Assay of catecholamines

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

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... 

P. Moleman, Preservation of urine samples for assay of catecholamines and their metabolites, Clin. Chem., 31, 653-654 (1985). 

Methods of biological, chemical, and fluorometric assay of catecholamines have been reviewed." Another review dealing with radioimmunoassay of drugs includes amphetamines." Numerous analytical methods and modifications have been applied to various types of phene thy lamine, and these are summarized in Table 2. A convenient derivative of phenylalanine for g.I.c. determination is 2-trifluoromethyl-... 

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. 

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. 

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... 

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. 

Catechol Amine Biosynthesis and MetaboUsm, Assay of Enzymes of Catecholamines and Catecholamine Metabolites, Estimation of Total (Free + Conjugated), in... 

Catecholamines. The quantitative determination of dopamine and noradrenaline in tissue samples of 0.1-10 mg at levels in the order of 0.5 pmol has been described [84]. These methods are based on extraction, formation of the pentafluorpropionyl derivatives, and the use of the homologues, a-methyidopamine and a-methylnoradrenaline as internal standards in SIM. Higher sensitivity than obtainable with fluorimetric or enzymic assays is reported [462J. Applications have been to amine determination in specific regions of rat brain [84] and to measurement of heart ventricle concentrations [463]. A combination of assays of this type with the use of synthesis inhibitors or radioisotope labelled precursors allows direct estimation of brain amine turnover in animals. 

The determination of catecholamines requires a highly sensitive and selective assay procedure capable of measuring very low levels of catecholamines that may be present. In past years, a number of methods have been reported for measurement of catecholamines in both plasma and body tissues. A few of these papers have reported simultaneous measurement of more than two catecholamine analytes. One of them utilized Used UV for endpoint detection and the samples were chromatographed on a reversed-phase phenyl analytical column. The procedure was slow and cumbersome because ofdue to the use of a complicated liquid-liquid extraction and each chromatographic run lasted more than 25 min with a detection Umit of 5-10 ng on-column. Other sensitive HPLC methods reported in the literature use electrochemical detection with detection limits 12, 6, 12, 18, and 12 pg for noradrenaline, dopamine, serotonin, 5-hydroxyindoleace-tic acid, and homovanillic acid, respectively. The method used very a complicated mobile phase in terms of its composition while whilst the low pH of 3.1 used might jeopardize the chemical stability of the column. Analysis time was approximately 30 min. Recently reported HPLC methods utilize amperometric end-point detection. 

Assay of ATP-dependent uptake of catecholamines or amino acid transmitters... 

In contrast to the catecholamines, measurements of urinary metanephrines and VMA are still based in some routine laboratories on the early spectrophotometric assays developed by Pisano, Crout, and others in the late 1950s and early 1960s. Despite subsequent development of a variety of preanalytical cleanup and extraction procedures, these assays remain susceptible to analytical interference. They are also restricted to measurements in urine. Another limitation for spectrophotometric or fiuorometric assays of urinary metanephrines is that these methods do not allow separate (fractionated) measurements of normetanephrine and metanephrine. 

Dietary constituents or drugs can either cause direct analytical interference in assays or influence the physiological processes that determine plasma and urinary levels of catecholamines and catecholamine metabolites. In the former circumstances, the interference can be highly variable depending on the particular measurement method. In the latter circumstances, interference is usually of a more general nature and independent of the measurement method (Table 29-6). 

Adenyi cyclase - The synthesis of cyclic AMP from ATP is catalyzed by the enzyme adenyi cyclase, which itself is responsive to hormone stimulation in various tissues . Catecholamines have been shown to stimulate the formation of cyclic AMP in a variety of tlssues (Table 1 ), and adenyi cyclase has been shown to be wide-spread in various tissues . Since procedures for the isolation and assay of this enzyme and measurement of the products of its reaction are available - , direct effects of analogues of ATP and/or cyclic AMP (or hormones) on the enzyme can be determined in an in vitro system. 

Since these basic works, many binding assays confirmed the existence of catecholamine-insensitive imidazoline specific binding sites in various tissues including the human brain. As an example, Bricca et al. showed that about 80% of the [3 H] clonidine bound to human brainstem membranes were resistant to the endogenous catecholamines [15]. 

In general, minimal sample handling offers many advantages and if the HPLC-conditions are well chosen for a particular analytical problem, very crude samples can often be analyzed without off-line manipulation. Assays involving direct injection of the specimen may eliminate the necessity for an internal standard if recoveries are high and reproducible. In some cases of catecholamine analysis, the compounds may also be injected directly for... 

Chan et developed an assay for the simultaneous determination of catecholamines and metanephrines using FMOC derivatization. The assay is convenient for the simultaneous analysis of NM, MN, E and DA in human urine sample without prior extraction procedures. In this study, urine was directly derivatized and subjected to a simple extraction step with... 

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