Magnesium Orotate and the Microbiome-Gut-Brain Axis Modulation: New Approaches in Psychological Comorbidities of Gastrointestinal Functional Disorders.

Magnesium orotate has been cited in the medical literature for the past three years as a possible adjuvant in some pediatric and adult gastroenterological disorders associated with dysbiosis. Studies also focus on the possibility of adding magnesium orotate in psychiatric disorders' treatment, such as major depression and anxiety. The most relevant element in these studies is the efficiency of magnesium orotate therapy in cases with both gastroenterological and psychiatric symptoms. This article proposes a literature review, focused on the studies published in the last three years, targeting magnesium orotate treatment and probiotic supplementation in patients with both digestive and psychiatric symptoms. Moreover, this review will compare the efficiency of magnesium orotate and probiotics within both the pediatric and adult communities, focusing on the possibility of gut-brain axis modulation and its involvement in the clinical evolution of these patients.

Orotic acid production by Yarrowia lipolytica under conditions of limited pyrimidine.

Orotic acid (OA) is an intermediate of the pyrimidine biosynthesis with high industrial relevance due to its use as precursor for production of biochemical pyrimidines or its use as carrier molecule in drug formulations. It can be produced by fermentation of microorganisms with engineered pyrimidine metabolism. In this study, we surprisingly discovered the yeast Yarrowia lipolytica as a powerful producer of OA. The overproduction of OA in the Y. lipolytica strain PO1f was found to be caused by the deletion of the URA3 gene which prevents the irreversible decarboxylation of OA to uridine monophosphate. It was shown that the lack of orotidine-5'-phosphate decarboxylase was the reason for the accumulation of OA inside the cell since a rescue mutant of the URA3 deletion in Y. lipolytica PO1f completely prevented the OA secretion into the medium. In addition, pyrimidine limitation in the cell massively enhanced the OA accumulation followed by secretion due to intense overflow metabolism during bioreactor cultivations. Accordingly, supplementation of the medium with 200 mg/L uracil drastically decreased the OA overproduction by 91%. OA productivity was further enhanced in fed-batch cultivation with glucose and ammonium sulfate feed to a maximal yield of 9.62 ± 0.21 g/L. Y. lipolytica is one of three OA overproducing yeasts described in the literature so far, and in this study, the highest productivity was shown. This work demonstrates the potential of Y. lipolytica as a possible production organism for OA and provides a basis for further metabolic pathway engineering to optimize OA productivity.

Dietary egg white protein hydrolysate improves orotic acid-induced fatty liver in rats by promoting hepatic phospholipid synthesis and microsomal triglyceride transfer protein expression.

We investigated the effects of egg white protein hydrolysates (EWH) on orotic acid (OA)-induced nonalcoholic fatty liver (NAFL) in rats. Effects of the egg white protein (EWP) and EWH were also compared. Four groups of male Sprague-Dawley rats were separately fed AIN-76-based diets, supplemented with 20% casein for control, or with 1% OA, together with either 20% casein (OA), 20% EWP, or 20% EWH, respectively, for 3 d (developing stage) and 14 d (developed stage). In both feeding periods, animals from the OA group showed higher accumulation hepatic triacylglycerol (TAG) compared with those from the control group. In the 14-d experiment, dietary EWP and EWH significantly reduced the hepatic TAG levels. Intake of EWP reduced liver fat in OA-fed rats by 61%, while EWH reduced it by 92%. In addition, EWH restored the OA-induced high serum-TAG level to that seen in the control group. The 3 d experiment showed that consumption of EWH improved the expression of hepatic MTP, that was reduced by OA, without changing Mttp gene expression. It also increased the hepatic synthesis of PC and PE by enhancing the transcription of Pcyt1 and Pemt genes. Inclusion of EWP and EWH in the diet improves the OA-induced NAFL. EWH reduces the liver TAG better than EWP, and works more rapidly. Dietary EWH ameliorates OA-induced NAFL by promoting the secretion of hepatic TAG.

Regulation effects of total flavonoids in Morus alba L. on hepatic cholesterol disorders in orotic acid induced NAFLD rats.

BACKGROUND: Mulberry leaves are the dried leaves of Morus alba L., flavonoids from mulberry leaves (MLF) has showed regulatory effect on abnormal lipid metabolism, but the regulatory mechanism of MLF on cholesterol metabolism is still missing. This study was designed to investigate the effect of MLF and its active metabolite quercetin on regulating cholesterol disorders. METHODS: The mechanism of MLF on alleviating liver injury and regulating cholesterol was examined in dyslipidemic SD rats. The regulatory mechanism of quercetin for cholesterol disorders have also been detected through lipid laden HepG2 cell model. RESULTS: Our results showed that MLF significantly inhibited lipid accumulation and alleviate hepatic injury in NAFLD rat model. The hepatic expression level of SREBP2, HMGCR and miR-33a were significantly down-regulated, while CYP7A1 was induced by MLF treatment. In vitro, Quercetin significantly decreased lipid accumulation in HepG2 cells. Mechanistically, quercetin could inhibit the mRNA and protein expression level of SREBP2 and HMGCR with or without LDL treatment. In addition, quercetin could also reduce the LXRß while induced SR-BI mRNA expression. CONCLUSION: Our findings indicate that MLF and quercetin could reduce the excessive cholesterol accumulation in vivo and in vitro. These cholesterol-regulating phenomenon might attribute to its effect on down-regulating the expression of lipid-related markers such as SREBP2 and HMGCR, which may exert a protective role in the NAFLD treatment.

Regulation effects of rosemary (Rosmarinus officinalis Linn.) on hepatic lipid metabolism in OA induced NAFLD rats.

Rosmarinus officinalis Linn. is a kind of medicinal and edible homologous plant, which is popular in the Mediterranean region with a significant effect on mind tranquilization, anti-oxidation, and metabolic improvement. However, the hypolipidemic effects and mechanism of rosemary ethanol extract (RO) and their metabolites are less known. In this study, the hypolipidemic effects of RO and its active compounds were clarified. The results showed that RO, rosmarinic acid (RA) and carnosic acid (CA) significantly reduced the contents of liver triglyceride (TG), total cholesterol (TC), free fatty acids (FFA) and improved cell hypertrophy, vacuolation, and cell necrosis in the liver of orotic acid induced non-alcoholic fatty liver disease (NAFLD) model rats. The mechanism and related pathways of RO and its main metabolites against lipid disorder were related to the up-regulation of the phosphorylation of adenosine 5'-monophosphate(AMP)-activated protein kinase (AMPK) and the inhibition of the sterol regulatory element binding protein-1c (SREBP-1c) cracking into the nucleus, following the down-regulation of fatty acid synthesis. In conclusion, our study demonstrates that RA and CA are active substances of RO, and provides scientific evidence to support functional food product development for improving NAFLD.

Antioxidant Potential and Angiotensin-Converting Enzyme (ACE) Inhibitory Activity of Orotic Acid-Loaded Gum Arabic Nanoparticles.

Orotic acid (OA) nanoparticles were prepared using the freeze-drying method. The antihypertensive activity and antioxidant capacity of OA and orotic acid-loaded gum arabic nanoparticles (OAGANPs) were examined using the angiotensin-converting enzyme (ACE), 1,1-diphenyl-2-picrylhydrazyl (DPPH), nitric oxide (NO), and ß-carotene assays, as well as the quantification of total phenolic content (TPC). The DPPH and NO scavenging activities of OAGANPs were significantly higher than those of the OA solution. The ß-carotene bleaching assay of OAGANPs showed a dose-dependent trend, while 500 µg/ml was significantly more effective than the other concentrations, which exerted 63.4% of the antioxidant activity. The in vitro antihypertensive assay revealed that the OAGANPs exhibited the most potent ACE inhibition activity, when compared to the OA solution. Hence, results revealed the potential of preparing the OA as a nanoparticle formulation in enhancing the antioxidant and antihypertensive properties compared to the OA solution.

Effects of extracellular orotic acid on acute contraction-induced adaptation patterns in C2C12 cells.

Dietary administration of orotic acid (OA), an intermediate in the pyrimidine biosynthetic pathway, is considered to provide a wide range of beneficial effects, including cardioprotection and exercise adaptation. Its mechanisms of action, when applied extracellularly, however, are barely understood. In this study, we evaluated potential effects of OA on skeletal muscle using an in vitro contraction model of electrically pulse-stimulated (EPS) C2C12 myotubes. By analyzing a subset of genes representing inflammatory, metabolic, and structural adaptation pathways, we could show that OA supplementation diminishes the EPS-provoked expression of inflammatory transcripts (interleukin 6, Il6; chemokine (C-X-C Motif) ligand 5, Cxcl5), and attenuated transcript levels of nuclear receptor subfamily 4 group A member 3 (Nr4A3), early growth response 1 (Egr1), activating transcription factor 3 (Atf3), and fast-oxidative MyHC-IIA isoform (Myh2). By contrast, OA had no suppressive effect on the pathogen-provoked inflammatory gene response in skeletal muscle cells, as demonstrated by stimulation of C2C12 myotubes with bacterial LPS. In addition, we observed a suppressive effect of OA on EPS-induced phosphorylation of AMP-activated protein kinase (AMPK), whereas EPS-triggered phosphorylation/activation of the mammalian target of rapamycin (mTOR) was not affected. Finally, we demonstrate that OA positively influences glycogen levels in EP-stimulated myotubes. Taken together, our results suggest that in skeletal muscle cells, OA modulates both the inflammatory and the metabolic reaction provoked by acute contraction. These results might have important clinical implications, specifically in cardiovascular and exercise medicine.

Comparative Analysis of EPA/DHA-PL Forage and Liposomes in Orotic Acid-Induced Nonalcoholic Fatty Liver Rats and Their Related Mechanisms.

Nonalcoholic fatty liver disease (NAFLD) has become one predictive factor of death from various illnesses. The present study was to comparatively investigate the effects of eicosapentaenoic acid-enriched and docosahexaenoic acid-enriched phospholipids forage (EPA-PL and DHA-PL) and liposomes (lipo-EPA and lipo-DHA) on NAFLD and demonstrate the possible protective mechanisms involved. The additive doses of EPA-PL and DHA-PL in all treatment groups were 1% of total diets, respectively. The results showed that Lipo-EPA could significantly improve hepatic function by down-regulating orotic acid-induced serum aspartate aminotransferase (AST) and alanine aminotransferase (ALT) levels by 55.6% and 34.2%, respectively (p < 0.01). Moreover, lipo-EPA exhibited excellent inhibition on the mRNA expression of SREBP-1c and FAS at the values of 0.454 ± 0.09 (p < 0.01) and 0.523 ± 0.08 (p < 0.01), respectively, thus ameliorating OA-induced NAFLD. Meanwhile, lipo-EPA could significantly suppress the SREBP-2 and HMGR levels (31.4% and 66.7%, p < 0.05, respectively). In addition, EPA-PL and lipo-DHA could also significantly suppress hepatic lipid accumulation mainly by enhancement of hepatic lipolysis and cholesterol efflux. Furthermore, DHA-PL played a certain role in inhibiting hepatic lipogenesis and accelerating cholesterol efflux. The results obtained in this work might contribute to the understanding of the biological activities of EPA/DHA-PL and liposomes and further investigation on its potential application values for food supplements.

Exploring pathogenesis in subjects with subjective Tinnitus having kidney deficiency pattern in terms of Traditional Chinese Medicine based on serum metabolic profiles.

OBJECTIVE: To investigate the metabolic pathogenesis in subjects with subjective tinnitus (ST) having kidney deficiency pattern (KDP) (ST/KDP) in terms of Traditional Chinese Medicine. METHODS: Three groups of subjects, including healthy individuals, subjects with ST/KDP, and subjects who were healthy initially and then developed ST/KDP one year later (healthy ¡ú ST/KDP), were recruited for this study. Serum metabolic profiles of all subjects were analyzed using ultra-performance liquid chromatography coupled with quadruple-time-of-flight mass spectrometry. The metabolic characteristics of the ST/KDP subjects were determined, and the corresponding biomarkers were predicted. The metabolomics data from the healthy ¡ú ST/KDP subjects were collected for further verification. RESULTS: Twelve metabolites in the ST/KDP subjects were different from those of the healthy control subjects. Of these metabolites, according to the prediction, except for octanoic acid, other metabolites might characterize ST/KDP. Ten metabolites at the outcome ST/KDP stage were different from those at the initial (control) stage. Through the comparison of these metabolites with the predicted metabolites, five common metabolites, including upregulated glutamate, serotonin, orotic acid and 8-oxoguanine, as well as downregulated taurine, were found. These common metabolites were significantly associated with canonical pathways including calcium signaling, ¦Ã-aminobutyric acid (GABA) receptor signaling, purine and pyrimidine biosynthesis, taurine biosynthesis, and serotonin receptor signaling. CONCLUSION: The metabolic pathogenesis in ST/KDP subjects was characterized by upregulated glutamate, serotonin, orotic acid and 8-oxoguanine, as well as downregulated taurine, additionally, perturbations of calcium signaling, GABA receptor signaling, purine and pyrimidine biosynthesis, taurine biosynthesis, and serotonin receptor signaling.

Targeted gene replacement at the URA3 locus of the basidiomycetous yeast Pseudozyma antarctica and its transformation using lithium acetate treatment.

The basidiomycetous yeast Pseudozyma antarctica is a remarkable producer of industrially valuable enzymes and extracellular glycolipids. In this study, we developed a method for targeted gene replacement in P. antarctica. In addition, transformation conditions were optimized using lithium acetate, single-stranded carrier DNA and polyethylene glycol (lithium acetate treatment), generally used for ascomycetous yeast transformation. In the rice-derived P. antarctica strain GB-4(0), PaURA3, a homologue of the Saccharomyces cerevisiae orotidine-5'-phosphate decarboxylase gene (URA3), was selected as the target locus. A disruption cassette was constructed by linking the nouseothricine resistance gene (natMX4) to homologous DNA fragments of PaURA3, then electroporated into the strain GB-4(0). We obtained strain PGB015 as one of the PaURA3 disruptants (Paura3Δ::natMX4). Then the PCR-amplified PaURA3 fragment was introduced into PGB015, and growth of transformant colonies but not background colonies was observed on selective media lacking uracil. The complementation of uracil-auxotrophy in PGB015 by introduction of PaURA3 was also performed using lithium acetate treatment, which resulted in a transformation efficiency of 985 CFU/6.8 µg DNA and a gene-targeting ratio of two among 30 transformants. Copyright © 2017 John Wiley & Sons, Ltd.

[Clinical diagnosis and treatment of three cases with hyperornithinemia-hyperammonemia-homocitrullinuria syndrome].

Objective: To study the clinical characteristics, methods of diagnosis and treatment of hyperornithinemia-hyperammonemia- homocitrullinuria (HHH) syndrome. Method: From July 2011 to August 2016, 3 Chinese patients with HHH syndrome were enrolled in this study. The clinical course, biochemical features, brain MRI findings, and gene mutations were analyzed. Result: The three patients' age at onset of symptoms was 3 months to 7 years, and the age of diagonosis was 3 years and 10 months to 9 years and 10 months. All of them presented with intolerance to protein-rich foods from the infant period, development retardation and abnormal posture. Case 1 and 2 had moderate mental retardation. Serum ammonia 25-276 µmol/L (reference range<60 µmol/L), alanine aminotransferase (ALT) 20-139 IU/L (reference range 9-50 IU/L), ornithine 29.12-99.44 µmol/L(reference range 15-100 µmol/L), urinary orotic acid 1.49-29.75 mmol/mol Cr (reference range 0-7 mmol/mol Cr), uracil 6.09-103.97 mmol/mol Cr (reference range 0-1.5 mmol/mol Cr). The cranial MRI revealed lesions in the basal ganglia, abnormal white matter signal, progressive demyelination and cerebral atrophy. On their SLC25A15 gene, a novel homozygous missense mutation c. 416A>G (p.E139G) was identified in case 1, a known pathogenic homozygous nonsense mutation c. 535C>T was found in case 2 and 3. Liver transplantation had been performed when case 1 was 6 years old. Significant improvements were observed in dietary habit, mental and motor functions, and biochemical parameters. After the dietary intervention with the supplements of arginine, L-carnitine, case 2 was improved, spastic paraplegia of case 3 had no mitigation. Liver transplant was recommended. Conclusion: HHH syndrome has an aversion to protein-rich food, and the patients have recurrent vomiting and progressive neurological dysfunction. Clinical diagnosis of HHH syndrome is difficult and patients may present with incomplete biochemical phenotype. The genetic analysis is key for the diagnosis. Depending on their condition, individuals with HHH syndrome can be treated with a low-protein diet, drugs and liver transplantation.

A combination of probiotics and magnesium orotate attenuate depression in a small SSRI resistant cohort: an intestinal anti-inflammatory response is suggested.

Approximately, one-third of those who develop major depression will have a poor response to treatment and over time can become treatment resistant. Intestinal dysbiosis has been implicated in depression with systemic inflammation and vagal and enteric nerve impairment. We report on a sequel pilot study (n = 12) with a combination probiotics/magnesium orotate formulation adjuvant administered with SSRIs for treatment resistant depression. At the end of an 8-week intervention mean changes for depression scores and quality of life in the group was clinically significantly improved (p < 0.001) with all but 4 participants experiencing a benefit. An intestinal anti-inflammatory response was suggested. At 16-weeks follow-up while still on SSRI medications, the group had relapsed after cessation of the test intervention.

Eicosapentaenoic Acid-Enriched Phosphatidylcholine Attenuated Hepatic Steatosis Through Regulation of Cholesterol Metabolism in Rats with Nonalcoholic Fatty Liver Disease.

Nonalcoholic fatty liver disease (NAFLD) is the most common chronic liver disease in the world. Disturbed cholesterol metabolism plays a crucial role in the development of NAFLD. The present study was conducted to evaluate the effects of EPA-PC extracted from sea cucumber on liver steatosis and cholesterol metabolism in NAFLD. Male Wistar rats were randomly divided into seven groups (normal control group, model group, lovastatin group, low- and high-dose EPA groups, and low- and high-dose EPA-PC groups). Model rats were established by administering a diet containing 1% orotic acid. To determine the possible cholesterol metabolism promoting mechanism of EPA-PC, we analyzed the transcription of key genes and transcriptional factors involved in hepatic cholesterol metabolism. EPA-PC dramatically alleviated hepatic lipid accumulation, reduced the serum TC concentration, and elevated HDLC levels in NAFLD rats. Fecal neutral cholesterol excretion was also promoted by EPA-PC administration. Additionally, EPA-PC decreased the mRNA expression of hydroxymethyl glutaric acid acyl (HMGR) and cholesterol 7α-hydroxylase (CYP7A), and increased the transcription of sterol carrying protein 2 (SCP2). Moreover, EPA-PC stimulated the transcription of peroxisome proliferators-activated receptor α (PPARα) and adenosine monophosphate activated protein kinase (AMPK) as well as its modulators, liver kinase B1 (LKB1) and Ca2+/calmodulin-dependent kinase kinase (CAMKK). Based on the results, the promoting effects of EPA-PC on NAFLD may be partly associated with the suppression of cholesterol synthesis via HMGR inhibition and the enhancement of fecal cholesterol excretion through increased SCP2 transcription. The underlying mechanism may involve stimulation of PPARα and AMPK.

Long-term fatty liver-induced insulin resistance in orotic acid-induced nonalcoholic fatty liver rats.

We investigated whether fatty liver preceded insulin resistance or vice versa using a long-term orotic acid (OA)-induced nonalcoholic fatty liver disease (NAFLD) model without the confounding effects of obesity and hyperlipidemia and explored the role of the liver in insulin resistance. Male Wistar rats were fed with or without OA supplementation for 30, 60, and 90 days. The NAFLD group showed increased liver lipid at 30, 60, and 90 days; glucose intolerance was noted at 60 and 90 days. Furthermore, partial liver proteins and gene expressions related to upstream signaling of insulin were decreased. However, the liver glycogen content was elevated, and gluconeogenesis genes expressions were obviously decreased at 90 days. The occurrence of fatty liver preceded insulin resistance in OA-induced NAFLD without the interference of obesity and hyperlipidemia, and hepatic insulin resistance may not play a conclusive role in insulin resistance in this model.

Accumulation of Pyrimidine Intermediate Orotate Decreases Virulence Factor Production in Pseudomonas aeruginosa.

The impact of orotate accumulation in the medically important bacterium Pseudomonas aeruginosa was studied by deleting pyrE, the gene encoding orotate phosphoribosyltransferase and responsible for converting orotate into orotate monophosphate within the de novo pyrimidine synthesis pathway. The pyrE mutant accumulated orotate and exhibited decreased production of hemolysin, casein protease, and elastase. Feeding orotate at a concentration of 51.25 µM to the wild type, PAO1, likewise decreased production of these factors except for hemolysin, which was not affected. A significant increase in the pigments pyocyanin and pyoverdin was also observed. Pyocyanin increase in the pyrE mutant was heightened when the mutant was supplemented with orotate. Although pyoverdin production in the wild-type PAO1 was unaffected by orotate supplementation, a decrease in the mutant's production was observed when supplemented with orotate. These results indicate a significant reduction in virulence factor production in the pyrE mutant and reduction in some virulence factors in the wild type when supplemented with orotate.

Dehydroepiandrosterone sulfate and cytochrome P450 inducers alleviate fatty liver in male rats fed an orotic acid-supplemented diet.

The effects of the peroxisome proliferator, dehydroepiandrosterone sulfate (DHEAS), and the typical cytochrome P450 (CYP) inducers phenobarbital (PB) and 3-methylcholanthrene (3-MC) on fatty liver were examined in rats. Treating rats with orotic acid caused marked accumulation of lipid droplets in the liver. This effect of orotic acid was almost eradicated by co-treatment with DHEAS and PB. While DHEAS or PB alone also alleviated fatty liver, treatment with 3-MC caused little effect on a reduction in lipid droplets. Histopathological examinations revealed numerous peroxisomes in the liver of rats treated with DHEAS. In addition, a significant increase in the expression on hepatic CYPs was observed in rats the fatty liver of which was attenuated. Regarding other enzymes associated with hepatic fatty acid oxidation, the expression levels of sirtuin 1, sirtuin 6, and carnitine palmitoyltransferase 1 were also upregulated most markedly by treatment with DHEAS alone. Thus, the attenuation in fatty liver observed in the present study is likely due to peroxisome proliferation and the induction of fatty acid-metabolizing enzymes by DHEAS and typical CYP inducers.

Towards newborn screening for ornithine transcarbamylase deficiency: fast non-chromatographic orotic acid quantification from dried blood spots by tandem mass spectrometry.

BACKGROUND: Orotic acid (OA) is the key parameter in the detection of ornithine transcarbamylase deficiency (OTC-D). Inclusion of OA into newborn screening compatibility with existing analytical procedures is necessary. METHODS: OA was eluted from dried blood spots with methanol containing deuterated [1,3-(15)N2] OA as internal standard. Quantification by tandem mass spectrometry was accomplished without chromatographic separation. Samples were measured in MRM mode for the masses m/z 155.1 → 111 for OA and 157.1 → 113 for d2 OA. RESULTS: OA was determined in a wide range of concentrations with high precision, LOD and LOQ being 0.21 and 0.65 µmol/L, respectively. Values correlated well with those obtained after chromatography. Pretreatment of samples with HCl-butanol regularly used for acylcarnitine measurement did not significantly affect quantitative results. Inclusion of the new method into the standard newborn screening procedure did not alter the results for acylcarnitines or amino acids; the total time per analysis, however, was increased from 1.15 to 1.85 min. OA levels of 707 unaffected newborns ranged from 0.28 to 3.73 µmol/L. Five newborns with OTC-D showed concentrations of 89.7-211.1 µmol/L. In newborns with severe citrullinaemia we found values in the range of 4.99-127.7 µmol/L. CONCLUSIONS: This new method can be used as a standalone measurement of OA but it can also easily be implemented into standard newborn screening techniques as a useful supplement. In this case the method allows detection of newborns with OTC deficiency without an extra analytical run.

[Magnesium orotate in treatment of chronic hypertension in pregnant women].

We examined 150 pregnant women with essential hypertension (EHT), EHT and connective tissue dysplasia (CTD), and healthy. Presence of CTD aggravated clinical picture of EHT and was associated with pronounced cardialgic, neurological, asthenic, vertebrogenic, visceral, and other syndromes. The use of antihypertensive, metabolic (magnesium orotate) drugs, sedative and uroseptic phytotherapy, application of other nondrug measures in conditions of multidisciplinary dynamic support of the gestational period facilitated regress of clinical symptoms of EHT and EHT+CTD, favorable course of pregnancy and successful delivery.

Trypanosoma brucei (UMP synthase null mutants) are avirulent in mice, but recover virulence upon prolonged culture in vitro while retaining pyrimidine auxotrophy.

African trypanosomes are capable of both de novo synthesis and salvage of pyrimidines. The last two steps in de novo synthesis are catalysed by UMP synthase (UMPS) - a bifunctional enzyme comprising orotate phosphoribosyl transferase (OPRT) and orotidine monophosphate decarboxylase (OMPDC). To investigate the essentiality of pyrimidine biosynthesis in Trypanosoma brucei, we generated a umps double knockout (DKO) line by gene replacement. The DKO was unable to grow in pyrimidine-depleted medium in vitro, unless supplemented with uracil, uridine, deoxyuridine or UMP. DKO parasites were completely resistant to 5-fluoroorotate and hypersensitive to 5-fluorouracil, consistent with loss of UMPS, but remained sensitive to pyrazofurin indicating that, unlike mammalian cells, the primary target of pyrazofurin is not OMPDC. The null mutant was unable to infect mice indicating that salvage of host pyrimidines is insufficient to support growth. However, following prolonged culture in vitro, parasites regained virulence in mice despite retaining pyrimidine auxotrophy. Unlike the wild-type, both pyrimidine auxotrophs secreted substantial quantities of orotate, significantly higher in the virulent DKO line. We propose that this may be responsible for the recovery of virulence in mice, due to host metabolism converting orotate to uridine, thereby bypassing the loss of UMPS in the parasite.