Aminoimidazole Carboxamide Ribotide Research Articles

Metabolites of De Novo Purine Synthesis: Metabolic Regulators and Cytotoxic Compounds.

Cytotoxicity of de novo purine synthesis (DNPS) metabolites is critical to the pathogenesis of three known and one putative autosomal recessive disorder affecting DNPS. These rare disorders are caused by biallelic mutations in the DNPS genes phosphoribosylformylglycineamidine synthase (PFAS), phosphoribosylaminoimidazolecarboxylase/phosphoribosylaminoimidazolesuccinocarboxamide synthase (PAICS), adenylosuccinate lyase (ADSL), and aminoimidazole carboxamide ribonucleotide transformylase/inosine monophosphate cyclohydrolase (ATIC) and are clinically characterized by developmental abnormalities, psychomotor retardation, and nonspecific neurological impairment. At a biochemical level, loss of function of specific mutated enzymes results in elevated levels of DNPS ribosides in body fluids. The main pathogenic effect is attributed to the accumulation of DNPS ribosides, which are postulated to be toxic to the organism. Therefore, we decided to characterize the uptake and flux of several DNPS metabolites in HeLa cells and the impact of DNPS metabolites to viability of cancer cell lines and primary skin fibroblasts. We treated cells with DNPS metabolites and followed their flux in purine synthesis and degradation. In this study, we show for the first time the transport of formylglycinamide ribotide (FGAR), aminoimidazole ribotide (AIR), succinylaminoimidazolecarboxamide ribotide (SAICAR), and aminoimidazolecarboxamide ribotide (AICAR) into cells and their flux in DNPS and the degradation pathway. We found diminished cell viability mostly in the presence of FGAR and AIR. Our results suggest that direct cellular toxicity of DNPS metabolites may not be the primary pathogenetic mechanism in these disorders.

Untargeted metabolomics confirms and extends the understanding of the impact of aminoimidazole carboxamide ribotide (AICAR) in the metabolic network of Salmonella enterica.

In Salmonella enterica, aminoimidazole carboxamide ribotide (AICAR) is a purine biosynthetic intermediate and a substrate of the AICAR transformylase/IMP cyclohydrolase (PurH) enzyme. When purH is eliminated in an otherwise wild-type strain, AICAR accumulates and indirectly inhibits synthesis of the essential coenzyme thiamine pyrophosphate (TPP). In this study, untargeted metabolomics approaches were used to i) corroborate previously defined metabolite changes, ii) define the global consequences of AICAR accumulation and iii) investigate the metabolic effects of mutations that restore thiamine prototrophy to a purH mutant. The data showed that AICAR accumulation led to an increase in the global regulator cyclic AMP (cAMP) and that disrupting central carbon metabolism could decrease AICAR and/or cAMP to restore thiamine synthesis. A mutant (icc) blocked in cAMP degradation that accumulated cAMP but had wild-type levels of AICAR was used to identify changes in the purH metabolome that were a direct result of elevated cAMP. Data herein describe the use of metabolomics to identify the metabolic state of mutant strains and probe the underlying mechanisms used by AICAR to inhibit thiamine synthesis. The results obtained provide a cautionary tale of using metabolite concentrations as the only data to define the physiological state of a bacterial cell.

Screening for adenylosuccinate lyase deficiency using tandem mass spectrometry analysis of succinylpurines in neonatal dried blood spots

ObjectivesStable isotope dilution coupled with liquid chromatography–tandem mass spectrometry (LC–MS/MS) is the sensitive method for screening for various inherited metabolic disorders using dried blood spots (DBSs). We present a method for LC–MS/MS determination of succinyladenosine (SAdo) and succinylaminoimidazole carboxamide riboside (SAICAr), biomarkers for adenylosuccinate lyase deficiency (dADSL), in DBS. Design and methodsSAICAr and SAdo were separated on a Symmetry-C18 column and detected using positive electrospray ionisation in selected reaction monitoring mode. The quantification was performed using the isotopically labelled internal standards SAdo-13C4 and SAICAr-13C4, which were prepared via ADSL-catalysed reactions of fumarate-13C4 with adenosine monophosphate and aminoimidazole carboxamide ribotide, respectively, and subsequent alkaline phosphatase-catalysed dephosphorylation of the resulting products. ResultsThe detection of SAICAr and SAdo in DBS was linear over the range of 0–25μmol/L. The respective intra-assay and inter-assay imprecision values were less than 10.7% and 15.2% for SAICAr and 4.7% and 5.7% for SAdo. The recoveries from DBS spiked with different concentrations of SAICAr and SAdo were between 94% and 117%. The concentrations of SAICAr and SAdo were higher in the archived DBS from dADSL patients (SAICAr, 0.03–4.7μmol/L; SAdo, 1.5–21.3μmol/L; n=5) compared to those of the control subjects (SAICAr, 0–0.026μmol/L; SAdo, 0.06–0.14μmol/L; n=31), even after DBSs from dADSL patients were stored for 2–23years. ConclusionsWe developed and validated a method of succinylpurine analysis in DBS that improves selective screening for dADSL in the paediatric population and may be used for retrospective diagnosis to aid the genetic counselling of affected families.

Low-Dose Methotrexate Results in the Selective Accumulation of Aminoimidazole Carboxamide Ribotide in an Erythroblastoid Cell Line

Therapeutic and toxic response to low-dose methotrexate (MTX) in the treatment of autoimmune disease continues to be highly variable, resulting in a critical need to identify predictive biomarkers of response. Biomarker development has been hampered by an incomplete understanding of the molecular pharmacology of low-dose MTX. To address this issue, accumulation of the substrates for aminoimidazole carboxamide ribonucleotide transformylase (AICART) and thymidylate synthase (TS) was measured as markers of pharmacological activity of MTX in an erythroblastoid cell line. A 115-fold increase in the AICART substrate and anti-inflammatory mediator, 5-aminoimidazole-4-carboxamide-1-β-d-ribofuranosyl 5'-monophosphate (ZMP), was observed following exposure to 10 nM MTX but subsequently decreased with increasing MTX concentrations, declining to baseline levels with 1000 nM MTX. In contrast, the TS substrate, 2'-deoxyuridine 5'-monophosphate disodium salt (dUMP), displayed concentration-dependent accumulation, increasing 29-, 342-, and 471-fold over baseline with 10, 100, and 1000 nM MTX, respectively. Cellular levels of dUMP correlated with levels of the parent drug (MTX-PG1; r = 0.66, P < 0.001) and its polyglutamates (MTX-PG2-6) (r = 0.81, P < 0.001), whereas cellular levels of ZMP were only moderately correlated with MTX-PG1 (r = 0.34, P < 0.01). In contrast, accumulation of ZMP at 10 nM MTX was associated with a 2.9-fold increase in the AICART inhibitor dihydrofolate (DHF), represented primarily by long-chain DHF polyglutamates. Selectivity, defined as the ratio of ZMP to dUMP, was maximal following exposure to 6 nM MTX. Characterizing the range of MTX concentrations that selectively promote ZMP accumulation while preserving pyrimidine biosynthesis may lead to optimization of low-dose MTX therapy.

Isolated Neonatal Seizures: Adenylosuccinase Deficiency is Another Cause!

I read with great interest the very useful review “Isolated Neonatal Seizures: When to Suspect Inborn Errors of Metabolism” by Ficicioglu and Bearden [ [1] Ficicioglu C. Bearden D. Isolated neonatal seizures: When to suspect inborn errors of metabolism. Pediatr Neurol. 2011; 45: 283-291 Abstract Full Text Full Text PDF PubMed Scopus (31) Google Scholar ]. I would like to add adenylosuccinase deficiency to their causes for suspicion. This genetic, mainly neurologic disorder of the de novo purine synthesis was first reported in 1984 [ [2] Jaeken J. van den Berghe G. An infantile autistic syndrome characterized by the presence of succinylpurines in body fluids. Lancet. 1984; 2: 1058-1061 PubMed Google Scholar ]. Most of the approximately 60 reported patients demonstrate variable associations of moderate to severe psychomotor retardation, epilepsy after their first years, autistic features, and growth retardation [ [3] van den Berghe G. Vincent M.-F. Marie S. Disorders of purine and pyrimidine metabolism. in: Saudubray J.-M. van den Berghe G. Walter J.H. Inborn metabolic diseases: Diagnosis and treatment. 5th ed. Springer-Verlag, Berlin2012: 499-518 Crossref Scopus (11) Google Scholar ]. However, a subgroup of patients presents with intractable seizures beginning within the first days or weeks of age [ [4] Mouchegh K. Zikánová M. Hoffmann G.F. et al. Lethal fetal and early neonatal presentation of adenylosuccinate lyase deficiency: Observation of 6 patients in 4 families. J Pediatr. 2007; 150: 57-61 Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar ]. Often severe hypotonia is associated such as seen, e.g., in nonketotic hyperglycinemia and γ-aminobutyric acid transaminase deficiency, as mentioned by Ficicioglu and Bearden [ [1] Ficicioglu C. Bearden D. Isolated neonatal seizures: When to suspect inborn errors of metabolism. Pediatr Neurol. 2011; 45: 283-291 Abstract Full Text Full Text PDF PubMed Scopus (31) Google Scholar ]. These seizures can lead to death or severe mental retardation. In some patients, intrauterine growth retardation, microcephaly, fetal hypokinesia, and a lack of fetal heart rate variability have also been observed. Adenylosuccinase catalyzes two steps in purine synthesis: the conversion of succinyl aminoimidazole carboxamide ribotide to aminoimidazole carboxamide ribotide (in the de novo pathway), and the conversion of adenylosuccinate to adenosine monophosphate. Its deficiency results in the accumulation in body fluids of succinyl purines, i.e., succinyl aminoimidazole carboxamide riboside and succinyl adenosine (the dephosphorylated substrates of the enzyme). A modified Bratton-Marshall (colorimetric) test performed on urine can be used as a screening test, but some medications such as sulfonamides cause a false-positive result. A final diagnosis requires high-pressure liquid chromatography with ultraviolet light detection, or high-pressure liquid chromatography with electrospray ionization tandem mass spectroscopy. Isolated Neonatal Seizures: When to Suspect Inborn Errors of MetabolismPediatric NeurologyVol. 45Issue 5PreviewNeonatal seizures are common, and often comprise the first clinical indicator of central nervous system dysfunction. Although most neonatal seizures are secondary to processes such as hypoxic-ischemic injury, infection, or cortical malformations (which are readily identifiable through routine testing and imaging), seizures secondary to inborn errors of metabolism can be much more difficult to diagnose, and thus a high index of suspicion is required. The early diagnosis of inborn errors of metabolism is crucial, considering that many can receive effective treatment (e.g., dietary supplementation or restriction) with favorable long-term outcomes. Full-Text PDF ResponsePediatric NeurologyVol. 47Issue 3PreviewWe thank Dr. Jaaken for his insightful discussion of adenylosuccinase deficiency as a cause of neonatal seizures. Our review was intended to offer an overview of metabolic disorders presenting with isolated neonatal seizures, i.e., neonatal seizures not associated with other characteristic abnormalities. Dr. Jaaken is correct in observing that neonatal seizures may constitute an atypical presentation of this rare disease, with six cases reported in the literature [1]. However, the severe form of adenylosuccinase deficiency that presents with neonatal seizures is often accompanied by severe hypotonia, intrauterine growth retardation, and magnetic resonance imaging abnormalities that are variable but that generally include dysmyelination [2]. Full-Text PDF

Molecular characterization of the AdeI mutant of Chinese hamster ovary cells: A cellular model of adenylosuccinate lyase deficiency

Adenylosuccinate lyase (ADSL, E. C. 4.3.2.2) carries out two non-sequential steps in de novo AMP synthesis, the conversion of succinylaminoimidazole carboxamide ribotide (SAICAR) to aminoimidazolecarboxamide ribotide (AICAR) and the conversion of succinyl AMP (AMPS) to AMP. In humans, mutations in ADSL lead to an inborn error of metabolism originally characterized by developmental delay, often with autistic features. There is no effective treatment for ADSL deficiency. Hypotheses regarding the pathogenesis include toxicity of high levels of SAICAR, AMPS, or their metabolites, deficiency of the de novo purine biosynthetic pathway, or lack of a completely functional purine cycle in muscle and brain. One important approach to understand ADSL deficiency is to develop cell culture models that allow investigation of the properties of ADSL mutants and the consequences of ADSL deficiency at the cellular level. We previously reported the isolation and initial characterization of mutants of Chinese hamster ovary (CHO-K1) cells ( AdeI) that lack detectable ADSL activity, accumulate SAICAR and AMPS, and require adenine for growth. Here we report the cDNA sequences of ADSL from CHO-K1 and AdeI cells and describe a mutation resulting in an alanine to valine amino acid substitution at position 291 (A291V) in AdeI ADSL. This substitution lies in the “signature sequence” of ADSL, inactivates the enzyme, and validates AdeI as a cellular model of ADSL deficiency.

Evidence for the hypothesis that 10-formyldihydrofolate is the in vivo substrate for aminoimidazolecarboxamide ribotide transformylase

We postulate that 10-formyl-7,8-dihydrofolate (10-HCO-H(2)folate), not 10-formyl-5,6,7,8-tetrahydrofolate (10-HCO-H(4)folate), is the predominant in vivo substrate for mammalian aminoimidazolecarboxamide ribotide (AICAR) transformylase, an enzyme in purine nucleotide biosynthesis de novo, which introduces carbon 2 (C(2)) into the purine ring. 10-HCO-H(2)folate exists in vivo as labeled 10-formyl-folic acid (10-HCO-folic acid: an oxidation product of 10-HCO-H(4)folate and 10-HCO-H(2)folate) and is found after doses of labeled folic acid in humans or laboratory animals. The bioactivity of the unnatural isomer, [6R]-5-formyltetrahydrofolate, in humans is explained by its in vivo conversion to 10-HCO-H(2)folate. The structure and active site of AICAR transformylase are not consistent with other enzymes that utilize 10-HCO-H(4)folate. Because 10-HCO-H(4)folate is rapidly oxidized in vitro to 10-HCO-H(2)folate by cytochrome C alone and in mitochondria, it is hypothesized that this process takes place in vivo. In vitro data indicate that 10-HCO-H(2)folate is kinetically preferred over 10-HCO-H(4)folate by AICAR transformylase and that this enzyme may not have access to sufficient supplies of 10-HCO-H(4)folate. Methotrexate blockage of the AICAR transformylase process in patients with rheumatoid arthritis suggests that dihydrofolate (H(2)folate) reductase is involved and is consistent with H(2)folate and 10-HCO-H(2)folate being the product and substrate for AICAR transformylase. The labeling of purine C(2) by an oral dose of [6RS]-5-H[(13)C]O-H(4)folate in a human subject is consistent with 10-H[(13)C]O-H(2)folate formation from unnatural isomer, [6R]-5-H[(13)C]O-H(4)folate, and it being a substrate for AICAR transformylase. In vitro exchange reactions of purine C(2) using H(4)folate coenzymes are not duplicated in vivo and is consistent with H(2)folate coenzymes being used in vivo by AICAR transformylase.

Exclusive use of ring 2‐[13C]L‐histidine for carbon 2 of the purine ring in purine nucleotide biosynthesis (PNB) de novo in an adult human

The ring 2‐carbon of histidine is a source of one‐carbon units (1‐C) carried by folate coenzymes. In theory, this would be metabolically available for carbons 2 (C2) and 8 (C8) of the purine ring in PNB. An adult male ingested 0.71 g of ring 2‐[13C]L‐histidine ?HCl ?H2O (98%, Cambridge Isotope), and the 13C enrichment of urinary uric acid at C2 and C8 of each void was measured by an LC/MS/MS method. The % 13C enrichment at C2 in 33 voids (3 days) ranged from 0.05 to 0.58 with rhythmicity. Means (±SD, number of voids) were 0.15 (±0.09, 12), 0.24 (±0.19, 10) and 0.28 (±0.19, 11) for days 1, 2, and 3, respectively and were greater than zero (P &lt; 0.01). In contrast, the % 13C enrichment at C8 varied from ‐0.12 to 0.09 with the means of 0.00 (±0.06), ‐0.01 (±0.02) and 0.00 (±0.02), respectively and these were not different from zero (P &gt; 0.05). Our data indicate that histidine, like formate, is a source of 1‐C utilized by aminoimidazolecarboxamide ribotide transformylase; thus, C2 is enriched. We reported that the 2‐carbon of glycine is utilized by glycinamide ribotide transformylase for C8 in humans. It is likely in humans that completion of PNB requires different carbon sources for C2 and C8 and that different organs play concerted roles. The seemingly wasteful catabolism of histidine, an essential amino acid, to glutamate, a non‐essential amino acid, may make metabolic sense, when 1‐C produced is used for an essential purpose such as PNB.

Clinical, biochemical and molecular findings in seven Polish patients with adenylosuccinate lyase deficiency

Adenylosuccinate lyase (ADSL) catalyzes two steps in purine nucleotide metabolism—the 8th step in the de novo pathway: conversion of succinylaminoimidazole carboxamide ribotide (SAICAR) to aminoimidazole carboxamide ribotide (AICAR), and conversion of adenylosuccinate (S-AMP) to adenylate (AMP) in the purine nucleotide cycle. To date, over 50 patients have been reported suffering from ADSL deficiency. We report all seven so far diagnosed Polish patients with this defect. Most of our patients shared intractable seizures and psychomotor retardation since the neonatal period and had biochemical evidence of severe (type I) deficiency. Two patients with type II suffered only from mild/moderate psychomotor retardation and showed a transientvisual contact disturbance. One patient had a fatal neonatal form of ADSL deficiency with lack of spontaneous movement, respiratory failure, severe encephalopathy and intractable seizures. Analysis of the ADSL gene showed that four apparently unrelated patients carried a R426H mutation (two homozygous and two compound heterozygous). With the exception of the latter mutation, a Y114H mutation that had been reported previously, and a novel mutation T242I, all other mutations (including D268H and three novel S23R, D215H and I351T mutations) were found only in single families in single alleles. A search for this disorder should be included in the screening program of all infants with unexplained neonatal seizures, severe infantile epileptic encephalopathy, developmental delay, hypotonia, and/or autistic features.

13C-enrichment at carbons 8 and 2 of uric acid after 13C-labeled folate dose in man

To evaluate folate-dependent carbon incorporation into the purine ring, we measured 13C-enrichment independently at C2 and C8 of urinary uric acid (the final catabolite of purines) in a healthy male after an independent oral dose of [6RS]-5-[13C]-formyltetrahydrofolate ([6RS]-5-H13CO–H4folate) or 10-H13CO-7,8-dihydrofolate (10-H13CO–H2folate). The C2 position was 13C-enriched more than C8 after [6RS]-5-H13CO–H4folate, and C2 was exclusively enriched after 10-H13CO–H2folate. The enrichment of C2 was greater from [6RS]-5-H13CO–H4folate than 10-H13CO–H2folate using equimolar bioactive doses. Our data suggest that formyl C of [6RS]-10-H13CO–H4folate was not equally utilized by glycinamide ribotide transformylase (enriches C8) and aminoimidazolecarboxamide ribotide (AICAR) transformylase (enriches C2), and the formyl C of 10-H13CO–H2folate was exclusively used by AICAR transformylase. 10-HCO–H2folate may function in vivo as the predominant substrate for AICAR transformylase in humans.

Methotrexate and erythro-9-(2-hydroxynon-3-yl) adenine therapy for rat adjuvant arthritis and the effect of methotrexate on in vivo purine metabolism

The objectives were: (1) to test the association of methotrexate (MTX) efficacy in rat adjuvant arthritis (rat AA) with interference of purine biosynthesis and adenosine metabolism and (2) to test the efficacy of erythro-9-(2-hydroxynon-3-yl) adenine (EHNA), an inhibitor of adenosine deaminase, and the efficacy of aminoimidazolecarboxamide (AICA) riboside plus MTX in rat AA. Radiographic and histologic examinations of the hind limbs were measures of efficacy. Urinary excretions of AICA and adenosine were makers of AICA ribotide transformylase inhibition (i.e., blockage of purine biosynthesis) and interference with adenosine metabolism, respectively. AICA and adenosine excretions increased during the day of MTX dosing (treatment day) compared to the previous baseline day in animals responding well to MTX (i.e., low radiographic and histologic scores). Based on radiographic and histologic scores, adjuvant injected rats were separated into two disease categories (i.e., no/mild and moderate/severe). Only AICA excretion was significantly elevated on the treatment day in rat AA with no/mild disease (i.e., those responding well to MTX therapy). AICA (not adenosine) excretion was significantly correlated with the above scores. EHNA was not efficacious, even at toxic levels, while AICA riboside potentiated the efficacy of MTX. The data suggests that efficacious MTX therapy in rat AA (1) blocks purine biosynthesis; (2) increases in in vivo AICA levels. Also adenosine accumulation and blockage of adenosine deaminase (i.e., by EHNA) appear to be less critical to MTX efficacy. Increased levels of AICA metabolites may suppress the immune response in rat AA.

Metabolic flux in both the purine mononucleotide and histidine biosynthetic pathways can influence synthesis of the hydroxymethyl pyrimidine moiety of thiamine in Salmonella enterica.

Together, the biosyntheses of histidine, purines, and thiamine pyrophosphate (TPP) contain examples of convergent, divergent, and regulatory pathway integration. Mutations in two purine biosynthetic genes (purI and purH) affect TPP biosynthesis due to flux through the purine and histidine pathways. The molecular genetic characterization of purI mutants and their respective pseudorevertants resulted in the conclusion that <1% of the wild-type activity of the PurI enzyme was sufficient for thiamine but not for purine synthesis. The respective pseudorevertants were found to be informational suppressors. In addition, it was shown that accumulation of the purine intermediate aminoimidazole carboxamide ribotide inhibits thiamine synthesis, specifically affecting the conversion of aminoimidazole ribotide to hydroxymethyl pyrimidine.

Mutation of a Nuclear Respiratory Factor 2 Binding Site in the 5′ Untranslated Region of the ADSL Gene in Three Patients with Adenylosuccinate Lyase Deficiency

Adenylosuccinate lyase (ADSL; also called "adenylosuccinase") catalyzes two steps in the synthesis of purine nucleotides: (1) the conversion of succinylaminoimidazolecarboxamide ribotide into aminoimidazolecarboxamide ribotide and (2) the conversion of adenylosuccinate into adenosine monophosphate. ADSL deficiency, a recessively inherited disorder, causes variable-but most often severe-mental retardation, frequently accompanied by epilepsy and/or autism. It is characterized by the accumulation, in body fluids, of succinylaminoimidazolecarboxamide riboside and succinyladenosine, the dephosphorylated derivatives of the two substrates of the enzyme. Analysis of the ADSL gene of three unrelated patients with ADSL deficiency, in whom one of the ADSL alleles displayed a normal coding sequence, revealed a -49T-->C mutation in the 5' untranslated region of this allele. Measurements of the amount of mRNA transcribed from the latter allele showed that it was reduced to approximately 33% of that transcribed from the alleles mutated in their coding sequence. Further investigations showed that the -49T-->C mutation provokes a reduction to 25% of wild-type control of promoter function, as evaluated by luciferase activity and mRNA level in transfection experiments. The mutation also affects the binding of nuclear respiratory factor 2 (NRF-2), a known activator of transcription, as assessed by gel-shift studies. Our findings indicate that a mutation of a regulatory region of the ADSL gene might be an unusually frequent cause of ADSL deficiency, and they suggest a role for NRF-2 in the gene regulation of the purine biosynthetic pathway.

Oxidation of 10-formyltetrahydrofolate to 10-formyldihydrofolate by complex IV of rat mitochondria.

We hypothesized that the unanticipated bioactivity of orally administered unnatural carbon-6 isomers, (6R)-5-formyltetrahydrofolate (5-HCO-THF) and (6S)-5,10-methenyltetrahydrofolate (5,10-CH-THF), in humans [Baggott, J. E., and Tamura, T. (1999) Biochim. Biophys. Acta 1472, 323-32] is explained by the rapid oxidation of (6S)-10-formyltetrahydrofolate (10-HCO-THF), which is produced by in vivo chemical processes from the above folates. An oxidation of 10-HCO-THF produces 10-formyldihydrofolate (10-HCO-DHF), which no longer has the asymmetric center at carbon-6 and is metabolized by aminoimidazole carboxamide ribotide (AICAR) transformylase forming bioactive dihydrofolate. Since cytochrome c (Fe(3+)) rapidly oxidizes both (6R)- and (6S)-10-HCO-THF [Baggott et al. (2001) Biochem. J. 354, 115-22], we investigated the metabolism of 10-HCO-THF by isolated rat liver mitochondria. We found that 10-HCO-THF supported the respiration of mitochondria without uncoupling ATP synthesis. The site of electron donation was identified as complex IV, which contains cytochrome c; the folate product was 10-HCO-DHF, and the reaction was saturable with respect to 10-HCO-THF. Both (6S)- (unnatural) and (6R)-10-HCO-THF supported the respiration of mitochondria, whereas (6S)-5-formyltetrahydrofolate (5-HCO-THF) was inactive. To our knowledge, this cytochrome c oxidation of 10-HCO-THF to 10-HCO-DHF in the mitochondrial intermembrane space represents a possible folate metabolic pathway previously unidentified and would explain the bioactivity of unnatural carbon-6 isomers, (6R)-5-HCO-THF and (6S)-5,10-CH-THF, in humans.

Metabolism of 10-formyldihydrofolate in humans

The metabolism of 10-formyldihydrofolate is reviewed in this article. It had been the dogma that only tetrahydrofolates participate in enzyme-catalyzed one-carbon transfer reactions, until we showed in 1986 that 10-formyldihydrofolate serves as a substrate for aminoimidazolecarboxamide ribotide (AICAR) transformylase. Our data from studies in humans, cultured cells and bacteria as well as in vitro experiments indicate that the oxidation of 10-formyltetrahydrofolate to 10-formyldihydrofolate takes place, and 10-formyldihydrofolate is subsequently converted to dihydrofolate by AICAR transformylase. Dihydrofolate is then reduced to tetrahydrofolate and further metabolized by the well-established enzyme reactions. We believe that a new folate metabolic map is needed which incorporates the oxidation of 10-formyltetrahydrofolate and the utilization of 10-formyldihydrofolate by AICAR transformylase.

Urinary adenosine and aminoimidazolecarboxamide excretion in methotrexate-treated patients with psoriasis.

We hypothesized that low-dose methotrexate treatment for patients with psoriasis would block purine biosynthesis at the step catalyzed by aminoimidazolecarboxamide (AICA) ribotide transformylase and would inhibit adenosine metabolism as evidenced by increased urinary levels of AICA and adenosine, respectively. Eight patients collected a 24-hour urine specimen on the day before their methotrexate dose and the next day during their methotrexate dose. Eight age- and sex-matched controls also collected a 24-hour urine sample. Urinary AICA and adenosine were assayed by spectrophotometric and radioimmune assays, respectively; means are reported as micromole per millimole of creatinine and were compared by the paired t test (1-tailed). Mean AICA excretion increased from 1.30 micromol/mmol on the day before to 1.85 micromol/mmol on the day during methotrexate dosing (P<.01). Mean adenosine values increased from 0.68 to 1.07 micromol/mmol, (P<.03). Controls had mean AICA and adenosine levels of 1.29 and 0.50 micromol/mmol, respectively. During the day of methotrexate dosing, patients had higher mean AICA and adenosine levels when compared with controls (P<.01). Mean AICA levels increased from 1.36 to 2.06 micromol/mmol (P<.025), and mean adenosine levels increased from 0.72 to 1.25 micromol/mmol (P<.025) in 5 patients showing improvement in clinical disease activity. In contrast, 3 patients with no change or worsening in clinical disease activity had smaller increases. Methotrexate treatment of patients with psoriasis inhibits AICA ribotide transformylase and adenosine metabolism. Since adenosine is a T-lymphocyte toxin, it may be partially responsible for the immunosuppressive effect.

10-Formyl-dihydrofolic Acid Is Bioactive in Human Leukemia Cells

The bioactivity of 10-formyl-7,8-dihydrofolic acid and 10-formyl-folic acid was determined in human leukemia (CCRF-CEM) cells grown in a folate-depleted medium containing methotrexate. Excess 10-formyl-7,8-dihydrofolic acid, (but not 10-formyl folic acid) supported the growth of these cells, but it was less potent than5-formyl-5,6,7,8-tetrahydrofolic acid (a control). 10-formyl-7,8-dihydrofolic acid (not 10-formyl folic acid) was active as substrate for aminoimidazole carboxamide ribotide transformylase and dihydrofolate reductase. This is the first experimental evidence that 10-formyl-7,8-dihydrofolic acid is a bioactive folate in mammalian cells. These experiments and several other lines of evidence in the literature suggest that 10-formyl-folic acid must be metabolized to bioactive folate by enteric bacteria before it can be utilized by the vertebrate host.

Iron compounds catalyze the oxidation of 10-formyl-5,6,7,8 tetrahydrofolic acid to 10-formyl-7,8 dihydrofolic acid

We have previously demonstrated that 10-formyl-7,8-dihydrofolic acid (10-HCO-H 2folate) is a better substrate for mammalian aminoimidazolecarboxamide ribotide transformylase (EC 2.1.2.3) than is 10-formyl-5,6,7,8-tetrahydrofolic acid (10-HCO-H 4folate) (J.E. Baggott, G.L. Johanning, K.E. Branham, C.W. Prince, S.L. Morgan, I. Eto, W.H. Vaughn, Biochem. J. 308, 1995, 1031–1036). Therefore, the possible metabolism of 10-HCO-H 4folate to 10-HCO-H 2folate was investigated. A spectrophotometric assay for the oxidation of 10-HCO-H 4folate to 10-HCO-H 2folate which measures the disappearance of reactant (decrease in absorbance at 356 nm after acidification of aliquots of the reaction solution), is used to demonstrate that iron compounds catalyze the oxidation of 10-HCO-H 4folate to 10-HCO-H 2folate in the presence and absence of ascorbate. Chromatographic separation of the 10-HCO-H 2folate product from the reaction mixture, its UV spectra, a microbiological assay and an enzymatic assay established that the iron-catalyzed oxidation product of 10-HCO-H 4folate was 10-HCO-H 2folate; without substantial side reactions. The inhibition of this iron-catalyzed oxidation by deferoxamine, apotransferrin and mannitol and the stimulation by citrate and EDTA indicated a mechanism involving a reaction of 10-HCO-H 4folate with hydroxyl radicals ( •OH) generated by Fenton chemistry. The presence of “free iron” (e.g. Fe 3+ citrate) in bile, cerebrospinal fluid and intracellularly suggest that this oxidation could occur in vivo and that 10-HCO-H 4folate may be a •OH scavenger.

Succinylpurines induce neuronal damage in the rat brain.

Adenylosuccinase (adenylosuccinate lyase, EC 4.3.2.2: ASase) catalyses two steps in the purine metabolic pathways: a) the eighth step in the de novo pathway of purine synthesis: conversion of succinylaminoimidazole carboxamide ribotide (SAICAR) into aminoimidazole carboxamide ribotide (AICAR) and b) the second step in the formation of adenyl nucleotides from IMP: conversion of adenylosuccinate (S-AMP) into AMP. As a result of the enzyme deficiency, there is a recognised accumulation of the two dephosphorylated substrates of the enzyme: SAICA riboside and succinyladenosine (S-Ado). Dephosphorylation of SAICAR and S-AMP is performed by cytosolic 5′nucleotidases. Neither SAICA riboside nor S-Ado are normally detectable in the blood or tissue samples of humans, but in patients with ASase deficiency, both compounds are present in the urine, blood and CSF. In the latter, concentrations are usually around 100-200 μM (Jaeken et al., 1993).

Inborn errors of the purine nucleotide cycle: adenylosuccinase deficiency.

Adenylosuccinase catalyses two reactions in purine metabolism: the conversion of succinylaminoimidazole carboxamide ribotide (SAICAR) into aminoimidazole carboxamide ribotide (AICAR) along the de novo synthesis of purine nucleotides, and the conversion of adenylosuccinate (S-AMP) into AMP in the conversion of IMP into AMP. The hallmarks of adenylosuccinase deficiency are the presence of succinylaminoimidazole carboxamide riboside (SAICAriboside) and succinyladenosine (S-Ado) in body fluids. These normally undetectable succinylpurines are the products of the dephosphorylation, by cytosolic 5'-nucleotidase, of the two substrates of adenylosuccinase. The clinical picture of the enzyme deficiency is markedly heterogeneous with, as a rule, a profound, but nevertheless variable degree of psychomotor delay, often convulsions and/or autistic features, sometimes growth retardation and muscular dystrophy. The diagnostic tests that can be used for diagnosis, the enzyme and gene defects that have been identified, and the hypotheses that have been put forward to explain the pathophysiology of the disorder are reviewed.