Uridine, pyrimidine metabolism

The broad spectrum of clinical presentation highlights the importance of particular steps in purine and pyrimidine metabolism to different cells and tissues and should have assisted in the development of appropriate treatment. Unfortunately, only three of the nineteen disorders described can be treated successfully hereditary orotic aciduria with life-long uridine, 2,8-di-hydroxyadenine lithiasis with allopurinol. ADA deficiency is treatable by bone marrow transplantation (BHT), or enzyme replacement with polyethylene glycol (PEG)-ADA, but the cost is prohibitive. Er/throcyte-encapsu-lated ADA is effective and less expensive. Oral ribose is reportedly beneficial in myoadenylate deaminase deficiency [1, 4] and also in adenylosucci-nase deficiency [1, 5]. PNP deficiency is also treatable by BMI. 

Fig. 23.1. Pyrimidine pathways Pathways for the de novo synthesis, interconversion, and breakdown of pyrimidine ribonucleotides, indicating their metabolic importance as the essential precursors of the pyrimidine sugars and, with purines, of DNA and RNA. Note that in contrast to purines salvage takes place at the nucleoside not the base level in human cells and pyrimidine metabolism normally lacks any detectable end-product. The importance of this network is highlighted by the variety of clinical symptoms associated with the possible enzyme defects indicated. 23.10, Uridine monophosphate synthase (UMPS), 23.11a, uridine monophosphate hydrolase 1 (UMPHl), 23.12, thymidine phosphorylase (TP), 23.13, dihydropyrimidine dehydrogenase (DPD), 23.14, dihydropyrimidine amidohydrolase (DHP), 23.15, y -ureidopropionase (UP) (23.11b, UMPH superactivity specific to fibroblasts is not shown). CP, carbamoyl phosphate. The pathological metabolites used as specific markers in differential diagnosis are highlighted...

The fact that many agents which interrupt the synthesis of pyrimidine nucleotides from orotic acid in animals can also inhibit the growth of experimental neoplasms suggests a search for additional antimetabolites whose locus of action is in this metabolic sequence. Two in vitro biological screening systems were developed for this purpose [202—207]. From a study of systems with oxidative energy sources, 5-bromo-[208—209] (Villa), 5-chloro-[210] (Vlllb) and 5-diazo-orotic acid [211] (IX) were found to inhibit the conversion of orotic acid to the uridine nucleotides by 40—100 per cent [202]. 

Leflunomide is a potent noncyto toxic inhibitor of the proliferation of stimulated B and T-lymphocytes, which depend on de novo pyrimidine synthesis to fulfill their metabolic needs (Breedveld and Dayer, 2000 Herrmann et al., 2000). This antiproliferative effect can be antagonized in vitro by the addition of uridine or cytidine to the cell culture medium, underscoring the significance of this mechanism of action (Cao et al., 1995 Williamson et al., 1995 Zielinski et al.,... 

In hereditary orotic aciduria, orotic acid is excreted in the urine because the enzymes that convert it to uridine monophosphate, orotate phosphoribosyl transferase and orotidine 5 -phosphate decarboxylase, are defective (see Figure 7-20). Pyrimidines cannot be synthesized, and therefore, normal growth does not occur. Oral administration of uridine bypasses the metabolic block and provides the body with a source of pyrimidines. 

Certain metabolites of the pyrimidine pathway are excreted in excess of the normal in inherited metabolic disorders of the urea cycle. They include orotic acid, uridine, and uracil. Of these substances, only uracil is a normal constituent of urine, the other two either being absent or present in very small amount. They are readily detected qualitatively as dark bands at the appropriate Rf values when a paper chromatogram of the urine is examined under ultraviolet light. They may be estimated by an ion exchange method similar to that for urinary pseudouridine (R13). 

Formation of starting materials for cell wall synthesis begins with two metabolic substances normally found in all life forms N-acetylglucosamine 1-phosphate and the pyrimidine nucleotide uridine triphosphate (UTP) (see Fig. 6-3). Condensation of these two compounds by elimination of pyrophosphate affords uridine-diphospho-N-acetylglucosamine (UDPNAG). Reaction with phosphoenolpyruvic acid (PEP, the activated form of the enol tautomer of pyruvic acid),5 catalyzed by a specific transferase, yields the 3-O-enolic ether. 

Consistent with the metabolic data, there is no dihydrofolate reductase/thymidylate synthase activity (75). Thymidine is salvaged by a phosphotransferase. Uracil PRTase, uridine phosphorylase, cytidine deaminase and uridine and cytidine phosphotransferases were found. The major pyrimidine salvaged is uracil via its PRTase. The lack of incorporation of salvaged uracil into DNA and the lack of thymidylate synthase indicates that this parasite cannot synthesize TMP from UMP. It is dependent on salvage for its thymidine requirements. This parasite possesses a hydroxyurea-resistant ribonucleotide reductase and can synthesize deoxycytidine nucleotides from cytidine nucleotides. 

Trypanosoma brucei complex. African trypanosomes of the Trypanosoma brucei complex metabolize pyrimidines in a manner similar to that of Leishmania and T. cruzi (Fig. 6.17). All six enzyme activities for the synthesis of UMP were detected in homogenates of blood trypomastigotes of T.b. brucei (87). In addition, uracil PRTase, cytidine deaminase, and pyrimidine cleavage activities have been detected in cell-free homogenates no uridine kinase activity was detected (94). 

Orotidine 5 -phosphate decarboxylase (ODCase, E. C. 4.1.1.23) catalyzes the decarboxylation of orotidine 5 -phosphate (OMP) to form uridine 5 -phos-phate in the sixth and final step of pyrimidine biosynthesis (Fig. 1) [1]. The discovery of ODCase in 1954 followed the identification, three years earlier, of orotic acid as the metabolic precursor of nucleic acids [2, 3]. ODCase is a distinct, monofunctional polypeptide in bacteria and fungi, whereas in mammals it combines with orotate phosphoribosyltransferase (OPRTase) to form the bifunctional enzyme UMP synthase. Human deficiencies in either OPRTase or ODCase activity result in an autosomal recessive disorder called hereditary orotic aciduria [4]. The disease is characterized by depleted levels of pyrimidine nucleotides in the blood and by the appearance of crystalline... 

Biosynthesis de novo is the initial regulatory level to be considered. The first complete nucleotide to be formed in the purine biosynthetic pathway is inosine-5 -phosphate (IMP), and that in the pyrimidine pathway is uridine-5 -phosphate (UMP). The two pathways are operationally separate and distinct and metabolically related only in that they share some common participants such as glutamine, CO2,... 

In summary, it is proposed that the most hopeful prospect for therapy of this condition would be to search for a specific inhibitor of orotate transport across cell membranes. This is likely to affect the metabolism of pyrimidines in only two tissues, liver and erythrocytes, and, unless it gives rise to toxic accumulations of orotate in the liver, could help to reduce pyrimidine nucleotide levels in pyrimidine 5 nucleotidase deficient erythrocytes. A trial of a specific inhibitor of uridine kinase might also be appropriate, and the final choice of a therapeutic approach will depend on the demonstration of which of these two alternative pathways amenable to therapeutic intervention contributes most to erythrocyte pyrimidine nucleotide accumulation. 

The final steps of pyrimidine biosynthesis novo which are catalyzed by two sequential enzymes, orotate phosphoribosyltransfer-ase (OPRT) and orotidylic decarboxylase (ODC), involve the PP-ribose P dependent conversion of orotic acid to orotidine-5 -monophosphate (OMP) followed by decarboxylation at the 7 position to form uridine 5 -monophosphate (UMP) (Fig. 1). UMP is then utilized further in the synthesis of nucleic acids and co-enzymes. Defects at this site in this metabolic pathway are important for they can result in "pyrimidine starvation" from depletion of the intracellular pool of pyrimidine nucleotides. In man the rare genetic disease, orotic aciduria, involves a deficiency of both OPRT and ODC (Type 1) (Smith, Sullivan and Huguley, 1961) or, less commonly, only ODC (Type II) (Fox, 0 Sullivan and Firken, 1969). 

In addition to the role of orotic acid and/or orotidine in pyrimidine biosynthesis, orotic acid may be involved in some other aspects of intermediary metabolism. This is indicated by the recent finding of Hulbert, who has shown that labeled orotic acid, in addition to forming labeled uridylic acid in rat liver, yielded another uridine-type compound which contains both stable and labile phosphorus. 

In the nucleic acids (DNA and RNA sections 9.2.1 and 9.2.2 respectively) it is the purine or pyrimidine that is important, carrying the genetic information. However, in the link between energy-yielding metabolism and the performance of physical and chemical work, what is important is the phosphorylation of the ribose. Although most reactions are linked to adenosine triphosphate, a small number are linked to guanosine triphosphate (GTP see, for example, sections 5.7 and 9-2.3-2) or uridine triphosphate (UTP section 5.5.3). 

In addition to their role as components of nucleoproteins, purines and pyrimidines are vital to the proper functioning of the cell. The bases are constituents of various coenzymes, such as coenzyme A (CoA), adenosine triphosphate (ATP), guanosine triphosphate (GTP), cytidine triphosphate (CTP), diphosphopyridine nucleotide (DPN), triphosphopyridine nucleotide (TPN), and flavin adenine dinucleotide (FAD). A pyrimidine derivative, cytidine diphosphate choline, is involved in phospholipid synthe another pyrimidine compound, uridine diphosphate glucose, is an important substance in carbohydrate metabolism. Cytidine diphosphate ribitol functions in the biosynthesis of a new group of bacterial cell-wall components, the teichoic acids. While mammals excrete nitrogen derived from protein catabolism in the form of urea, birds eliminate their nitrogen by synthesizing it into the purine compound, uric acid. 

Relatively little is known about the metabolism and biological action of PF, mainly because of the lack of adequate analytical methodology. In reversal studies of the PF-induced inhibition of vaccinia virus vitro (2), it was revealed that the activity of PF is reversed by uridine and uridylic acid, but not by cytidine or orotidine. This led to the suggestion (5) that the mode of action of PF might be the inhibition of the dje novo biosynthetic pathway of pyrimidine nucleotides. The most likely mode of action of PF is shown in Fig. 1. 

Orotidine and its derivatives play an important role as intermediates in the metabolism of pyrimidine-nucleotides [91]. Its structure is shown in Figure 8 (top, left) it is closely related to uridine (see Figure 1), but due to the (C6)-carboxylate group it exists in solution mainly in the syn conformation [89]. The (C6)COOH group is very acidic for aqueous solution it was estimated that pATa = 0.5 0.3 [92]. Consequently, the stability constants of the orotidinate (Or ) complexes of Mg ", Cu ", and Zn " (only these metal ions have been studied [92]) are somewhat below of those measured for the corresponding M(Ac) complexes (see Table 1, column 7). There is no evidence for any significant chelate formation in aqueous solution [92]. Therefore, one may assume that all this also holds for the Cd(Or) complex, which gives as an estimate for its stability log cd(Or) = 1-0 0-3-... 

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