Amines extraction from tissues

Although water hydrolyses acetic anhydride, amino groups and phenolic hydroxyls react much faster, and can therefore be acetylated in aqueous solution. This is the basis of the Schotten-Baumann conditions the reaction is driven by addition of sodium bicarbonate to remove the acid formed. The stirred aqueous solution of amines (or amine extract from tissues or biological fluids), totalling 4 ml, is treated with acetic anhydride (0.3 ml). A slight molar excess of sodium bicarbonate is added in small portions, letting effervescence subside each time. At the end of the reaction the products are extracted with dichloromethane (2x5 ml), and the combined extracts are blown down in a stream of nitrogen [25-27]. Similar procedures have been described for phenols [28, 29]. 

As an alternative to RP columns some authors used cation-exchange columns. Salazar et al. [301] used an Alkion column (4x 150mm) heated at 40°C to separate OPA derivate of biogenic amines extracted from complete feeds and animal tissues, while Saccani et al. [302] used an lonPac CS17 column and a gradient elution of methanesulfonic acid to separate biogenic amines in meat products. 

Having accomplished the homogenization of the sample, extraction of free amino acids is usually a simple process of stirring the sample in an appropriate solvent. This is typically a dilute solution (0. IN) of hydrochloric acid (2,7). Elevated temperatures may be used to assist dissolution, but care must be taken not to damage the more acid-labile amino acids. More recently, Moret and Conte (8) report that 0.1N HC1 was not a suitable solvent for all types of food samples. For some types of meat samples, 5% trichloroacetic acid is reported to afford superior performance (for biogenic amines, also amino acids ). It is not unusual to see perchloric acid employed for extraction from meat tissues (also more commonly for biogenic amines). 

Departing from these results, Eritamann and Melik-Sarkissyan (123) succeeded in reproducing the amination of FU in cell-free extracts from fresh or acetone-dried liver tissue of various animala. The complete system includes diluted (or dialysed) liver ensyme extract, ammonium pyruvate, cosymase, small amounts of fumarate or OA, phosphate buffer and some ccmcentrate of co-os-aph. The amounts of NHrN synth ... 

Tyramine, p-hydroxy phenylethylamine, HO.CgHj.CHjj.CHa.NHg, the amine derived from tyrosine, is the chief pressor base found in some extracts of ergot, in putrefied animal tissues, and in ripe cheese. 

The problem with the silylation of different biogenic amines is that it is difficult to prepare uniform derivatives as these compounds usually contain amino groups of different reactivity, hydroxyl and other functional groups. Several procedures have been proposed for solving this problem. The determination of norepinephrine and dopamine in brain tissue was described by Maruyama and Takemori [90]. Dried residue from the extraction... 

Follicle-Stimulating hormone-releasing hormone (FSH-RH). FSH-RH was discussed at this meeting by Dr. McCann and Dr. Jutisz. Our preparations of porcine and bovine FSH-RH seem to be considerably more potent than their preparations of ovine FSH-RH and are also free of LH> H. A complete separation of FSH-RH from LH-RH was achieved by preparative column electrophoresis (3, 18, 19). Thus fractions which release LH and have no effect on FSH secretion and conversely fractions which release FSH and have no effect on IM secretion. Highly purified FSH-RH at doses of 4-10 nanog depletes pituitary FSH content in castrated male rats pretreated with testosterone (3, 19). FSH-RH will also deplete pituitary FSH in normal male rats (3, 19). Some amines such as putrescine are also capable of depleting pituitary FSH content in vivo (20). Mittler and Meites (36) were the first to demonstrate the stimulatory effect of rat hypothalamic extracts on the release of FSH in vitro. e have shown by in vitro experiments that purified porcine FSH-RH stimulates the release of FSH by a direct action on pituitary tissue (21). Thus, addition of partially purified FSH-RH, but not of histamine, greatly enhanced the release of FSH. 

The previously described methods necessitate the extraction of the catecholamines from the tissue prior to assay. The histochemical fluorescence technique allows the visualization of the amines in situ, but it is not an accurate quantitative procedure. Freeze-dried sections of tissue are exposed to formaldehyde vapour at SO C for 1 hr or more. The catecholamines are thereby converted to hydroxyiso-quinoline derivatives (Fig. 1), which fluoresce strongly under U-V light in the fluorescence microscope (Fig. 1, p. 110). ADR and other secondary amines can be distinguished from NA, DA and DOPA by their slower rate of reaction with formaldehyde. 

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