Siyang Li1, Tomoko Nakayama2, Akin Akinc2, Shiaw-Lin Wu3, Barry L Karger4. 1. Barnett Institute, Northeastern University, Boston, MA 02115, United States. 2. Alnylam Pharmaceuticals, Inc., Cambridge, MA 02138, United States. 3. Barnett Institute, Northeastern University, Boston, MA 02115, United States. Electronic address: si.wu@neu.edu. 4. Barnett Institute, Northeastern University, Boston, MA 02115, United States. Electronic address: b.karger@neu.edu.
Abstract
INTRODUCTION: 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. METHODS: 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. RESULTS: Initially, 40 normal cynomolgus monkeys and 40 normal mice were studied to determine the normal endogenous levels of hepcidin, and an average of 50ng/mL was found in the monkeys and 46ng/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 24h and 74% after 48h, demonstrating to effectively reduce serume level of hepcidin. CONCLUSIONS: 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.
INTRODUCTION: 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. METHODS: 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. RESULTS: Initially, 40 normal cynomolgus monkeys and 40 normal mice were studied to determine the normal endogenous levels of hepcidin, and an average of 50ng/mL was found in the monkeys and 46ng/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 24h and 74% after 48h, demonstrating to effectively reduce serume level of hepcidin. CONCLUSIONS: 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.
Authors: Ahmed Moghieb; Lia Tesfay; Song Nie; Marina Gritsenko; Thomas L Fillmore; Jon M Jacobs; Richard D Smith; Frank M Torti; Suzy V Torti; Tujin Shi; Charles Ansong Journal: Sci Rep Date: 2019-05-13 Impact factor: 4.379