Li S, Nakayama T, Akinc A, Wu SL, Karger BL.
Journal of Pharmacological and Toxicological Methods (2014), doi: 10.1016/j.vascn.2014.09.008
A requisite step in developing a therapeutic to modulate the levels of hepcidin is the
development of a quantitative method for measuring the concentration of serum hepcidin. To
this end, an LC-MS method, based on selected reaction monitoring (SRM) with a triple
quadrupole MS and an isotopically labeled hepcidin as internal standard, was developed to
measure hepcidin in mouse and monkey sera. Initially, 40 normal cynomolgus monkeys and 40
normal mice were studied to determine the normal endogenous levels of hepcidin, and an
average of 50 ng/mL was found in the monkeys and 46 ng/mL in the mice. Next, experiments
were conducted where an siRNA, targeting hepcidin, was administered to cynomolgus monkeys,
resulting in effective hepcidin reduction (inhibition rate) of 87% after 24 hr and 74% after 48 hr,
demonstrating to effectively reduce serume level of hepcidin. For better sensitivity, especially
for the low volumes available for mouse sera, a second LC-MS method, based on parallel
reaction monitoring (PRM) using a Orbitrap MS was developed and shown to be at least 10 fold
lower in detection limits (or consumption of serum volume) than the SRM approach.
Northeastern University, Alnylam
United States
