Abstract
BackgroundAdiponectin is an adipocyte-derived hormone that acts as a marker of insulin sensitivity. Bloodspot sampling by fingerstick onto filter paper may increase the feasibility of large-scale studies of the determinants of insulin sensitivity. We first describe the validation of an enzyme-linked immunoassay (ELISA) for quantifying adiponectin from dried blood spots and then demonstrate its application in a large trial (PROBIT).MethodsWe quantified adiponectin from 3-mm diameter discs (≈3 µL of blood) punched from dried blood spots obtained from: i) whole blood standards (validation); and ii) PROBIT trial samples (application) in which paediatricians collected blood spots from 13,879 children aged 11.5 years from 31 sites across Belarus. We examined the distribution of bloodspot adiponectin by demographic and anthropometric factors, fasting insulin and glucose.ResultsIn the validation study, mean intra-assay coefficients of variation (n = 162) were 15%, 13% and 10% for ‘low’ (6.78 µg/ml), ‘medium’ (18.18 µg/ml) and 'high’ (33.13 µg/ml) internal quality control (IQC) samples, respectively; the respective inter-assay values (n = 40) were 23%, 21% and 14%. The correlation coefficient between 50 paired whole bloodspot versus plasma samples, collected simultaneously, was 0.87 (95% CI: 0.78 to 0.93). Recovery of known quantities of adiponectin (between 4.5 to 36 µg/ml) was 100.3–133%. Bloodspot adiponectin was stable for at least 30 months at −80°C. In PROBIT, we successfully quantified fasting adiponectin from dried blood spots in 13,329 of 13,879 (96%) children. Mean adiponectin (standard deviation) concentrations were 17.34 µg/ml (7.54) in boys and 18.41 µg/ml (7.92) in girls and were inversely associated with body mass index, fat mass, triceps and subscapular skin-fold thickness, waist circumference, height and fasting glucose.ConclusionsBloodspot ELISA is suitable for measuring adiponectin in very small volumes of blood collected on filter paper and can be applied to large-scale studies.
Highlights
We first describe the validation of an enzyme-linked immunoassay (ELISA) for quantification of adiponectin from dried blood spots, which offers the important advantage that results can be read on universally available microtitre plate readers, without the need for regulatory approvals to use radioisotopes or more specialised and costly measuring equipment
We show the successful practical application of our dried blood spot assay in a large-scale, multicentre trial by describing the distribution of fasting adiponectin levels by demographic and anthropometric factors, fasting insulin and glucose measured in over 13,000 children aged 11.5 years from 31 polyclinics distributed across the Republic of Belarus
In linear regression analyses, adjusted for lot number, there was no evidence of any association between storage time at 280uC and levels of adiponectin for the low, medium (0.05 mg/ml; 95% CI: 20.12 to 0.22; p = 0.53), or high (0.07 mg/ ml; 95% CI: 20.30 to 0.44; p = 0.70) internal quality control samples
Summary
There is substantial interest in large-scale epidemiology studies of the genetic and environmental determinants of insulin resistance,[1,2,3,4,5,6,7,8,9,10] which may inform strategies for the prevention of insulin resistance and its sequelae. [11] Adiponectin is an adipocyte-derived hormone that circulates in high concentrations in humans, moderating glycemia, lipidemia, endothelial dysfunction and proinflammatory mechanisms.[12,13,14,15] Higher levels of circulating adiponectin are inversely associated with obesity, especially central obesity, as well as hyperlipidemia, insulin resistance, b–cell dysfunction, and intramyocellular lipid accumulation,[16,17,18,19,20,21,22,23] both in children and in adults with risk of cardiovascular events.[13,24,25,26] These relationships, the fact that adiponectin levels are not materially affected by time of day or eating, and the low long-term intra-individual variation, [27] indicate that adiponectin could act as an integrated quantitative measure of insulin sensitivity for use in epidemiology studies. [20,28].Important challenges in large-scale epidemiology include nonacceptance of venepuncture especially by children and/or their parents; the costs, safety and logistics of serum or plasma separation by centrifugation; and frozen storage and transport of aliquots. [29,30] The major advantages of dried blood spot sampling are minimal training, lower cost than venepuncture, acceptability to parents and children, [30] negligible processing requirements (cards must be air dried), low biohazard risk because samples cannot shatter or leak, and the ease of storage and transport of filter paper cards. As well as large-scale use of dried blood spots in paediatric screening programs for rare inherited disorders, there is widespread interest in developing and validating a wide range of bloodspot assays, [32] including adiponectin, [33] for population research. Development and validation of an ELISA blood spot assay offers a simple, convenient and novel alternative method of measuring adiponectin in large-scale epidemiology studies in a wide range of settings. We first describe the validation of an enzyme-linked immunoassay (ELISA) for quantifying adiponectin from dried blood spots and demonstrate its application in a large trial (PROBIT)
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