Effects of storage and homogenization methods on the hepatic recovery of dextrans determined by size-exclusion chromatography.
The effects of storage and homogenization methods on the analytical recovery of dextran macromolecules from rat livers were investigated using a high-performance size-exclusion chromatographic (HPSEC) method. Livers were collected from rats dosed with fluorescein-labeled dextrans with molecular weights of 150 or 70 kD. Subsequently, the livers were subjected to different methods to study the effects of the following parameters on the hepatic recovery of dextrans: storage method (freezing the livers before homogenization or freezing the homogenates); contents of the homogenization buffer (addition of 1% Triton X-100); and sample type (HPSEC analysis of the whole homogenate or the supernatant after centrifugation). It is shown that in the absence of Triton in the homogenization buffer, the hepatic recovery of dextrans is substantially affected by all the factors studied. However, in the presence of 1% Triton in the buffer, the hepatic recoveries were maximal and independent of the storage method or sample type. These studies suggest that for optimal recovery of dextran macromolecules from the liver, a sample preparation method capable of disrupting the subcellular membranes should be used.
Mehvar
R
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College of Pharmacy and Health Sciences, Drake University, Des Moines, IA 50311 USA.
D000704:Analysis of Variance; D000818:Animals; D002021:Buffers; D002850:Chromatography, Gel; D002851:Chromatography, High Pressure Liquid; D003911:Dextrans / Q000378:metabolism*; D004356:Drug Storage / Q000592:standards*; D005456:Fluorescent Dyes; D008099:Liver / Q000378:metabolism*; D008297:Male; D008970:Molecular Weight; D017830:Octoxynol / Q000737:chemistry; D051381:Rats; D017207:Rats, Sprague-Dawley
D002021:Buffers; D003911:Dextrans; D005456:Fluorescent Dyes; D017830:Octoxynol