Vanna Micheli

Demonstration of induction of erythrocyte inosine monophosphate dehydrogenase activity in Ribavirin-treated patients using a high performance liquid chromatography linked method

The activity of inosine monophosphate dehydrogenase (IMPDH: EC 1.2.1.14) was measured in erythrocyte lysates using a non-radiolabelled method linked to reversed-phase liquid chromatography (RPLC). The mean activity in erythrocytes from healthy controls using this sensitive method was extremely low (mean 85 pmol/h per mg protein, range 4-183). The elevated erythrocyte IMPDH activity reported previously in hypoxanthine-guanine phosphoribosyltransferase (HPRT) deficiency was confirmed (mean 234 pmol/h per mg protein). Erythrocyte IMPDH activity of patients with other disorders of purine metabolism, or with leukaemias and lymphomas, showed no marked difference from controls, except in one instance--an immunodeficient child with purine nucleoside phosphorylase (PNP) deficiency, treated with Ribavirin, where a 30-fold increase in activity was found (2670 pmol/h per mg protein). Investigation of erythrocyte IMPDH in other immunodeficient children with normal PNP activity demonstrated that this grossly elevated erythrocyte activity was attributable to induction of IMPDH by Ribavirin therapy.

Cytosolic 5′-nucleotidase hyperactivity in erythrocytes of Lesch–nyhan syndrome patients

Lesch–Nyhan syndrome is a metabolic–neurological syndrome caused by the X-linked deficiency of the purine salvage enzyme hypoxanthine-guanine phosphoribosyltransferase (HGPRT). Metabolic consequences of HGPRT deficiency have been clarified, but the connection with the neurological manifestations is still unknown. Much effort has been directed to finding other alterations in purine nucleotides in different cells of Lesch–Nyhan patients. A peculiar finding was the measure of appreciable amount of Z-nucleotides in red cells. We found significantly higher IMP-GMP-specific 5′-nucleotidase activity in the erythrocytes of seven patients with Lesch–Nyhan syndrome than in healthy controls. The same alteration was found in one individual with partial HGPRT deficiency displaying a severe neurological syndrome, and in two slightly hyperuricemic patients with a psychomotor delay. Since ZMP was a good substrate of 5′-nucleotidase producing Z-riboside, we incubated murine and human cultured neuronal cells with this nucleoside and found that it is toxic for our models, promoting apoptosis. This finding suggests an involvement of the toxicity of the Z-riboside in the pathogenesis of neurological disorders in Lesch–Nyhan syndrome and possibly in other pediatric neurological syndromes of uncertain origin.

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.

Severe pyridine nucleotide depletion in fibroblasts from Lesch-Nyhan patients.

The relationship between a complete deficiency of the purine enzyme hypoxanthine-guanine phosphoribosyltransferase and the neurobehavioural abnormalities in Lesch-Nyhan disease remains an enigma. In vitro studies using lymphoblasts or fibroblasts have evaluated purine and pyrimidine metabolism with conflicting results. This study focused on pyridine nucleotide metabolism in control and Lesch-Nyhan fibroblasts using radiolabelled salvage precursors to couple the extent of uptake with endocellular nucleotide concentrations. The novel finding, highlighted by specific culture conditions, was a marked NAD depletion in Lesch-Nyhan fibroblasts. ATP and GTP were also 50% of the control, as reported in lymphoblasts. A 6-fold greater incorporation of [(14)C]nicotinic acid into nicotinic acid- adenine dinucleotide by Lesch-Nyhan fibroblasts, with no unmetabolized substrate (20% in controls), supported disturbed pyridine metabolism, NAD depletion being related to utilization by poly(ADP-ribose) polymerase in DNA repair. Although pyrimidine nucleotide concentrations were similar to controls, Lesch-Nyhan cells showed reduced [(14)C]cytidine/uridine salvage into UDP sugars. Incorporation of [(14)C]uridine into CTP by both was minimal, with more than 50% [(14)C]cytidine metabolized to UTP, indicating that fibroblasts, unlike lymphoblasts, lack active CTP synthetase, but possess cytidine deaminase. Restricted culture conditions may be neccesary to mimic the situation in human brain cells at an early developmental stage. Cell type may be equally important. NAD plus ATP depletion in developing brain could restrict DNA repair, leading to neuronal damage/loss by apoptosis, and, with GTP depletion, affect neurotransmitter synthesis and basal ganglia dopaminergic neuronal systems. Thus aberrant pyridine nucleotide metabolism could play a vital role in the pathophysiology of Lesch-Nyhan disease.

HPLC determination of oxidized and reduced pyridine coenzymes in human erythrocytes

The nucleotide concentrations in acid and alkaline erythrocyte extracts have been measured by RP-HPLC in healthy controls and in patients bearing different inherited disorders, with altered erythrocyte NAD(P) levels. The objective was the simultaneous determination of the nucleotide profile and of the oxidative state of pyridine coenzymes by the most suitable extraction method. Both alkaline and acid extractions were necessary to obtain the complete pattern, due to defective recovery of the oxidized or reduced coenzymes, respectively, during the extraction procedures. Purine nucleotide quantification seemed to be reliable by all methods. High NADP+ levels were confirmed in two glucose-6-phosphate dehydrogenase deficient patients, coupled with raised NAD levels, lowered NADPH/NADP+ ratio and increased NADH/NAD+ ratio. Higher NAD+ and normal or lower NADH/NAD+ ratios were found in two hypoxanthine-phosphoribosyltransferase deficient patients, while a patient with superactive phosphoribosylpyrophosphate synthetase showed a decreased NADH level in addition to the low NAD+ level previously found.

Guanine nucleotide depletion triggers cell cycle arrest and apoptosis in human neuroblastoma cell lines

Mycophenolic acid (MPA) specifically inhibits inosine‐5′‐monophosphate dehydrogenase, the first committed step toward GMP biosynthesis. In its morpholinoethyl ester pro‐drug form it is one of the most promising immunosuppressive drugs recently developed. The aim of the present study was to investigate the in vitro effects of MPA, at concentrations readily attainable during immunosuppressive therapy, on 3 human neuroblastoma cell lines (LAN5, SHEP and IMR32). Mycophenolic acid (0.1–10 μM) caused a decrease of intracellular levels of guanine nucleotides, a G1 arrest and a time‐ and dose‐dependent death by apoptosis. These effects, associated with an up‐regulation of p53, p21 and bax, a shuttling of p53 protein into the nucleus and a down‐regulation of bcl‐2, survivin and p27 protein, were reversed by the simultaneous addition of guanine or guanosine and were more evident using nondialysed serum containing hypoxanthine. These results suggest that in neuroblastoma cell lines clinically attainable concentrations of mycophenolic acid deplete guanine nucleotide pools triggering G1 arrest and apoptosis through p53‐mediated pathways, indicating a potential role of its morpholinoethyl ester pro‐drug in the management of patients with neuroectodermal tumors.

Importance of nicotinamide as an NAD precursor in the human erythrocyte.

The effect of variation in the concentration of inorganic phosphate and of the pyridine precursors nicotinamide (NAm) and nicotinic acid (NA) on pyridine nucleotide synthesis was studied using intact human erythrocytes. A wide range of incubation times was employed. The results showed that under physiological conditions the rate of synthesis of NAD from NAm exceeded that from NA twofold, while the reverse situation pertained at higher and unphysiological substrate levels. The two pathways had different regulation points. For NAm the rate-limiting factor was the initial step, namely its conversion into the mononucleotide, while for NA it lay at the second step, conversion of NA mononucleotide (NAMN) to its adenine dinucleotide. At physiological substrate levels the uptake of NA and conversion to NAMN were rapid, while the uptake and conversion of NAm were time dependent. This process was stimulated significantly by inorganic phosphate only for NAm. These results indicate that while NA is the predominant precursor of human erythrocyte NAD at high (unphysiological) substrate and phosphate levels, NAm is more efficient as an NAD precursor under physiological conditions, suggesting an important and hitherto unrecognized role for nicotinamide in NAD synthesis in vivo.

Late-onset MNGIE without peripheral neuropathy due to incomplete loss of thymidine phosphorylase activity

Mitochondrial NeuroGastroIntestinal Encephalomyopathy (MNGIE) is an autosomal recessive disorder characterized by severe gastrointestinal dysmotility, cachexia, peripheral neuropathy, ptosis, ophthalmoplegia, and leukoencephalopathy with early onset and severe prognosis. Mutations in the TYMP/ECGF1 gene cause a loss of thymidine phosphorylase catalytic activity, disrupting the homeostasis of intramitochondrial nucleotide pool. We report a woman with a very late onset of MNGIE, lacking peripheral neuropathy. Thymidine phosphorylase activity was markedly reduced in cultured fibroblasts, but only mildly reduced in buffy coat, where the defect is usually detected, and plasma thymidine was mildly increased compared to typical MNGIE patients. TYMP/ECGF1 analysis detected two heterozygous mutations, including a novel missense mutation. These findings indicate that a partial loss of thymidine phosphorylase activity may induce a late-onset and incomplete MNGIE phenotype.

HYPOXANTHINE-GUANINE PHOSPHORIBOSYLTRANSFERASE DEFICIENCY AND ERYTHROCYTE SYNTHESIS OF PYRIDINE COENZYMES

Purine and pyridine metabolism were studied in ten Lesch-Nyhan patients, with virtually no hypoxanthine-guanine phosphoribosyltransferase (HPRT) activity in erythrocytes. Increased NAD erythrocyte concentrations were found in all patients. Raised activities of two enzymes catalysing NAD synthesis from nicotinic acid (nicotinic acid phosphoribosyltransferase: NAPRT, and NAD synthetase: NADs) was found in erythrocyte lysates from all patients. The two enzymes had normal apparent Km for their substrates and increased Vmax. The rate of synthesis of pyridine nucleotides from nicotinic acid by intact erythrocytes in vitro was also increased in most patients. These findings suggest that raised NAD concentrations in HPRT- erythrocytes are due to enhanced synthesis as a result of increased enzyme activities.

Simple non-radiochemical HPLC-linked method for screening for purine metabolism disorders using dried blood spot.

BACKGROUND Pathologies associated with rare inherited disorders affecting purine metabolic pathways range from renal failure to neurological dysfunction and immunodeficiency. The disorders are usually diagnosed by measuring enzyme activities in hemolysates. A non-radiochemical HPLC-linked method is described for simultaneous determination of the activities of hypoxanthine-guanine phosphoribosyltransferase (HPRT: E.2.4.2.8.), adenine phosphoribosyltransferase (APRT: E.2.4.2.7.), adenosine deaminase (ADA: E.3.5.4.4.) and purine nucleoside phosphorylase (PNP: E.2.4.2.1.) in dried blood spots. METHOD 7-mm-diameter blood spots stored at 4 degrees C or room temperature were transferred to an Eppendorf tube and eluted with 500-microl 0.1 mol/l Tris-HCl buffer, pH 7.4. The eluate was added to substrate solutions and incubated at 37 degrees C. Reaction products were analysed by HPLC. RESULTS AND CONCLUSIONS The enzyme activities tested in spot eluates were similar to those in erythrocyte lysates from the same subjects. None of the enzymatic activities tested were significantly affected by different storage temperatures. The main advantages of the proposed method are small blood volume required, easy sample collection and transfer, and accurate results. The method is therefore suitable for screening inborn errors of purine metabolism even in newborns.