Catecholamine Assay

The oxidations in dilute solution discussed in this section mostly involve molecular oxygen as the oxidizing agent. The oxidative step in several catecholamine assay procedures, which usually involves the participation of an inorganic oxidizing agent, and which also occurs in dilute solution, is considered in Section V, E. 

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). 

Cook FJ, Chandler DW, Snyder DK. Effect of bus-pirone on urinary catecholamine assays. N Engl J Med 1995 332 401. 

Davidson DF. Urinary catecholamine assay by HPLC in vitro interference by some drugs. Ann Clin Biochem 1988 25 (Pt 5) 583-4. 

O. Nozaki, H. Kawamoto and H. Moriyama, Total free catecholamines assay by identiflcation of its two functional groups and micro-flow injection chemiluminescence. Luminescence, 14, 369-374 (1999). 

M. Israel and M. Tomasi, A chemiluminescent catecholamine assay its application for monitoring adrenergic transmitter release, J. Neurosci. Methods., 91, 101-107 (1999). 

Measurement of catecholamine metabolites can provide insight into the rate of release or turnover of catecholamines in the brain. In clinical studies, metabolites of catecholamines are generally assayed in the CSF because the large quantities derived from the peripheral sympa-thomedullary system obscure the small contribution from the brain to urinary concentrations. However, acid metabolites are actively excreted from the CSF more reliable estimates of turnover in the brain are obtained when this transport process is blocked by pretreatment with the drug probenecid. 

Chan ECY, Wee PY, Ho PY, Ho PC. 2000. High-performance liquid chromatographic assay for catecholamines and metanephrines using fluorimetric detection with pre-column 9-fl.uorenylmethyloxycarbonyl chloride derivatiza-tion. J Chromatogr B 749 179... 

Vaarman A, Kask A, Maeorg U. 2002. Novel and sensitive high-performance liquid chromatographic methods based on electrochemical coulometric assay detection for simultaneous determination of catecholamines, kynure-nine and indole derivatives of tryptophan. J Chrom B 769 145-153. 

Colorimetric assays used in endocrinological procedures are also often subject to drug interference. We have observed an interesting interference in a patient with carcinoid. The patient excreted 400 mg of 5-hydroxyindoleacetic acid (5-HIAA) and when a vanillylmandclic acid (VMA) determination was performed by a nonspecific diazo method, the value was reported to be 375 mg. The catecholamines were just above normal. There was an immediate suggestion that the patient also had a pheochromocytoma. However, when a specific chromatographic VMA method was used, the value was found to be within normal limits. Subse-... 

Assay of catecholamines in urine by ion exchange chromatography with electrochemical detection... 

The initial report of sustained, lower urinary cortisol levels in PTSD highlighted the disassociation between cortisol and catecholamine levels in PTSD. Norepinephrine and epinephrine levels assayed from the same urine specimens revealed elevations in both of these catecholamines, while cortisol levels in PTSD fell within the normal range of 20-90 pg/day, indicating that the alteration was not in the hypoadrenal or endocrinopathologic range (Mason et al. 1986). This finding established the expectation that alterations in basal levels of cortisol might be subtle, and not easily differentiated from normal values (Mason et al. 1986). 

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. 

McGregor, D.B., Riach, C.G., Brown, A.., Edwards, I., Reynolds, D., West, K. Willington, S. (1988) Reactivity of catecholamines and related substances in the mouse lymphoma L5178Y cell assay for mutagens. Environ, mol. Mutag., 11, 523-544... 

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. 

Baumgartner, H., Ridl, W., Klein, G., and Preindl, S., Improved radioenzymic assay for the determination of catecholamines in plasma, Clin. Chim. Acta, 132, 111, 1983 Chem. Abs., 99, 99459k, 1983. 

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