Technical Note Validation of an adiponectin immunoassay in human skeletal muscle biopsies An M. Van Berendoncks a,b,c, ⁎, Viviane M. Conraads a,b,c , Wendy Van Leuven d , Viviane Van Hoof e , Sylvia De Wilde d , Christiaan J. Vrints a,b,c , Vicky Y. Hoymans a,b,c a Department of Cardiology, Antwerp University Hospital, Edegem, Belgium b Laboratory for Cellular and Molecular Cardiology, Antwerp University Hospital, Edegem, Belgium c University of Antwerp, Antwerp, Belgium d Department of Biomedical Sciences, University of Antwerp, Antwerp, Belgium e Department of Clinical Chemistry, Antwerp University Hospital, Edegem, Belgium article info abstract Article history: Received 14 July 2010 Received in revised form 23 August 2010 Accepted 1 September 2010 Available online 15 September 2010 Adiponectin is an insulin-sensitizing adipocytokine that circulates in plasma as multimeric isoforms, including trimers, hexamers and high molecular weight complexes. Although adiponectin multimers have previously been measured by Western blot, this remains a relatively delicate technique that is often hampered by high background or multiple bands. We validated a commercially available ELISA, designed to measure total (low, middle and high molecular weight) human adiponectin concentration in cell culture supernatants, serum and plasma, for its proper use in skeletal muscle biopsies obtained in patients with chronic heart failure and healthy controls. © 2010 Elsevier B.V. All rights reserved. Keywords: Adiponectin Immunoassay Skeletal muscle 1. Introduction Recently, adiponectin has raised interest as a potential player in the pathophysiology of several chronic diseases. Adiponectin is a main regulator of glucose and lipid metabolism at the level of the skeletal muscle and in the liver, and mediates anti-diabetic, anti-inflammatory and anti- atherosclerotic effects. In plasma, the adipocytokine circu- lates in several multimeric forms involving trimer, hexamer and high molecular weight adiponectin. These different forms have been advocated to act through distinct signaling path- ways, and to have different biological functions (Tsao et al., 2003). Adiponectin is secreted by adipocytes, but also by other cells like cardiomyocytes and skeletal muscle myocytes (Delaigle et al., 2004; Krause et al., 2008; Skurk et al., 2008). We have previously described the existence of functional adiponectin resistance at the level of the skeletal muscle in patients with chronic heart failure (CHF) (Van Berendoncks et al., 2010a). Particularly, and in parallel to increased total adiponectin concentrations in plasma, adipo- nectin mRNA and protein expression were significantly raised in skeletal muscle biopsies of these patients compared to healthy controls (Van Berendoncks et al., 2010a, 2010b). Disturbances in skeletal muscle metabolism were supported by deactivation of the peroxisome proliferator-activated receptor alpha (PPAR-alpha)/AMP-activated protein kinase (AMPK) pathway and downregulation of several target genes involved in both fatty acid oxidation and glucose metabolism. Quantification of total adiponectin protein concentration is mainly performed by semi-quantitative Western blot under several (non)reducing and (non)heating denaturing condi- tions. The difficulties and the impact of reduction and denaturation on adiponectin multimer formation have been extensively discussed by Waki et al. (2003). Indeed, Western blot remains a relatively troublesome and time-consuming approach that is often hampered by high background and/or multiple bands. Compared to Western blot, enzyme linked immunosorbent assay (ELISA) is a more sensitive, easy to use, and precise technology. Notably, the ELISA methodology has been optimized to measure protein concentrations from as Journal of Immunological Methods 362 (2010) 209–212 ⁎ Corresponding author. Department of Cardiology, Antwerp University Hospital, Wilrijkstraat 10, 2650 Edegem, Belgium. Tel.: + 32 3 821 40 12; fax: +32 3 821 39 74. E-mail address: An.VanBerendoncks@ua.ac.be (A. M. Van Berendoncks). 0022-1759/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.jim.2010.09.012 Contents lists available at ScienceDirect Journal of Immunological Methods journal homepage: www.elsevier.com/locate/jim