SAT-456 Heterogeneity of Macroprolactin in Samples Suspected of False Hyperprolactinemia

Abstract

Circulating prolactin (PRL) exists in several molecular forms with different biological and immunological activities, in some instances making PRL measurements and its diagnostic value unreliable. Macroprolactin (macroPRL), a 150 kDa molecular weight (MW) form, is known to have low biological activity which may lead to misdiagnosis and inappropriate treatment in patients with suspected hyperprolactinemia. Polyethylene glycol precipitation (PEGP) is widely used to identify the presence of macroPRL, but up to 20% of monomeric PRL (23 kDa) is lost during this procedure and PEG itself can interfere with some immunoassays. Additionally, PEGP has been reported to give false positive results for macroPRL in patients with increased serum globulins (IgG myeloma and HIV patients). The aim of this study is to identify different PRL variants in samples suspected of macroPRL using gel filtration chromatography (GFC) Thirteen samples with prolactin levels measured prior to and following PEGP were analyzed using GFC. Briefly, 100 uL of sample or protein markers (MWs ranging from 12.4 to 200 kDa) were applied to a Superdex 200 column (Pharmacia). Forty fractions of 0.5 mL each were collected per patient sample at a flow rate of 0.5 mL/min using PBS containing 0.1% (w/w) bovine serum albumin as the mobile phase. Elution of marker proteins was detected by recording absorbance at 280 nm. To pull down PRL-IgG complexes from our samples, 100 uL of each sample was applied to NAb protein G spin columns (Thermo-Scientific) according to the manufacture’s protocol. The elution and flow-through fractions were collected and analyzed by GFC as described above. PRL concentrations of all GFC fractions were measured using ELISA (RnD systems) according to the manufacture’s protocol. PEGP suggested 8 of our 13 samples were positive for macroPRL, 3 samples had no macroPRL, and 2 samples are within the indeterminate zone (recovery between 40-60%). GFC analyses corelated well with those results, and no macroPRL was present in samples from the indeterminate zone according to GFC. In samples containing macroPRL, 30.8 - 99% of total PRL was bound to protein G columns, while only 0 - 4% of PRL was pulled down from samples negative for macroPRL. Of 8 samples positive for macroPRL, 4 had a fraction of macroPRL that was not bound to protein G columns suggesting the existence of non-IgG-bound macroPRL. GFC analysis showed marked molecular heterogeneity for macroPRL. Mid-molecular weight-PRLs (30-150kDa) were present in every sample, and both high-molecular weight-PRL (>150kDa) and mid-MW-PRL had diverse patterns, but their clinical significance and physiological roles remained unclear. This is the first study to report molecular heterogeneity for macroPRL.