A COMPARISON OF ANALYTIC PROCEDURES FOR MEASUREMENT OF FRACTIONAL DEXTRAN CLEARANCES

Fractional dextran clearances have been extensively used to study glom erular size selectivity. We report on an analysis of different laborat ory procedures involved in measuring fractional dextran clearances. Th e deproteinization of plasma samples by 20% trichloroacetic acid (TCA) revealed a protein contamination of 0.2% +/- 0.3%, whereas both 5% TC A and zinc sulfate deproteinization revealed a significantly higher re maining sample protein content (2.5% +/- 0.4% and 3.4% +/- 0.1%, respe ctively). Only zinc sulfate revealed incomplete deproteinization of ur ine samples (0.6% +/- 0.2%). Dextran recovery in plasma and urine supe rnatants was significantly lower after 5% TCA and zinc sulfate deprote inization when compared with 20% TCA deproteinization. Gel permeation chromatography (GPC) and high-performance liquid chromatography (HPLC) showed a variance of calibration smaller than 5% over 1 year. The use of 3 different sets of standard dextrans revealed significant differe nces in calibration. GPC and HPLC followed by anthrone assay showed a comparable variance in dextran concentration in plasma, from 3 to 6 nm (14% to 25%), whereas the variance in urine was lower for the GPC and anthrone assay, especially from 5.4 to 6 nm (23% to 43% versus 50% to 78%). HPLC and online refractometry showed the lowest variance of dex tran concentration in plasma, from 3 to 6 nm (<4%), and in urine, from 3 to 5.2 nm (<7%), whereas it showed a higher variance in urine, from 5.4 to 6 nm, in comparison with GPC and HPLC with the anthrone assay. The GPC and anthrone assay revealed higher fractional dextran clearan ces in comparison with the HPLC and anthrone assay in healthy subjects (3 to 5.4 nm) as well as in patients with nondiabetic proteinuria (4. 2 to 5.8 nm), and lower clearances in patients from 3 to 3.4 nm. The H PLC and anthrone assay revealed higher clearances in comparison with H PLC and online refractometry in healthy subjects (3.6 to 5.4 nm) and i n patients (3.6 to 5.2 nm). The GPC and anthrone assay revealed charac teristic differences in fractional dextran clearances between healthy subjects and patients. The HPLC and anthrone assay showed no significa nt differences between both groups, whereas HPLC and online refractome try showed only an increased clearance of dextrans from 4.6 to 5.2 nm in patients. Fractional clearances of dextran 5.6 nm as estimated by a ll 3 dextran assays were not significantly related to the fractional i mmunoglobulin G clearance or the immunoglobulin-to-albumin clearance i ndex in our patients. Quantitative and qualitative differences in frac tional dextran clearances may be induced by differences in laboratory procedures. We recommend sample preparation by 20% TCA deproteinizatio n, frequent calibration with 1 set of dextran standards with low polyd ispersity, size-exclusion chromatography by GPC, and dextran detection by anthrone assay for optimal measurement of fractional dextran clear ances. Even with such an approach, however, the variability in the mea surement remains extremely high in the important range of dextrans gre ater than 5 nm.