Tyrosine, fluorometric determination

Amines and amino acids were extracted as described by Tachiki and Aprison (1975). Tissues were homogenized in 2 ml of acidified-n-butanol and centrifuged at 1500 x g for 30 min. (4 C). A 1.0 ml aliquot of supernatant was returned to a second set of tubes containing 4.0 ml n-heptane and 0.4 ml O.IN HCl. This mixture was shaken for 10 min. and the layers cleanly separated by centrifugation at 1000 x g for 5 min. The organic layer was removed and 5-HIAA content was determined by the OPT fluorometric method. Serotonin (in the O.IN HCL) was assayed by the OPT fluorometric method while tryptophan was determined fluorometrically as described by Denckla and Dewey (1967). Tyrosine was determined flurometrically by the method of Waalkes and Undenfriend (1957). 

Determination of oxidized amino acids in urine is usually performed by isotope dilution gas chromatography-mass spectrometry (L9). DOPA is estimated by HPLC separation of acid protein hydrolysates with fluorescence detection (excitation 280 nm, emission at 320 nm) (A15). Other methods are based on borate-hydrochloric acid difference spectroscopy (this method suffers interference from tyrosine and tryptophan) (W2), derivatization of DOPA with nitrite and subsequent coulometric determination (W3), and fluorometric detection after derivatization with ethylenediamine (A15). 3-Hydroxylysine is quantitated by HPLC with 9-fluorenylmethyl chloroformate precolumn derivatization (M25) of amino acids obtained by gas-phase hydrolysis of proteins (F21). Other general methods to detect amino acid damage are mass spectometry methods applied to protein hydrolysates, such as tandem mass spectrometry (F6). 

One of the most useful applications of fluorescence is in the routine determination of certain important molecules in body fluids for diagnostic purposes. Some such molecules are naturally fluorescent, but others must be chemically treated to form fluorescent products. For example, the amino acids tyrosine, tryptophan, and phenylalanine are all measured fluorometrically. Both tyrosine and tryptophan possess aromatic rings that absorb intensely and therefore have an intense natural fluorescence. Tyrosine is excited at both 225 and 280 nm, and emits at 303 nm tryptophan is excited at 220 and 280 nm and emits at 438 nm [34]. 

The enzymatic method described above has two disadvantages (1) trapping of CO2 is a cumbersome procedure, and (2) the use of a radioactive substrate requires special precautions for use and disposal of reagents. Measurement of the primary amine formed by decarboxylation of the amino acid can also be exploited to monitor the PLP-dependent, enzyme-catalyzed reaction. This principle has been applied by Allenmark et al. (106), who used L-3,4-dihydroxyphenyl-alanine (L-DOPA) as substrate for tyrosine decarboxylase the dopamine produced by the decarboxylation reaction was determined by HPLC followed by amperometric detection. Both Hamfelt (107) and Lequeu et al. (108) utilized apo-tyrosine decarboxylase with tyrosine as substrate. The tyramine produced by the decarboxylation reaction was separated from the substrate (tyrosine) by HPLC and quantitated by either amperometric (108) or fluorometric (107) detection. The procedures discussed above are still subject to the main disadvantage of enzymatic methods possible interference by other materials present in the PLP containing extract which could either inhibit reconstitution of the holoenzyme or alter the reaction rate of enzyme catalysis. Moreover, HPLC with amperometric detection can hardly be described as less cumbersome than CO2 trapping difficulties in baseline-stabilization encountered with these detectors are well known. 

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See also in sourсe #XX -- [ ]

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