Accepted Manuscript Orotic Acid, More Than Just an Intermediate of Pyrimidine de novo Synthesis Monika Löffler, Elizabeth A. Carrey, Elke Zameitat PII:

S1673-8527(15)00060-0

DOI:

10.1016/j.jgg.2015.04.001

Reference:

JGG 359

To appear in:

Journal of Genetics and Genomics

Received Date: 30 October 2014 Revised Date:

4 April 2015

Accepted Date: 9 April 2015

Please cite this article as: Löffler, M., Carrey, E.A., Zameitat, E., Orotic Acid, More Than Just an Intermediate of Pyrimidine de novo Synthesis, Journal of Genetics and Genomics (2015), doi: 10.1016/ j.jgg.2015.04.001. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

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ACCEPTED MANUSCRIPT Orotic Acid, More Than Just an Intermediate of Pyrimidine de novo Synthesis.

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Monika Löffler a, *,1, Elizabeth A. Carreyb,1 , Elke Zameitat a

Institute of Physiological Chemistry, Faculty of Medicine，Philipps University Marburg,

35032 Marburg, Germany.

UCL Institute of Child Health, University College London, London WC1N 1EH, United

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Kingdom

Email: [email protected]

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The authors are retired.

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*Corresponding author: Tel: +49 6422 92016, fax +49 6422 92017.

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ACCEPTED MANUSCRIPT ABSTRACT

It is timely to consider the many facets of the small molecule orotic acid (OA), which is wellknown as an essential intermediate of pyrimidine de novo synthesis. In addition, it can be taken up by erythrocytes and hepatocytes for conversion into uridine and for use in the

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pyrimidine recycling pathway. We discuss the link between dietary orotate and fatty liver in rats, and the potential for the alleviation of neonatal bilirubinaemia. We address the

development of orotate derivatives for application as anti-pyrimidine drugs, and of complexes with metal ions and organic cations to assist therapies of metabolic syndromes. Recent genetic

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data link human Miller syndrome to defects in the dihydroorotate dehydrogenase (DHODH) gene, hence to depleted orotate production. Another defect in pyrimidine biosynthesis, the orotic aciduria arising in humans and cattle with a deficiency of UMP synthase (UMPS), has

regulating gene transcription.

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KEY WORDS

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different symptoms. More recent work leads us to conclude that OA may have a role in

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Orotic acid; pyrimidines; orotic aciduria; Miller syndrome; gene defects

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ACCEPTED MANUSCRIPT Introduction Since its chemical identification in 1905, the molecule orotic acid (OA, uracilcarboxylic acid) has had a long and colourful scientific history, which we anticipate will continue into the twenty first century. As the name suggests (Gk oros = whey), OA is a

normal component of mammalian milk and thus valuable for suckling young: it has also been

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hailed as a vitamin, and as a muscle-building product (“a truly remarkable new supplement we hope will reveal its genuine potential in enhancing your athletic performance and body composition” http://www.bodybuilding.com/fun/jrod10.htm). Other features of orotate in the diet include the potential to alleviate hyperuricosuria in gout, and to aid the establishment of a

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healthy gut flora in neonates.

Orotate (orotic acid, OA) is the product of dihydroorotate dehydrogenase (DHODH),

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the fourth enzyme of pyrimidine de novo synthesis (Fig. 1). It is abundant in the serum and particularly urine of patients suffering from hereditary orotic aciduria or from enzyme defects in the urea cycle (Webster et al., 2001). Theoretically, severe impairment of each one of the consecutive six steps in this unbranched pathway should cause similar or even identical metabolic disturbance through a lack of newly formed UMP, and finally should result in comparable biochemical and clinical phenotypes. However, while oral uridine therapy has

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been proven to be clinically necessary for patients with defective UMP synthase, its benefit for patients with deficient DHODH activity - as the putative cause for the malformation disorder of Miller syndrome - has not been reported to date. Several recent studies in experimental animals have emphasised the importance of

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UMP in the developing organism and it is becoming apparent that OA itself also has a role in

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regulating gene transcription.

OA IN PYRIMIDINE BIOSYNTHESIS Six enzyme steps are required to make UMP, the product from which other

pyrimidine nucleotides are derived (Fig 1). 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) (review Jones, 1980). 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 (Grisham et al., 2008; Voet and Voet, 2011). In higher eukaryotes as well as in

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ACCEPTED MANUSCRIPT most unicellular organisms, DHODH is an integral protein of the inner mitochondrial membrane, and it is ubiquinone-dependent and by its connection to the respiratory chain absolutely oxygen-dependent (Jones, 1980; Löffler et al., 1997). Different soluble forms of

DHODH have been characterized in microorganisms (Jensen and Björnberg, 1998) and yeast. In situ analysis of mouse and zebrafish embryos has shown high expression of all pyrimidine

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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 (Willer et al., 2005; Rainger et al., 2012).

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As seen in Fig. 1, the signposted paths of urea cycle and orotate production start with carbamoyl phosphate but in different cell compartments, and are thus distinct, except in liver

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cells with defective ornithine transcarbamoylase (OTCase) or deficient ornithine. In this case a large excess of carbamoyl phosphate will accumulate in the mitochondrial matrix and thus can spill into the cytosol and be used for the production of orotate, leading to orotic aciduria and orotic acidaemia. In the fully-developed organism, recycling of OA (e.g., from food intake) can occur normally in the liver, where UMP synthase acts to fill the pyrimidine nucleotide pool for all kinds of metabolism, but predominantly for the production of UDP-

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sugars (Weed et al., 1950; Cortes et al., 1979) as is exemplified through the formation of UDP-glucuronate (UDP-GlcUA) in Fig. 2. The uptake of orotate from blood circulation by erythrocytes to produce UMP and re-donate uridine to circulation – which then is available

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for salvage by lymphocytes and peripheral organs - seems to be a special feature of these cells (Harley et al., 1986). The permanent consumption of orotate by erythrocytes may be the cause

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for its low concentration in serum/plasma. In a review on physiological concentrations of all purines and pyrimidines in animal and human cells and tissues (Traut, 1994), the average intracellular concentration of OA is 62 µmol/L, and the extracellular concentration is 0.1 µmol/L. The highest value, of 132 µmol/L orotate, was reported for mouse leukemia L1210 cells compared with less than10 µmol/L in human lymphoblasts, while in human serum orotate levels are less than 0.045 µmol/L, which is far below 1-1.8 µmol/L uracil and 3 - 8 µmol/L uridine (review Connolly and Duley, 1999). If in excess, OMP (orotidylate), the intracellular product of the first step of UMP synthase, could be available for conversion by 5′nucleotidase, and orotidine for pyrimidine phosphorylase, but the pyrimidine ring of OA is not opened for degradation, as is that of

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dihydrouracil in the pyrimidine catabolic pathway. In contrast, OA and orotidine are disposed of by renal excretion only (Jeevanadam et al., 1985; Webster et al., 2001; Miura et al., 2011). Historical aspects of OA as a nutrient Long before the detailed knowledge of cellular pyrimidine biosynthesis, OA was

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discovered as an ingredient of mother liquor after lactose isolation from milk (Biscaro and Belloni, 1905). The identification of pyrimidine carboxylic acids as functional components of nucleic acids and the chemical search for naturally occurring heterocyclic compounds led to the structural elucidation of OA itself (Bachstez, 1930 and references therein), and, likewise,

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to the development of protocols in organic synthesis to make heterocyclic constructions easily available for future research (Ballard and Johnson, 1942). The expanding field of

biochemistry and medicinal chemistry was occupied with the novel discovery of vitamins,

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hormones and putative antagonists, and orotate was identified as an early antitumor principle (Hitchings et al., 1948). At about the same time intensive efforts were made by agriculture and industrial farming to improve animal nutrition. An unknown factor present in distillers’ dried solubles (DDS) from grains and other sources was named as vitamin B13 (Novak and Hauge, 1948) because of its apparently growth-enhancing effect on poultry and other animals.

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Only in the 1950s were vitamin B13 and OA reported to be identical, based on the similarity of their absorption spectra, maximum at 278 and minimum at 240 nm, and microbiological assays (Manna and Hauge, 1953). Concentrates from DDS were less effective than had been hoped (Ott et al., 1957), but pure orotic acid was later obtained by chemical synthesis or

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fermentation (Kinoshita and Tanaka, 1963) for use in various applications such as farming (livestock), food industry, biochemical and biomedical research. OA is only slightly soluble in

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water and presents mainly as orotate anion between pH 3 and 9. It forms strong complexes with divalent transition metal cations and trivalent rare earth cations (Fig.3). When the existence of OA as an intermediate of pyrimidine biosynthesis in eukaryotes

was outlined in Neurospora (Mitchell and Houlahan, 1947), incorporation of authentic OA by rat liver slices proved its precursor role for pyrimidine nucleotides of RNA (Weed et al., 1950); similarly, incorporation of 14C-labelled aspartic acid into OA confirmed its biogenesis (Reichard and Lagerkvist, 1953).

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

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ACCEPTED MANUSCRIPT of sensitive techniques to measure orotate concentration in biological samples without interference by other compounds (Kesner et al., 1975). Earlier microbiological, enzymatic,

polarographic and colorimetric assays were critically considered in reports on improvement of high performance liquid chromatography (HPLC) methodology (Brusilow and Hauser, 1989; Ferrari et al., 1989). It is evident that the detection systems of HPLC were mainly based on

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the characteristic UV absorbance of OA and orotidine; therefore dihydroorotic acid and other pyrimidine metabolites without the double bond were not considered for analysis. The isotope dilution method (Jacobs et al., 1984) and further advanced technology, such as differential pulse polarography (Blazquez et al. 1990), 1H-NMR spectroscopy (Wevers et al., 1999),

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capillary zone electrophoresis (Salerno et al., 1999), fluorescence sensing (Fabrizzi et al., 2002) 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

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introduced with gas chromatography-mass spectrometry (GC-MS) (Carpenter et al., 1997). Ultimately, the parallel determination of nonaromatic intermediates of pyrimidine biosynthesis, dihydroorotic acid and carbamoyl aspartate or dihydrouracil from catabolism, became feasible for routine clinical analysis by HPLC-electrospray tandem mass spectrometry (Van Kuilenburg et al., 2005), eliminating the previously obligatory chemical derivatisation

et al., 2002).

Experimental uses of OA

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process for metabolites in samples for highly sensitive fluorimetric detection (Hayek-Ouassini

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From the mid twentieth century, OA became a compound of considerable interest as a tool for studying pyrimidine metabolism in cells, tissues and animals: monitoring precursors, nucleotide pools and the rate of RNA synthesis, differentiating between de novo and

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salvage/recycling pathways, searching for antimetabolites and anticancer drugs (reviewed by Heidelberger, 1965; Kaneti and Golovinsky, 1971; Lewan et al., 1975; Cortes et al., 1979). Concurrently, the growth-promoting features of OA were still of interest, and OA was used on animals and humans, with various biochemical rationales, assumptions and expectations (review O’Sullivan, 1973; review Falk, 1985). When OA was an additive to pharmaceutical preparations, its benefits were attributed to its being an intermediate of pyrimidine biosynthesis and therefore augmenting uridine nucleotide pools which are required for nucleic acid synthesis and for all pyrimidine nucleotide-dependent biosynthetic processes (Grisham et al., 2008; Voet and Voet, 2011).

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Even if orotate is known to be easily taken up by liver from the portal vein, it may not be equally well transported and taken up by other cells and tissues in the body (Itzhaki, 1972; Hinkel and Le Petit, 1973; Miura et al., 2011). A lack of uptake and conversion was explained by low UMP synthase activity detectable in some organs, absent especially in gut and brain (overview in Raisonnier et al., 1981). A later study on rat small intestine revealed

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considerably different uptake of OA and uridine in different parts of the rat small intestine (Uddin et al., 1984). The adult brain’s requirement for UMP was thought to be predominantly satisfied by uridine transport from the blood in rats as well as in humans (reviewed by

Cansev, 2006). However, more recent studies clearly demonstrated the gene expression of

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UMP synthase and the other enzymes of pyrimidine biosynthesis in the central nervous

system (Schaefer et al., 2010; Gerlach et al., 2012). Therefore, other reasons for the low uptake of the negatively charged OA in some tissues should be considered (Wohlhueter et al.,

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1980). To solve the problem of limited transportation and uptake, chemical derivatives such as OA ethyl ester were applied, e.g., to improve transport through the blood brain barrier (Akiho et al., 1998a), but also salts and complexes with divalent metal ions (Fig. 3) (Kumberger et al.,1991) and formulas with organic cations (choline-, lysine-, arginineorotate) were investigated (overview Falk, 1985). A great number of coordination

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compounds of OA with metal ions of group 8 to 10 elements, e.g., palladium and platinum, were screened for their usefulness in chemotherapy (overview in Bach et al., 1990; Butour et al., 1997 and references therein). This was also the rationale for studying structural derivatives of OA as antipyrimidine agents with parasites, e.g., 5′-fluorodihydroorotic acid

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and 5′fluoroorotic acid (Heidelberger, 1965; Rathod and Khatri, 1990; Javaid et al., 1999). 5′Fluoroorotic acid was investigated by several laboratories in directed selection of auxotroph mutants of various yeasts (Mentel et al., 2006; Wellington et al., 2006). As pharmaceutical

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interest was directed to carriers for divalent metals, the excellent features of orotate as a ligand to metals with loss of its acid function were seen in preparations of zinc-, magnesium-, calcium-orotate complexes. The increased bio-availability of OA as a complex supports the promotion of such preparations for metal ion replacement therapies in diabetes, heart disease, and geriatrics (Nieper, 1974; Geiss et al., 1998; Rosenfeldt et al., 1998). A combination of metformin with carnitine-orotate complex has been used to get good effect in a randomised study of 52 patients with diabetes-associated fatty liver (Hong et al., 2014). The carrier function of orotate has also been used for novel development of tin-complexes as anticancer drugs (Nath et al., 2013).

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ACCEPTED MANUSCRIPT It is not easy to distinguish between the benefits of orotate for improving the supply

of pyrimidines, or of its various ligands improving vital functions through other mechanisms; this is evident from the outlandish biochemical explanations accompanying orotate complexes for sale to laymen as mentioned in the introduction. Early commercially motivated studies of OA in humans in the 1970s gave embarrassingly poor results. In the German Democratic

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Republic, sports medicine programmes were established to test compounds for pharmacological improvement in performance (Latzel, 2009). The results obtained with the nootropicum OA were greatly inferior to those of anabolic steroids. High hopes had been raised by animal studies: it was reported that administration of the RNA precursor OA to

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young adult rats as well as to suckling rats improved the retention of learned responses such as active avoidance reaction, brightness discrimination and raised the learning performances of neurotoxically impaired animals (Rüthrich et al., 1983 and references therein). However,

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preclinical and clinical studies to improve the concentration and long-term memory of learning-disabled school children by the pro-drug methylglucamine orotate (1-deoxy-1methylaminosorbitol OA) apparently did not meet these great expectations (Latzel, 2009). As early as the 1950s the induction of fatty livers in rats by a diet including 1% OA (equivalent to 10 g/L, compare with the contents in milk, Table 1), had been observed and

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intensively studied (overview Robinson, 1981). An accumulation of triglycerides, a lower 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 (overview in Buang, 2011). Liver steatosis was similarly induced when OA was replaced by

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dihydroorotic acid, but was not observed with uridine and other pyrimidines (Durschlag and Robinson, 1980), nor could it be seen when other rodents, chicken, rabbits, pigs and monkeys were tested. On the other hand, when cow calves were fed a high orotate diet, triglycerides

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accumulated in the liver while total cholesterol and triglycerides in blood plasma decreased (Motyl, et al., 1993). 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 (PRPP) in hepatocytes thus re-balancing the pyrimidine and purine nucleotide pools which were thought to be related to the alterations in lipoprotein metabolism (overviews in Raisonnier et al.,1981; Visek, 1992; Buang, 2011). The study mentioned above is one of the few which addressed metabolic effects of exogenous dihydroorotic acid in animals. The general low interest in this metabolite (DHO) may have been due to the lack of simple and accurate methods to analyse dihydroorotate in the past, or perhaps it had been overshadowed by the early work on OA.

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ACCEPTED MANUSCRIPT New studies on the development of fatty-liver in rats on dietary orotate point to a higher level of control (Wang et al 2011): the activity and mRNA concentration of mitochondrial carnitine palmitoyl transferase were found to be depressed, those of fatty acid synthase were upregulated. A 3.2-fold induction in SREBP-1c (sterol regulatory element

binding protein-1) made the effect of OA likely to occur at the transcriptional level. In another

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study on primary rat hepatocytes and rats (Jung et al., 2011), the mediation by the AMPK/SREBP-1 pathway for OA induced lipogenesis was more specifically detailed: an exposure to OA decreased the phosphorylation of adenosine monophosphate activated protein kinase-1 and increased the maturation of the AMPK-sterol regulatory element binding

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protein-1 and the expression of SREBP-responsive genes in the rat liver. The increased

acetyl-CoA carboxylase protein expression observed in this study could underlie alterations in fatty acid metabolism. Since the OA-induced lipid accumulation was completely inhibited by

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rapamycin, the involvement of mTOR was suspected but this interpretation remains to be verified. Because a response similar to that in rats was also seen in human hepatoma cell lines but not in mouse hepatocytes and mice, Jung et al. (2011) came to the conclusion that humans could be a species that may be sensitive to OA-induced fatty liver under certain circumstances. Normal consumption of dietary orotate, however, is not expected to cause liver

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steatosis in humans, nor would OA crystalluria and blocking of collecting tubules in kidney occur as observed in experimental rodents and in patients with orotic aciduria (Webster et al., 2001).

Experimental rats have a marked tendency for induction of liver carcinogenesis and

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development of larger mammary and other tumors by chemical carcinogens (Rao et al., 1984; overview Visek, 1992). Therefore, although the negative effect of high orotate diet in rats was shown not to happen with other animals, the European Food Safety Authority (EFSA)

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advised restraint in the dosage of several mineral salts of OA (EFSA, 2009). “The Panel concludes that in the light of the tumour-promoting effect of OA in animal experimentation, the small margin of safety to this effect from foreseeable exposure, and the absence of any relevant studies on genotoxicity and of any developmental studies, the use of orotate as a source of the eight other minerals and choline at the proposed levels of use is of safety concern”. Since several OA formulas are widely available to the public, the caution expressed by the EFSA seems reasonable. On the other hand beneficial features of OA have been addressed through distinct biochemical studies and are outlined in the following sections.

OA in milk

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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 (Table 1). The content of OA in human milk was found to be low (< 10 mg/L) (Hallanger et al., 1953; Karatas, 2002), or not detectable (Ahmed et al., 1978). Milk from ruminant species contains high orotate levels, especially that of cows in the first week of

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lactation (range of 100-400 mg/L versus 40-90 mg/L) and on grass-rich grazing; other biological factors may be important (Gil and Sànchez-Medina, 1981; Tiemeyer et al., 1984; Akalin and Gönc, 1996; Indyk and Woollard, 2004). Holstein cattle can be carriers of a

defective UMP synthase gene and thus the calves of heterozygote cows are exposed to higher

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orotate levels in the milk (range of 346 to 958 mg/L versus 59 to 251 mg/L (Robinson et

al.,1983)); 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

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entirely dependent on milk as a food (Motyl et al., 1993). 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 (Table 1) (Karatas, 2002).

Since changed orotate excretion during milk production was not paralleled by other

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pyrimidine metabolites, purine nucleosides, uric acid and creatinine as also analysed in milk (Gil and Sànchez-Medina, 1981; Tiemeyer et al., 1984; Indyk and Woollard, 2004), it can be concluded that OA is not diffused from plasma in circulation like other ingredients, but it stems from targeted production in the mammary gland, implying a high rate of UMP

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synthesis. Biosynthesis of OA has been shown in mammary tissue from cattle (Ehle et al., 1981) and overproduction can be a marker of UMP synthase deficiency (Robinson et al., 1983). This raises the question of which special function OA could serve to the newborn

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mammal, distinct from pyrimidine and purine nucleosides to support RNA synthesis. We propose that orotate has a valuable function connected to the role of UDP sugars.

In the 1970s, a most remarkable approach to reduce neonatal jaundice was made by treatment of newborn babies with orotate (Kintzel et al., 1971; Vazzoler et al., 1974). These, and especially premature infants, have a transient deficiency in the conjugating ability of the liver at the level of UDP-glucuronyltransferase (Fig. 2), resulting in elevated levels of nonconjugated bilirubin in blood from neonatal catabolism of fetal hemoglobin. There is a high risk of abnormal brain development in "Kernikterus", a brain disorder induced by high levels of unconjugated bilirubin. The positive effect of orotate (interestingly uridine was not reported) in babies as observed in these clinical studies was explained by enhanced formation

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of (UDP-GlcUA for a better saturation of the transferase, resulting in more efficient disposal of bilirubin. This treatment has been superseded by phototherapy to reduce hyperbilirubinemia (Voet and Voet, 2011). The increased content of OA in milk during early lactation in cows may thus be viewed in the light of a natural precaution evolved for the benefit of newborn calves. By analogy with the formation of UDP-GlcUA (Fig. 2), it is

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feasible that orotate is also used to make UDP-glucose and hence helps suckling calves to lay down glycogen, the storage form of glucose in the liver, at a time when milk is the only source of food. It remains a matter of debate whether the uptake of OA could improve

glycogen synthesis in human cardiac muscle (Ferdinandi et al.,1998; Rosenfeldt et al., 1998)

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and whether effects on important metabolic steps in hearts of rat fed with 1% OA can be extrapolated to humans (Porto et al., 2012). The elevated activity of lipoprotein lipase in

cardiac tissue (increasing the uptake of fatty acids) and the increase of the mass of GLUT4

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glucose transporter and hence glucose consumption may be considered as hormonal and metabolic effects of liver steatosis, but may equally have been of great advantage when animals were exposed to ischemia and reperfusion.

It is vitally important for newborns to establish effective intestinal microbiota, and enteral OA may also be important here. Gram-positive Bifido bacteria species, which grow

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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 (Hinkel and Le Petit, 1973); their analysis of the orotate level in blood of human newborns showed that only 6 % of 100 mg/kg orotate were absorbed when given after 4 h without food.

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Anaerobic Clostridiae can take OA as nitrogen source and degrade it by means of NADPH to dihydroorotate and further to aspartate and ammonia (Lieberman and Kornberg, 1953). This use for OA could improve the nitrogen and carbon balance of developing anaerobic gut

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bacteria in the neonatal intestine. Earlier observations that daily uptake of bovine milk by adult humans could lower

serum cholesterol and also urate (Boguslawski and Wrobel, 1974; Kelley et al., 1979; Robinson and Dombrowski, 1983) were substantiated by studies of the components of milk. OA was shown to be a non-competitive inhibitor of acetyl-CoA synthetase from several organisms (Bernstein et al., 1977). Whereas the yeast enzyme was 50% inhibited at 66 micromolar orotate, this was achieved at 7 micromolar orotate for the rat liver enzyme. An effect on 3-hydroxy-3-methyl-glutaryl-CoA reductase was excluded. Also in newer studies, the beneficial role of dairy ingestion (preferentially skim milk) in gout prevention has been again attributed to OA (Dalbeth et al., 2010). The decrease in serum urate level could result

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from impaired reabsorption of uric acid from the ultrafiltrate by OA which is also transported by the anion exchanger URA1 of the nephron (Miura et al., 2011). Interestingly, sodium and potassium salts of OA were applied empirically for uricosuria in the early 1970s (Kumberger et al., 1991, and references therein) and a review on urate excretion by different transporters (Lipkowitz, 2012) mentioned that a milk diet to treat gout has been known since the 18th

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century (Stephens, 1732).

OA and the central nervous system.

Orotate’s benefit for the brain, mentioned earlier, deserves closer attention. It can be

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assumed that a continuous supply of pyrimidine nucleotides is essential not only for the

developing central nervous system (Connolly and Duley, 1999), but equally for its plasticity, regenerations and neurotransmission: for example, some metabotropic P2Y receptor subtypes

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are sensitive to uridine nucleotides (von Kügelen, 2006). In situ hybridization analysis of adult rat brain revealed that all three genes of the pyrimidine de novo synthesis pathway were expressed in many brain regions, though to different extents (Gerlach et al., 2011). High signal intensities were seen in cortical regions with high density of neurons, e.g. hippocampus, neocortex and cerebellar cortex. The distribution of DHODH mRNA was in

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accordance with the histochemical localization of DHODH activity in neurons (Schaefer et al., 2010). Taking these findings into consideration, and the oxygen-dependent mitochondrial DHODH as producer of orotate, we may have the answer to the search for a neuroprotective agent to stabilize pyrimidine nucleotide pools during and after anoxia: for example, in gerbils

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after transient cerebral ischemia, treatment with OA protected hippocampus subfields from damage, and the application of OA ethyl ester to cats in a focal cerebral ischemia model reduced the volume of ischemic damage (Akiho et al., 1997, 1998b). In these and other

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studies the putative protective effects of OA - improvement of pyrimidine nucleotide pools, RNA synthesis, receptor saturation, repairing of ischemia-induced membrane damage – is explained by the function of OA as an intermediate in de novo UMP synthesis.

Other functions of OA There are very few studies investigating specialized functions of OA. On isolated buckwheat cotyledons (Shopova and Moskova-Simeonova, 2000), OA, phenylorotate and other derivatives were assayed for plant growth regulating activity observed with some nitrogen-containing heterocycles. A cytokinine-like activity of these compounds, especially of phenylorotate (Fig. 3), was deduced from inhibition of root-formation, increase in growth of

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ACCEPTED MANUSCRIPT cotyledons, and stimulation of anthocyanin biosynthesis: it was proposed that their cyclic ureide structure promoted the biosynthesis of aromatic amino acids in plants. Orotate has also been implicated as a tumor promoter in rat liver, and its mechanism elucidated first in studies of mitoinhibition in cultured cells. On treatment of epidermal growth factor-stimulated rat hepatocytes with OA, inhibition of DNA synthesis, mRNA

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transcription and depression of both subunits M1 and M2 of ribonucleotide reductase (RNR) was observed, whereas the expression of early cell cycle related genes was unimpaired

(Manjeshwar et al., 1993). The effect of OA on mRNA transcripts was confirmed by a later in vivo study with normal rats with partial hepatectomies, which provoked tissue regeneration.

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However, the authors finally came to the conclusion that the treatment with orotate may lead to an imbalance in nucleotide pools (an increase in pyrimidines and a decrease in purines),

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which affected the stability or degradation of RNR mRNA transcripts rather than to a specific effect of orotate on transcriptional processes (Manjeshwar et al., 1999). Genetic defects of pyrimidine de novo synthesis enzymes.

Novel findings on inherited defects of enzymes of pyrimidine de novo synthesis have

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provoked this re-consideration of the natural history of OA. Theoretically, severe impairment of any one of the consecutive six steps in the unbranched pathway of de novo UMP synthesis should result in a similar phenotype if the resulting effects were caused only by lack of UMP. However, mutations in the UMPS gene cause a considerably different clinical phenotype in

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patients (hereditary orotic aciduria), compared with Miller syndrome (malformations) which has only recently been described as arising from mutation of the DHODH gene (Ng et al.,

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2010). The question arises, whether such discrepancy is the result of differences in availability of intermediates, such as an overload with OA (UMPS deficiency) versus a lack of OA (DHODH deficiency), or is this due to hitherto unknown functions of the respective enzymes aside from their roles in the de novo synthesis of UMP? The first known genetic disorder of pyrimidine biosynthesis was described as crystalluria of OA in urine and was later diagnosed with mutations in either of the two enzymically-active regions of the bifunctional UMP synthase gene (Fig. 1). Three different subtypes of UMPS deficiency in patients have been reported to date (Balasubramaniam et al., 2014). The most comprehensive review by Webster et al. (2001) looks at disorders of pyrimidine metabolism from biochemical, genetic and clinical perspectives. It listed

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ACCEPTED MANUSCRIPT individually patients/cases diagnosed with orotic aciduria suffering from anemia and bone marrow megaloblastosis, some of these from obstructive uropathy, developmental delay, growth deficiency, immunodeficiency, weakness, failure to thrive, congential cardiac

malformation as clinical manifestation. Affected children would die in early childhood, if they had no access to oral uridine replacement therapy of several grams of uridine daily.

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Studies in the nematode Caenorhabditis elegans revealed that mutants with defective CAD gene pyr-1(-), did not phenotypically resemble those of umps-1(-) mutants (Levitte et al., 2010). Defects of UMP synthase caused synthetic lethality, enlargement of gut granules

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and increase in volume of this organism. These were not detected in pyr-1(-) mutants, in fact this deletion could even suppress the effect seen in umps-1(-) mutants, presumably by limiting the production of orotate as described in the earlier studies of similar mutants in the

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fruit fly Drosophila (Conner and Rawls, 1982). Disrupting the activity of pyr-1 and umps-1 genes using RNA-mediated interference (RNAi) by feeding the worms with bacteria that express double-stranded RNA (Levitte et al., 2010), the authors came to the conclusion that the reduction in UMP levels (as a consequence of CAD or UMPS deficiency) appears to play only a minor role in promoting gut granule changes and embryonic lethality, but the increased

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level of OA was of great importance in embryos. Not only a decline in both metabolic rate and lifespan, but also UV-C-hypersensitivity and resistance to cytotoxic effect of 5fluororotic acid were observed in C. elegans mutants of the rad-6 gene, which was identified as UMP synthase (Merry et al., 2014). In contrast with human cases, uridine replacement was

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of limited efficacy in rad-6 mutants (P. Kuwabara, personal communication). The inherited disorder is different from secondary or metabolic orotic aciduria

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observed in patients with enzyme defects of the urea cycle (Webster et al., 2001), mainly OTCase, lysinuric protein intolerance, and arginine deficiency, which was studied to a great extent in animal models (Fico et al., 1984; Alonso and Rubio, 1989; Brosnan and Brosnan, 2007). More recently, it has been found that deficiency of arginase or the ornithine/citrulline antiporter (HHH syndrome) is systematically associated with increased circulating levels of orotate in humans (reviewed by Häberle and Rubio, 2014). In all these cases of metabolic orotic aciduria, it is assumed that an excess of carbamoyl phosphate from the urea cycle in liver mitochondria (Fig. 1) would spill into the cytoplasm and fuel pyrimidine de novo synthesis to form OA in abundance. Orotic aciduria occurring in patients under allopurinol therapy was explained by considerable conversion of allopurinol to oxipurinol-ribotide by

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orotate-phosphoribosyl transferase, emptying the PRPP pool (Reiter et al., 1986). On the other hand, an allopurinol load test analysing the rate of OA and orotidine formation can be applied to identify urea cycle and other mitochondrial disease in children (Bonham et al., 1999). It is remarkable that the description of known cases of inherited or metabolic orotic aciduria so far do not include body malformation disorders nor tumour promotion (to our

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knowledge), although increased evidence for structural heart defects may be deduced from some cases (Webster et al, 2001). In contrast, marked dysmorphology of face, feet and hands are seen with patients diagnosed as Miller syndrome (formerly Genée-Wiedemann Syndrome)

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which was causatively related to mutations in the DHODH gene (Ng et al., 2010). Exome or genome sequencing has been very effective in identification of new genes in such rare but clinically well-defined Mendelian diseases (Gilissen et al., 2011). Miller syndrome is

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characterized by a pattern of craniofacial dysmorphology, symmetric post-axial limb anomalies, hypoplasia of the 4th and 5th fingers and toes, sometimes orofacial clefts, eyelid coloboma and ear dysplasia. Most patients have normal intelligence and development. Further study of the patients, typically compound heterozygotes, identified disease-associated missense mutations in the DHODH coding region which resulted in moderate, low or nearly

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no residual DHODH activity (Kinoshita et al., 2011; Fang et al., 2012; Rainger et al., 2012). Thus it was concluded that Miller syndrome is associated with a defect that disrupts the conversion of DHO to OA, although analysis of urine from only two patients revealed unexpectedly elevated orotate and non-detectable dihydoorotate levels; other intermediates

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were not measured (Rainger et al., 2012). Only recently, elevated DHO has been confirmed in plasma of a Miller syndrome patient (Balasubramaniam et al., 2014). Many more analyses of

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body fluids from affected patients – not only urine - would be necessary to find an explanation for the paradoxical results. Since orotate can be obtained from the diet, and from residual DHODH activity within the cell, any protocol for identification of a biomarker for Miller syndrome and related disorders should take this into account. At any rate, it appears that the known inborn errors of metabolism leading to orotic aciduria in humans are associated with generalised symptoms that are clearly distinct from the developmental dysfunction, seen in Miller syndrome. Some indication of a direct link between orotate and tissue-specific growth can be obtained from studies on experimental animals. Mouse embryos exposed to leflunomide (widely used clinically as an immunosuppressive agent, and a known inhibitor of DHODH) develop limb and craniofacial

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malformations that can be partially prevented by the co-administration of uridine (Fukushima et al., 2007, 2009). In zebrafish it was found (White et al., 2011) that leflunomide prevents the transcriptional elongation of crucial genes in the neural crest, thus DHODH inhibition prevented the developmental pathways in neural crest formation that might lead to melanoma in this system. The corollary is that production of orotate is required to promote transcription:

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this is presumably linked with natural production of orotate by DHODH, and contrasts with the putative link between exogenous orotate and the inhibition of RNR described earlier, in the rat.

We therefore speculate that endogenously-produced orotate has an important role in

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early transcriptional events in development of crucial tissues such as the neural crest, and thus deficiency of orotate underlies the mechanism for the dysmorphology seen in patients with

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Miller syndrome. If the production of orotate is an essential metabolic and regulatory step, then mutations in the earlier steps (encoded by the CAD gene in mammals) could have similar phenotypes. We can also predict that while uridine can be recycled into nucleotides for UMPS-deficient orotic aciduria patients, neither uridine nor orotate can be usefully applied exogenously to compensate for DHODH deficiency in the control of gene translation. Hereditary defects of the CAD enzyme have to date not been found in humans. This

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may be because mutations in CAD are not compatible with life or from a lack of appropriate diagnostics for screening heterozygotes with partial deficiency of these early steps. None of the Miller syndrome patients described by Rainger et al. (2012) have mutations in the coding region of the CAD gene (D.R. Fitzpatrick, personal communication). However, animal models

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have provided suggestive evidence: zebrafish perplexed mutants are deficient in cell proliferation and differentiation during growth and morphogenesis of retina, jaw, and pectoral

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fins (Willer et al., 2005), arising from a single base mutation in the CAD gene. The study confirmed the importance of pyrimidine synthesis in de novo DNA synthesis and UDP-sugar dependent protein glycosylation, but did not eliminate the possibility that orotate had another, specific role in controlling transcription of genes important for development. The earliest investigations of the genes of pyrimidine biosynthesis in animals used

Drosophila as the model organism, and identified three genes: rudimentary (r), encoding the CAD protein; Dhod; and rudimentary-like (r-1) which encoded the bifunctional UMPS protein. The phenotypes of the mutations had much in common, and could be partially resolved by feeding uridine to the embryos, but the accumulation of OA (in the eyes and body fluid) was a unique feature of organisms lacking one or other of the UMPS activities (Conner and Rawls, 1982; Eisenberg et al., 1993).

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The distinctive arrangement of the enzymes of pyrimidine biosynthesis contributes to the concept of distinct pools of orotate within the mammalian cell. Small and labile intermediates are kept within the multifunctional enzymes CAD and UMP synthase, the close proximity of enzyme active sites giving the advantage of metabolic channelling (Christopherson and Jones, 1980; Evans and Guy, 2004). Because DHODH resides within the

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mitochondrion, dihyhydroorotic acid enters from the cytosol and the product, OA, is removed to the cytosol, each by crossing the outer mitochondrial membrane (Fig. 1). Efficient

channelling of orotate from mitochondrion to the cytosolic compartment was deduced from experiments on the preferential use by UMPS of orotidylate (OMP) produced from

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endogenously-produced orotate and PRPP over exogeneously added OMP even at a 100-fold excess (reviewed by Keppler and Holstege, 1982; Traut and Jones, 1996). Even though the majority of the cytosolic enzymes CAD and UMP synthase are located in close vicinity to the

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mitochondrion (as observed with sperm cells and fibroblasts (Carrey et al., 2002)), both intermediates - dihydroorotate and orotate - could escape into solution in the cytoplasm and therefore, theoretically could serve other tasks. Excess dihydroorotic acid may not be found in solution, because the equilibrium of the uncoupled dihydroorotase catalyzed reaction favours the reverse conversion to carbamoyl aspartate (Christopherson and Jones, 1980). Excess

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(‘endogenous’) OA is, of course, known to be released into the cytoplasm when the enzyme activities in UMP synthase are inadequate (see above). It is also now apparent that the mitochondria are highly mobile within the cell, moving towards the nucleus by virtue of attachments to the cytoskeleton, and a model has been proposed (Evans and Guy, 2004) in

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which a small proportion of CAD and UMPS molecules are able to detach from the mitochondrial surface and could enter the nucleus. In the case of CAD, this occurs as a result of activation by MAP kinase, in parallel with increased pyrimidine biosynthesis in the cell

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(Sigoillot at al., 2005) and we have proposed that the sensitivity to phosphorylation is linked to depletion of UTP supplies in the cell (Löffler et al., 2015). The presence of CAD within the nucleus has been connected with a DNA replication centre (Angeletti and Engler, 1998). More recently, CAD has been found to interact with the androgen receptor in the nucleus (Morin et al, 2012). Since we also know that other cytosolic enzymes in nucleotide biosynthesis have additional roles in the nucleus (such as GMP synthetase in transcriptional regulation of ecdysteroid target genes (Van der Knaap et al., 2010), and IMP dehydrogenase of D. melanogaster as a DNA-binding trancription repressor (Kozhevnikova et al., 2012)), we might also suspect that UMPS molecules in the nucleus may be used in a non-biosynthetic

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function. Mutations in these enzymes may therefore have effects on several functions within the cells. At the same time, responding to the demand for increased transcription in the cell nucleus, it is feasible that the small molecule OA may enter the nucleus from its synthesis in adjacent mitochondria, there to act in a novel way on transcription of some early genes implicated in

Concluding remarks and perspectives

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cell proliferation.

OA was identified in 1905 as a component of milk from cattle and other ruminants: it can

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probably no longer justify the name of vitamin B13, but it evidently has an important

nutritional role for suckling calves and in dairy products through its conversion to UMP and hence into the pathway for all pyrimidine nucleotides, predominantly in liver. Investigations

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have suggested that OA has an important neuroprotective role through the repletion of pyrimidines. Numerous chemical complexes with orotate have been used to introduce metal ions and other moieties into cells through the OA uptake mechanism. It is evident that the idiosyncratic metabolism in some animal models has hindered the full adoption of orotate formulas as useful supplementations to assist therapies.

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For many years, patients with orotic aciduria arising from defects in the gene for UMP synthase have been helped by oral uridine therapy which can compensate for insufficient de novo pyrimidine biosynthesis: this rescue by uridine has not been the case for newlyidentified patients with defects in DHODH, and no living mutants have been found in the

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earlier biosynthesis steps catalysed by CAD. The important conclusion is that the production of orotate itself is required for development of the organism. We have proposed that OA has a role in regulation of transcription of crucial genes; the enzyme UMP synthase may also have a

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regulatory role in the nucleus. We anticipate that our remaining questions may soon be answered by genetic manipulation in novel animal models such as C. elegans and zebrafish.

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ACCEPTED MANUSCRIPT REFERENCES

Ahmed, A.A., Porter, G.A., McCarthy, R.D., 1978. Isolation, quantification, and biosynthetic origin of orotic acid in milk. J. Dairy Sci. 61, 39-43.

Akiho, H., Iwai, A., Katoh-Sudoh, M., Tsukamoto, S., Koshiya, K., Yamaguchi, T., 1997.

RI PT

Post-ischaemic treatment with orotic acid prevented neuronal injury in gerbil brain ischaemia. Neuroreport 8, 607-610.

Akiho, H., Iwai, A., Katoh-Sudoh, M., Tsukamoto, S., Koshiya, K., Yamaguchi, T., 1998a.

SC

Neuroprotective effect of Y-39558, orotic acid ethylester, in gerbil forebrain ischemia. Jpn. J. Pharmacol. 76, 441-444.

M AN U

Akiho, H., Iwai, A., Tsukamoto, S., Koshiya, K., Yamaguchi, T., 1998b. Neuro-protective effect of YM-39558 in focal cerebral ischemia in cats. Neuropharmacology 37, 159-168.

Akalin, A.S., Gönc, S., 1996. The determination of orotic acid in ruminant milk using HPLC.

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Milchwissenschaft 51, 554-556.

Alonso, E., Rubio, V., 1989. Orotic aciduria due to arginine deprivation: changes in the levels of carbamoyl phosphate and of other urea cycle intermediates in mouse liver. J. Nutr. 119,

EP

1188-1195.

Angeletti, P.C., Engler, J.A., 1998. Adenovirus preterminal protein binds to the CAD enzyme

AC C

at active sites of viral DNA replication on the nuclear matrix. J. Virol. 72, 2896-2904.

Bach I., Kumberger O., Schmidbaur H., 1990. Synthesis and crystal structure of lithium orotate (-1) monohydrate and magnesium bis [orotate(-1)] octahydrate. Chem. Ber. 123, 22672271.

Bachstez, M., 1930. Über die Konstitution der Orotsäure. Berichte der Chemie 63,1000-1007.

Ballard, E., Johnson, T.B., 1942. Synthesis of derivatives of pyrimidine-5-carboxylic acid. J. Am. Chem. Soc. 64, 795-796.

20

ACCEPTED MANUSCRIPT Balasubramaniam, S., Duley, J.A., Christodoulou, J., 2014. Inborn errors of pyrimidine metabolism: clinical update and therapy. J. Inherit. Metab. Dis. 37, 687-698.

Bernstein, B.A., Richardson, T., Amundson, C.H., 1977. Inhibition of cholesterol biosynthesis and acetyl-coenzyme A synthetase by bovine milk and orotic acid. J. Dairy Sci. 60, 1846-

RI PT

1853.

Biscaro, G., Belloni E., 1905. About a new compound of the milk. Annuario della Soc. Chimica di Milano 11, 15.

SC

Blázquéz, L.C., Flores, J.r., Jara, F.V., Misiego, A.S., 1990. Determination of orotic acid (Vitamin B13) in milk by differential pulse polarography (DPP). Fresenius J. Anal. Chem.

M AN U

338, 80-81.

Boguslawski, W., Wrobel, J., 1974. An inhibitor of sterol biosynthesis in cow’s milk. Nature 247, 210 - 211.

Bonham J.R., Guthrie, P., Downing, M., Allen, J.C., Tanner, M.S., Sharrard, M., Rittey, C.,

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Land, J.M., Fensom, A., O’Neill, D., Duley, J.A., Fairbanks, L.D., 1999. The allopurinol load test lacks specificity for primary urea cycle defects but may indicate unrecognized mitochondrial disease. J. Inherit. Metab. Dis. 22, 174-184.

EP

Brosnan, M.E., Brosnan, J.T., 2007. Orotic acid excretion and arginine metabolism. J. Nutr.

AC C

137, 1656S-1661S.

Brusilow A.W., Hauser E., 1989. Simple method of measurement of orotic acid and orotidine in urine. J. Chromatogr. 493, 388-391.

Buang, Y., 2010. Dietary adenine alleviates fatty liver induced by orotic acid. Indo. J. Chem. 10, 363-369.

Butour, J.L., Wimmer, S., Wimmer, F., Castan, P., 1997. Palladium (II) compounds with potential antitumour properties and their platinum analogues: a comparative study of the reaction of some orotic derivatives with DNA in vitro. Chem. Biol. Interact. 104,165-178.

21

ACCEPTED MANUSCRIPT

Cansev, M., 2006. Uridine and cytidine in the brain: their transport and utilzation. Brain Res. Rev. 52, 390-397.

Carpenter, K.H., Potter, M., Hammond, J.W., Wilcken, B., 1997. Benign persistent orotic aciduria and the possibility of misdiagnosis of ornithine carbamoly transferase deficiency. J.

RI PT

Inherit. Metab. Dis. 20, 354-358.

Carrey, E.A., Dietz, C., Glubb, D.M., Löffler, M., Lucocq, J.M., Watson, P.F., 2002.

Detection and location of the enzymes of de novo pyrimidine biosynthesis in mammalian

SC

spermatozoa. Reproduction 123, 757-768.

Christopherson, R.I., Jones, M.E., 1980. The overall synthesis of L-5,6-dihydroorotate by

M AN U

multienzymatic protein pyr1-3 from hamster cells. Kinetic studies, substrate channeling, and the effects of inhibitors. J. Biol. Chem. 255, 11381-11395.

Conner, T.W., Rawls J.M., 1982. Analysis of the phenotypes exhibited by rudimentarylike mutants of Drosophila melanogaster. Biochem. Genet. 20, 607-619.

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Connolly, G.P., Duley, J.A., 1999. Uridine and its nucleotides: biological actions, therapeutic potentials. Trends Pharmacol. Sci. 20, 218-225.

Cortes, P., Levin, N.W., Dumler F., Venkatachalam K.K., Verghese, C.P., Bernstein, J., 1979.

EP

Incorporation of exogenous precursors into uridine nucleotides and ribonucleic acid. Nucleotide compartmentation in the renal cortex in vivo. Biochem. J. 182, 677-686.

AC C

Dalbeth, N., Wong, S., Gamble, G.D., Horne, A., Mason, B., Pool, B., Fairbanks, L., McQueen, F.M., Cornish, J., Reid, I.R., Palmano, K., 2010. Acute effect of milk on serum urate concentration: a randomized control cross-over trail. Ann. Rheum. Dis. 69, 1677-1682.

Durschlag, R.P., Robinson, J.L., 1980. Orotic acid-induced metabolic changes in the rat. J. Nutr. 110, 816-821. Ehle, F.R., Robinson, J.L., Clark, J.H., Baumrucker, C.R., 1981. Effect of amino acid concentration on orotic acid production by bovine mammary tissue. J. Dairy Sci. 64, 2192 2196

22

ACCEPTED MANUSCRIPT Eisenberg, M., Kirkpatrick, R., Rawls, J., 1993. Structure of the rudimentary-like gene and UMP synthase in Drosophila melanogaster. Gene 124, 263-267.

European Food Safety Authority, 2009. Orotic acid salts as sources of orotic acid and various

RI PT

minerals added for nutritional purposes to food supplements. EFSA J. 1187, 1-25.

Evans, D.R., Guy, H.I., 2004. Mammalian pyrimidine biosynthesis: fresh insights into an ancient pathway. J. Biol. Chem. 279, 33035-33038.

SC

Fabbrizzi, L., Licchelli, M., Mancin, F., Pizzeghello, M., Rabaioli, G., Taglietti, A., Tecilla, P., Tonellato, U., 2002. Fluorescence sensing of ionic analytes in water: from transition metal

M AN U

ions to vitamin B13. Chem-Eur. J. 8, 94-101.

Fang, J.X., Uchiumi, T., Yagi, M., Matsumoto S., Amamoto, R., Saito, T., Takazaki, S., Kanki, T., Yamaza, H., Nonaka, K., Kang, D., 2012. Protein instability and functional defects caused by mutations of dihydroorotate dehydrogenase in Miller syndrome patients. Biosci.

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Rep. 32, 631-639.

Falk, M., 1985. Orotsäure. Die Pharmazie 40, 377-383.

Ferdinandy, P., Fazekas, T., Kádár, E., 1998. Effects of orotic acid on ischaemic/ reperfused

111-114.

EP

myocardial function and glycogen content in isolated working rat hearts. Pharmacol Res. 37,

AC C

Ferrari, V., Giordano, G., Cracco, A.T., Chiandeti, L., Zacchello F., 1989. Determination of urinary orotate excretion by high-performance liquid chromatography. J. Chromatogr. 497, 101-107.

Fico, M.E., Motyl, T., Milner, J.A., 1984. Species comparison of the influence of ammonia on orotic acid and urea biosynthesis in liver. J. Nutr. 114, 613-621.

Fukushima, R., Kanamori, S., Hirashiba, M., Hishikawa, A., Muranaka, R.I., Kaneto, M., Nakamura, K., Kato, I., 2007. Teratogenicity study of the dihydroorotate dehydrogenase

23

ACCEPTED MANUSCRIPT

inhibitor and protein tyrosine kinase inhibitor leflunomide in mice. Reprod. Toxicol. 24, 310316.

Fukushima, R., Kanamori, S., Hirashiba, M., Hishikawa, A., Muranaka, R., Kaneto, M., Kitagawa, H., 2009. Inhibiting the teratogenicity of the immunosuppressant leflunomide

RI PT

in mice by supplementation of exogenous uridine. Toxicol. Sci. 108, 419-426.

Geiss, K.R., Stergiou, N., Neuenfeldt, H.U., Jester, H.G., 1998. Effects of magnesium orotate on exercise tolerance in patients with coronary heart disease. Cardiovasc. Drug Ther. 12, 153-

SC

156.

Gerlach, J., Löffler, M., Schäfer, M.K.H., 2011. Gene expression of enzymes required for the

Nucleot. Nucl. 30, 1147-1154.

M AN U

de novo synthesis and degradation of pyrimidines in rat peripheral tissues and brain. Nucleos.

Gil, A., Sànchez-Medina, F., 1981. Acid-soluble nucleotides of cow’s, goat’s and sheep’s

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milk at different stages of lactation. J. Dairy Res. 48, 35-44.

Gilissen, C., Hoischen, A., Brunner H.G., Veltman, J.A., 2011. Unlocking Mendelian disease using exome sequencing. Genome Biol. 12, 228-238.

EP

Griffin, J.L., Bonney, S.A., Mann, C., Hebbachi, A.M., Gibbons, G.F., Nicholson, J.K., Shoulders, C.C., Scott, J., 2004. An integrated reverse functional genomic and metabolic

AC C

approach to understanding orotic acid-induced fatty liver. Physiol. Genomics 17, 140-149.

Grisham, C.M., Garrett, R.H., Garrett, R., 2008. Biochemistry. Brooks Cole Publishing, Pacific Grove, USA.

Häberle, J., Rubio, V., 2014. Hyperammonemia and related disorders. In: Blau, N., Duran, R., Gibson, K.M., Blaskovics, M., Dionisi-Vici V. (Eds.), Physician’s Guide to the Diagnosis, Treatment and Follow-up of Inherited Metabolic Diseases. Springer-Verlag, Heidelberg, Germany, pp. 47-62.

24

ACCEPTED MANUSCRIPT

Hallanger,L.E., Laakso, J.W., Schultze, M.O., 1953. Orotic acid in milk. J. Biol. Chem. 202, 83-89.

Harley, E.H., Sacks, S., Berman, P., Cohen, L., Simmonds, H.A., Fairbanks, L.D., Black D.,

RI PT

1986. Source and fate of circulating pyrimidines. Adv. Exp. Med. Biol. 195A, 109-113.

Hayek-Ouassini, M., Henseling, J., Löffler, M., 2002. Separation of N-carbamoyl aspartate and L-dihydroorotate from serum with high-performance liquid chromatography with

SC

fluorimetric detection. Chromatographia 55, 411-415.

Heidelberger, C., 1965. Fluorinated pyrimidines. Prog. Nucleic Acid Res. Mol. Biol. 4, 1-50.

M AN U

Hinkel, G.H., Le Petit, G., 1973. Untersuchungen über die enterale Resorption der Orotsäure bei Neugeborenen. Padiatr. Grenzgeb. 12, 63-69.

Hitchings, G.H., Elion G.B., VanderWerff, H., Falco E.A., 1948. Pyrimidine derivatives as

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antagonists of pteroylglutamic acid. J. Biol. Chem. 174, 765-766.

Hong, E.S., Kim, E.K., Kang, S.M., Khang, A.R., Choi, S.H., Park, K.S., Jang, H.C., Lim, S., 2014. Effect of carnitine-orotate complex on glucose metabolism and fatty liver: a double-

EP

blind, placebo-controlled study. J. Gastroen. Hepatol. 29, 1449-57.

Itzhaki S., 1972, Regulation of uracil utilization for RNA synthesis in Ehrlich ascites tumor

AC C

cells by glucose metabolism. Life Sci. 11, 649-655.

Indyk, H.E., Woollard, D.C., 2004. Determination of orotic acid, uric acid, and creatinine in milk by liquid chromatography. J. AOAC Int. 87, 116-122.

Jacobs, C., Sweetman, L., Nyhan, W.L., Gruneke, L., Craig, J.C., Wadman, S.K., 1984. Stable isotope dilution analysis of orotic acid and uracil in amniotic fluid. Clin. Chim. Acta 143, 123-133.

Javaid, Z.Z., el Kouni, M.H., Iltzsch, M.H., 1999. Pyrimidine nucleobase ligands of orotate phosphoribosyl transferase from Toxoplasma gondii. Biochem. Pharmacol. 58, 1457-1465.

25

ACCEPTED MANUSCRIPT

Jeevanadam, M., Shoemaker, J.D., Horowitz, G.D., Lowry, S.F., Brennan, M.F., 1985. Orotic acid excretion during starvation and refeeding in normal men. Metabolism 34, 325-329.

Jensen, K.F., Björnberg, O., 1998. Evolutionary and functional families of dihydroorotate

RI PT

dehydrogenase. Path Pyrimidines 6, 20-28.

Jones, M.E. 1980. Pyrimidine nucleotide biosynthesis in animals: genes, enzymes, and

SC

regulation of UMP biosynthesis. Ann. Rev. Biochem. 49, 253-279.

Jung, E.J., Kwon, S.W., Jung, B.H., Oh, S.H., Lee, B.H., 2011. Role of the AMPK/SREBP-1

M AN U

pathway in development of orotic acid-induced fatty liver. J. Lipid Res. 52, 1617-1625.

Kaneti, J.J., Golovinsky, E.V., 1971. Quantitative relationships between the electronic structure and biological activity of some analogues of orotic acid. Chem. Biol. Interact. 3, 421-428.

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Karatas, F., 2002. An investigation of orotic acid levels in the breastmilk of smoking and nonsmoking mothers. Eur. J. Clin. Nutr. 56, 958-960. Kelley, W.N., Greene, M.L., Fox, I.H., Rosenbloom, F.M., Levy R.I., Seegmiller, J.E., 1979.

1035.

EP

Effects of orotic acid on purine and lipoprotein metabolism in man. Metabolism 19, 1025-

AC C

Keppler, D., Holstege, A., 1982. Pyrimidine nucleotide metabolism and its compartmentation. In: Sies, E. (Ed.), Metabolic Compartmentation. Academic Press, London, UK, pp. 148-203. Kesner, L., Aronson F.L., Silverman, M., Chan, P.C., 1975. Determination of orotic and dihydroorotic acids in biological fluids and tissues. Clin. Chem. 21, 353-355. Kinoshita, S., Tanaka, K., 1963. Method for producing orotic acid by fermentation process. US Patent 3086917. Kinoshita, F., Kondoh, T., Komori, K., Matsui, T., Harada, N., Yanai, A., Fukuda, M., Morifuji, K., Matsumoto, T., 2011. Miller syndrome with novel dihydroorotate dehydrogenase gene mutations. Pediatr. Int. 53, 587-591.

26

ACCEPTED MANUSCRIPT

Kintzel, H.W., Hinkel, G.K., Schwarze, R., 1971. The decrease in the serum bilirubin level in premature infants by orotic acid. Acta Paediatr. Scand. 60, 1-5. Kozhevnikova, E.N., van der Knaap, J.A., Pindyurin, A.V., Ozgur, Z., van Ijcken W.F.J., Moshkin, Y.M., Verrijzer, C.P., 2012. Metabolic enzyme IMPDH is also a transcription

RI PT

factor regulated by cellular state. Mol. Cell 47, 133-139.

Kumberger, O., Riede, J., Schmidbaur, H., 1991. Preparation and crystal structure of calcium and zinc orotate(2-)hydrates. Chem. Ber. 124, 2739-2742.

M AN U

dairy products. J. Dairy Sci. 62, 1641-1644.

SC

Larson, B.L., Hegarty, H.M., 1979. Orotic acid in milks of various species and commercial

Latzel, K., 2009. Staatsdoping. Der VEB Jenapharm im Sportsystem der DDR. Böhlau Verlag, Wien, Austria.

Levitte, S., Selesky, R., King, B., Smith, S.C., Depper, M., Cole, M., Hermann, G.J., 2010. A Caenorhabditis elegans model of orotic aciduria reveals enlarged lysosome-related organelles

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in embryos lacking umps-1 function. FEBS J. 277, 1420-1439.

Lewan, L., Petersen, I., Ynger, T., 1975. Incorporation of orotic acid into nucleotides and

EP

RNA in mouse organs during 60 minutes. Hoppe-Seyler’s Z. Biol. Chem. 356, 425-429.

Lieberman, I., Kornberg, A., 1953. Enzymatic synthesis and breakdown of a pyrimidine,

AC C

orotic acid. I. Dihydroorotic dehydrogenase. Biochim. Biophys. Acta 12, 223-234.

Lipkowitz, M.S., 2012. Regulation of uric acid excretion by the kidney. Curr. Rheumatol. Rep. 14, 179-188.

Löffler, M., Carrey, E.A., Zameitat, E., 2015. Essential role of mitochondria in pyrimidine metabolism. In Mazurek, S., Shoshan M. (Eds.), Tumor Cell Metabolism: Pathways, Regulation and Biology. Springer, Wien, Austria, pp. 287-312.

27

ACCEPTED MANUSCRIPT Löffler, M., Jöckel, J., Schuster, G., Becker, C. 1997. Dihydroorotate-ubiquinone oxidoreductase links mitochondria in the biosynthesis of pyrimidine nucleotides. Mol. Cell. Biochem. 174, 125-129.

Manjeshwar, S., Pichiri-Coni, G., Coni, P., Rao, P.M., Rajalakshmi, S., Sarma, D.S.R., 1993.

hepatocytes in primary culture. Cancer Lett. 73, 149-154.

RI PT

Ribonucleotide reductase: a possible target for orotic acid mitoinhibition in normal

Manjeshwar, S., Rao P.M., Rajalakshmi, S., Sarma D.S.R., 1999. The regulation of

SC

ribonucleotide reductase by the tumor promotor orotic acid in normal rat liver in vivo. Mol. Carcinogen. 24, 188-196.

M AN U

Manna, L., Hauge, S.M., 1953. A possible relationship of Vitamin B13 to orotic acid. J. Biol. Chem. 202, 91-96.

Mentel, M., Spirek, M., Jorck-Ramberg, D., Piskur, J., 2006. Transfer of genetic material

TE D

between pathogenic and food-borne yeasts. Appl. Environ. Microbiol. 72, 5122-5225.

Merry, A., Quiao, M., Hasler, M., Kuwabara P.E., 2014. RAD-6: pyrimidine synthesis and radiation sensitivity in Caenorhabditis elegans. Biochem. J. 458, 343-353.

EP

Mitchell, K.J., Houlahan, M.B., 1947. Investigations on the biosynthesis of pyrimidine nucleosides in Neurospora. Fed. Proc. 6, 506-509.

AC C

Miura, D., Anzai, N., Jutabha, P., Chanluang S., He, X., Toshiyuki, F., Endou, H., 2011. Human urate transporter 1 (hURAT1) mediates the transport of orotate. J. Physiol. Sci. 61, 253-257.

Morin, A., Fritsch, L., Mathieu, J.R.R., Gilbert, C., Guarmit, B., Firlej, V., Gallou-Kabani, C., Viellefond, A., Delongchamps, N.B., Cabon, F., 2012. Identification of CAD as an androgen receptor interactant and an early marker of prostate tumor recurrence. FASEB J. 26, 460-467.

28

ACCEPTED MANUSCRIPT Motyl, T., Wincenciak, M., Blachowski, S., Kukulska, W., Grzelkowska, Podgurniak, M.,

Kasterka, K., Krzeminski, J., Bartnikowska, E., Skalska, E., 1993. Metabolic effect of orotic acid in calves. J. Vet. Med. A 40, 679-689.

Nath, M., Vats, M., Roy, P., 2013. Tri- and diorganotin(IV) complexes of biologically

RI PT

important orotic acid: synthesis, spectroscopic studies, in vitro anti-cancer, DNA fragmentation, enzyme assays and in vivo anti-inflammatory activities. Eur. J. Med. Chem. 59, 310-21.

SC

Ng, S.B., Buckingham, K.J., Lee, C., Bigham, A.W., Tabor, H.K., Dent, K.M., Huff, C.D., Shannon, P.T., Jabs, E.W., Nickerson, D.A., Shendure, J., Bamshad, M.J., 2010. Exome

M AN U

sequencing identifies a mendelian disorder. Nat. Genet. 42, 30‒35.

Nieper, H.A., 1974. Die Wirkung von Zink-Orotat. Z. Präklin. Geratrie 6, 127-131.

Novak, A.F., Hauge S.M., 1948. Isolation of an unidentified growth factor (vitamin B13) in

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distillers’ dried solubles. J. Biol. Chem. 174, 647-651.

Ott, H.W., Dickinson, A.M., van Inderstine, A., Bazemore, A.W., Page, A.C., Folkers, K. 1957. Studies related to “vitamin B13”. J. Nutr. 64, 525-531.

EP

O’Sullivan, W.J. 1973. Orotic acid. Aust. N. Z. J. Med. 3, 417-423.

AC C

Pôrto, L.C., de Castro, C.H., Savergnini, S.S., Santos S.H., Ferreira, A.V., Cordeiro, L.M., Sobrinho, D.B., Santos, R.A., de Almeida, A.P., Botion, L.M., 2012. Improvement of the energy supply and contractile function in normal and ischemic rat hearts by dietary orotic acid. Life Sci. 90, 476-483.

Rathod, O.K., Khatri, A., 1990. Synthesis and antiproliferative activity of threo-5-fluoro-ldihydroorotate. J. Biol. Chem. 265, 14242-14249.

Rainger, J., Bengani, H., Campbell, L., Anderson, E., Sokhi, K., Lam, W., Riess, A., Ansari, M., Smithson, S., Lees, M., Mercer, C., McKenzie, K., Lengfeld, T., Gener Querol, B. , Branney, P., McKay, S., Morrison, H., Medina, B., Robertson, M., Kohlhase, J.,

29

ACCEPTED MANUSCRIPT Gordon, C., Kirk, J., Wieczorek, D., Fitzpatrick, D.R., 2012. Miller (Genée–Wiedemann)

syndrome represents a clinically and biochemically distinct subgroup of postaxial acrofacial dysostosis associated with partial deficiency of DHODH. Hum. Mol. Genet. 21, 3969-3983.

Raisonnier, A., Bouma, M.E., Salva, C., Infante, R., 1981. Metabolism of orotic acid: lack of

RI PT

orotate phosphoribosyltransferase in rat intestinal mucosa. Eur. J. Biochem. 118, 65-569.

Rao, P.M., Nagamine, Y., Roomi, M.W., Rajalakshmi, S., Sarma, D.S.R., 1984. Orotic acid, a

SC

new promoter for experimental liver carcinogenesis. Toxicol. Pathol. 12, 173-178.

Reichard P., Lagerkvist, U., 1953. The biogenesis of orotic acid in liver slices. Acta Chem.

M AN U

Scand. 7, 1207-1217.

Reiter, S., Löffler, W., Gröbner, W., Zöllner, N., 1986. Urinary oxipurinol-1-riboside excretion and allopurinol-induced orotic aciduria. Adv. Exp. Med. Biol. 195A, 453-460.

Robinson, J.L., 1981. Adenine-induced orotic aciduria in rats fed diets with and without orotic

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acid. Nutr. Res. 1, 253-259.

Robinson J.L., Dombrowski, D.B., 1983. Effects of orotic acid ingestion on urinary and blood

EP

parameters in humans. Nutr. Res. 3, 407-415.

Robinson J.L., Drabik, M.R., Dombrowski, D.B., Clark, J.H., 1983. Consequences of UMP

AC C

synthase deficiency in cattle. Proc. Natl. Acad. Sci. USA 80, 321-323.

Rosenfeldt, F.L., Richards, S.M., Lin, Z., Pepe, S., Conyers, R.A., 1998. Mechanism of cardioprotective effect of orotic acid. Cardiovasc. Drugs Ther. 12, 159-170.

Rüthrich, H.L., Wetzel, W., Matthies, H., 1983. Memory retention in old rats: improvement by orotic acid. Psychopharmacology 79, 348-351.

Salerno, C., Celli, P.D.M., Finocchiaro R., Crifó, C., Giradinin, O., 1999. Determination of urinary orotic acid and uracil by capillary zone electrophoresis. J. Chromatogr. 734, 175-178.

30

ACCEPTED MANUSCRIPT Schaefer C., Schäfer M.K.H., Löffler, M., 2010. Region specific distribution of

dihydroorotate dehydrogenase in the rat CNS points to pyrimidine de novo synthesis in brain. Nucleos. Nucleot. Nucl. 29, 476-481.

Shopova, M.D., Moskova-Simeonova, D., 2000. Plant growth regulating activity of orotic

RI PT

acid and its 1-cyclohexyl and 1-phenylderivatives. Biol. Plantarum. 43, 437-439.

Sigoillot, F.D., Kotsis, D.H., Serre, V., Sigoillot, S.M., Evans, D.R., Guy, H.I., 2005. Nuclear localisation and mitogen-activated protein kinase phosphorylation of the multifunctional

SC

protein CAD. J. Biol. Chem. 280, 25611- 25620.

Stephens, W., 1732. Dolaeus upon the Cure of Gout by Milk-diet. Osborn and Longman,

M AN U

London.

Tiemeyer, W., Stohrer, M., Giesecke, D., 1984. Metabolites of nucleic acids in bovine milk. J. Dairy Sci. 67, 723-728.

Biochem. 140, 1-22.

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Traut, T.W., 1994. Physiological concentrations of purines and pyrimidines. Mol. Cell

Traut, T.W., Jones, M.E., 1996. Uracil metabolism – UMP synthesis from orotic acid or

AC C

53, 1-78.

EP

uridine and conversion to ß-alanin: enzymes and cDNA. Progr. Nucleic Acids Res. Mol. Biol.

Uddin, M., Altmann, G.G., Leblond, C.P., 1984. Radioautographic visualization of differences in the pattern of [3H]uridine and [3H]orotic acid incorporation into the RNA of migrating columnar cells in the rat small intestine. J. Cell Biol. 98, 1619-1629. Van der Knaap, J.A., Kozhevnikova, E.N., Langenberg, K., Moshkin, Y.M., Verrijzer, C.P., 2010. Regulation of ecdysteroid target genes. Mol. Cell Biol. 30, 736-744.

Van Kuilenburg, A., van Lenthe, H., Löffler, M., van Gennip, A.H., 2004. Analysis of pyrimidine synthesis de novo intermediates in urine and dried urine filter-paper strips with HPLC-electrospray tandem mass spectrometry. Clin. Chem. 50, 2117-2124.

31

ACCEPTED MANUSCRIPT Vazzoler G, Bassani N, Menin, A., 1974. Treatment of hyperbilirubinemia in the newborn with orotic acid. Minerva Pediatr. 26, 1046-1048.

Visek, W., 1992. Nitrogen-stimulated orotic acid synthesis and nucleotide imbalance. Cancer

RI PT

Res. 152, 2082-2084.

Voet, D., Voet, J.G., 2011. Biochemistry. John Wiley and Sons, New York, USA.

subtypes. Pharmacol. Therapeut. 110, 414-432.

SC

Von Kügelen, I., 2006. Pharmacological profiles of cloned mammalian P2Y-receptor

M AN U

Wang, Y.M., Hu, X.Q., Xue, Y., Li, Z.J., Yanagita T., Xue C.H., 2011. Study on possible mechansim of orotic acid-induced fatty liver in rats. Nutrition 27, 571-575.

Webster, D.R., Becroft, D.M., van Gennip, A.H., van Kuilenburg, A.B.P., 2001. Hereditary orotic aciduria and other disorders of pyrimidine metabolism. In: Scriver, C.R., Beaudet,

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A.L., Sly, W.S., Valle, D. (Eds.), The Metabolic and Molecular Bases of Inherited Disease. Medical Publishing Division, McGraw-Hill, New York, USA, Vol. II, pp. 2663-2702.

Weed, L.L., Edmonds, M., Wilson, D.W., 1950. Conversion of radioactive orotic acid into

EP

pyrimidine nucleotides of nucleic acid by slices of rat liver. P. Soc. Expt. Biol. Med. 75, 192193.

AC C

Wellington, M., Kabir, M.A., Rutschenko, E., 2006. 5-Fluoro-orotic acid induces chromosomal alterations in genetically manipulated strains of Candida albicans. Mycologia 98, 393-398.

Wevers, R.A., Engelke, U.F.H., Moolenaar, S.H., Bräutigam, C., deJong, J.G.N., Duran, R., deAbreu, R.A., van Gennip, A.H., 1999. 1H-NMR Spectroscopy of body fluids: inborn errrors of purine and pyrimidine metabolism. Clin. Chem. 45, 539-548.

White, R.M., Cech, J., Ratanasirintrawoot, S., Lin, C.Y., Rahl, P.B., Burke, C.J., Langdon, E., Tomlinson, M.L., Mosher, J., Kaufman, C., Chen, F., Long, H.K., Kramer, M., Datta, S.,

32

ACCEPTED MANUSCRIPT Neuberg, D., Granter, S., Young, R.A., Morrison, S., Wheeler, G.N., Zon, L.I., 2011.

DHODH modulates transcriptional elongation in the neural crest and melanoma. Nature 471, 518-522.

Willer, G.B., Lee, V.M., Gregg R.G., Link, B.A., 2005. Analysis of the

during development. Genetics 170, 1827-37.

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Zebrafish perplexed mutation reveals tissue-specific roles for de novo pyrimidine synthesis

Wohlhueter, R.M., McIvor, R.S., Plagemann, P.G.W., 1980. Facilitated transport of uracil and

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5-fluorouracil and permeation of orotic acid into cultured mammalian cells. J. Cell. Physiol.

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104, 309-319.

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Species

Results and S.E (mg/L)

Number of samples

Analytical method

Reference

15 Microbiological Hallanger et al., 86.1 ± 2.8 assay 1953 9 324 ± 35.3 7 63.1 ± 12.3 4 17.7 ± 2.9 6 5 ± n.d 6 7 ± n.d 8 Colorimetric Larson and 88 ± 19 Hegarty, 1979 6 13 ± 7 9 26 ± 9 3 < 1 ± n.d. Commercial dairy (cow) products 6 Whole milk 75 ± 2 Skim milk 3 79 ± 6 b Dried skim milk 3 802 ± 65 Yoghurtb 3 66 ± 18 Ice creamb 3 98 ± 7 Cow: normal Robinson et al., 10 Colorimetric 121.1 ± 20.3 1983 and HPLC Cow: UMPS-deficient 4 630 ± 134 Cow Akalin and 6 HPLC 82.43 ± 9.64 Gönc, 1996 Goat 6 30.59 ± 1.74 6 Sheep 23.87 ± 1.36 Human: HPLC Karatas, 2002 non-smoking 20 1.66 ± 0.15 smoking 17 3.92 ± 0.20 a Further ruminant species are reported in the reference. b(mg/kg). n.d. , not determined.

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Cow Sheep Goat Horse Swine Human Cow Goat Sheepa Human

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LEGENDS TO THE FIGURES

Fig. 1 Pyrimidine de novo synthesis and urea cycle. Dihydroorotate (dihydroorotic acid, DHO), and orotate (orotic acid, OA) are marked as wellknown intermediates of pyrimidine de novo synthesis in all cells. Only liver is specialized for production of urea. The interrupted arrow indicates that in hepatocytes with defective ornithine transcarbamoylase or deficient ornithine/citrulline antiporter a large excess of carbamoyl phosphate will accumulate in the mitochondrial matrix and thus can be transported to the cytosol and used for the production of orotate in liver. Other metabolic causes are explained in the text.

Fig. 2 Detoxification of bilirubin in hepatocytes.

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ACCEPTED MANUSCRIPT Uptake of exogeneous orotate in liver to fill the intracellular UMP pool. The formation of UDP-glucuronate (UDP-GlcUA) exemplifies the specialized functional role of uridine nucleotides for activation of sugars: for synthesis of glycogen, and further glycoconjugation of protein molecules.

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Fig. 3 The chemical structure of orotic acid and its derivatives. Chemical structure of orotic acid (A), orotic acid ethylester (B), N1-phenyl-orotic acid (C),

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Zn-orotate tetrahydrate (D).

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O

HN O

N H

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A

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O H2O H2O

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