Disorders of purine biosynthesis metabolism.

Purines are essential molecules that are components of vital biomolecules, such as nucleic acids, coenzymes, signaling molecules, as well as energy transfer molecules. The de novo biosynthesis pathway starts from phosphoribosylpyrophosphate (PRPP) and eventually leads to the synthesis of inosine monophosphate (IMP) by means of 10 sequential steps catalyzed by six different enzymes, three of which are bi-or tri-functional in nature. IMP is then converted into guanosine monophosphate (GMP) or adenosine monophosphate (AMP), which are further phosphorylated into nucleoside di- or tri-phosphates, such as GDP, GTP, ADP and ATP. This review provides an overview of inborn errors of metabolism pertaining to purine synthesis in humans, including either phosphoribosylpyrophosphate synthetase (PRS) overactivity or deficiency, as well as adenylosuccinate lyase (ADSL), 5-aminoimidazole-4-carboxamide ribonucleotide formyltransferase/IMP cyclohydrolase (ATIC), phosphoribosylaminoimidazole succinocarboxamide synthetase (PAICS), and adenylosuccinate synthetase (ADSS) deficiencies. ITPase deficiency is being described as well. The clinical spectrum of these disorders is broad, including neurological impairment, such as psychomotor retardation, epilepsy, hypotonia, or microcephaly; sensory involvement, such as deafness and visual disturbances; multiple malformations, as well as muscle presentations or consequences of hyperuricemia, such as gouty arthritis or kidney stones. Clinical signs are often nonspecific and, thus, overlooked. It is to be hoped that this is likely to be gradually overcome by using sensitive biochemical investigations and next-generation sequencing technologies.

New insights in research on purine and pyrimidine metabolism.

In June 2021, the Purine and Pyrimidine Society (PPS) organized the 19th biennial symposium on Purine and Pyrimidine metabolism (PP21). Due to the ongoing pandemic, the conference was organized as a webinar over 3 days with sessions dealing with enzymes, cancer, inborn errors, gout among others. The current issue of Nucleosides, Nucleotides & Nucleic Acids is a special issue covering proceedings from PP21-presentations and other PPS-related manuscripts, and in this editorial, we will give an overview of the scientific program of the meeting.

Partial Purine Nucleoside Phosphorylase Deficiency Helps Determine Minimal Activity Required for Immune and Neurological Development.

Introduction: Complete or near complete absence of the purine nucleoside phosphorylase (PNP) enzyme causes a profound T cell immunodeficiency and neurological abnormalities that are often lethal in infancy and early childhood. We hypothesized that patients with partial PNP deficiency, characterized by a late and mild phenotype due to residual PNP enzyme, would provide important information about the minimal PNP activity needed for normal development. Methods: Three siblings with a homozygous PNP gene mutation (c.769C>G, p.His257Asp) resulting in partial PNP deficiency were investigated. PNP activity was semi-quantitively assayed by the conversion of [14C]inosine in hemolysates, mononuclear cells, and lymphoblastoid B cells. PNP protein expression was determined by Western Blotting in lymphoblastoid B cells. DNA repair was quantified by measuring viability of lymphoblastoid B cells following ionizing irradiation. Results: A 21-year-old female was referred for recurrent sino-pulmonary infections while her older male siblings, aged 25- and 28- years, did not suffer from significant infections. Two of the siblings had moderately reduced numbers of T, B, and NK cells, while the other had near normal lymphocyte subset numbers. T cell proliferations were normal in the two siblings tested. Hypogammaglobulinemia was noted in two siblings, including one that required immunoglobulin replacement. All siblings had typical (normal) neurological development. PNP activity in various cells from two patients were 8-11% of the normal level. All siblings had normal blood uric acid and increased PNP substrates in the urine. PNP protein expression in cells from the two patients examined was similar to that observed in cells from healthy controls. The survival of lymphoblastoid B cells from 2 partial PNP-deficient patients after irradiation was similar to that of PNP-proficient cells and markedly higher than the survival of cells from a patient with absent PNP activity or a patient with ataxia telangiectasia. Conclusions: Patients with partial PNP deficiency can present in the third decade of life with mild-moderate immune abnormalities and typical development. Near-normal immunity might be achieved with relatively low PNP activity.

Clinical, biochemical, mitochondrial, and metabolomic aspects of methylmalonate semialdehyde dehydrogenase deficiency: Report of a fifth case.

Methylmalonate semialdehyde dehydrogenase deficiency (MMSDD; MIM 614105) is a rare autosomal recessive defect of valine and pyrimidine catabolism. Four prior MMSDD cases are published. We present a fifth case, along with functional and metabolomic analysis. The patient, born to non-consanguineous parents of East African origin, was admitted at two weeks of age for failure to thrive. She was nondysmorphic, had a normal brain MRI, and showed mild hypotonia. Gastroesophageal reflux occurred with feeding. Urine organic acid assessment identified excess 3-hydroxyisobutyrate and 3-hydroxypropionate, while urine amino acid analysis identified elevated concentrations of ß-aminoisobutyrate and ß-alanine. Plasma amino acids showed an elevated concentration of ß-aminoisobutyrate with undetectable ß-alanine. ALDH6A1 gene sequencing identified a homozygous variant of uncertain significance, c.1261C > T (p.Pro421Ser). Management with valine restriction led to reduced concentration of abnormal analytes in blood and urine, improved growth, and reduced gastroesophageal reflux. Western blotting of patient fibroblast extracts demonstrated a large reduction of methylmalonate semialdehyde dehydrogenase (MMSD) protein. Patient cells displayed compromised mitochondrial function with increased superoxide production, reduced oxygen consumption, and reduced ATP production. Metabolomic profiles from patient fibroblasts demonstrated over-representation of fatty acids and fatty acylcarnitines, presumably due to methylmalonate semialdehyde shunting to ß-alanine and subsequently to malonyl-CoA with ensuing increase of fatty acid synthesis. Previously reported cases of MMSDD have shown variable clinical presentation. Our case continues the trend as clinical phenotypes diverge from prior cases. Recognition of mitochondrial dysfunction and novel metabolites in this patient provide the opportunity to assess future patients for secondary changes that may influence clinical outcome.

Modeling the Outcome of Systematic TPMT Genotyping or Phenotyping Before Azathioprine Prescription: A Cost-Effectiveness Analysis.

BACKGROUND: Thiopurine S-methyltransferase (TPMT) testing, either by genotyping or phenotyping, can reduce the incidence of adverse severe myelotoxicity episodes induced by azathioprine. The comparative cost-effectiveness of TPMT genotyping and phenotyping are not known. OBJECTIVE: Our aim was to assess the cost-effectiveness of phenotyping-based dosing of TPMT activity, genotyping-based screening and no screening (reference) for patients treated with azathioprine. METHODS: A decision tree was built to compare the conventional weight-based dosing strategy with phenotyping and with genotyping using a micro-simulation model of patients with inflammatory bowel disease from the perspective of the French health care system. The time horizon was set up as 1 year. Only direct medical costs were used. Data used were obtained from previous reports, except for screening test and admission costs, which were from real cases. The main outcome was the cost-effectiveness ratios, with an effectiveness criterion of one averted severe myelotoxicity episode. RESULTS: The total expected cost of the no screening strategy was €409/patient, the total expected cost of the phenotyping strategy was €427/patient, and the total expected cost of the genotyping strategy was €476/patient. The incremental cost-effectiveness ratio was €2602/severe myelotoxicity averted in using the phenotyping strategy, and €11,244/severe myelotoxicity averted in the genotyping strategy compared to the no screening strategy. At prevalence rates of severe myelotoxicity > 1%, phenotyping dominated genotyping and conventional strategies. CONCLUSION: The phenotype-based strategy to screen for TPMT deficiency dominates (cheaper and more effective) the genotype-based screening strategy in France. Phenotype-based screening dominates no screening in populations with a prevalence of severe myelosuppression due to azathioprine of > 1%.

Effects of AMPD1 common mutation on the metabolic-chronotropic relationship: Insights from patients with myoadenylate deaminase deficiency.

PURPOSE: Current evidence indicates that the common AMPD1 gene variant is associated with improved survival in patients with advanced heart failure. Whilst adenosine has been recognized to mediate the cardioprotective effect of C34T AMPD1, the precise pathophysiologic mechanism involved remains undefined to date. To address this issue, we used cardio-pulmonary exercise testing data (CPX) from subjects with myoadenylate deaminase (MAD) defects. METHODS: From 2009 to 2013, all the patients referred in our laboratory to perform a metabolic exercise testing, i.e. a CPX with measurements of muscle metabolites in plasma during and after exercise testing, were prospectively enrolled. Subjects that also underwent an open muscle biopsy for diagnosis purpose were finally included. The metabolic-chronotropic response was assessed by calculating the slope of the linear relationship between the percent heart rate reserve and the percent metabolic reserve throughout exercise. MAD activity was measured using the Fishbein's technique in muscle biopsy sample. The common AMPD1 mutation was genotyped and the AMPD1 gene was sequenced to screen rare variants from blood DNA. RESULTS: Sixty-seven patients were included in the study; 5 had complete MAD deficiency, 11 had partial MAD deficiency, and 51 had normal MAD activity. Compared with normal MAD activity subjects, MAD deficient subjects appeared to have a lower-than-expected metabolic-chronotopic response during exercise. The metabolic-chronotropic relationship is more closely correlated with MAD activity in skeletal muscle (Rs = 0.57, p = 5.93E-7, Spearman correlation) than the presence of the common AMPD1 gene variant (Rs = 0.34, p = 0.005). Age-predicted O2 pulse ratio is significantly increased in MAD deficient subjects, indicating a greater efficiency of the cardiovascular system to deliver O2 (p < 0.01, Scheffé's post hoc test). CONCLUSION: The metabolic-chronotropic response is decreased in skeletal muscle MAD deficiency, suggesting a biological mechanism by which AMPD1 gene exerts cardiac effect.

Thiopurine S-Methyltransferase as a Pharmacogenetic Biomarker: Significance of Testing and Review of Major Methods.

BACKGROUND: Thiopurine S-methyltransferase (TPMT) enzyme metabolizes thiopurine drugs which are widely used in various disciplines as well as in leukemias. Individual enzyme activity varies depending on the genetic polymorphisms of TPMT gene located at chromosome 6. Up to 14% of population is known to have a decreased enzyme activity, and if treated with standard doses of thiopurines, these individuals are at a high risk of severe Adverse Drug Reactions (ADR) as myelosuppression, gastrointestinal intolerance, pancreatitis and hypersensitivity. However, TPMT-deficient patients can successfully be treated with decreased thiopurine doses if enzyme status is identified by a prior testing. TPMT status identification is a pioneering experience in application of pharmacogenetic testing in clinical settings. 4 TPMT (*2, *3A, *3B, *3C) alleles are known to account for 80-95% of a decreased enzyme activity, and therefore, identifying the presence of these alleles supported by phenotypic measurement of the enzyme activity can reveal patient's TPMT status. Evaluation of the levels of thiopurine metabolites further supports the practice of appropriate dose adjustment by providing the efficient monitoring of drug cytotoxicity. CONCLUSION: We hereby review the thiopurine pharmacogenetics and the methods applied in common practice to evaluate patient's TPMT status.

Diagnostic Algorithm for Glycogenoses and Myoadenylate Deaminase Deficiency Based on Exercise Testing Parameters: A Prospective Study.

AIM: Our aim was to evaluate the accuracy of aerobic exercise testing to diagnose metabolic myopathies. METHODS: From December 2008 to September 2012, all the consecutive patients that underwent both metabolic exercise testing and a muscle biopsy were prospectively enrolled. Subjects performed an incremental and maximal exercise testing on a cycle ergometer. Lactate, pyruvate, and ammonia concentrations were determined from venous blood samples drawn at rest, during exercise (50% predicted maximal power, peak exercise), and recovery (2, 5, 10, and 15 min). Biopsies from vastus lateralis or deltoid muscles were analysed using standard techniques (reference test). Myoadenylate deaminase (MAD) activity was determined using p-nitro blue tetrazolium staining in muscle cryostat sections. Glycogen storage was assessed using periodic acid-Schiff staining. The diagnostic accuracy of plasma metabolite levels to identify absent and decreased MAD activity was assessed using Receiver Operating Characteristic (ROC) curve analysis. RESULTS: The study involved 51 patients. Omitting patients with glycogenoses (n = 3), MAD staining was absent in 5, decreased in 6, and normal in 37 subjects. Lactate/pyruvate at the 10th minute of recovery provided the greatest area under the ROC curves (AUC, 0.893 ± 0.067) to differentiate Abnormal from Normal MAD activity. The lactate/rest ratio at the 10th minute of recovery from exercise displayed the best AUC (1.0) for discriminating between Decreased and Absent MAD activities. The resulting decision tree achieved a diagnostic accuracy of 86.3%. CONCLUSION: The present algorithm provides a non-invasive test to accurately predict absent and decreased MAD activity, facilitating the selection of patients for muscle biopsy and target appropriate histochemical analysis.

Novel developments in metabolic disorders of purine and pyrimidine metabolism and therapeutic applications of their analogs.

The biennial 15th symposium on Purine and Pyrimidine metabolism was held in Madrid, June 2013 (PP13). During the meeting, several novel developments on the diagnosis, pathophysiology, and treatment of several inborn errors of purine and pyrimidine metabolism were presented. These ranged from new drugs for gout to enzyme replacement therapies for mitochondrial diseases. A relatively novel aspect in this meeting was the interest in purine and pyrimidine metabolism in nonmammalian systems, such as parasites, mycoplasms, and bacteria. Development of novel analogs for parasite infections, cardiovascular diseases, inflammatory diseases, and cancer were also discussed.

Metabolic disorders of purine metabolism affecting the nervous system.

The purines are a group of molecules used by all cells for many vital biochemical processes including energy-requiring enzymatic reactions, cofactor-requiring reactions, synthesis of DNA or RNA, signaling pathways within and between cells, and other processes. Defects in some of the enzymes of purine metabolism are known to be associated with specific clinical disorders, and neurological problems may be a presenting sign or the predominant clinical problem for several of them. This chapter describes three disorders for which the clinical features and metabolic basis are well characterized. Deficiency of adenylosuccinate-lyase (ADSL) causes psychomotor retardation, epilepsy, and autistic features. Lesch-Nyhan disease is caused by deficiency of hypoxanthine-guanine phosphoribosyltransferase (HPRT) and is characterized by hyperuricemia, motor and cognitive disability, and self-injurious behavior. Deficiency of myoadenylate deaminase (mAMPD) is associated with myopathic features. In addition to these disorders, several other disorders are briefly summarized. These include defects of phosphoribosylpyrophosphate synthase, adenosine deaminase (ADA), purine nucleoside phosphorylase (PND), deoxyguanosine kinase (dGK), or IMP dehydrogenase (IMPDH). Each of these disorders provides an unusual window on the unique importance of purine metabolism for function of different parts of the nervous system.

Genetic and metabolomic analysis of AdeD and AdeI mutants of de novo purine biosynthesis: cellular models of de novo purine biosynthesis deficiency disorders.

Purines are molecules essential for many cell processes, including RNA and DNA synthesis, regulation of enzyme activity, protein synthesis and function, energy metabolism and transfer, essential coenzyme function, and cell signaling. Purines are produced via the de novo purine biosynthesis pathway. Mutations in purine biosynthetic genes, for example phosphoribosylaminoimidazole carboxylase/phosphoribosylaminoimidazole succinocarboxamide synthetase (PAICS, E.C. 6.3.2.6/E.C. 4.1.1.21), can lead to developmental anomalies in lower vertebrates. Alterations in PAICS expression in humans have been associated with various types of cancer. Mutations in adenylosuccinate lyase (ADSL, E.C. 4.3.2.2) or 5-aminoimidazole-4-carboxamide ribonucleotide formyltransferase/IMP cyclohydrolase (ATIC, E.C. 2.1.2.3/E.C. 3.5.4.10) lead to inborn errors of metabolism with a range of clinical symptoms, including developmental delay, severe neurological symptoms, and autistic features. The pathogenetic mechanism is unknown for these conditions, and no effective treatments exist. The study of cells carrying mutations in the various de novo purine biosynthesis pathway genes provides one approach to analysis of purine disorders. Here we report the characterization of AdeD Chinese hamster ovary (CHO) cells, which carry genetic mutations encoding p.E177K and p.W363* variants of PAICS. Both mutations impact PAICS structure and completely abolish its biosynthesis. Additionally, we describe a sensitive and rapid analytical method for detection of purine de novo biosynthesis intermediates based on high performance liquid chromatography with electrochemical detection. Using this technique we detected accumulation of AIR in AdeD cells. In AdeI cells, mutant for the ADSL gene, we detected accumulation of SAICAR and SAMP and, somewhat unexpectedly, accumulation of AIR. This method has great potential for metabolite profiling of de novo purine biosynthesis pathway mutants, identification of novel genetic defects of purine metabolism in humans, and elucidating the regulation of this critical metabolic pathway.

High prevalence of polymorphism and low activity of thiopurine methyltransferase in patients with inflammatory bowel disease.

BACKGROUND: Gene polymorphism of thiopurine methyltransferase (TPMT) correlates with decreased enzyme activity which determines a significant risk of adverse effect reactions (ADR) in patients treated with thiopurines. The aim of this study was to investigate TPMT genotype and phenotype status in patients with inflammatory bowel diseases (IBD). METHODS: Fifty-one consecutive out-patients with IBD were genotyped for the following allelic variants: rs1800462 (referred as TPMT 2 allele), rs1800460 (referred as TPMT 3B allele), and 1142345 (referred as TPMT 3C allele). Red blood cell TPMT activity was measured using a competitive micro-well immunoassay for the semi-quantitative determination of TPMT activity in red blood cells (RBC) by means of a 6-MP substrate. RESULTS: Polymorphism of TPMT was found in 5 out of 51 patients (10%; 95% CI 2%-18%), three heterozygous and two homozygous carriers. Six patients (11.8%; 95% CI 2.4%-19.5%) displayed very low, 12 (23.5%; 95% CI 11.4%-34.5%) intermediate, and 33 (64.7%; 95% CI 52%-78%) normal/high TPMT activity. There were no differences between TPMT genotype and phenotype groups according to age, type of disease, smoking, and chronic medications. A 71% (95% CI 61%-81%; κ=0.45) concordance rate was found between genotype and phenotype status. Six out of 27 (22%) current or past users of azathioprine developed ADR, with three (50%) displaying TPMT genotype and/or phenotype alterations. CONCLUSION: Compared to the general population, IBD patients may have significantly higher prevalence of TPMT polymorphism and, even more, low activity. Phenotypic more than genotypic TPMT analysis could be useful to better manage IBD therapy with thiopurines.

Neurological disorders of purine and pyrimidine metabolism.

Purines and pyrimidines, regarded for a long time only as building blocks for nucleic acid synthesis and intermediates in the transfer of metabolic energy, gained increasing attention since genetically determined aberrations in their metabolism were associated clinically with various degrees of mental retardation and/or unexpected and often devastating neurological dysfunction. In most instances the molecular mechanisms underlying neurological symptoms remain undefined. This suggests that nucleotides and nucleosides play fundamental but still unknown roles in the development and function of several organs, in particular central nervous system. Alterations of purine and pyrimidine metabolism affecting brain function are spread along both synthesis (PRPS, ADSL, ATIC, HPRT, UMPS, dGK, TK), and breakdown pathways (5NT, ADA, PNP, GCH, DPD, DHPA, TP, UP), sometimes also involving pyridine metabolism. Explanations for the pathogenesis of disorders may include both cellular and mitochondrial damage: e.g. deficiency of the purine salvage enzymes hypoxanthine-guanine phosphoribosyltransferase and deoxyguanosine kinase are associated to the most severe pathologies, the former due to an unexplained adverse effect exerted on the development and/or differentiation of dopaminergic neurons, the latter due to impairment of mitochondrial functions. This review gathers the presently known inborn errors of purine and pyrimidine metabolism that manifest neurological syndromes, reporting and commenting on the available hypothesis on the possible link between specific enzymatic alterations and brain damage. Such connection is often not obvious, and though investigated for many years, the molecular basis of most dysfunctions of central nervous system associated to purine and pyrimidine metabolism disorders are still unexplained.

Pediatric neurological syndromes and inborn errors of purine metabolism.

This review is devised to gather the presently known inborn errors of purine metabolism that manifest neurological pediatric syndromes. The aim is to draw a comprehensive picture of these rare diseases, characterized by unexpected and often devastating neurological symptoms. Although investigated for many years, most purine metabolism disorders associated to psychomotor dysfunctions still hide the molecular link between the metabolic derangement and the neurological manifestations. This basically indicates that many of the actual functions of nucleosides and nucleotides in the development and function of several organs, in particular central nervous system, are still unknown. Both superactivity and deficiency of phosphoribosylpyrophosphate synthetase cause hereditary disorders characterized, in most cases, by neurological impairments. The deficiency of adenylosuccinate lyase and 5-amino-4-imidazolecarboxamide ribotide transformylase/IMP cyclohydrolase, both belonging to the de novo purine synthesis pathway, is also associated to severe neurological manifestations. Among catabolic enzymes, hyperactivity of ectosolic 5'-nucleotidase, as well as deficiency of purine nucleoside phosphorylase and adenosine deaminase also lead to syndromes affecting the central nervous system. The most severe pathologies are associated to the deficiency of the salvage pathway enzymes hypoxanthine-guanine phosphoribosyltransferase and deoxyguanosine kinase: the former due to an unexplained adverse effect exerted on the development and/or differentiation of dopaminergic neurons, the latter due to a clear impairment of mitochondrial functions. The assessment of hypo- or hyperuricemic conditions is suggestive of purine enzyme dysfunctions, but most disorders of purine metabolism may escape the clinical investigation because they are not associated to these metabolic derangements. This review may represent a starting point stimulating both scientists and physicians involved in the study of neurological dysfunctions caused by inborn errors of purine metabolism with the aim to find novel therapeutical approaches.

Nephrolithiasis related to inborn metabolic diseases.

Nephrolithiasis associated with inborn metabolic diseases is a very rare condition with some common characteristics: early onset of symptoms, family history, associated tubular impairment, bilateral, multiple and recurrent stones, and association with nephrocalcinosis. The prognosis of such diseases may lead to life threatening conditions, not only because of unabated kidney damage but also because of progressive extra-renal involvement, either in a systemic form (e.g. primary hyperoxaluria type 1, requiring combined liver and kidney transplantation), or in a neurological form (Lesch-Nyhan syndrome leading to auto-mutilation and disability, phosphoribosyl pyrophosphate synthetase superactivity, which is associated with mental retardation). Patients with other inborn metabolic diseases present only with recurrent stone formation, such as cystinuria, adenine phosphoribosyl-transferase deficiency, xanthine deficiency. Finally, nephrolithiasis may be secondarily part of some other metabolic diseases, such as glycogen storage disease type 1 or inborn errors of metabolism leading to Fanconi syndrome (nephropathic cystinosis, tyrosinaemia type 1, fructose intolerance, Wilson disease, respiratory chain disorders, etc.). The diagnosis is based on highly specific investigations, including crystal identification, biochemical analyses and DNA study. The treatment of nephrolithiasis requires hydration as well as specific measures. Compliance is a major issue regarding the progression of renal damage, but the overall outcome mainly depends on extra-renal involvement in relation to the metabolic defect.

Children's toxicology from bench to bed--Liver injury (1): Drug-induced metabolic disturbance--toxicity of 5-FU for pyrimidine metabolic disorders and pivalic acid for carnitine metabolism.

Congenital disorders of metabolism show a wide spectrum of symptoms as a consequence of impairment of a certain metabolic pathway by mutated enzymes resulting in abnormal accumulation of enzyme substrates, deficiency of expected products, and abnormal burden to collateral metabolic pathways, etc. However, in some occasions, depending on which pathway up to what degree of disturbance, it can be asymptomatic until a certain kind of burden is placed on to the patient. Enzyme deficiency involved in pyrimidine degradation, such as Dihydropyrimidine dehydrogenase (DPD) and Dihydropyrimidinase (DHP), has been reported with convulsion or autism as symptoms, but many asymptomatic cases are also reported. However, when the patients are treated with 5-fluorouracil, a pyrimidine analogue anticancer drug, lethal side-effects can be seen even in asymptomatic patients. Some oral cephem antibiotics have pivalic acid side chain to increase absorption rate at intestine. These antibiotics degrade into active antibiotics and pivalic acid at the intestinal wall. This pivalic acid is carnitine-conjugated and excreted into urine. Carnitine acts as a carrier of long chain fatty acid to mitochondria and to beta-oxidation, thus an important molecule for energy production by beta-oxidation and maintenance of mitochondrial function. Because of this, long term administration of such antibiotics could induce depletion of carnitine from the body and lead to low ketotic hypoglycemia, convulsion and consciousness disturbance. This paper reports some possible serious side effects closely linked to drug metabolism.

Efficacy and safety of allopurinol in patients with hypoxanthine-guanine phosphoribosyltransferase deficiency.

Hypoxanthine-guanine phosphoribosyltransferase (HPRT) deficiency is a genetic disease of purine metabolism resulting in uric acid overproduction. Allopurinol, which inhibits the enzyme xanthine oxidase and reduces uric acid synthesis, is widely used for the treatment of gout and uric acid overproduction. The aim of the study was to analyze the long-term efficacy and safety of allopurinol in patients with HPRT deficiency. Nineteen patients (13 with Lesch-Nyhan syndrome and 6 with partial HPRT deficiency) were treated with allopurinol (mean dose, 6.4 mg/kg body weight per day; range, 3.7-9.7 mg/kg body weight per day) and followed up for at least 12 months (mean follow-up, 7.6 years). The efficacy of allopurinol was evaluated by serial measurement of purine metabolic parameters and renal function as well as by clinical manifestations. Safety was assessed by recording adverse events. Treatment with allopurinol normalized serum urate level in all patients and resulted in a mean reduction in serum urate of 47%. Allopurinol treatment was associated with a mean 74% reduction in urinary uric acid-to-creatinine ratio. In contrast, allopurinol treatment increased mean hypoxanthine and xanthine urinary excretion rates 5.4- and 9.5-fold, respectively, compared with baseline levels. The decrease in uric acid excretion in complete and partial HPRT-deficient patients was not accompanied by a stoichiometric substitution of hypoxanthine and xanthine excretion rates. Allopurinol-related biochemical changes were similar in patients with either complete or partial HPRT deficiency. Renal function remained stable or improved with treatment. Three patients had urolithiasis during allopurinol treatment. In 2 patients, xanthine stones were documented and they required allopurinol dose adjustments aimed at reducing excessive oxypurine excretion rates. No allopurinol hypersensitivity reactions occurred. Neurologic manifestations were not influenced by allopurinol therapy. In conclusion, allopurinol is efficacious and generally safe for the treatment of uric acid overproduction in patients with HPRT deficiencies. Xanthine lithiasis, developing as a consequence of allopurinol therapy, should be preventable by adjustment of allopurinol dose.

Pyrimidine pathways in health and disease.

Genetic defects involving enzymes essential for pyrimidine nucleotide metabolism have provided new insights into the vital physiological functions of these molecules in addition to nucleic acid synthesis. Such aberrations disrupt the haematological, nervous or mitochondrial systems and can cause adverse reactions to analogue therapy. Regulation of pyrimidine pathways is also known to be disrupted in malignancies. Nine genetic defects have now been identified but only one is currently treatable. Diagnosis is aided by the accumulation of specific metabolites. Recently, progress has been made in understanding the molecular mechanisms underlying inborn errors of pyrimidine metabolism, together with the key clinical issues and the implications for the future development of novel drugs and therapeutic strategies.

Preparation of 5-amino-4-imidazole-N-succinocarboxamide ribotide, 5-amino-4-imidazole-N-succinocarboxamide riboside and succinyladenosine, compounds usable in diagnosis and research of adenylosuccinate lyase deficiency.

The enzyme adenylosuccinate lyase (ADSL) intervenes twice in the biosynthesis of adenine nucleotides. ADSL deficiency is an inherited metabolic disease characterized by various degrees of psychomotor retardation and accumulation of dephosphorylated enzyme substrates 5-amino-4-imidazole-N-succinocarboxamide riboside (SAICAr) and succinyladenosine (SAdo) in body fluids. Severity of symptoms seems to correlate with residual activity of mutant enzyme and with SAdo/SAICAr concentration ratio in cerebrospinal fluid. To better understand the pathogenetic mechanisms of the disease symptoms, studies of catalytic properties of mutant enzymes together with in vitro and in vivo experiments utilizing SAICAr and SAdo must be performed. Such studies require availability of both ADSL substrates, 5-amino-4-imidazole-N-succinocarboxamide ribotide (SAICAR) and succinyladenosine 5'-monophosphate (SAMP) and their dephosphorylated products in sufficient amounts and purity. Except for SAMP, none of these compounds is commercially available and they must therefore be synthesized. SAICAR was prepared by recombinant human ADSL-catalysed reaction of AICAR (5-aminoimidazole-4-carboxamide) with fumarate and isolated by thin-layer chromatography. SAICAr and SAdo were prepared by calf intestine alkaline phosphatase-catalysed dephosphorylation of SAICAR and SAMP and isolated on cation- and anion-exchange resin columns. The procedures described are easily scalable and provide high yields of sufficiently pure products for use in experiments related to studies of pathogenetic mechanisms in ADSL deficiency.

Recombinant Rhodobacter capsulatus xanthine dehydrogenase, a useful model system for the characterization of protein variants leading to xanthinuria I in humans.

Rhodobacter capsulatus xanthine dehydrogenase (XDH) forms an (alphabeta)2 heterotetramer and is highly homologous to homodimeric eukaryotic XDHs. The crystal structures of bovine XDH and R. capsulatus XDH showed that the two proteins have highly similar folds. We have developed an efficient system for the recombinant expression of R. capsulatus XDH in Escherichia coli. The recombinant protein shows spectral features and a range of substrate specificities similar to bovine milk xanthine oxidase. However, R. capsulatus XDH is at least 5 times more active than bovine XDH and, unlike mammalian XDH, does not undergo the conversion to the oxidase form. EPR spectra were obtained for the FeS centers of the enzyme showing an axial signal for FeSI, which is different from that reported for xanthine oxidase. X-ray absorption spectroscopy at the iron and molybdenum K-edge and the tungsten LIII-edge have been used to probe the different metal coordinations of variant forms of the enzyme. Based on a mutation identified in a patient suffering from xanthinuria I, the corresponding arginine 135 was substituted to a cysteine in R. capsulatus XDH, and the protein variant was purified and characterized. Two different forms of XDH-R135C were purified, an active (alphabeta)2 heterotetrameric form and an inactive (alphabeta) heterodimeric form. The active form contains a full complement of redox centers, whereas in the inactive form the FeSI center is likely to be missing.