Fluorescent technique, catecholamines

Bioassay methods are often both sensitive and specific. Some are indeed so sensitive that they can be used for the assay of extremely small amounts of circulating catecholamines (see Fig. 1. p. 117). For most purposes, however, chemical methods of assay are now widely used. Of the chemical methods available, only fluorescence techniques offer sufficient sensitivity for application to biological materials. For assay, the catecholamines must be converted to suitable fluorescent derivatives, since their native fluorescence is inadequate. There are two chemical procedures in use, both of which have given rise to a host of methods varying only in experimental detail. 

The previously described methods necessitate the extraction of the catecholamines from the tissue prior to assay. The histochemical fluorescence technique allows the visualization of the amines in situ, but it is not an accurate quantitative procedure. Freeze-dried sections of tissue are exposed to formaldehyde vapour at SO C for 1 hr or more. The catecholamines are thereby converted to hydroxyiso-quinoline derivatives (Fig. 1), which fluoresce strongly under U-V light in the fluorescence microscope (Fig. 1, p. 110). ADR and other secondary amines can be distinguished from NA, DA and DOPA by their slower rate of reaction with formaldehyde. 

The DA neurons were initially identified by Sulston and coworkers, who used the catecholamine-specific technique of formaldehyde-induced fluorescence (FIF) (23). DA cell bodies and processes were visualized using fluorescence microscopy, confirmed as DA by aluminaabsorptionandthin-layerchromatography (TLC). The precursor L-DOPA was also identified but not the catecholamines norepinephrine and epinephrine. Based on FIF micrographs and worm DA content as well as the estimated volume of the cell bodies and processes, the concentration of DA in the nerve endings is predicted to be very similar to the concentration within mammalian varicosities (23). We and others [(6) R. Nass, unpublished data] have confirmed via high-perfomance liquid chromatography (HPLC) that DA and its metabolites dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA) are present in the animal. 

Harrison and Whisler have studied the mushroom tyrosinase catalysed oxidations of a number of catecholamines using fluorescence spectroscopy and tritium tracer techniques [44, 52, 53], The oxidation of adrenaline and noradrenaline to the open-chain quinone (12) was monitored by the loss of the native fluorescence of the substrate [44], The cyclisation step for this... 

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