I. Basic Introduction

Orotic acid, historically known as vitamin B??, is an normal organic acid with the molecular formula C?H?N?O?. Orotic acid was first identified and isolated in the context of biological metabolite research in 1905, underscoring its significance in metabolic pathways related to nucleotide biosynthesis. This compound is synthesized in the body via a mitochondrial enzyme, dihydroorotate dehydrogenaseor a cytoplasmic enzyme of pyrimidine synthesis pathway. It is sometimes used as a mineral carrier in some dietary supplements (to increase their bioavailability), most commonly for lithium orotate. Orotic acid and its derivatives are widely used in the medical field. Oral acid is an important drug for the treatment of liver diseases for the treatment of jaundice-type liver disease and general liver dysfunction. The drug molecules involved in the orotic acid group play an important and positive role in the treatment of myocardial infarction, gastric ulcer, and Mycobacterium tuberculosis. In the life sciences, orotic acid plays an important role in the metabolic transformation of pyrimidine nucleotides. Biochemists have studied the expression and clinical significance of orotate phosphoribosyltransferase, and found that the expression of this enzyme in different cancer tissues shows its close relationship with fluorouracil in the treatment of cancer, and provides clinical treatment and prognosis for cancer. The guidance has certain application prospects. In addition, orotic acid is widely used in the food, health care, cosmetics and feed industries. [1]Due to the wide range of uses of orotic acid, the research and application of its synthesis methods have received more and more attention.

II. Synthesis and Production

Orotic acid can be chemically synthesized through different routs. Here we list a bromine-free synthesis route, using diethyl oxalate as a raw material, condensed with ethyl acetate, and cyclized to obtain 5-ethoxymethylene hydantoin, followed by ring expansion and acid precipitation.

Control the temperature in the reaction channel at 110-115 °C, adjust the flow rate of the double pump, and simultaneously pump 1.5 kg of hydantoin, 10 kg of water, 2.5 kg of 50% mixed solution of glyoxylic acid and 7.5 liters of 8N sodium hydroxide. After the solution is heated and reacted for 2 hours, the heating is stopped. When the temperature is lowered to 70-80 °C, the reaction solution is transferred to a 50-liter purification reactor, the internal temperature is maintained at 60-65 °C, and concentrated hydrochloric acid is added dropwise to adjust the pH to 9-9.5, the precipitated solid is centrifuged to obtain a crude white crude sodium orotate, the crude product is added to 5 liters of water, kept at 60-65 °C, concentrated hydrochloric acid is added dropwise to adjust the pH to 1-2, and after stirring for half an hour, The precipitated solid was centrifuged to obtain 2.95 kg of crude orotic acid.[2]

III. Biological Functions

1.     Nucleotide Synthesis

Six enzyme steps are required to make UMP, the product from which other pyrimidine nucleotides are derived. The first three enzymes form a single trifunctional polypeptide: CAD, glutamine-dependent carbamoyl phosphate synthetase 2 (CPS2) + aspartate transcarbamoylase (ATCase) + dihydroorotase (DHOase); the fifth and sixth enzymes – orotate phosphoribosyl transferase (OPRTase) and orotidine monophosphate (OMP) decarboxylase – are combined in the bifunctional uridine monophosphate synthase (UMPS) (reviewed by Jones, 1980). [3]In addition to the de novo synthesis of uridine monophosphate (UMP), salvage/recycling of uridine can contribute to the supply of pyrimidine nucleotides in animal cells. In higher eukaryotes as well as in most unicellular organisms, DHODH is an integral protein of the inner mitochondrial membrane, and it is ubiquinone-dependent. Through its connection to the respiratory chain, DHODH is absolutely oxygen-dependent. Different soluble forms of DHODH have been characterized in microorganisms and yeast. In situ analysis of mouse and zebrafish embryos has shown high expression of all pyrimidine biosynthesis enzymes in specific tissues and developing regions, correlating with site- and stage-specific requirements for de novo pyrimidine biosynthesis during embryonic development, so that homozygous deficiencies in de novo synthesis would not be compatible with life. Simultaneously, the growth-promoting properties of OA remained of interest, leading to its use in both animals and humans, supported by various biochemical rationales and assumptions. When incorporated as an additive in pharmaceutical formulations, OA's benefits were ascribed to its role as an intermediate in pyrimidine biosynthesis, thereby enhancing uridine nucleotide pools necessary for nucleic acid synthesis and other pyrimidine nucleotide-dependent biosynthetic processes.

2.     Liver Function Support

Orotic acid has been shown to have positive effects on liver function. It plays a role in regulating liver enzymes and metabolic pathways, facilitating detoxification processes and promoting liver cell regeneration. As early as the 1950s the induction of fatty livers in rats by a diet with 1% OA, had been observed and intensively studied. An accumulation of triglycerides, a low mitochondrial capacity of fatty-acid oxidation, and a decreased secretion of very low and low density lipoproteins from hepatocytes were the main changes in lipid metabolism of rat liver. Liver steatosis was similarly induced when OA was replaced by dihydroorotic acid, but was not observed with uridine and other pyrimidines, nor could it be seen when other rodents, chicken, rabbits, pigs and monkeys were tested. Since an adenine-enriched diet may stimulate urine excretion of OA and minimize the orotate-induced effect in rat liver, it was concluded that orotate and adenine can compete for 5-phosphoribosyl-1-pyrophosphate in hepatocytes thus re-balancing the pyrimidine and purine nucleotide pools which were thought to be related to the alterations in lipoprotein metabolism. This is particularly important when the liver is damaged due to factors such as alcohol overconsumption or toxin exposure. By enhancing liver function, orotic acid indirectly supports overall physiological balance and homeostasis.

IV. Medical and Nutritional Applications

In view of the high content of OA in cow milk and the assumption of its biogenic function for newborns, and adults likewise, extensive studies were made to compare milk from different mammals. The content of OA in human milk was found to be low (<10 mg/L), or not detectable. Milk from ruminant species contains high orotate levels, especially that of cows in the first week of lactation and on grass-rich grazing; other biological factors may be important. Holstein cattle can be carriers of a defective UMP synthase gene, and thus the calves of heterozygote cows are exposed to higher orotate levels in the milk (346–958 mg/L versus 59-251 mg/L; other work has shown that these levels can have an unfavourable effect on the metabolism of polyamines, purines, and lipids in the tissues of calves, who of course are entirely dependent on milk as food. The breast milk of human mothers with UMP synthase deficiency would be unlikely to provide such high concentrations of orotate, since human milk contains very much less orotate than that of cows.

It is vitally important for newborns to establish effective intestinal microbiota, and enteral OA may also be important in intestine. Gram-positive Bifido bacteria species, which grow under fairly anaerobic conditions in the gut, could make excellent use of OA since this was not as easily absorbed in the upper intestine as galactose, glucose and amino acids from milk. An analysis of the orotate level in blood of human newborns showed that only 6% of 100 mg/kg orotate was absorbed when given after 4 h without food. Anaerobic Clostridia can take OA as nitrogen source and degrade it by means of NADPH to dihydroorotate and further to aspartate and ammonia. This use for OA could improve the nitrogen and carbon balance of developing anaerobic gut bacteria in the neonatal intestine.[4]

V. Analysis and measurement of OA

The studies on dairy products, and also the detection of OA in human body fluids caused by inborn errors of pyrimidine metabolism induced the development and optimisation of sensitive techniques to measure orotate concentration in biological samples without interference by other compounds. [1]Earlier microbiological, enzymatic, polarographic and colorimetric assays were critically considered in reports on improvement of high performance liquid chromatography (HPLC) methodology. It is evident that the detection systems of HPLC were mainly based on the characteristic UV absorbance of OA and orotidine; dihydroorotic acid and other pyrimidine metabolites without the double bond were not considered for analysis. The isotope dilution method and further advanced technology, such as differential pulse polarography, 1H-NMR spectroscopy, capillary zone electrophoresis, and fluorescence sensing received little application in clinical chemistry. For screening of a great number of patients (7500) for organic acids including elevated OA, automated selected ion-search was introduced with gas chromatography-mass spectrometry (GC-MS).

References

1.  Liffler, M., Carrey, E. A. & Zameitat, E. Orotic Acid, More Than Just an Intermediate of Pyrimidine de novo Synthesis. Journal of Genetics and Genomics 42, 207–219 (2015).

2.  Improved synthesis method of orotic acid: CN109761916A

3.  Pepe, S. et al. Targeting oxidative stress in surgery: Effects of ageing and therapy. Experimental Gerontology 43, 653–657 (2008).

4.  Chiara, F. et al. The Strange Case of Orotic Acid: The Different Expression of Pyrimidines Biosynthesis in Healthy Males and Females. Journal of Personalized Medicine 13, 1443 (2023).