A comparison of analytic procedures for measurement of fractional dextran clearances

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Abstract

Fractional dextran clearances have been extensively used to study glomerular size selectivity. We report on an analysis of different laboratory procedures involved in measuring fractional dextran clearances. The deproteinization of plasma samples by 20% trichloroacetic acid (TCA) revealed a protein contamination of 0.2% ± 0.3%, whereas both 5% TCA and zinc sulfate deproteinization revealed a significantly higher remaining sample protein content (2.5% ± 0.4% and 3.4% ± 0.1%, respectively). Only zinc sulfate revealed incomplete deproteinization of urine samples (0.6% ± 0.2%). Dextran recovery in plasma and urine supernatants was significantly lower after 5% TCA and zinc sulfate deproteinization 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 differences 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 dextran 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 clearances 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 HPLC and anthrone assay revealed higher clearances in comparison with HPLC and online refractometry in healthy subjects (3.6 to 5.4 nm) and in patients (3.6 to 5.2 nm). The GPC and anthrone assay revealed characteristic differences in fractional dextran clearances between healthy subjects and patients. The HPLC and anthrone assay showed no significant differences between both groups, whereas HPLC and online refractometry 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 all 3 dextran assays were not significantly related to the fractional immunoglobulin G clearance or the immunoglobulin-to-albumin clearance index in our patients. Quantitative and qualitative differences in fractional dextran clearances may be induced by differences in laboratory procedures. We recommend sample preparation by 20% TCA deproteinization, frequent calibration with 1 set of dextran standards with low polydispersity, size-exclusion chromatography by GPC, and dextran detection by anthrone assay for optimal measurement of fractional dextran clearances. Even with such an approach, however, the variability in the measurement remains extremely high in the important range of dextrans greater than 5 nm.

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