Measurement of monoamines in murine tissue.

Abstract

Monoamines play a critical role as neurotransmitters and hormones in the functioning of brain and body of all mammals and are therefore often connected to diseases like Parkinson’s Disease or Diabetes. Up until now, catecholamines and related monoamines are routinely measured only in blood and plasma, as well as in the brain, for diagnostics and research in mouse models, respectively. But, as the monoamine pathways are complex and are present throughout the whole body, it would be important to have the means to analyze them in other body parts as well. Therefore, we, the group of Molecular EXposomics (MEX) at the Helmholtz Center Munich, focused on the development of a method for the measurement of these compounds in other tissues than brain. For this, the targets were different muscles (gastrocnemius, soleus, EDL), liver, pancreas, and brown and white adipose tissue. A simple clean up and extraction was developed for each of these tissues. The instrument for this method was a HPLC, fitted with a new generation solid core particle C-18 column and an amperometric electrochemical detector from Dionex. The sensitivity of this setup ranged from 0.625 pg/µl to 2.5 pg/µl. The analytes which could be differentiated, are the catecholamine dopamine, its metabolites 3,4‑dihydroxyphenylacetic acid, 3‑methoxytyramine, and homovanillic acid, the catecholamines norepinephrine, epinephrine, their metabolite 3‑methoxy‑4‑hydroxyphenylglycol, as well as the monoamine serotonin and its degradation product 5-hydroxyindoleacetic acid. These compounds could be found with different occurrence and concentrations in the analyzed tissues. However, for some of these tissues a few of the analytes were close to or below the limit of detection. To improve upon this, a new UHPLC instrument with a coulometric detector was procured. This instrument allows for greater sensitivity ranging from 0.25 pg/µl to 0.5 pg/µl. This system is now tested on other samples, e.g. brain perfusates, which have a very small sample volume and therefore need the greater sensitivity. Also completely new tissues like placenta, which could be useful in the diagnostics of early onset or predisposition of diseases, are currently tested