S. Sestini

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.

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.

Purine and pyridine nucleotide production in human erythrocytes.

Human erythrocyte adenyl and pyridine nucleotide production has been tested in cell-free lysates and in intact cells. The main products obtained in cells incubated with adenine and nicotinic acid are adenosine triphosphate and nicotinate mononucleotide, respectively, under any experimental condition used (incubation time, base concentration). Adenine-phosphoribosyltransferase activity determined in crude lysates is about 100 times higher than nicotinate-phosphoribosyltransferase activity, while cellular adenyl nucleotide production is only three times higher than that of pyridine nucleotide. A strong intracellular regulation for the former, but not latter, synthetic process is thus suggested. Intact erythrocyte nicotinate nucleotide production is inhibited by adenine, while nicotinate-phosphoribosyltransferase activity is not. The possible regulation by adenyl nucleotides is discussed in light of the modulating action of ATP on nicotinate-phosphoribosyltransferase activity. The kinetic characteristics of both adenine- and nicotinate-phosphoribosyltransferases, determined on crude lysates, are reported.

Pyridine Nucleotide Metabolism: Purine and Pyrimidine Interconnections

The role of the pyridine coenzymes NAD and NADP in oxidation-reduction reactions, leading to energy production and to reductive synthesis, is well documented, and the characteristics of the proteins involved have been extensively studied. Since the balance between the oxidised and reduced form of pyridine coenzymes modulates both catabolic and synthetic pathways, their differing levels in different tissues, organs and cellular compartments indicate metabolic differentiations1. In addition to this role, pyridine coenzymes take part in a number of other cellular processes occurring in prokaryotes or eukaryotes or both2. These processes include the utilization of NAD for: protein ADP-ribosylations, mainly involved in the mechanism of action of bacterial toxins, affecting protein synthesis or some processes mediated by cAMP and G-proteins; poly-ADPribose synthesis, involved in the regulation of DNA repair and replication and in cellular differentiation; DNA ligase reactions, active in prokaryotes. NAD(P) involvement in the production of cytotoxic compounds and in phagocytosis process has also been described. Such findings revealed the versatility of the NAD(P) molecule in cell function, suggesting that its biological role is not yet fully appreciated.

PARP Activity and NAD Concentration in PMC from Patients Affected by Systemic Sclerosis and Lupus Erythematosus

The enzyme poly(ADP-ribose) polymerase (PARP-1, EC 2.4.2.30) is activated by DNA strand breaks caused by several agents and utilizes NAD to form polyADPR, bound to acceptor proteins. The involvement of PARP-1 in autoimmune diseases has been suggested: antiPARP autoantibodies are described in systemic lupus erythematosus (SLE), DNA strand breaks have been evidenced in systemic sclerosis (SSc). We tested poly(ADP-ribosyl)ation activity and NAD concentration in PMC from patients affected by SLE or SSc and from controls. Lower PARP-1 activity and higher NAD concentration were observed in pathological conditions than controls, supporting the role of PARP-1 activation in modulating NAD concentration.

Altered Pyridine Metabolism in the Erythrocytes of a Mentally Retarded Infant with Partial HPRT Deficiency

Alterations in the erythrocyte NAD(P) concentration have been reported in inherited defects of purine metabolism, such as PNP and HPRT deficiency, and phosphoribosylpyrophosphate synthetase (PRPS) superactivityl. The neurological disturbances associated with these disorders, as well as the utilization of nicotinate (NA) or nicotinamide (NAm) for the treatment of psychotic states, schizophrenia and depression2 and the findings on the role of NAD in the synaptic modulation3, suggested a correlations between neurological disorders and pyridine nucleotides.

Inborn Errors of Purine and Pyrimidine Metabolism: How Much We Owe to H. Anne Simmonds

Purines and pyrimidines, regarded for a long time merely as building blocks for nucleic acid synthesis and intermediates in the transfer of metabolic energy, have attracted increasing attention after genetically determined aberrations in their metabolism were linked to a range of symptoms from hyperuricemia and immunodeficiency to neurological disorders. The pathogenesis of such disorders involves cell or mitochondrial damage, but the molecular mechanisms underlying symptoms is often unclear. H. Anne Simmonds made major contributions to the metabolic, clinical, and molecular aspects of these disorders and the Purine Research Laboratory, which she established in London, became the world center for clinical and experimental studies in the field. We owe her gratitude not only for this direct contribution but also for her enthusiasm for purine and pyrimidine research that she transmitted to generations of scientists. Our research in this field stemmed from expertise in pyridine metabolism and its connection with purines, and from clinical involvement with biochemical diagnosis of enzyme deficiencies. We joined H. Anne Simmonds in studying the biochemical basis of altered NAD content in erythrocytes of PNP- and HPRT-deficient patients, discovering some alterations in NAD synthesis and breakdown.

Are Allopurinol and Metabolites Found in HPRT Deficient Erythrocytes Responsible for Increased NAD Synthesis

Aim of this study was to ascertain whether allopurinol, usually administered to hypoxanthine‐guanine phosphoribosyltransferase (HPRT) deficient patients, or metabolites abnormally increased in HPRT deficient erythrocytes (NAD, PPribP) could be directly responsible for the reported increased activities of nicotinic acid phosphoribosyltransferase (NAPRT) and NADsynthetase (NADs) in these patients. No direct effect of the mentioned metabolites was demonstrated.

NAD synthesis in human erythrocytes: determination of the activities of some enzymes.

The oxido-reductive properties of pyridine coenzymes (NAD and NADP) are well known. In the last fifteen years different roles have been pointed out for NAD as substrate in ADP-ribosylation and other reactions involved in DNA repair and replication, and in protein synthesis. Such findings suggested that hitherto unrevealed functions and biological roles may exist for NAD.