonium rayon (CUP-R) membrane (CL-15SE, MW cut-off 60 kDa; Terumo, Italy), a polyamide (POLY) membrane (Polyflux®, MW cut-off 30 kDa; Gambro-Hospal, UK), or a Hemophane® (HEM) membrane (MW cut-off 20 kDa; Gambro-Hospal, UK), and their recovery of [125I]SP (Bachem, UK) was compared. Membrane fibre outer diameter dimensions were 0.226mm, 0.270mm and 0.208mm respectively. Probes were perfused using a CMA 100 syringe pump with a push-pull adaptor (CMA/Microdialysis, Sweden) at flow rates of 1, 2, 3 and 5 µl.min-1 with an artificial extracellular fluid solution (aECF). Its composition was (mM): NaCl, 138; KCl, 3; NaH2PO4, 1; MgCl2, 0.9; NaHCO3, 11; CaCl2, 1.2 (pH 7.4), and contained 0.2% bovine serum albumin and 0.03% bacitracin (Sigma, UK), The probe membranes were immersed in an Eppendorf containing 1.5 ml aECF and [125I]SP (average counts per minute (CPM) was 20,000-23,000 CPM/100µl). A volume equivalent to twice the dead volume of the probe and associated tubing were collected, followed by two 20 min samples. The radioactive [125I]SP content of the dialysate samples and an equivalent volume of the external medium were then determined using a gamma counter, and the relative recovery calculated (see Table 1).
| Membrane |
Flow
rate µl/min %
|
Relative
Recovery
|
CUP-R
|
1
|
11.6
± 1.7
|
|
2
|
6.9
± 0.8
|
|
|
3
|
5.3
± 0.8
|
|
|
5
|
3.5
± 0.2
|
|
|
POLY |
1
|
9.1
± 0.4
|
|
2
|
6.9
± 0.5
|
|
|
3
|
4.3
± 0.2
|
|
|
5
|
2.9
± 0.5
|
|
|
HEM |
1
|
5.6
± 0.7
|
|
2
|
4.3
± 0.8
|
|
|
3
|
2.8
± 0.6
|
|
|
5
|
2.0
± 0.3
|
Table 1. The relative recovery of [125I]SP for microdialysis membranes at varying perfusate flow rates.Data are presented as mean ± s.e. mean, n= 4.
The recovery of SP for all the membranes characterised here is greater than previous reports (Kendrick, 1990). The rank order of recovery was in line with the nominal MW cut-off of the membranes employed. Perfusate ultrafiltration was not seen, demonstrating push-pull perfusion as a successful strategy to negate perfusate ultrafiltration common to 'high flux' dialysis membranes. The CUP-R membrane recorded the highest recovery of [125I]SP, and together with a finer outer diameter should make it ideal for recovery of high molecular weight substances in discrete areas of the CNS.
Snijdelaar D.G. et
al., (2000) Eur J. Pain 4, 121-135.
Kendrick K.M. (1990) J. Neurosci. Methods 34, 35-46.