Hereditary Orotic Aciduria and the Excretion of Orotidine.

Objective Orotic aciduria and deficiency of uridine monophosphate synthetase have been observed in a patient, studied over 10 years, who had no megaloblastic anemia. Excretion of orotic acid and orotidine were 8.24 and 0.52 mmol/mol of creatinine. The ratio of 15.85 differed appreciably from that of 6 patients reported with no megaloblastic anemia. Methods The analysis of orotidine by gas chromotography mass spectrometry was conducted. Conclusion Patients with orotic aciduria with and without megaloblastic anemia cannot be distinguished by ratio of orotic acid to orotidine.

[THE DIAGNOSTICS OF HEREDITARY DISORDERS OF METABOLISM OF PURINES AND PYRIMIDINES IN CHILDREN USING HIGH PERFORMANCE LIQUID CHROMATOGRAPHY OF ELECTRO-SPRAY TANDEM MASS-SPECTROMETRY].

The article presents data concerning new technique of diagnostic of diseases of metabolism of purines and pyrimidines using high performance liquid chromatography combined with electro-spray mass-spectrometry. The procedure of analysis is described in detail: from pre-analytical stage to interpretation of data of liquid chromatography mass-spectrometry, control of quality of data analysis, mass-spectrometry parameters and chromatographic conditions of analysis of purines, pyrimidines and their metabolites. The reference values are presented for purine and pyrimidine nucleosides and bases in urine of healthy individuals. The chemical structure of purines, pyrimidines and their metabolites and examples of chromato-mass-spectrograms under various hereditary disorders of metabolism of purines and pyrimidines are presented as well. The article is targeted to pediatricians of all profiles, medical geneticists and physicians of laboratory diagnostic.

Early diagnosis of adenylosuccinate lyase deficiency using a high-throughput screening method and a trial of oral S-adenosyl-l-methionine as a treatment method.

AIM: The aim of this study was to develop a high-throughput urine screening technique for adenylosuccinate lyase (ADSL) deficiency and to evaluate S-adenosyl-l-methionine (SAMe) as a potential treatment for this disorder. METHOD: Testing for succinyladenosine (S-Ado), a marker of ADSL deficiency, was incorporated into a screening panel for urine biomarkers for inborn errors of metabolism using electrospray tandem mass spectrometry. Liquid chromatography-mass spectrometry and high-performance liquid chromatography were used to confirm and monitor the response of metabolites to oral SAMe treatment. RESULTS: Increased levels of S-Ado were detected in a 3-month-old male infant with hypotonia and seizures. ADSL gene sequencing revealed a previously described c.-49T>C mutation and a novel c.889_891dupAAT mutation, which was likely to disrupt enzyme function. After 9 months of SAMe treatment, there was no clear response evidenced in urine metabolite levels or clinical parameters. INTERPRETATION: These results demonstrate proof of the principle for the high-throughput urine screening technique, allowing earlier diagnosis of patients with ADSL deficiency. However, early treatment with SAMe does not appear to be effective in ADSL deficiency. It is suggested that although SAMe treatment may ameliorate purine nucleotide deficiency, it cannot correct metabolic syndromes in which a toxic nucleotide is present, in this case presumed to be succinylaminoimidazole carboxamide ribotide.

Quantitation of Purine in Urine by Ultra-Performance Liquid Chromatography-Tandem Mass Spectrometry.

Inborn errors of purine metabolism, either deficiencies of synthesis or catabolism pathways, lead to a wide spectrum of clinical presentations: urolithiasis (adenine phosphoribosyltransferase), primary immune deficiency (adenosine deaminase deficiency and purine nucleoside phosphorylase deficiency), severe intellectual disability, and other neurological symptoms (Lesch-Nyhan disease, adenylosuccinase deficiency, and molybdenum cofactor deficiency). A rapid quantitative purine assay was developed using UPLC-MS/MS to determine purine nucleoside and base concentrations in urine. Taking advantages of ultra-performance liquid chromatography, we achieved satisfactory analyte separation and recovery with a polar T3 column in a short run time with no requirement of time-consuming sample preparation or derivatization. This targeted assay is intended for diagnosis and management of purine diseases, newborn screening follow-up of SCID, and evaluation of autism spectrum disorders.

[A case of Xanthinuria in a patient with marked hypouricemia]. / Un caso di xantinuria in una paziente con marcata ipouricemia.

Xanthinuria is a rare autosomal recessive disorder associated with a deficiency of xanthine oxidoreductase (XOR), which normally catalyzes the conversion of hypoxanthine to uric acid. The effects of this deficit are an elevated concentration of hypoxanthine and xanthine in the blood and urine, hypouricemia, and hypouricuria. The deficit in XOR can be isolated (type I xanthinuria) or associated with a deficit in aldehyde oxidase (type II xanthinuria) and sulfite oxidase (type III xanthinuria). While the first two variants have a benign course, are often asymptomatic (20%), and clinically indistinguishable, type III xanthinuria is a harmful form that leads to infant death due to neurological damage. The clinical symptoms (kidney stones, CKD, muscle and joint pain, peptic ulcer) are the result of the accumulation of xanthine, which is highly insoluble, in the body fluids. We describe a case of type I xanthinuria in a 52-year-old woman who presented with hypouricemia, hypouricuria and kidney stones. The diagnosis was based on purine catabolite levels in urine and serum measured by 3 nonroutine methods: high-pressure liquid chromatography, mass spectrometry, and magnetic resonance imaging. To identify the type of xanthinuria the allopurinol test was used. We believe that these tests will facilitate the diagnosis of xantinuria especially in asymptomatic patients without the need for a biopsy of the liver or intestines, which is useful only for scientific purposes.

In vivo proton MR spectroscopy findings specific for adenylosuccinate lyase deficiency.

Adenylosuccinate lyase (ADSL) deficiency is an inherited metabolic disorder affecting predominantly the central nervous system. The disease is characterized by the accumulation of succinylaminoimidazolecarboxamide riboside and succinyladenosine (S-Ado) in tissue and body fluids. Three children presented with muscular hypotonia, psychomotor delay, behavioral abnormalities, and white matter changes on brain MRI. Two of them were affected by seizures. Screening for inborn errors of metabolism including in vitro high resolution proton MRS revealed an ADSL deficiency that was confirmed genetically in all cases. All patients were studied by in vivo proton MRS. In vitro high resolution proton MRS of patient cerebrospinal fluid showed singlet resonances at 8.27 and 8.29 ppm that correspond to accumulated S-Ado. In vivo proton MRS measurements also revealed a prominent signal at 8.3 ppm in gray and white matter brain regions of all patients. The resonance was undetectable in healthy human brain. In vivo proton MRS provides a conclusive finding in ADSL deficiency and represents a reliable noninvasive diagnostic tool for this neurometabolic disorder.

A case of dihydropyrimidinase deficiency incidentally detected by urine metabolome analysis.

Dihydropyrimidinase deficiency is a rare autosomal recessive disease affecting the second step of pyrimidine degradation. It is caused by mutations in the DPYS gene. Only approximately 30 cases have been reported to date, with a phenotypical variability ranging from asymptomatic to severe neurological illness. We report a case of dihydropyrimidinase deficiency incidentally detected by urine metabolome analysis. Gas chromatography-mass spectrometry-based urine metabolomics demonstrated significant elevations of dihydrouracil and dihydrothymine, which were subsequently confirmed by a quantitative analysis using liquid chromatography-tandem mass spectrometry. Genetic testing of the DPYS gene revealed two mutations: a novel mutation (c.175G > T) and a previously reported mutation (c.1469G > A). Dihydropyrimidinase deficiency is probably underdiagnosed, considering its wide phenotypical variability, nonspecific neurological presentations, and an estimated prevalence of 2/20,000. As severe 5-fluorouracil-associated toxicity has been reported in patients and carriers of congenital pyrimidine metabolic disorders, urinary pyrimidine analysis should be considered for those who will undergo 5-fluorouracil treatment.

Extended diagnosis of purine and pyrimidine disorders from urine: LC MS/MS assay development and clinical validation.

BACKGROUND AND AIMS: Inborn errors of purine and pyrimidine metabolism are a diverse group of disorders with possible serious or life-threatening symptoms. They may be associated with neurological symptoms, renal stone disease or immunodeficiency. However, the clinical presentation can be nonspecific and mild so that a number of cases may be missed. Previously published assays lacked detection of certain diagnostically important biomarkers, including SAICAr, AICAr, beta-ureidoisobutyric acid, 2,8-dihydroxyadenine and orotidine, necessitating the use of separate assays for their detection. Moreover, the limited sensitivity for some analytes in earlier assays may have hampered the reliable detection of mild cases. Therefore, we aimed to develop a liquid chromatography-tandem mass spectrometry (LC-MS/MS) assay that allows the simultaneous and sensitive detection of an extended range of purine and pyrimidine biomarkers in urine. METHODS: The assay was developed and validated using LC-MS/MS and clinically tested by analyzing ERNDIM Diagnostic Proficiency Testing (DPT) samples and further specimens from patients with various purine and pyrimidine disorders. RESULTS: Reliable determination of 27 analytes including SAICAr, AICAr, beta-ureidoisobutyric acid, 2,8-dihydroxyadenine and orotidine was achieved in urine following a simple sample preparation. The method clearly distinguished pathological and normal samples and differentiated between purine and pyrimidine defects in all clinical specimens. CONCLUSIONS: A LC-MS/MS assay allowing the simultaneous, sensitive and reliable diagnosis of an extended range of purine and pyrimidine disorders has been developed. The validated method has successfully been tested using ERNDIM Diagnostic Proficiency Testing (DPT) samples and further clinical specimens from patients with various purine and pyrimidine disorders. Sample preparation is simple and assay duration is short, facilitating an easier inclusion of the assay into the diagnostic procedures.

Clinical and genetic analysis of 7 Chinese patients with ß-ureidopropionase deficiency.

ß-Ureidopropionase (ßUP) deficiency is an autosomal recessive disease caused by abnormal changes in the pyrimidine-degradation pathway. This study aimed to investigate the mutation of ß-ureidopropionase gene (UPB1) gene and clinical features of 7 Chinese patients with ßUP deficiency.We reported 7 Chinese patients with ßUP deficiency who were admitted at Tianjin Children's Hospital. Urine metabolomics was detected by gas chromatography-mass spectrometry (GC-MS). Then genetic testing of UPB1 was conducted by polymerase chain reaction (PCR) method.The patients presented with developmental delay, seizures, autism, abnormal magnetic resonance imaging, and significantly elevated levels of N-carbamyl-ß-alanine and N-carbamyl-ß-aminoisobutyric acid in urine. Subsequent analysis of UPB1 mutation revealed 2 novel missense mutations (c.851G>T and c.853G>A), 3 previously reported mutations including 2 missense mutations (c.977G>A and c.91G>A) and 1 splice site mutation (c.917-1 G>A).The results suggested that the UPB1 mutation may contribute to ßUP deficiency. The c.977G>A is the most common mutation in Chinese population.

Xanthine urolithiasis: Inhibitors of xanthine crystallization.

OBJECTIVE: To identify in vitro inhibitors of xanthine crystallization that have potential for inhibiting the formation of xanthine crystals in urine and preventing the development of the renal calculi in patients with xanthinuria. METHODS: The formation of xanthine crystals in synthetic urine and the effects of 10 potential crystallization inhibitors were assessed using a kinetic turbidimetric system with a photometer. The maximum concentration tested for each compound was: 20 mg/L for 3-methylxanthine (3-MX); 40 mg/L for 7-methylxanthine (7-MX), 1-methylxanthine (1-MX), theobromine (TB), theophylline, paraxanthine, and caffeine; 45 mg/L for 1-methyluric acid; 80 mg/L for 1,3-dimethyluric acid; and 200 mg/L for hypoxanthine. Scanning electron microscopy was used to examine the morphology of the crystals formed when inhibitory effects were observed. RESULTS: Only 7-MX, 3-MX, and 1-MX significantly inhibited xanthine crystallization at the tested concentrations. Mixtures of inhibitors had an additive effect rather than a synergistic effect on crystallization. CONCLUSION: Two of the inhibitors identified here-7-MX and 3-MX-are major metabolites of TB. In particular, after TB consumption, 20% is excreted in the urine as TB, 21.5% as 3-MX, and 36% as 7-MX. Thus, consumption of theobromine could protect patients with xanthinuria from the development of renal xanthine calculi. Clinical trials are necessary to demonstrate these effects in vivo.

Hereditary xanthinuria is not so rare disorder of purine metabolism.

Hereditary xanthinuria (type I) is caused by an inherited deficiency of the xanthine oxidorectase (XDH/XO), and is characterized by very low concentration of uric acid in blood and urine and high concentration of urinary xanthine, leading to urolithiasis. Type II results from a combined deficiency of XDH/XO and aldehyde oxidase. Patients present with hematuria, renal colic, urolithiasis or even acute renal failure. Clinical symptoms are the same for both types. In a third type, clinically distinct, sulfite oxidase activity is missing as well as XDH/XO and aldehyde oxidase. The prevalence is not known, but about 150 cases have been described so far. Hypouricemia is sometimes overlooked, that´s why we have set up the diagnostic flowchart. This consists of a) evaluation of uric acid concentrations in serum and urine with exclusion of primary renal hypouricemia, b) estimation of urinary xanthine, c) allopurinol loading test, which enables to distinguish type I and II; and finally assay of xanthine oxidoreductase activity in plasma with molecular genetic analysis. Following this diagnostic procedure we were able to find first patients with hereditary xanthinuria in our Czech population. We have detected nine cases, which is one of the largest group worldwide. Four patients were asymptomatic. All had profound hypouricemia, which was the first sign and led to referral to our department. Urinary concentrations of xanthine were in the range of 170-598 mmol/mol creatinine (normal < 30 mmol/mol creatinine). Hereditary xanthinuria is still unrecognized disorder and subjects with unexplained hypouricemia need detailed purine metabolic investigation.

Identification of two mutations in human xanthine dehydrogenase gene responsible for classical type I xanthinuria.

Hereditary xanthinuria is classified into three categories. Classical xanthinuria type I lacks only xanthine dehydrogenase activity, while type II and molybdenum cofactor deficiency also lack one or two additional enzyme activities. In the present study, we examined four individuals with classical xanthinuria to discover the cause of the enzyme deficiency at the molecular level. One subject had a C to T base substitution at nucleotide 682 that should cause a CGA (Arg) to TGA (Ter) nonsense substitution at codon 228. The duodenal mucosa from the subject had no xanthine dehydrogenase protein while the mRNA level was not reduced. The two subjects who were siblings with type I xanthinuria were homozygous concerning this mutation, while another subject was found to contain the same mutation in a heterozygous state. The last subject who was also with type I xanthinuria had a deletion of C at nucleotide 2567 in cDNA that should generate a termination codon from nucleotide 2783. This subject was homozygous for the mutation and the level of mRNA in the duodenal mucosa from the subject was not reduced. Thus, in three subjects with type I xanthinuria, the primary genetic defects were confirmed to be in the xanthine dehydrogenase gene.

Uric acid changes in urine and plasma: an effective tool in screening for purine inborn errors of metabolism and other pathological conditions.

Purine inborn errors of metabolism (IEM) are serious hereditary disorders, which should be suspected in any case of neonatal fitting, failure to thrive, recurrent infections, neurological deficit, renal disease, self-mutilation and other manifestations. Investigation usually starts with uric acid (UA) determination in urine and plasma. UA, the final product of purine metabolism in humans, may be altered not only in purine IEM, but also in other related pathologies and clinical conditions. However, data and information about abnormal UA levels are scattered in the literature, often being controversial and confusing. A comprehensive overview has been elaborated, according to abnormal UA levels in urine and plasma, which associates these alterations with purine IEM. Other possible diseases, clinical conditions, diet and drug intake, related to the metabolism of uric acid, are also presented. The article includes tables that classify the disorders according to different patterns of UA alterations, with pertinent enzymes, clinical symptoms, inheritance and comments. Additionally, summarized pathophysiological mechanisms of important disorders are described. The overview is intended to assist in the interpretation of the results of UA analyses. It demonstrates that variation of UA concentrations in urine and plasma may constitute an effective tool in screening for purine IEM and other related pathological conditions.

[Recurrent urinary lithiasis revealing hereditary xanthinuria]. / Lithiases urinaires récidivantes révélant une xanthinurie héréditaire.

INTRODUCTION: Hereditary xanthinuria, due to a purine metabolism disorder, is a rare cause of urinary lithiasis in children. CASE: We report the case of a child aged 3 and a half years, who presented recurrent urinary lithiasis that led to destruction of the right kidney. Infrared spectrophotometric analysis of the calculus concluded that it was composed of 100% xanthine. Laboratory tests showed hypouricemia and hypouricosuria with elevated urinary excretion of oxypurines. These findings led to a diagnosis of hereditary xanthinuria. CONCLUSION: Early diagnosis of this rare disease is essential to avoid its complications. Metabolic causes must be sought in children with lithiasis.

Methods for the diagnosis of creatine deficiency syndromes: a comparative study.

The increasing number of patients with creatine deficiency syndromes (CDS) stresses the need to develop screening procedures for the identification these inherited disorders. Guanidinoacetate (GAA) and creatine (Cr) are reliable biochemical markers of CDS and several analytical methods to measure both metabolites have been developed. High-pressure liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) is quick and sensitive but, unlike HPLC and gas chromatography-mass spectrometry (GC/MS), it is unavailable in most laboratories. Thus, we decided to evaluate comparatively HPLC-MS/MS, GC/MS and HPLC methods, as well as to establish reference values in a healthy paediatric population. According to our results, these three methods may be suitable for analysing GAA in urine. Furthermore, Passing-Bablock plots showed good agreement among all three. However, when comparing the Cr/Crn ratio, our results revealed that while HPLC-MS/MS data were in agreement with those of GC/MS, a constant and proportional error was observed when compared with those of HPLC. Consequently, the Cr/Crn ratio obtained by the last method should be evaluated with caution. Our reference values for GAA and Cr/Crn ratio in urine negatively correlate with age. Concerning GAA and Cr measurements in plasma, it is interesting to note that in contrast to what was occurring in urine, GAA concentration increased significantly with age, while we did not find any significant difference for Cr values within the same age group.

NMR-based urinalysis for rapid diagnosis of ß-ureidopropionase deficiency in a patient with Dravet syndrome.

BACKGROUND: Beta-ureidopropionase deficiency is a rare inborn error of metabolism (IEM) affecting pyrimidine metabolism. To-date, about 30 genetically confirmed cases had been reported. The clinical phenotypes of this condition are variable; some patients were asymptomatic while some may present with developmental delay or autistic features. In severe cases, patients may present with profound neurological deficit including hypotonia, seizures and mental retardation. Using NMR-based urinalysis, this condition can be rapidly diagnosed within 15 min. CASE: An 11-month-old Chinese boy had dual molecular diagnoses, ß-ureidopropionase deficiency and Dravet syndrome. He presented with intractable and recurrent convulsions, global developmental delay and microcephaly. Urine organic acid analysis using GC-MS and NMR-based urinalysis showed excessive amount of ß-ureidopropionic acid and ß-ureidoisobutyric acid, the two disease-specific markers for ß-ureidopropionase deficiency. Genetic analysis confirmed homozygous known disease-causing mutation UPB1 NM_016327.2: c.977G>A; NP_057411.1:p.R326Q. In addition, genetic analysis for Dravet syndrome showed the presence of heterozygous disease-causing mutation SCN1A NM_001165963.1:c.4494delC; NP_001159435.1:p.F1499Lfs*2. CONCLUSIONS: The differentiation between Dravet syndrome and ß-ureidopropionase deficiency is clinically challenging since both conditions share overlapping clinical features. The detection of urine ß-ureidoisobutyric and ß-ureidopropionic acids using NMR or GC-MS is helpful in laboratory diagnosis of ß-ureidopropionase deficiency. The disease-causing mutation, c.977G>A of ß-ureidopropionase deficiency, is highly prevalent in Chinese population (allele frequency=1.7%); ß-ureidopropionase deficiency screening test should be performed for any patients with unexplained neurological deficit, developmental delay or autism.

Hereditary disorders of purine and pyrimidine metabolism: identification of their biochemical phenotypes in the clinical laboratory.

OBJECTIVE: To describe a laboratory approach to the diagnosis of hereditary diseases of purine and pyrimidine metabolism and emphasize clinical situations in which these disorders should be considered in the differential diagnosis. DESIGN: Disease-specific patterns were identified in random specimens of ultrafiltered urine by using gradient high-performance liquid chromatography with diode-array detection, and reference ranges were established for uric acid, hypoxanthine, xanthine, and uracil expressed per creatinine in random specimens of urine. MATERIAL AND METHODS: Diagnostically significant purines and pyrimidines were separated with use of a Supelco LC-18-S nucleoside column eluted with 25 mmol/L ammonium acetate buffer and acetonitrile-methanol-water. Biologic fluids were prepared by ultrafiltration after addition of 3-methyluridine as internal standard. We used specimens negative for screening of metabolic disorders to establish reference ranges. RESULTS: Disease-specific patterns were identified in specimens with purine and pyrimidine disorders and several urea cycle disorders characterized by increased production of pyrimidine. CONCLUSION: The approach described identified disease-specific patterns of purine and pyrimidine disorders and several urea cycle disorders. We suggest that testing for purine and pyrimidine disorders be done in specimens evaluated in metabolic laboratories for "screening for inborn errors of metabolism."

Identification of a new point mutation in the human molybdenum cofactor sulferase gene that is responsible for xanthinuria type II.

A 43-year-old xanthinuric female was referred to our department because of hypouricemia. Routine laboratory data showed hypouricemia, a high level of plasma oxypurines, decreased urinary uric acid excretion, and increased urinary oxypurine excretion, with xanthine dehydrogenase activity in the duodenal mucosa below the limits of detection. In addition, allopurinol was not metabolized. From these findings, the patient was diagnosed with xanthinuria type II. To investigate the properties of xanthine dehydrogenase/xanthine oxidase (XDH/XO) deficiency, a cDNA sequence encoding XDH/XO, aldehyde oxidase (AO), and molybdenum cofactor sulferase (MCS), as well as immunoblotting analysis for XDH/XO protein, obtained from duodenal mucosa samples were performed. The XDH/XO cDNA and AO cDNA sequences of the xanthinuric patient were consistent with previously reported ones, whereas the MCS cDNA sequence revealed a point mutation of G to C in nucleotide 466, which changed codon 156 from GCC (Ala) to CCC (Pro). In addition, the MCS genomic DNA sequence including the site of the mutation revealed the same, suggesting that the xanthinuric patient was homozygous for this mutation. Such findings have not been previously reported for patients with xanthinuria type II.

Screening and diagnosis of beta-ureidopropionase deficiency by gas chromatographic/mass spectrometric analysis of urine.

Dihydropyrimidine dehydrogenase (DHPDase), dihydropyrimidinase (DHPase) and beta-ureidopropionase (betaUPase) are the enzymes that catalyze the first, second, and third steps of the degradation of pyrimidines, respectively. beta-Ureidopropionate (betaUP) and beta-ureidoisobutyrate (betaUIB) are increased in the urine of patients with betaUPase deficiency. The original case in which betaUPase deficiency was discovered by NMR spectroscopy was an 11-month-old patient who presented with hypotonia and dystonic movement. We detected a second but asymptomatic case during a pilot study of neonatal screening with filter-paper urine, urease pretreatment and gas chromatography/mass spectrometry (GC/MS). The urease pretreatment of urine without fractionation resulted in a high recovery of these polar ureide compounds and allowed the highly sensitive GC/MS detection and diagnosis of betaUPase deficiency. betaUP and betaUIB were identified using GC/MS techniques. In the urine of the neonate with betaUPase deficiency, betaUP and betaUIB were persistently increased. Thymine, 5,6-dihydrothymine and 5,6-dihydrouracil were increased only moderately but significantly. It is known that thymine and uracil increase markedly in DHPDase deficiency, and 5,6-dihydrothymine and 5,6-dihydrouracil increase in DHPase deficiency. Therefore, betaUPase deficiency can be differentially diagnosed from the first and second enzyme deficiencies. Application of this specific and sensitive diagnostic procedure will lead to an understanding of the clinical heterogeneity of betaUPase deficiency. Furthermore, the identification of patients with defects in pyrimidine metabolism will enable doctors to avoid cancer chemotherapy with pyrimidine analogues such as 5-fluorouracil, which could be dangerous for these patients.

Dihydropyrimidine dehydrogenase deficiency leading to thymine-uraciluria. An inborn error of pyrimidine metabolism.

Three unrelated patients with excessive thymine-uraciluria due to dihydropyrimidine dehydrogenase deficiency are described. Excretory values (mmol/g creatinine) were: uracil 2.0-10.5, thymine 2.3-7.5, 5-hydroxymethyluracil 0.2-0.9. Orally administered (index patient) uracil and thymine were excreted for the greater part whilst dihydrouracil and S-dihydrothymine were mainly metabolised. Dihydropyrimidine dehydrogenase activities (nmol X h-1 X mg-1 protein) in leucocytes were 0.04, 0.01 and less than 0.01 in the patients, 0.31-1.66 in their parents, and 1.01-4.46 in controls (n = 4). The patients presented with a non-specific clinical picture of cerebral dysfunction.