Age-related changes in plasma leptin binding activity in rats: A comparison of a simple acid-ethanol precipitation technique with column chromatography

A novel assay for measuring the free leptin fraction was developed and vali dated against a chromatographic technique, The assay used acid-ethanol extr action (AEE) for separation of bound/free leptin moieties, The interassay c oefficient of variation was 3.9%, The specificity for leptin binding was co nfirmed by incubation with 1 mu g of unlabeled rat leptin that effectively competed with radiolabeled leptin whereas human growth hormone and interleu kin-6 were ineffective in competing with radiolabeled leptin binding. Scatc hard analysis of competitive binding experiments with rat plasma demonstrat ed a linear relationship with a binding affinity of 0.3-0.6 x 10(9) M-1. Th is novel assay was used to determine if age-related insensitivity to leptin action is secondary to altered serum leptin binding, Rats at various age g roups were studied for changes in body adiposity and serum total and free l eptin concentrations, Serum free leptin concentrations (ng/ml mean +/- SEM) were significantly increased in 24-month-old rats (5.56 +/- 0.21) compared with 18-month-old rats (4.76 +/- 0.17) (P < 0.01) despite similar body wei ght and adiposity of the two age groups. The increase in plasma free leptin concentrations in 12-month-old rats (3.86 +/- 0.28) and B-month-old rats ( 2.05 +/- 0.06) relative to 3-month-old rats (1.37 +/- 0.06) (P < 0.001) was out of proportion to the increase in body adiposity in aging rats, It is c oncluded that aging in rats is associated with relative insensitivity to le ptin, This change cannot be attributed to increased plasma binding or to a reduction in the leptin free fraction.