Filter Paper Blood Spot Enzyme Linked Immunoassay for Adiponectin and Application in the Evaluation of Determinants of Child Insulin Sensitivity

Background: Adiponectin 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). Methods: We 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. Results: In 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. Conclusions: Bloodspot ELISA is suitable for measuring adiponectin in very small volumes of blood collected on filter paper and can be applied to large-scale studies

Filter Paper Blood Spot Enzyme Linked Immunoassay for Insulin and Application in the Evaluation of Determinants of Child Insulin Resistance

In large-scale epidemiology, bloodspot sampling by fingerstick onto filter paper has many advantages, including ease and low costs of collection, processing and transport. We describe the development of an enzyme-linked immunoassay (ELISA) for quantifying insulin from dried blood spots and demonstrate its application in a large trial.) to quantify insulin from two 3-mm diameter discs (≈6 µL of blood) punched from whole blood standards and from trial samples. Paediatricians collected dried blood spots in a follow-up of 13,879 fasted children aged 11.5 years (interquartile range 11.3–11.8 years) from 31 trial sites across Belarus. We quantified bloodspot insulin levels and examined their distribution by demography and anthropometry.Mean intra-assay (n = 157) coefficients of variation were 15% and 6% for ‘low’ (6.7 mU/L) and ‘high’ (23.1 mU/L) values, respectively; the respective inter-assay values (n = 33) were 23% and 11%. The intraclass correlation coefficient between 50 paired whole bloodspot versus serum samples, collected simultaneously, was 0.90 (95% confidence interval 0.85 to 0.95). Bloodspot insulin was stable for at least 31 months at −80°C, for one week at +30°C and following four freeze-thaw cycles. Paediatricians collected a median of 8 blood spots from 13,487 (97%) children. The geometric mean insulin (log standard deviation) concentrations amongst 12,812 children were 3.0 mU/L (1.1) in boys and 4.0 mU/L (1.0) in girls and were positively associated with pubertal stage, measures of central and peripheral adiposity, height and fasting glucose.Our simple and convenient bloodspot assay is suitable for the measurement of insulin in very small volumes of blood collected on filter paper cards and can be applied to large-scale epidemiology studies of the early-life determinants of circulating insulin

Transcending the replacement paradigm of solid-state lighting

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Effects of promoting increased duration and exclusivity of breastfeeding on adiposity and insulin-like growth factor-I at age 11.5 years:a randomized trial

IMPORTANCE: Evidence that increased duration and exclusivity of breastfeeding reduces child obesity risk is based on observational studies that are prone to confounding. OBJECTIVE: To investigate effects of an intervention to promote increased duration and exclusivity of breastfeeding on child adiposity and circulating insulin-like growth factor (IGF)-I (which regulates growth). DESIGN: Cluster-randomized controlled trial. SETTING: 31 Belarusian maternity hospitals and their affiliated polyclinics, randomized to usual practices (n=15) or a breastfeeding promotion intervention (n=16). PARTICIPANTS: 17,046 breastfeeding mother-infant pairs enrolled in 1996/7, of whom 13,879 (81.4%) were followed-up between January 2008 and December 2010 at a median age of 11.5 years. INTERVENTION: Breastfeeding promotion intervention modeled on the WHO/UNICEF Baby Friendly Hospital Initiative. MAIN OUTCOME MEASURES: Body mass index (BMI), fat and fat-free mass indices (FMI and FFMI), percent body fat, waist circumference, triceps and subscapular skinfold thicknesses, overweight and obesity, and whole-blood IGF-I. Primary analysis was based on modified intention-to-treat (without imputation), accounting for clustering within hospitals/clinics. RESULTS: The experimental intervention substantially increased breastfeeding duration and exclusivity (43% vs. 6% and 7.9% vs. 0.6% exclusively breastfed at 3 and 6 months, respectively) versus the control intervention. Cluster-adjusted mean differences in outcomes at 11.5 years between experimental vs. control groups were: 0.19 kg/m(2) (95% 4 CI: −0.09, 0.46) for BMI; 0.12 kg/m(2) (−0.03, 0.28) for FMI; 0.04 kg/m(2) (−0.11, 0.18) for FFMI; 0.47% (−0.11, 1.05) for % body fat; 0.30 cm (−1.41, 2.01) for waist circumference; −0.07 mm (−1.71, 1.57) for triceps and −0.02 mm (−0.79, 0.75) for subscapular skinfold thicknesses; and −0.02 standard deviations (−0.12, 0.08) for IGF-I. The cluster-adjusted odds ratio for overweight / obesity (BMI ≥85(th) percentile v

Recovery of adiponectin from five different dried blood spot concentrations.

<p>Recovery of adiponectin from five different dried blood spot concentrations.</p

Typical calibration curve.

<p>Whole bloodspot adiponectin standards at 3.75, 7.5, 15.0, 30.46 µg/ml.</p

Adiponectin levels in boys and girls by demographic and clinical characteristics, N = 13,329.

<p>SD = standard deviation; CI = confidence interval.</p>a<p>Linear regression coefficient and 95% CI (age-adjusted, accounting for clustering by polyclinic).</p>b<p>Normal weight, overweight and obese as defined by the International Obesity Task Force BMI cut-offs. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0071315#pone.0071315-Cole1" target="_blank">[44]</a>.</p>c<p>Centiles derived from NHANES European age and sex-specific thresholds (reference) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0071315#pone.0071315-Fernandez1" target="_blank">[45]</a>.</p>d<p>Centile derived from sample.</p>e<p>Quartiles derived from NHSR age-and sex-specific thresholds <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0071315#pone.0071315-McDowell1" target="_blank">[46]</a>.</p>f<p>Quartiles derived from sample.</p

Comparison of adiponectin levels measured in PROBIT with other studies in children of a similar age.

<p>SD = standard deviation; m = months;</p>a<p>median (interquartile range);</p>b<p>interquartile range;</p>c<p>95% CI.</p