Isobarbituric Acid Research Articles

UV-Photolyse von 5-Joduracil / UV Photolysis of 5-Iodouracil

UV Photosylis (254 nm) of 5-iodouracil in aqueous, oxygenated solution in presence of KI, FeSO4, KBr and K2SO4 was investigated over a pH-range of 1—6. Uracil, isodialuric acid, and isobarbituric acid were identified as photoproducts. At a constant pH-value (pH=1) at high KI-concentrations (10-1 mole/1) the main product is uracil. With decreasing KI-concentrations the yield of uracil decreases and isobarbituric acid appears as a photoproduct. On further decreasing KI-concentrations (< 10-2 mole/1) the yield of isobarbituric acid decreases and the formation of isodialuric acid commences. UV Photolysis of iodouracil at constant KI-concentrations (10—1 mole/1) and increasing pH (pH = 1—6) yields similar results with one exception. At higher pH-values on account of isodialuric acid at least four further compounds are formed. The yield of these products increases with increasing pH value. Addition of KBr and K2SO4 has no effect on the product distribution. The possible mechanisms for the formation of the reaction products are discussed.

Inhibition of methotrexate resistant mutant of Streptococcus faecalis by analogs of purine and pyrimidine.

Purine analogues, 8-azaguanine (2.5 µg/ml); 6-thio- guanine (5 µg/ml); 6-mercaptopurine (50 µg/ml); 6- chloropurine (50 µg/ml); and 2,6-diaminopurine (50 µg/ml); inhibited the thymine growth response of Streptococcus faecalis Akt., a Methotrexate (Lederle trade name for 4-amino-N10-CH3-folic acid) resistant mutant of S. faecalis. However, these same concentrations of the purine analogues did not influence the thymine growth response of the parent strain of S. faecalis. The inhibitory effect of 8-azaguanine or of 6-thioguanine on S. faecalis Akt., was of a competitive nature and was reversed by 5 µg/ml each of adenine or guanine or 5 µg/ml each of the nucleosides or nucleotides of adenine or guanine. Pyrimidine analogs 2-thiouracil (100 µg/ml); dithiothymine (500 µg/ml); and isobarbituric acid (100 µg/ml); inhibited the thymine growth response of S. faecalis Akt., but did not influence the thymine growth response of S. faecalis. The inhibitory effect of isobarbituric acid on the thymine growth response was reversed by cytosine (50 µg/ml); uracil (50 µg/ml); deoxycytidine (50 µg/ml); thymine (50 µg/ml); or thymidine (50 µg/ml). The results suggest that the S. faecalis Akt. has acquired de novo purine and pyrimidine biosynthetic pathways and is therefore inhibited by the analogs at lower concentrations. It may be speculated that clinical resistance to methotrexate may be associated with the acquisition of de novo purine biosynthetic capabilities in methotrexate-resistant cells and this opens a new avenue of attack on methotrexate-resistant leukemia.

Radiolysis of 4-thiouracil and of the 4-thiouracil moieties of 4-thiouridine and transfer ribonucleic acid from Escherichia coli in aqueous solutions

4-Thiouracil, 4-thiouridine and tRNA from Escherichia coli were irradiated with X-rays in air-saturated solutions and the radiolysis of the 4-thiouracil moieties was determined spectrophotometrically. The dose-effect curves were exponential, giving initial yields of destruction ( G value) of 3.22 for thiouracil, 2.45 for thiouridine and 0.31 for the thiouracil residues in tRNA. The thiopyrimidine residues were more radiolabile than the corresponding oxygen analogues, as the G values found for uracil and uridine under the same conditions were 2.28 and 1.83, respectively. The following radiolysis products were demonstrated in irradiated thiouracil solutions: uracil, thiouracil disulfide, isobarbituric acid, di-isobarbituric acid and inorganic sulfate. Radiolysis products identified in irradiated thiouridine solutions were uridine, thiouridine disulfide and sulfate.

The Response of Uracil-2- 14 C to X-Irradiation under Nitrogen and Oxygen and to Treatment with Ascorbic Acid

The following compounds were produced when solutions of $\text{uracil-}{}^{14}{\rm C}$ were irradiated with X-rays: dihydrouracil, 6-hydroxy-5-hydrouracil (under nitrogen but not oxygen), isobarbituric acid, cis- and trans-glycols of uracil, alloxan, alloxantin, and dialuric acid. When uracil was treated with ascorbic $\text{acid}-{\rm FeSO}_{4}$, the results were similar in many respects to those obtained after X-irradiation except that more dihydrouracil was produced relative to the more oxidized products (glycols, etc.), as compared to the results after X-irradiation. Auto-irradiation of an aged sample of $\text{uracil-}{}^{3}{\rm H}$ also mimicked in certain respects the effect of X-irradiation on uracil. The major products identified were the cis- and trans-glycols of uracil.

Polarographic study of the effects of γ-radiation on cytosine

Polarographic studies of the effects of γ-radiation on cytosine supplementing the Ekert-Monier scheme showed that isobarbituric acid undergoes further radiation-induced reactions. The temporary yellow-green coloration and the green fluorescence were ascribed to the formation of 4,4′-diisobarbituric acid. Further radiolytic products of isobarbituric acid were found to be isodialuric and dialuric acids. The very easy oxidation of dialuric acid leads to the formation of alloxan and subsequently to the system dialuric acid + alloxan ⇌ alloxantin. With increasing radiation dose, parabanic acid is also formed, probably as an oxidative product of alloxanic acid formed by a benzilic acid type of rearrangement of alloxan. However, parabanic acid undergoes hydrolysis, giving rise to oxaluric acid. Above a radiation dose of 5 × 10 20 ev ml −1, the over-all effects are ring opening and hydrolytic formation of simple aliphatic compounds, the solution again turning colorless.

Decomposition of pyrimidines by Nocardia corallina.

SUMMARY: A bacterial species which degrades the pyrimidines, uracil, thymine and cytosine by induced enzymes has been characterized as Nocardia corallina (strain S). All other strains of N. corallina investigated oxidized thymine, but varied in their abilities to oxidize uracil and cystosine. Organisms adapted to pyrimidines converted uracil to barbituric acid and thymine to 5-methylbarbituric acid. Oxidation of uracil by thymine-grown organisms was almost entirely by a pathway in which barbituric acid was an intermediate. Oxidation of thymine by uracil-grown organisms was similarly almost entirely via 5-methylbarbituric acid. Oxidation of uracil by uracil-grown organisms and of thymine by thymine-grown organisms occurred, at least in part, through the respective barbituric acids. Discrepancies between the theoretical and observed values for O2 uptake suggested however that other pathways may also occur in these cases. Pyrimidine-grown organisms oxidized 2-thiouracil to 2-thiobarbituric acid and 2-thiothymine to a compound which was probably 5-methyl-2-thiobarbituric acid. These products were not further degraded by the organism. Barbituric acid was oxidized by uracil-grown organisms to CO2 , NH3ss and urea with concurrent oxidative assimilation. The oxidation of barbituric acid was inhibited by isobarbituric acid and sodium azide although barbiturase activity in cell-free extracts was not affected by these substances. Barbiturase preparations converted barbituric acid anaerobically to malonic acid, CO2 and NH3, but barbituric acid was not degraded by whole organisms under anaerobic conditions. Whole organisms, grown on uracil, degraded urea but did not oxidize malonic acid. Acetic and propionic but not malonic or barbituric acids were activated by cell-free extracts as judged by hydroxamate formation. From the evidence presented, it is unlikely that free malonic acid is an intermediate in the breakdown of barbituric acid.

A cytological analysis of the antimetabolite activity of 5-hydroxyuracil in Vicia faba roots.

The effects of 5-hydroxyuracil (5-HU) (isobarbituric acid) upon cell elongation, mitosis, and DNA synthesis were studied in Vicia faba roots. 5-HU had no consistent effect upon root elongation. It blocked DNA synthesis (analyzed by photometric measurements of Feulgen dye in nuclei) during the first 6 hours of treatment; the block spontaneously disappeared by the 12th hour of treatment. Uracil and thymine had no effect upon this block of synthesis. Both thymidine and uridine reversed the block in 6 and 9 hours respectively. In all cases blockage of DNA synthesis was followed by inhibition of mitosis (determined by changes in the percentage of cells in mitosis) and resumption of DNA synthesis was followed by resumption of mitosis. Inhibition indices calculated from the mitotic data indicated a competitive relationship between 5-HU and thymidine and 5-HU and uridine. 5-HU is considered to block DNA synthesis by competing with thymidine for sites on enzymes involved in the synthesis. It is suggested that uridine reverses the block in synthesis by undergoing a conversion to thymidine.

Physiology of Pyrimidines. VIII. Metabolism of Isobarbituric Acid in the Rabbit

As a preliminary to a study of the origin of ethereal sulfates in the rabbit, we thought it worth while to investigate the metabolism of isobarbituric acid in this species. In the adult dog1 and in man2 this pyrimidine is partly broken down to urea, partly conjugated with sulfuric acid.Four adult rabbits were placed in metabolism cages, and kept on a diet consisting of equal parts of alfalfa and oats. The amount of food given each animal was 10 gm. less than that eaten at will. This was found to be sufficient to permit a slight gain in weight over a period of 3 months. The animals were allowed to drink water ad libitum. The freely voided urine was collected every 24 hours.Isobarbituric acid was prepared according to Davidson and Baudisch.3 The purity of the compound was checked by analysis.The following analytical methods were used: Total N, Kjel-dahl; urea, Van Slyke's gasometric method4; inorganic and ethereal sulfur, Folin's method5; total sulfur, Denis modification of Benedict's method.6Isobarbituric ...

Effects of Certain Pyrimidines on the Sulfur Metabolism of Dogs

It was observed by Cerecedo that after feeding isobarbituric acid to dogs there was a distinct decrease in the urinary output of inorganic sulfates and a corresponding rise in the ethereal sulfur fraction. These findings led him to assume that isobarbituric acid was partly excreted in the urine in conjugation with sulfuric acid, as an ethereal sulfate. The present investigation represents an extension of the previous work. After feeding isobarbituric acid in doses of 2.5 gm. to dogs, which were maintained on a nitrogen equilibrium, we found that 60-80% of the substance was metabolized to urea, 20-25% excreted as ethereal sulfate, and the remainder eliminated unchanged. Very striking is the effect of the ingestion of isobarbituric acid on the neutral sulfur fraction in the urine. We observed that after the feeding of the substance there was no detectable amount of neutral sulfur excreted. The disappearance of the neutral sulfur was also observed after the ingestion of isodialuric acid. These observations lead us to assume that in the eatabolism of these compounds a sulfur containing substance is involved, which is normally present in the neutral sulfur fraction of the urine. From the urine of dogs which had ingested isobarbituric acid, we have been able to isolate a compound in form of its xanthydrol derivative. The analytical results so far obtained indicate that we are dealing with dixanthydryl isobarbituric acid sulfate.

Studies on the Intermediary Metabolism of Purines and Pyrimidines

Previous experiments indicated that in the metabolism of uracil in the dog we are dealing with the following sequence of reactions: Uracil → Isobarbituric Acid → Isodialuric Acid → Urea + an unknown carbon compound. Offe has shown that formyloxaluric and oxaluric acids are intermediate products in the oxidation of isobarbituric acid in vitro by means of permanganate. Quite recently, Johnson and Flint have found that these compounds are also formed in the course of the oxidation of uracil by ozone. Formyloxaluric acid and oxaluric acid were fed to dogs maintained in nitrogen equilibrium. In every case there was an increase in the urea output, suggesting that these substances were metabolized. Injection experiments with oxaluric acid yielded the same results. Formyloxaluric acid, on the other hand, when injected, was found to be toxic. These findings seem to indicate that in the metabolism of uracil in the dog we are dealing with an oxidation resembling that in vitro as follows: They also suggest that oxaluric acid might be an intermediate product in purine metabolism. The formation of this substance as an end-product of the oxidation of uric acid by various oxidizing agents is a well-known fact. Experiments were also carried out with alloxan and parabanic acid to determine whether these substances might be intermediary products of purine breakdown in the animal body. Parabanic acid, when fed to dogs in nitrogen equilibrium, is excreted unchanged. The compound was isolated from the urine as the dixanthydryl derivative. Feeding experiments with alloxan show that this substance is excreted only to a small extent in the urine; the greater part is apparently excreted in the bile. After feeding alloxan we observed a distinct decrease in the output of inorganic sulfur in the urine.

Studies on the Physiology of Pyrimidines. The Intermediary Metabolism of Uracil.

In an earlier paper experiments were reported which showed that uracil and thymine, when fed in small amounts to dogs, are metabolized to urea. It was also shown that thymine glycol which is obtained on oxidation of thymine in vitro is probably an intermediate step in the oxidation of this pyrimidine in the animal body. These results led the writer to investigate the fate of isobarbituric and isodialuric acids which are oxidation products of uracil in vitro as possible intermediate metabolic products of uracil. Steudel reported feeding experiments with these two substances. He found that they were apparently completely oxidized. Isobarbituric acid and isodialuric acid were fed to dogs maintained on a nitrogen equilibrium. In every case when isodialuric acid was administered, there was an increase in the urea output, suggesting that this substance was metabolized. In the case of isobarbituric acid, the increase in the urea output after feeding the substance was not as pronounced as after administering isodialuric acid. This may have been due to a lesser absorption of isobarbituric acid on account of its being less soluble. These experiments seem to indicate that in the metabolism of uracil we are dealing with an oxidation resembling that in. vitro as follows : Uracil → Isobarbituric acid → Isodialuric acid → urea + 3 C-atom compound. In the course of these experiments we have observed a peculiar effect of these substances on the sulfur metabolism of the dogs. We found a distinct decrease in the output of inorganic sulfur on the day after feeding these pyrimidines. In some cases this decrease amounted to one-half of the amount ordinarily excreted. The writer is investigating this point further.