A Rapid and Sensitive Determination of Histamine in Mast Cells Using a Didansyl Derivatization Method

Abstract

<b><i>Background:</i></b> Mast cells play a central role in allergic responses such as food allergy, asthma, allergic rhinitis, and allergic conjunctivitis. Symptoms in the early phase of these allergic diseases are primarily caused by histamine. However, due to the high histidine content in the cytosol and low histamine content in secretory granules, separating and quantifying histamine from histidine is often difficult. <b><i>Objectives:</i></b> We studied a method for rapid and sensitive quantitation of mast cell-derived histamine and evaluated its application to allergic disease research. <b><i>Methods:</i></b> Bone marrow-derived mouse mast cells (BMMCs) were employed in this study. IgE-sensitized BMMCs were activated by FcεRI cross-linking. After activation, both the histamine released to the supernatant and histamine remaining in BMMCs were didansylated and then analyzed by high-performance liquid chromatography with fluorescence detection (HPLC-FD). Didansyl histamine was synthesized as a standard material. <b><i>Results:</i></b> Synthetic didansyl histamine was detected by HPLC-FD with a peak retention time of 18.5 min. Very high linearity of the standard curve was maintained at concentrations of 10 pg/μL or less when the didansyl histamine method was used. This method enables detection of histamine released from 1 × 10⁵ BMMCs. In addition, the histamine concentration in the supernatant due to spontaneous release was also determined. Finally, the ratio of histamine release was highly correlated with the degranulation ratio. <b><i>Conclusion:</i></b> These results indicate that the proposed method using didansylated histamine to determine mast cell-derived histamine is highly useful for allergy research applications.