Catecholamine fluorescence

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... 

As with the catecholamines, fluorescence methods have also been reported for urinary metanephrine analysis. Fluorescent derivatization of the metanephrines NM and MN by chemical oxidation was based on modification of the trihydroxyindole reaction used for catecholamines. The individual metanephrines were measured following chromatographic separation and fluorescent derivatization or through the formation of differential fluorescent compounds by oxidation at different pH levelsSince the stability of the fluorescent products was variable, with some products decomposing within 10 min,this method has limited application in current practice. Other early methods for analysis of NM and MN included electrophoresis and paper and thin-layer chromatography. These assays were technically complex and had poor analytical sensitivity. 

Kusakabe T, Kawakami T, Tanabe Y, Fuji S, Takenaka T. Distribution of substance P-containing and catecholaminergic nerve fibers in the rabbit carotid body an immunohistochemical study in combination with catecholamine fluorescent histochemistry. Arch Histol Cytol 1994 57 193-199. 

Note The pre- and post-treatment of the chromatograms with the basic tri-ethylamine solution, which can be replaced by an alcoholic solution of sodium hydroxide [1,4] or a phosphate buffer solution pH = 8.0 (c = 0.2 mol/1) [5], serves to stabilize the fluorescence of the amino derivatives [2]. A final spraying with methanolic hydrochloric acid (chci = 5 mol/1) or 70% perchloric acid renders the detection reaction highly specific for histamine [4] and for catecholamines and indolamines [5]. 

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

The derivatives have an optimum fluorescence at an excitation wavelength of 340 nm and an emission wavelength of 455 nm. The adduct is relatively stable at a pH of 9-11 but it rapidly degrades to a non-fluorescent residue at low pH values. Consequently, when used as a pre-column derivatizing reagent the pH of the mobile phase should be kept fairly high, o-phthalaldehyde has been employed for derivatization in the analysis of dopamine (29), catecholamines (30) and histamines (31). 

Excess of the reagent hydrolyses to a non-fluorescent residue and the reagent itself does not fluoresce. The optimum wavelength of the excitation light is 390 nm and that of the emitted light 475 nm. This regent is, however, less sensitive than Fluoropa and the derivative is unstable consequently, it must be injected onto the column immediately after formation if used in pre-column derivatization. It has been used successfully in the separation and analysis of polyamines (32), catecholamines (33) and amino acids (34). 

Falck, B. Hillarp, N.-A. Thieme, G. and Torp, A. Fluorescence of catecholamines and related compounds condensed with formaldehyde. 

Mitsui, A., Nohta, H., and Ohkura, Y., High-performance liquid chromatography of plasma catecholamines using 1,2-diphenylethylenediamine as precolumn fluorescence derivatization reagent, /. Chromatogr., 344, 61, 1985. 

The most commonplace substrates in energy-transfer analytical CL methods are aryl oxalates such as to(2,4,6-trichlorophenyl) oxalate (TCPO) and z s(2,4-dinitrophenyl) oxalate (DNPO), which are oxidized with hydrogen peroxide [7, 8], In this process, which is known as the peroxyoxalate-CL (PO-CL) reaction, the fluorophore analyte is a native or derivatized fluorescent organic substance such as a polynuclear aromatic hydrocarbon, dansylamino acid, carboxylic acid, phenothiazine, or catecholamines, for example. The mechanism of the reaction between aryl oxalates and hydrogen peroxide is believed to generate dioxetane-l,2-dione, which may itself decompose to yield an excited-state species. Its interaction with a suitable fluorophore results in energy transfer to the fluorophore, and the subsequent emission can be exploited to develop analytical CL-based determinations. 

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.

Lindvall 0, Bjorklund A. (1974). The organization of the ascending catecholamine neuron systems in the rat brain as revealed by the glyoxylic acid fluorescence method. Acta Phys Scand Suppl. 412 1-48. 

An important area of direct biochemical interference is that caused by fluorescent or fluorescent quenching materials in the blood or urine after the administration of a drug. These interferences may be observed during catecholamine analysis in urine from patients receiving -methyldopa, tetracyclines, chlortetracyclines, oxytetracycline, erythromycin, chlorpro-mazine, or quinidine (A2, G5). 

Drug/Lab test interactions Methyidopa may interfere with tests for Urinary uric acid by phosphotungstate method serum creatinine by alkaline picrate method AST by colorimetric methods. Because methyidopa causes fluorescence in urine samples at the same wavelengths as catecholamines, falsely high levels of urinary catecholamines may occur and will interfere with the diagnosis of pheochromocytoma. 

The presence of a-methyldopa and its metabolites in the urine reduces the diagnostic value of urinary catecholamine measurements as an indicator of pheochro-mocytoma, since these substances interfere with the fluorescence assay for catecholamines. 

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... 

Assay procedures for dopamine which are superficially similar to the lutin procedure described above have been reported recently.266-268 The chemistry of the production of the fluorophore from dopamine is, however, somewhat different since the fluorophore is not a 5,6-dihydroxyindoxyl, it is incorrect to refer to the trihy-droxyindole fluorophore of dopamine (cf. ref. 252). Oxidation of the extracted catecholamine is usually carried out with iodine,266-268 presumably with the formation of 7-iodonorepinochrome. The aminochrome is subsequently rearranged to 5,6-dihydroxyindole (it is probable that deiodination accompanies the rearrangement in this case) by a solution of sodium sulfite in aqueous alkali the solution is acidified before measuring the fluorescence of the product (which is said to form relatively slowly and to be very stable).266-268 Irradiation of the reaction mixture with ultraviolet light accelerates the maximal development of fluorescence.266 Since acidification will produce sodium bisulfite in the reaction mixture, it is probable that the fluorophore is a 5,6-dihydroxyindole-sodium bisulfite addition complex. Complexes of this type are known to be both fluorescent and relatively stable in dilute acid solution.118 123,156 265 They also form relatively slowly.255... 

The assay procedure for catecholamines based on the formation of fluorescent products with ethylenediamine, originally described by Weil-Malherbe and Bone, 197, 198, 270 and considered by some workers to be the most sensitive method, suffers to some extent by a lack of specificity since catechol and 3,4-dihydroxymandelic acid give the same fluorophore as noradrenaline. However, most of the interfering compounds can be eliminated by use of suitable extraction procedures. 

In contrast, the content of norepinephrine is substantially lower than the content of dopamine (28). Catecholamine-containing neurons do not innervate the parenchymal cells of the bovine parathyroid gland only an occasional norepinephrine-containing neuron terminating upon a blood vessel is demonstrated by fluorescence histochemistry (22). 

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