Gabriella Jacomelli

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.

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.

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.

Thiopurine methyltransferase activity in the erythrocytes of adults and children: an HPLC-linked assay

A non-radioactive method that uses reverse-phase high performance liquid chromatography is described for the determination of thiopurine methyltransferase (E.C. 2.1.1.67) activity in human erythrocytes. The method is based on the direct quantitation of 6-methyl-mercaptopurine produced from 6-mercaptopurine by crude erythrocyte lysates. The method is accurate and reliable and suitable for diagnostic use. Activity values in control adults ranged from 5 to 32 pmol/h/mg haemoglobin. The activity in the erythrocytes of adult males was significantly higher compared to females (21 +/- 5 and 15 +/- 8 pmol/h/mg haemoglobin, respectively). The activity measured in the erythrocytes of children (22 +/- 5 pmol/h/mg haemoglobin) did not show any significant difference compared to adults. Thiopurine methyltransferase activity was measured in a female patient with systemic sclerosis who developed severe bone marrow depression after treatment with azathioprine and allopurinol. Activity (6.3 +/- 0.5 pmol/h/mg haemoglobin) was found in the lowest range of controls thus supporting the hypothesis that it could be responsible for increased azathioprine cytotoxicity.

Molecular basis of hypoxanthine-guanine phosphoribosyltransferase deficiency in Italian Lesch-Nyhan patients: Identification of nine novel mutations

Summary: Lesch-Nyhan syndrome (LSN, McKusick 300322) is an X-linked genetic disease due, in its typical form, to the complete absence of hypoxanthine-guanine phosphoribosyltransferase (HPRT, EC 2.4.2.8) enzyme activity. It is characterized by hyperuricaemia, leading to gout and kidney stones, accompanied by severe neurological dysfunction with self-injurious behaviour, choreoathetosis and spasticity. Based on a worldwide birth incidence estimate of about 1:380000, one or two new cases are expected every year in Italy. We performed biochemical and molecular genetic studies on 28 Italian patients from 25 families who are likely to represent most living individuals with the syndrome in the country. They all had absent HPRT activity and a typical LNS phenotype. Genetic analysis identified 24 HPRT mutations, 9 of which had not been previously reported: 74C>G (P25R), IVS2+1G>C, 194-195delTC, 329-332delCAAC insTCTs, IVS9-1G>A, 506insC, IVS8-1G>C, 606G>T (L202F), 418G>C (G140R). No mutation hotspots were identified. Only two mutations were found in more than one family, indicating the lack of any major mutation causing LNS in Italy. Three mutations arose de novo, two in the probands mother, one in the maternal grandmother. The virtual complete absence of HPRT activity was related to deletions, nonsense, or missense mutations leading to nonconservative amino acid changes.

Identification of the Nucleotidase Responsible for the AMP Hydrolysing Hyperactivity Associated with Neurological and Developmental Disorders

Nucleoside monophosphate phosphohydrolases comprise a family of enzymes dephosphorylating nucleotides both in intracellular and extracellular compartments. Members of this family exhibit different sequence, location, substrate specificity and regulation. Besides the ectosolic 5′-nucleotidase, several cytosolic and one mitochondrial enzymes have been described. Nevertheless, researchers refer any AMP-dephosphorylating activity to as 5′-nucleotidase, lacking a more accurate identification. Increase of AMP hydrolysing activity has been associated with neurological and developmental disorders. The identification of the specific enzyme involved in these pathologies would be fundamental for the comprehension of the linkage between the enzyme activity alteration and brain functions. We demonstrate that the described neurological symptoms are associated with increased ectosolic 5′-nucleotidase activity on the basis of radiochemical assays and immunoblotting analysis. Furthermore, present data evidence that the assay conditions normally applied for the determination of cytosolic 5′-nucleotidases activity in crude extracts are affected by the presence of solubilised ectosolic nucleotidase.

Saccharomyces Cerevisiae as a Tool to Evaluate the Effects of Herbicides on Eukaryotic Life

1.1 Herbicides and their toxicity Agricultural herbicides play undoubted beneficial roles in preserving crop yields, although they may pose serious concerns for the environment and humans because of their widespread and intensive use/misuse (due to careless applications, high and repeated application rate, accidental spillage). It is also evident the potential to injure non-target cultivars and microorganisms, in particular those contributing to soil quality, and to cause adverse side-effects in mammals, including humans (Cabral et al.,2003; Cabral et al.,2004). Herbicide-related toxicity may be often underestimated because little information is provided on the effects of herbicides as complete formulations. As a matter of fact, acute or chronic toxicity tests are generally carried out using pure active ingredients (AIs). Herbicide-induce damaging effects are proportional to the absorbed substance and to the time of exposure, but also to the inherent characteristics of each compound. Agriculturists are most likely exposed to acute intoxication, by inhalation or direct contact with toxic substances, though each person can be subjected to possible toxic effects due to accumulation of pesticide residues in the body, being the diet the most important source of exposure. Typically, pure herbicide molecules are of limited value to end users. To give them practical value and make them usable, most herbicides are combined with appropriate solvents or surfactants to obtain a formulation. A given chemical may be formulated in a variety of differing formulations and sold under different trade names. For this reason, pure AIs are mixed with coformulants, also called ‘inert’ or auxiliary substances, to allow their use in common and convenient vehicles, such as water, and to obtain uniform and effective distributions. Several substances fall within the definition of coformulant, including: carrier substances, solvents, surfactants, dispersing agents, adhesives, absorption-promoting agents, antioxidants, bactericides, dyes, fillers, and perfumes. Coformulants can have various technical and physico/chemical properties in relation to their function in the pesticide formulation. These chemicals can be expected to have various toxicological profiles, some of them harmless (e.g., water) and some with serious toxicological properties (e.g., the organic solvent isophorone, which is a suspected carcinogen). Because

NAD metabolism in HPRT-deficient mice

The activity of hypoxanthine-guanine phosphoribosyltransferase (HPRT) is virtually absent in Lesch-Nyhan disease (LND), an X-linked genetic disorder characterized by uric acid accumulation and neurodevelopmental dysfunction. The biochemical basis for the neurological and behavioral abnormalities have not yet been completely explained. Prior studies of cells from affected patients have shown abnormalities of NAD metabolism. In the current studies, NAD metabolism was evaluated in HPRT gene knock-out mice. NAD content and the activities of the enzymes required for synthesis and breakdown of this coenzyme were investigated in blood, brain and liver of HPRT- and control mice. NAD concentration and enzyme activities were found to be significantly increased in liver, but not in brain or blood of the HPRT- mice. These results demonstrate that changes in NAD metabolism occur in response to HPRT deficiency depending on both species and tissue type.

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.

Quick Diagnosis of Alkaptonuria by Homogentisic Acid Determination in Urine Paper Spots

OBJECTIVES Two methods are described for homogentisic acid (HGA) determination in dried urine spots (DUS) on paper from Alkaptonuria (AKU) patients, devised for quick early diagnosis. AKU is a rare autosomal recessive disorder caused by deficiency of homogentisate 1,2-dioxygenase, yielding in accumulation of HGA. Its massive excretion causes urine darkening by exposure to air or alkalinization, and is a diagnostic marker. The deposition of polymers produced after HGA oxidation within the connective tissues causes ochronotic arthritis, a degenerative joint disease manifesting in adulthood and only rarely in childhood. No early diagnosis is usually accomplished, awareness following symptom development. DESIGN AND METHODS Two methods were designed for HGA determination in DUS: (1) a rapid semi-quantitative reliable method based on colour development in alkali and quantification by comparison with dried paper spots from HGA solutions of known concentration and (2) a quantitative and sensitive HPLC-linked method, previously devised for purine and pyrimidine analysis in urine and plasma. RESULTS Colour intensity developed by DUS after alkali addition was proportional to HGA concentration, and calculated amounts were in good agreement with quantitative analysis performed by RP-HPLC on DUS and on urines as such. CONCLUSIONS DUS, often used for different diagnostic purpose, are easily prepared and safely delivered. The simple and quick colour method proposed provides reliable HGA assessment and is fit for large screening. HGA concentration determined in 10 AKU patient DUS by both methods 1 and 2 was in agreement with direct urine assay and in the range reported by literature.A reliable HGA quantification based on colour development in paper urine spots is validated by HPLC-linked HGA quantification, and proposed as a quick diagnostic tool for AKU patients.