Adrenaline fluorescence

Fig. 1 Fluorescence scan of the catecholamine derivatives (each ca. 10 ng) of noradrenaline (1), adrenaline (2), dopamine (3), dopa (4).

Fig. II Fluorescence scan of a chromatogram track with SOO ng each of noradrenaline (1), adrenaline (2), serotonin (3), vanilmandelic add (4), 5-hydroxyindoleacetic add (6), homovanillic acid (7) and vanillic acid (8) together with 230 ng creatinine, all per chromatogram zone measurement at X(3,c = 313 nm and > 390 nm (cut off filter FI 39 (A)), X = 365 nm and Xj, >430 nm...

Figure 4.10 Direct analysis of catecholamines in urine sample. Column, Asahipak ES-502C eluent, 75 mM succinic acid + 25 mM borate buffer (pH 6.10) containing 0.5 mM EDTA flow rate, 1.0 min-1 detection, fluorescence reaction detection Ex. 350 nm. Peaks-. 1, adrenaline-, 2, noradrenaline-, and 3, dopamine.

In 1937 Arnow showed that tyrosine could be converted into DOPA by ultraviolet radiation51 and that the DOPA produced in this manner was subsequently destroyed by further irradiation, the solutions becoming red-brown in color (presumably due to the formation of dopachrome).51 In 1939 Konzett and Weis reported that the blood pressure-raising effect of adrenaline solutions was lost on ultraviolet irradiation and that the solutions became colored and fluorescent the initial red color fades to reddish yellow.62 This phenomenon suggests the initial formation of adrenochrome, followed by its isomerization to adrenolutin, both of these compounds being virtually void of pressor activity. Similarly to the radiation-induced hydroxylation of tyrosine mentioned above, synephrine was first... 

When adrenaline was oxidized in the presence of Cu++ ions, the loss of the native catecholamine fluorescence was again detected, but in this case the initial oxidation products were also fluorescent. Addition of ascorbic acid did not increase the fluorescence. However, addition of ferricyanide ions destroyed the fluorescence, but it could be regenerated by the addition of ascorbic acid. Noradrenaline behaved somewhat differently in that the initial oxidation product had little fluorescence, probably due to the quenching effect of the Cu++ ions, since reduction of the Cu++ ion concentration increased... 

It appears that the intermediates formed from different catecholamines are of different stability. The intermediate open-chain quinones derived from catecholamines with a primary amino group in the side chain do not appear to undergo intramolecular cyclization very readily and consequently would be able to take part in competing reactions this would account for the fact that in general it is difficult to obtain efficient conversions of such catecholamines (e.g. noradrenaline) into the corresponding aminochromes. This factor is important in catecholamine assay procedures (see Section V, E) and probably explains the wide variability in the apparent efficiency of the noradrenaline oxidation procedures used (as measured by the intensity of the fluorescence of the noradrenolutin obtained by the particular method). The fact that noradrenaline-quinone is relatively more stable than adrenaline-quinone accounts for the formation of entirely different types of fluorescent products from adrenaline and noradrenaline, respectively, in the Weil-Malherbe assay procedure for catecholamines (see Sections IV, H and V, E, 5). 

The characteristic transient yellow-green fluorescence exhibited by adrenaline solutions that were undergoing oxidation in the presence of alkali was first reported in 1918.182 This phenomenon was later shown to be general, and similar (but usually weaker) fluorescences were observed when other catecholamines were oxidized in alkaline solution.133 Many years were to elapse before the correct explanation of this phenomenon was forthcoming, i.e. that the fluorescent product derived from adrenaline was a rearrangement product of adrenochrome (the red oxidation product of adrenaline). 

The formation of relatively stable fluorescent products by the reaction of adrenaline with ethylenediamine (and certain other primary amines) in air, first reported in 1948 by Natelson et was adapted by Weil-Malherbe and Bone in 1952 for the assay of catecholamines.197 198 Since 1952 much work, largely of an empirical nature, has been carried out to improve the analytical procedure since often apparently minor variations of the reaction conditions have a significant effect on the fluorescence observed (see Section V, E, 4). Paper chromatographic examination of the reaction mixtures obtained from adrenaline and noradrenaline suggested that more than one product could be formed in each case.199-205 The main fluorescent product of the interaction of adrenochrome (1) (obtained by oxidation of adrenaline) and ethylenediamine in air has been obtained as a crystalline solid by Harley-Mason and Laird and shown to be 2,3-dihydro-3-hydroxy-l-methylpyrrolo[4,5-g]quinoxaline (94) (7% yield).206,207 This compound has two hydrogen atoms less than... 

There are vast differences in the quoted relative fluorescences of the fluorophores obtained from adrenaline and noradrenaline (i.e. adrenolutin and noradrenolutin). With one exception (Anton and Sayre252), noradrenolutin is reported to be less fluorescent (on a w/w basis) then adrenolutin. This, however, is not true, since experiments with crystalline noradrenolutin70,71 have shown that (i) noradrenolutin is approximately twice as fluorescent as adrenolutin,255 and (ii) it is somewhat more stable than adrenolutin in aqueous solution 255 (cf. ref. 256). The use of internal standards has, however, allowed the method to function, more or less satisfactorily, in most cases. 

HPLC analysis of polycyclic aromatic hydrocarbons (PAH) in drinking water is one of the current and classical applications of fluorescence. In this case, the detector contains a fluorescence flow cell placed after the chromatographic column. This mode of detection is specifically adapted to obtain threshold measurements imposed by legislation. The same process allows the measurement of aflatoxins (Fig. 12.11) and many other organic compounds (such as adrenaline, quinine, steroids and vitamins). 

Upon degradation, adrenaline solution first turns into pink, then red, and finally brown. Adrenaline is first oxidised into adrenochrome, which is consecutively oxidised into the fluorescent adrenolutin and brown melanin products [18]. The oxidation rates increase with increasing pH. The stability is the best at pH 3.2-3.6, by virtue of the relatively low reaction rate of the first step at this pH [19]. At pH 7.4, the rate of the second step is relatively high, whilst at pH 6.9, accumulation of adrenochrome occurs [18]. 

The colour reactions resulting from the oxidation of adrenaline and related catecholamines formed the basis of early qualitative and quantitative assay procedures for adrenaline [8-11]. The characteristic yellow-green fluorescence, which develops rapidly when alkaline solutions of adrenaline are allowed to stand in air, was first reported in 1918 [12], and this has become the basis of one of the most widely-used methods for the estimation of catecholamines (i.e. the so-called lutin or trihydroxyindole method... 

Harrison [44] observed the direct formation of a fluorescent product when adrenaline or noradrenaline was oxidised in the presence of Cu or Fe ions. Althou the identity of this fluorescent product was at first uncertain, it is probably the corresponding dihydroxyindoxyl derivative, adrenolutin (10) in the case of adrenaline oxidation or noradrenolutin (11) in the case of noradrenaline oxidation [45, 46]. Whisler concluded that the cyclisation of the catecholamine to the fluorescent product proceeds via the formation of a non-fluorescent intermediate [46]. 

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... 

The rates of the tyrosinase catalysed oxidations of a number of catecholamine-type substrates has been studied and found to be in the following order catechol > l(—)-DOPA > dopamine > d(- -)-DOPA > L(+)-adrena-line > d(—)-adrenaline > L(- -)-noradrenaline > d(—)-noradrenaline. The velocities of formation of the fluorescent products decreased in the order L(+)-adrenaline > d(—)-adrenaline > L(-l-)-noradrenaline > d(—)-noradrenaline. To explain the differences in the initial oxidation rates, it was proposed that the side chain of the catecholamine binds to a control site of the enzyme through chelation with the copper present in the enzyme and this attachment moderates the rate, the actual rate depending upon the... 

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