Adiponectin is a recently found anti-atherogenic plasma protein secreted by adipocytes. Plasma adiponectin level is reduced in patients with coronary artery disease [1], type 2 diabetes mellitus [1] and obesity [2]. In contrast to these high-risk groups, plasma adiponectin has been reported to be elevated in haemodialysis patients [3]. Since uraemic plasma is known to contain not only intact forms but also fragments of some peptide hormones such as parathyroid hormone, it is an important question whether adiponectin in uraemic plasma is intact or not. To answer the question, we analysed its molecular forms.
Plasma samples were taken from two patients (patients A and B) on maintenance haemodialysis and a healthy volunteer; both 42-year-old men without diabetes, obesity or coronary artery disease. Fresh plasma was fractioned by gel filtration using a 10 x 300 mm column of Superose 6 HR (Amarsham Biosciences, Tokyo) and 31 mM TrisHCl buffer (pH 7.2). An aliquot of each 0.5 ml fraction was assayed for adiponectin by ELISA [2]. Subsequently, another aliquot of the fractioned plasma was subjected to SDSpolyacrylamide gel electrophoresis (PAGE) in reducing condition. Western blotting was done using anti-adiponectin monoclonal antibody ANOC 4908 (a generous gift from Dr Funahashi, Osaka University Graduate School of Medicine) as the first antibody and peroxidase-labelled rabbit anti-mouse IgG polyclonal antibody as the second antibody. Plasma adiponecitin levels of the haemodialysis patients and the healthy subject were 25.1, 9.1 and 5.1 µg/ml, respectively. Reference range of adiponectin was 5.5±2.0 µg/ml (mean±SD, n = 51) for healthy men in our laboratory.
Upon gel filtration chromatography of plasma from the healthy subject, immunoreactive adiponectin migrated as macromolecules larger than IgG (150 kDa) showing three peaks. A similar gel filtration pattern was found for the uraemic plasma samples. No adiponectin immunoreactivity was detected in fractions corresponding to monomeric adiponectin or smaller fragments.
Western blotting of the fractioned healthy plasma (Figure 1) showed one major band at 30 kDa and another faint band at 80 kDa in reducing condition. In contrast, the 80 kDa band predominated in non-reducing condition. The extra band noticed at 28 kDa was non-specific staining for IgG light chain due to the known cross-reactivity of the second antibody (labelled polyclonal rabbit anti-IgG antibody) with human IgG light chain [2]. No other bands corresponding to adiponectin fragments were detected. These results agreed well with a previous report [2], and fit the proposed structure of adiponectin in the circulation. Adiponectin monomers form homo-trimers by disulfide bond, which further assemble as larger complex forms. The immunoblotting patterns of uraemic plasma samples were not different from that of the normal plasma, showing no adiponectin fragments.
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Conflict of interest statement. None declared.
Department of Metabolism, Endocrinology and Molecular Medicine Osaka City University Graduate School of Medicine 1-4-3, Asahi-machi Abeno-ku Osaka 545-8585 Japan Email: t-shoji{at}med.osaka-cu.ac.jp
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