Adrenaline assay

The name adrenochrome was proposed in 1937 by Green and Richter for the unstable red crystalline product obtained on enzymatic oxidation of adrenaline [22], In the same year Richter and Blashko obtained a crystalline iodo-derivative of adrenochrome by the oxidation of adrenaline with potassium iodate [23]. Thus the product responsible for the violet chromogen utilised in one of the original adrenaline assay procedures [10], was only isolated and characterised 30 years after the procklure was first described. 

Stopped-flow measurements with superoxide in aqueous solution at physiological pH are not possible due to its fast self-dismutation under these conditions. Therefore, the indirect assays such as McCord-Fridovich, adrenalin and nitroblue tetrazolium (NET) assays are widely used in the literature, not only for qualitative but also for quantitative detection of SOD activity of small molecular weight mimetics 52). Not going into details, we just want to stress that the indirect assays have very poor even qualitative reliability, since they can demonstrate the SOD activity of the complexes which does not react with superoxide at all. It has been reported in the literature that this is caused by the interference of hydrogen peroxide 29). We have observed that the direct reaction between complexes and indicator... 

Non-aqueous titration with acetous perchloric acid is used in the pharmacopoeial assays of adrenaline, metronidazole, codeine, chlorhexidine acetate, chlorpromazine.HCl, amitriptyline.HCl, propranolol.HCl, lignocaine.HCl and quaternary amine salts such as neostigmine bromide and pancuronium bromide. 

Adrenaline in bupivacaine/adrenaline injection is assayed by complex formation with iron (II). 20 ml of the injection is mixed with 0.2 ml of reagent and 2 ml of buffer and a reading is taken in a 4 cm pathlength cell. A reading of a solution containing 5.21 pg/ml of adrenaline is taken under the same conditions. 

Assay of adrenaline by chromatography with an anionic ion-pairing reagent... 

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 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... 

In the assay by Trocewicz et al. (1982), the enzyme phenylthanolamine N-methyltransferase catalyzes the conversion of noradrenaline (NA) to adrenaline (AD). 

In the assay described by Beaudouin et al. (1993), the phenylethanolamine AT-methyltransferase catalyzes the transfer of a methyl group from S-adenosyl-L-methionine to noradrenaline to form adrenaline and S-adenosyl-L-homocys-teine as the final step of adrenaline biosynthesis. Adrenaline is mainly synthesized in the adrenal medulla. 

Another possibility is that some other clotting factor is increased or activated in such a way that the assay system responds fortuitously to it in a way indistinguishable from the usual response to factor VIII, e.g., factors XI and XII factor XII is known to rise on exercise (II). That this might occur over a limited range of the dose-response curve in the thromboplastin generation test system was shown by experiments in which the addition of activation product (Wl) simulated an increased factor VIII concentration (author s unpublished observations, 1960 FI), although statistical invalidity would probably be detectable over a series of experiments if this were the explanation. This also was looked for, but was not found (14). It is interesting that, in a patient with severe factor VIII deficiency and partial factor XI deficiency (SI), adrenaline infusion was followed by a marked rise in factor XI concentration and the appearance of a trace of factor VIII (K. Schulz, personal communication, 1964). Furthermore, the confusion that arose some years ago over factor IX assay now seems to have been due to activation of the contact factors (P4), hence... 

However, the exercise effect has been demonstrated with assays involving (supposedly maximal) activation of factors XI and XII with kaolin (13), and a system to which activation product was added (VI) duly registered the adrenaline effect. Furthermore, a rise in factor VIII concentration has now been produced by exercise in patients with severe deficiencies of factors XI and XII. Egeberg (E13) tested two patients with gross deficiencies of factor XI, and Goudemand et al. (G2) and A. Parquet-Gemez (personal communication, 1964) tested factor Xll-deficient patients. 

Evidence was given above (Section 3.4.1) that the exercise or adrenaline effect was demonstrated by assay systems in which precautions had been taken to provide full surface activation. The comparative data of both Ikkala (12, 13) and Ingram (15) show, however, that the effects were more marked when these precautions were not taken, and Ikkala (12) also showed that the effect was more marked when phospholipid was not added to the system, although it was still clearly seen in its presence. The reduction in the effect either by surface contact or by phospholipid, or both (12), raises the suspicion that if even more efficient agents had been added to the assay the effect would have been eliminated altogether, but so far this has not been achieved. 

Proof of the generation of Or during electrolysis of aqueous solutions was obtained in the laboratory of Fridovich (206). The OI catalysed oxidation of adrenaline served as a monitor which could be inhibited by superoxide dismutase. Ultrasonication of buffered aerated solutions gave rise to the formation of Or which could be detected using the cytochrome-c reductase assay (207). This sonication induced cyto-chrome-c reduction was also inhibited by native erythrocuprein. Another sensitive superoxide dismutase assay using the reduction of nitro blue tetrazolium by Or was developed by Beauchamp and Fridovich (208). This assay allowed the detection of erythrocuprein in the ng/ml region. During the metalloenzyme conference in Oxford, 1972, Fridovich summarized the basic facts on superoxide dismutase (erythrocuprein) (209). 

The oxidation of catecholamines like epinephrine has been widely used as source for superoxide dismutase assays. Upon oxidation the catecholamines are transformed to the coloured product adrenochrome. The rate of oxidation by superoxide is inhibited in the presence of superoxide dismutases Likewise the autoxidation of catecholamines at alkaline pH-values is diminished Intriguingly, low molecular mass copper complexes which display superoxide dismutase activity accelerate the autoxidation Therefore, the interaction between superoxide and catecholamines and its inhibition by SOD is thought not to be a simple chemical reactionRecently, this reaction was investigated in more detail Whilst adrenalin autoxidation is very specifically inhibited by SOD, the reaction with other catecholamines like noradrenalin or dihydroxyphenylalanine, having no free amino group, is much less specific. Only 20 % inhibition by CujZnjSuperoxide dismutase are observed. The autoxidation reaction itself is very complex (Scheme 2) and still not fully understood. 

The detection limit of the autoxidation assay is 0.5 nM superoxide dismutase The oxidation of adrenalin is followed at 480 nm spectrometrically. 850 pi 100 mM carbonate buffer, pH 10 and 100 pi water or sample are mixed. The reaction is started with 50 pi catecholamine stabilized at pH 2. AA/min is followed. Due to its convenience, this method can be employed in homogenates and other biological samples However, the assay can interfere with reduced glutathione, causing an... 

In a series of studies, Sliwa [146-149, 151] demonstrated mouse sperm accumulation in acetylcholine, adrenaline, calcitonin, -endorphin, oj ocin, and substance P. Negative mouse sperm accumulation (i.e., apparent repulsion) was demonstrated with glucagon and vasopressin. However, since only a single assay was used in these studies (a choice assay that did not distinguish between chemotaxis and trapping), the significance of these observations with respect to chemotaxis is not clear. 

This A-methyltransferase is responsible for the conversion of noradrenaline to adrenaline. It may be assayed radiochemically using labelled 5-adenosylmethionine. 

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