Pyrimidines, urinary excretion

Several examples of prodrugs are found in the purine and pyrimidine analogs that substitute for natural nucleotides and inhibit nucleic acid formation. For example, 5-fluorouracil is essentially harmless to mammalian host and tumor cells. Upon administration, the drug is subject to one of two opposingmetabolicfates (10). Inactivation and elimination are accomplished by catabolism (about 80% of the dose) and by urinary excretion of unchanged drug... 

Classical orotic aciduria is a rare autosomal recessive disorder which is characterized by retarded growth and excretion of large quantities of orotic acid in the urine [221,222]. The disease was described in 1959 as an inborn error of pyrimidine biosynthesis in patients with crystals of orotic acid in the urine [223]. The urinary excretion of orotic acid by these patients was 1.34 g per day in contrast to approximately 0.014 g per day excreted by normal individuals [222,224]. When the diet of patients was supplemented with uridine, clinical remission and a remarkable reduction in orotic acid excretion took place [221,225,226]. 

The reduction in urinary excretion of both compounds following uridine therapy reflects the utilization of uridine for the formation of UMP by the salvage pathway. A similar phenomenon was observed in hereditary orotic aciduria following uridine replacement therapy which bypasses the congenital enzyme defect (Chapter 5). The reversal of 6-azauridine-induced orotic aciduria by hydroxyurea, methotrexate and cyclophosphamide [251] (i.e. by the drugs affecting the synthesis of DNA without any effect on orotic acid synthesis) suggests that the control of pyrimidine synthesis de novo is linked to DNA synthesis. 

The accumulation in blood and the excessive urinary excretion of orotic acid are postulated to result from the absence of either decarboxylase or the pyrophos-phorylase. These enzyme activities could not be directly assayed in the patient s tissues, but assays in the parents and two of the siblings indicated that the activities of both decarboxylase and phosphorylase were defective. In the patients described, the orotic aciduria and the megaloblastic anemia that it causes could be relieved by injecting nucleotides (cytidylic and uridylic acid). And in addition to its therapeutic significance, this observation also provides some invaluable information on the functioning of the pyrimidine metabolic pathway in vivo. 

At regular intervals the immune status of the patient was assessed as well as the serum content and the urinary excretion of purine- and pyrimidine metabolites according to methods described in detail previously (6,8,10). The (deoxy)ribonucleotide content of the erythrocytes was analyzed by HPLC Ul)- Perchloric acid extracts of freshly withdrawn blood were made according to Cohen et al. (2) with minor modifications. In order to analyze deoxyribo-nucleotides the neutralized perchloric acid extracts were treated with sodium periodate according to Garret and Santi (12). 2,3 Di-phosphoglycerate (2,3-DPG) in the erythrocytes was determined as previously described (13). Ecto-5 -nucleotidase on intact lymphocytes was determined as described(14). Adenosine deaminase activity of the lymphocytes was determined essentially according to van Laar-hoven et al. (15). 

The number of inherited defects of the pyrimidine metabolism described so far is small, compared to that of the purine metabolism. Combined deficiency of orotate phosphoribosyltransferase (OPRT) (EC 2.4.2.10) and orotidine 5 -monophosphate decarboxylase (ODC) (EC 4.1.1.23), designated as type I hereditary orotic aciduria, presents with characteristic clinical features such as hypochromic anemia with a megaloblastic bone marrow and crystalluria. Only six patients have been described and, as far as we know, new cases have not been discovered recently. ODC deficiency with similar clinical phenomena and leading to increased urinary excretion of orotate and orotidine has been detected in only one patient (1). A third defect, a deficiency of pyrimidine 5 -nucleotidase (Py-5NX (EC 3.1.3.5.) in erythrocytes, is associated with chronic hemolytic anemia and prominent basophylic stippling of the erythrocytes due to accumulated pyrimidine nucleotides. An increasing number of patients have been reported, their detection being facilitated by the typical phenomena. We do not know whether the urinary pyrimidine profile in this condition is abnormal. 

J.W. Stoop and S.K. Wadman, Urinary excretion of orotic acid, orotidine and other pyrimidines in a patient with purine nucleoside phosphorylase deficiency, Clin. Chim. Acta, 93 ... 

At the present time, we just report some experimental results of a study on the mechanism of action of allopurinol (U-hydroxy-pyrazolo (3, -d ) pyrimidine) and thiopurinol k thiopyrazolo (3, d) pyrimidine) on de novo biosynthesis of uric acid. In this present work, we have compared effect of alio and thiopurinol on oxypurine (xanthine and hypoxanthine) urinary excretion with their rate of synthesis of ribonucleotides in vitro by erythrocyte hemolysate in some particular enzymatic deficiencies (hypoxanthine-guanine phosphoribosyltransferase HGPRT, adenine phosphoribosyl-transferase APRT and xanthinuria). 

Loading tests, based on the increment in the urinary excretion of different pyrimidines have been used to confirm DPD, DHP or UP deficiency before methods for determination of the activity of the relative enzymes were available. Moreover, they still are important to determine the in vivo status, and to determine the carrier status for urea cycle disorders, but are not helpful in any other disorders in this group [17]. 

Thiamine, or vitamin Bi, is a water-soluble compound which is rapidly broken down by moist heat in neutral or alkaline solutions into its constituent pyrimidine and thiazole rings. The ready destructability of thiamine is important in human nutrition, since much may be lost in the preparation of food. Some of the biochemical methods used in evaluating thiamine nutrition are based on reactions with the thiazole and pyrimidine portions of the thiamine molecule. The thiochrome method is widely used in assaying biological materials for thiamine, while determination of the urinary excretion of pyramine (a pyrimidine-like compound) has been used to assist in assessment of nutritional status. 

Since allopurinol blocks xanthine conversion to uric acid, urinary xanthine excretion is increased, creating a risk of xanthine crystal formation in the urinary system or even in muscles this can result in nephrolithiasis (12). It is still an open question whether a predisposition to renal disease or renal disease itself is required to precipitate these adverse effects. It is also not known whether increased excretion of orotic acid, due to an interaction of allopurinol with pyrimidine formation, has any consequences for these adverse effects or for its role in reducing glucose tolerance. 

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). 

Carriers of OCT deficiency (estimated to be several thousand women in the U.S. A.) can be identified by administration of a single oral dose of allopurinol, a purine analogue, followed by measurement of urinary orotidine excretion. The underlying principle of this assay is that when the intramitochondrial carbamoyl phosphate accumulates in OCT heterozygotes, it diffuses into the cytoplasm stimulating the biosynthesis of pyrimidines. One of the intermediates in this pathway—orotidine—accumulates, leading to orotidinuria (Figure 17-8). 

Elevated concentrations of orotic acid and uracil may be found in the urine of heterozygote carriers for urea cycle disorders, most commonly ornithine carbamoyltransferase (OCT) deficiency. This is due to the increased flux through the pyrimidine pathway which occurs, especially where the relevant enzymes are expressed only in liver tissue, as is the case when urea cycle enzymes are defective. A protein load was used previously to stress this route and the elevation in orotic acid excretion used as a diagnostic marker. However, the test frequently failed to detect known carriers. The al-lopurinol loading test (measurement of the increment in urinary orotic acid and orotidine in three separate 8 hour urine collections over the 24 hours following a 300 mg allopurinol tablet) is the most reliable test so far for carrier detection for such disorders [17]. 

Urinary pyrimidines and uric acid. Administration of PF was followed by the excretion of a large amount of orotic acid and orotidine. Fig. 2 shows the total ion chromatogram of the urine of a patient 2 days after PF infusion started (250 mg/m ). The peaks corresponding to mass spectrayA 230 and 258 on the abscissa are hippuric and citric acid, respectively. In the control urine... 

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