ESPE Abstracts

Background: Recently, patients with severe obesity have been described due to functional leptin deficiency. This new entity is characterized by high immunoreactive levels of circulating leptin (Lep), but a reduced bioactivity of the hormone due to defective receptor binding (N Engl J Med 2015;372:48–54). Since these patients can be successfully treated with human recombinant leptin (metreleptin), a diagnostic tool to detect functional leptin deficiency is needed.

Aim: We hypothesize that the measurement of immunofunctional leptin (bioLEP) by a new analytical approach is appropriate to estimate bioactivity of leptin.

Methods: Microtiterplates were coated with the recombinant extracellular domain of the human leptin receptor. Added serum leptin molecules bound to the immobilized receptor and were detected by a highly specific polyclonal, biotin-conjugated antibody and a streptavidin-peroxidase conjugate.

Results: The analytical range of the bioLEP assay was 1–120 ng/ml with intra and interassay coefficients of variation below 10%. Recovery of leptin international standard (NIBSC 97/594) or metreleptin was 102 and 109%, respectively. Physiological concentrations of soluble leptin receptor up to 100 ng/ml did not interfere with bioLEP measurement. In a clinical cohort (n=444; age: 3–70 years, BMI-SDS: −2.1–5.3) with total Lep levels of 1.0–117.2 ng/ml, mean±SD for the ratio of bioLEP/Lep was 1.07±0.13 (range 0.75 to 1.66). Serum samples of patients with non-functional leptin due to homozygous amino acid exchanges like p.D100Y or p.N103K revealed high Lep levels but non-detectable levels of bioLEP. Upon treatment of these patients with metreleptin, Lep levels decreased while levels of bioLEP increased continuously. Individuals heterozygous for p.D100Y or p.N103K had ratios of bioLEP/Lep of 0.5 (0.42–0.67) making them clearly distinguishable from individuals homozygous for the wild type sequence.

Conclusion: The bioLEP assay is able to detect only those leptin molecules capable of binding to the extracellular domain of the leptin receptor. Accordingly, this assay is a reliable method to identify patients with reduced leptin bioactivity resulting from either homozygous or heterozygous mutations in the LEP gene.