title: Age-related changes in plasma leptin binding activity in rats: A comparison of a simple acid-ethanol precipitation technique with column chromatography.
abstract: A novel assay for measuring the free leptin fraction was developed and validated against a chromatographic technique. The assay used acid-ethanol extraction (AEE) for separation of bound/free leptin moieties. The interassay coefficient of variation was 3.9%. The specificity for leptin binding was confirmed by incubation with 1 microg of unlabeled rat leptin that effectively competed with radiolabeled leptin whereas human growth hormone and interleukin-6 were ineffective in competing with radiolabeled leptin binding. Scatchard analysis of competitive binding experiments with rat plasma demonstrated a linear relationship with a binding affinity of 0.3-0.6 x 109 M-1. This novel assay was used to determine if age-related insensitivity to leptin action is secondary to altered serum leptin binding. Rats at various age groups were studied for changes in body adiposity and serum total and free leptin 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 weight and adiposity of the two age groups. The increase in plasma free leptin concentrations in 12-month-old rats (3.86 +/- 0.28) and 6-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 concluded that aging in rats is associated with relative insensitivity to leptin. This change cannot be attributed to increased plasma binding or to a reduction in the leptin free fraction.
authors.affiliation: Division of Endocrinology, Diabetes, and Metabolism, Department of Internal Medicine, Saint Louis University School of Medicine, St. Louis, Missouri 63104, USA. mooradad@slu.edu
authors.forename: A D
authors.initials: AD
authors.lastname: Mooradian
authors.forename: R
authors.initials: R
authors.lastname: Hurd
authors.forename: J
authors.initials: J
authors.lastname: Chehade
authors.forename: K
authors.initials: K
authors.lastname: Pun
authors.forename: M J
authors.initials: MJ
authors.lastname: Haas
mesh_terms: D000273:Adipose Tissue / Q000737:chemistry; D000375:Aging / Q000502:physiology*; D000818:Animals; D001667:Binding, Competitive; D001823:Body Composition; D001835:Body Weight; D002352:Carrier Proteins / Q000032:analysis / Q000097:blood* / Q000378:metabolism; D002850:Chromatography, Gel; D000431:Ethanol / Q000378:metabolism*; D005590:Fractional Precipitation; D006863:Hydrogen-Ion Concentration; D020738:Leptin / Q000032:analysis / Q000097:blood / Q000378:metabolism; D008297:Male; D011485:Protein Binding; D051381:Rats; D011916:Rats, Inbred F344; D011956:Receptors, Cell Surface; D054411:Receptors, Leptin; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity
chemical_list: D002352:Carrier Proteins; C515448:LEPR protein, human; D020738:Leptin; D011956:Receptors, Cell Surface; D054411:Receptors, Leptin; D000431:Ethanol