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Leptin Binding and Binding Capacity in Serum

Methods: A new method to quantify the free and bound forms was developed, based on HPLC separation and RIA quantification in chromatography fractions. Reanalysis of specimens after addition of exogenous leptin allowed direct determination of leptin-binding capacity and the degree of saturation of leptin-binding capacity.

Results: HPLC chromatography fractionated serum leptin into both the free form and as a broad peak of 59–130 kDa. Several experiments were conducted to validate the new method. The concentrations of bound leptin in serum were 0.45–3.94 µg/L, and they increased as total leptin (reflecting adiposity) increased in 24 lean and obese volunteers. Leptin was readily dissociated from the bound fraction by competition from exogenous leptin. Rechromatography of the bound fraction led to dissociation of leptin, which was promoted by warming the sera before chromatography. Leptin-binding capacity was 1.8–5.3 µg/L; binding capacity was nearly constant over a range of total leptin concentrations of 2–10 µg/L, and slowly increased at higher total leptin concentrations. Saturation of binding capacity was low (15%) at very low total leptin concentrations (<5 µg/L), but rose quickly to a plateau near 80% at higher total leptin concentrations.

Conclusions: The new method facilitates measurement of free and bound fractions of serum leptin, and is the first method measuring leptin-binding capacity. These experiments demonstrate that the concentration of bound leptin and leptin-binding capacity vary physiologically, with binding/binding capacity increasing with adiposity. Except in very lean individuals, binding capacity is nearly completely saturated.

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