Tyrosine-ascorbate system

The most common cause of an elevation of the serum phenylalanine level about 3 mg dl is the transient neonatal delay in the development of the tyrosine oxidizing system (transient neonatal tyrosinemia). This condition, while far more common in low-birth-weight and premature infants, does occur occasionally in full-term infants as weU. The elevation of phenylalanine is secondary to a block in the metabolism of tyrosine. Administration of 100 mg of ascorbic acid will reduce the tyrosine levels, but whether this is necessary is an area of some controversy (Cohn and Roth 1983). 

L-Tyrosine metabohsm and catecholamine biosynthesis occur largely in the brain, central nervous tissue, and endocrine system, which have large pools of L-ascorbic acid (128). Catecholamine, a neurotransmitter, is the precursor in the formation of dopamine, which is converted to noradrenaline and adrenaline. The precise role of ascorbic acid has not been completely understood. Ascorbic acid has important biochemical functions with various hydroxylase enzymes in steroid, dmg, andhpid metabohsm. The cytochrome P-450 oxidase catalyzes the conversion of cholesterol to bUe acids and the detoxification process of aromatic dmgs and other xenobiotics, eg, carcinogens, poUutants, and pesticides, in the body (129). The effects of L-ascorbic acid on histamine metabohsm related to scurvy and anaphylactic shock have been investigated (130). Another ceUular reaction involving ascorbic acid is the conversion of folate to tetrahydrofolate. Ascorbic acid has many biochemical functions which affect the immune system of the body (131). 

Dopamine -hydroxylase is a copper-containing enzyme involved in the synthesis of the catecholamines noradrenaline and adrenaline from tyrosine in the adrenal medulla and central nervous system (see Figure 13.4). The active enzyme contains Cu+, which is oxidized to Cu + during the hydroxylation of the substrate. Reduction back to Cu+ specifically requires ascorbate, which is oxidized to monodehydroascorbate. 

Uchida, Suzuki, and Ichihara (878) isolated a soluble enzyme system (thereby possibly excluding mitochondrial participation) from rabbit liver, and partially purified it. Two enzymes were involved. The first of these converted p-hydroxyphenylpyruvic acid to 2,5-dihydroxyphenylpyruvic acid. If this enzyme was resolved, vitamin C alone did not restore the activity, but vitamin C and vitamin B12 did. The amount of B12 required was very low, and they suggested that the true enzyme was a Bw derivative, possibly aquocobalamin hydroxide bound to enzyme protein, and that the function of the ascorbic acid was solely to stabilize the reactive form of the coenzyme. This agrees with the work of La Du and Greenberg (524), who considered the role of ascorbic acid to be quite unspecific. Ascorbate increased the rate of tyrosine oxidation in liver preparations but the net consumption was zero, and moreover numerous ene-diols were just as effective on a molar basis. La Du and Greenberg considered that ascorbic acid participates in a cyclic oxidation-reduction and happens to be a substance of the correct oxidation-reduction potential either to participate directly or to protect some other participant. 

Noradrenergic and adrenergic neurons also contain the enzyme dopamine (i-hydroxylase. Like tyrosine hydroxylase, this enzyme is a mixed function oxygenase. The electron donor in this case is ascorbic acid rather than tetrahydrobiopterin, and dopamine is the primary substrate. The enzyme has been well characterized and is a tetramer of 75,000-Da subunits, which are copper-containing glycoproteins. No major regulatory systems are known for this enzyme. It appears that there are sufficient enzyme molecules present in cells in which this enzyme is expressed to completely convert all the dopamine that is formed into norepinephrine. 

Effect of coexisting species. The tolerable concentration ratios of foreign species on the determination of 10.0 pg of BSA in 10 mL of BSA-4MRASP system were as follows (the relative error less than 5 % fold) L-histidine, DL-aminoisovaleric acid, D-phenylalanine (60) L-leucine, DL-alanine (40) L-tryptophan, L-cysteine, L-tyrosine, DL-methionine (50) glycine (60) L-arginine (45) L-glutamic acid, L-lysine, L-methionine (70) DL-phenylalanine (100) ascorbic acid (200) Zn2+, Al3+ (20) K+, Fe2+ (30) Fe3+(5) Ca2+(20). 

Vitamin C is another enol-based redox system like the hydroquinones and tyrosine, but it has no aromatic character. The enediol component is stabilized by a conjugated lactone group. The oxidation (Ej = +58 mV) occurs in two one-electron steps, but the ascorbate radical is not as stable as the semiquinone radical (Bielski, and Richter, 1977). The anion radical disproportionates via an initial dimerization in a similar way as semiquinone radicals (Bielski et al., 1981 Sawyer, et al., 1982). The lifetime of the radical is in the order of microseconds. The acidity of ascorbic acid (pk = 4.2) stems from the OH group in the P position to the lactone carbonyl group. It corresponds to the OH group of a vinylogous carboxylic acid (Scheme 7.2.11). Its UV maximum occurs at 260 nm (e = 1 x 1(T). 

Ascorbic acid in this system is acting as an immediate hydrogen donator to the quinone and, in conjunction with the oxidase, confers the properties of a hydrogen carrier (reversible oxidation and reduction) on substances such as 3,4-dihydroxyphenylalanine. Tyrosine under such conditions serves as a reservoir for the supply of the dihydric phenolic derivative. 

The same reaction has been studied in a flow system coupled with a sensor-reference pair of iodide ion-selective electrodes but with the more efficient peroxidase catalyst substituted for molybdenumfVl) [386]. Maltose and cello-biose interfere while samples containing the more serious ascorbic acid, tyrosine or uric acid interferents require pre-treatment [386]. 

Ascorbic acid is required for many hydroxylase enzymes in the human body. Ascorbic acid is needed for conversion of tyrosine to the neurotransmitter dopamine and further hydroxylation to adrenaline and noradrenaline, for synthesis of carnitine from lysine, and probably for hydroxylation of steroid hormones. Ascorbate is also known to participate in hydroxylation of aromatic drugs and carcinogens via microsomal mono-oxygenase systems of Uver endoplasmic reticulum (31,32). Its role in the formation of collagen is thought to be to maintain iron in its ferrous state for an iron-dependent proline hydroxylase, or to act as a direct source of electrons for reduction of O2 (31). 

After purification of the enzyme preparation by treatment with chloroform, ascorbic acid is no longer effective, although the system is still activated by reduced dye. Thus the vitamin must act indirectly and ascorbic acid is not a true coenzyme for tyrosine oxidation. 

An example of a transient deficit that is the result of an immature enzyme system is a disorder in which the identification of increased concentrations of tyrosine and 4-hydroxyphenylpyruvic acid (4-HPPA) in urine of a premature newborn would suggest reduced activity of 4-HPPA oxidase. Administration of pharmacological doses of ascorbic acid, the cofactor required for activity of this enzyme, may overcome the temporary oxidase deficit and stimulate catabolism of the accumulating 4-HPPA. Figure 4 is an illustration of this disorder, which is termed transient neonatal tyrosinaemia. 

Fig. 11. Action of catalase on tyrosine hydroxylation by the Udenfriend system. Total volume 6 ml., containing 40 /imoles of ascorbic acid, and 20 fxmoles of tyrosine. Temperature 20°C., pH 7, 30 minutes. On the abscissae milligrams of catalase added. On the ordinates imoles of dopa formed (open circles) and /moles of ascorbic acid oxidized (solid circles). From Zito and Kertesz (1962).

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