Extraction catecholamines

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... 

Eiquid- or solid-phase extraction methods have been adopted for the isolation of catecholamines and their metabolites from urine samples. The liquid extraction system is ordinarily based on the formation of a complex, in alkaline medium, between diphenylborate and the diol group in the catecholamines. However, the liquid extraction methods reported in the literature are relatively tedious and often involved multiple extraction steps.For the more widely used solid-phase extraction methods, catecholamines may be selectively isolated from the urine sample by adsorption with activated alumina," " phenylboronic acid or cation-exchange resins. All the specimen preparative procedures are specific for the free catecholamines, i.e. the extracted catecholamines do not include the conjugated fraction. 

The modern usage of P2" go Asts for the treatment of asthma dates to 1903 when the effect of injected epinephrine [51-43-4] (adrenaline) C2H23NO2, (1 R = CH3) was investigated (see Epinephrine and norepinephrine) (33). As in some other modem treatments, eg, xanthines and anticholinergics, the roots of P2" go Ast therapy for asthma can be found in historical records which document the use of herbal extracts containing ephedrine [299-42-3] C qH NO, (2) as bronchodilators. Epinephrine and ephedrine are stmcturaHy related to the catecholamine norepinephrine [51-41-2] CgH NO, (1, R = H), a neurotransmitter of the adrenergic nervous system (see Neuroregulators). 

The ion-exchange mechanism of exfracfion does nof occur only for amino acids. We observed if also for cafecholamines [26]. These compounds are efficiently extracted into ILs in the cationic form, af pH 1-8. Af fhese pH, the primary (dopamine) or secondary (adrenaline and dobutamine) amino groups are protonated (catecholamines are oxidized in alkaline solutions at pH > 8). By analogy with amino acids, extraction may be described by the cation-exchange reaction ... 

Shvedene, N.V, Nemilova, M.Yu., Khachatryan, K.S., Mamonov, N.A., Shukhaev, A.V, Formanovsky, A.A., Pletnev, I.V, Extraction-voltammetric determination of catecholamines with the use of new-class solvents, ionic liquids, Moscow Univ. Chem. Bull., 45, 324-332, 2004. 

Tyramine acts as an indirect sympathomimetic to cause release of catecholamines from nerve terminals. It is present in a number of foods mature cheese, yeast extracts, some red wines, hung game, pickled herrings, broad bean pods. Normally, MAO-A in the intestinal mucosa will metabolise tyramine absorbed from the gut. In patients on the older MAOls, considerable amounts of tyramine will enter the circulation and this will lead to increased release of catecholamines stored in nerve terminals because the MAOI prevents their metabolism. For patients on RIMA drugs, high concentrations of tyramine can compete for MAO-A, thus mitigating some of the effects, and MAO-B is still available to metabolise noradrenaline (norepinephrine). MAO-B, however, has relatively much less effect on 5-HT and thus 5-HT function is still enhanced. 

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. 

URINE EXTRACTION. Neonatal urine (15 cm3 if the 24-hr volume was greater than 100 cm3 or 10 cm3 if less), normal adult urine (10 cm3), or urine of patients with suspected catecholamine secreting... 

Another naturally occurring drug that is similar to amphetamine can be found in the cactus Lophophora williamsii. Extracts are used to prepare a drink called peyote that contains 3,4,5-trimethoxyphenyl-ethylamine(the meth and phenyl point to a molecule that is quite lipid soluble). Known as mescaline, this compound is structurally similar to the catecholamines dopamine and norepinephrine but seems to act more directly upon serotonin receptors because of the presence of the meth-oxy groups on the molecule. This feature of the compound s structure would make the compound more fat-soluble and therefore better able to enter the brain quickly and may explain... 

Plasma malondialdehyde-like material, an indicator of lipid peroxidation, is increased in conditions of ischaemia, such as stroke [83, 84] and myocardial infarction [85]. Mitochondria extracted from hearts of vitamin-E-deficient rabbits showed a decreased mitochondrial function and an increased formation of oxygen radicals associated with a reduced superoxide dismutase activity. This was partially reversed by addition of vitamin E in vitro [86]. Measurement of in vitro susceptibility to lipid peroxidation in cardiac muscle from vitamin-E-deficient mice showed a highly significant negative correlation between the concentration of vitamin E and in vitro lipid peroxidation. The results indicate that short-term vitamin E deficiency may expose cardiac muscle to peroxidation injuries [ 87 ]. In rats, treatment for 2 days with isoprenaline increased lipid peroxide activity, as measured by malondialdehyde levels, in the myocardium. Vitamin-E-deficient animals were even more sensitive to this effect, and pretreatment with a-tocopheryl acetate for 2 weeks prevented the effect induced by isoprenaline. The authors [88] propose that free-radical-mediated increases in lipid peroxide activity may have a role in catecholamine-induced heart disease. 

In many cases, both a number of factors are varied in experiments and many response variables are measured. In such situations, there are often correlations between response variables. This suggests that it may be useful to try to find out to what extent the response variables can be independently affected by the factors and interactions between the factors. One way to analyse this problem is to use PCA or PLS and determine how many significant dimensions that can be extracted in designed studies. As an example, the influence of various mobile phase factors in a HPLC system for separating catecholamines and some catechol- and indolamine metabolites was investigated measuring the retention... 

Catecholamine neurotransmitters are subsequently inactivated by enzymic methylation of the 3-hydroxyl (via catechol-O-methyltransferase) or by oxidative removal of the amine group via monoamine oxidase. Monoamine oxidase inhibitors are sometimes used to treat depression, and these drugs cause an accumulation of amine neurotransmitters. Under such drug treatment, simple amines such as tyramine in cheese, beans, fish, and yeast extracts are also not metabolized and can cause dangerous potentiation of neurotransmitter activity. 

Fig. 3.11. HPLC of the hydroxylated products of salicylate (from Halliwell et al., 1988) 2,3-DHB, 2,3-dihydroxybenzoate 2,5-DHB, 2,5-dihydroxybenzoate RS, resorcinol SA, salicylate SU, salicylurate (a major metabolite of aspirin) CA, catecholamines, (a) Separation of a standard mixture of 2,3-dihydroxybenzoate, 2,5-dihydroxybenzoate and resorcinol (internal standard) (b) Separation of an extract of a plasma sample from a healthy individual not consuming aspirin (c) Separation of an extract of a plasma sample from a healthy individual consuming aspirin (the large peaks to the left of the chromatogram are probably catecholamines - marked CA).

Lee, M. et al. Selective sohd-phase extraction of catecholamines by chemically modified polymeric adsorbents with crown ether, J. Chromatogr. A. 2007, 1160, 340-344. 

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. 

Solid phase extraction. With the availability of pre-prepared cartridges of silica-based adsorbents, the use of solid phase extraction has increased in the last few years although the technique has been in use for many years for the isolation of many biochemicals, e.g. amino acids, catecholamines. In essence it is a version of chromatography conditions for the selective adsorption of the analytes (column, solvent, pH, etc.) are chosen, the sample is applied to a column, washed and the analytes selectively eluted with appropriate solvents. Since the columns are disposable there is no need to worry about protein contamination or infection. The adsorbents available cover an even wider range than HPLC materials since they are not required to withstand high back pressures. It is possible... 

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. 

P-Adrenergic receptors ((i-ARs) are members of the superfamily of G protein-coupled receptors that are stimulated by the catecholamines epinephrine and norepinephine (1). As part of the sympathetic nervous system, P-ARs have important roles in cardiovascular, respiratory, metabolic, central nervous system, and reproductive functions. Mice lacking one or more of the three p-AR subtype genes (P, p2, and p3) have been generated to elucidate the physiological role of individual subtypes. Moreover, cells and tissues extracted from these mice have been utilized as tools to understand the molecular and cellular basis of subtype-specific receptor function. These studies are summarized in this chapter. 

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. 

HPLC measurements of plasma catechols are usually limited to dopamine, norepinephrine, and epinephrine. However, with an alumina adsorption extraction procedure it is also possible to simultaneously measure several other catechols by HPLC or microchip electrophoresis. These catechols include DHPG, the deaminated metabolite of norepinephrine and epinephrine DOPAC, the deaminated metaboHte of dopamine and 3,4-dihydroxyphenylalanine (r-dopa), the immediate precursor of dopamine. All are present in plasma at concentrations many fold higher than the catecholamines, making their detection relatively simple once appropriate chromatographic separation is achieved. 

Earlier fluorometric methods for analysis of urinary free catecholamines have been replaced by HPLC methods that allow selective quantitation of epinephrine, norepinephrine, and dopamine. Preliminary extraction of urine is stid required and numerous preanalytical cleanup techniques are available. An alumina extraction procedure is typically coupled with ion-exchange or adsorption chromatography. Alumina pretreatment usually involves a batch extraction technique in which catechols are first adsorbed at pH 8.6 and then eluted with boric acid, which forms a complex with cis-diol groups. Purification on boric acid affinity gels provides an alternative procedure for selective adsorption of catecholamines. 

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