Luana Peruzzi

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.

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.

Early activation of the hypothalamic-pituitary-adrenal-axis in very-low-birth-weight infants with small thymus at birth

Background. An acute thymic involution in human fetuses and newborns has been described in very-low-birth-weight (VLBW) infants with histological chorioamnionitis. However, the mechanisms of thymic involution remain to be clarified. Here, we tested the hypothesis that an activation of the hypothalamic-pituitary-adrenal (HPA) axis occurs in VLBW infants with acute thymic involution at birth. Methods. A total of 180 randomly selected VLBW newborns (28.8 ± 3.15 wk gestation; 1093 ± 305 g) entered the study. Thymic size was measured on standard chest radiographs at birth, and expressed as the ratio between the transverse diameter of the cardiothymic image at the level of the carina (CT) and that of the thorax (T). CT/T < 0.28 was considered to indicate a small thymic size. Plasma cortisol and adrenocorticotropic hormone (ACTH) concentrations were determined on days 1 (d-1) and 7 (d-7), and at 1 month (mo-1). Results. A total of 66 (36.7%) newborns had CT/T < 0.28. Infants with small thymus had significantly increased cortisol on d-1 (∼5.2-folds) [median: 18.95 (95% CI: 11.20–39.4) μg/dl vs. 3.66 (1.94–6.82) μg/dl, p < 0.0001)] and d-7(∼1.7-folds) [12.0 (4.39–22.97) μg/dl vs. 7.8 (3.63–12.8) μg/dl, p = 0.0384)], as compared with those with normal thymic size, together with higher adrenocorticotropic hormone (ACTH) concentrations on d-1 (∼1.9-folds) [28 (15.6–61.07) pg/ml vs. 14.9 (9.0–23.42) pg/ml, p = 0.0005)], while no significant differences for cortisol at mo-1 or ACTH concentrations on d-7 and mo-1 were evidenced (p > 0.50). From a multivariate logistic regression analysis, a small thymus at birth was a significant independent predictor of plasma cortisol concentrations in the top-quartile (OR = 14.4; 95% CI: 6.079–34.11), and plasma ACTH concentrations in the top-quartile (OR = 4.40 (95% CI: 1.99–9.74) on d-1 (results adjusted for variables significant at univariate analysis). Conclusions. Our data indicated the presence of a previously unrecognized, early activation of the HPA axis in VLBW newborns with a small thymus at birth.

Compound heterozygosity in the GALC gene in a late onset Iranian patient with spastic paraparesis, peripheral neuropathy and leukoencephalopathy

Dear Sir, Krabbe disease (galactocerebrosidase deficiency) is an inherited leukodystrophy resulting in severe neurological defects due to altered myelination. Classically, the disease onset is within the first year of life. Juvenileand adult-onset cases may have less severe presentations, making difficult and often delayed the diagnosis. Cases of polyneuropathy or spastic paraparesis with late onset have been reported [1–4]. We describe an Iranian girl, 23 years old age, with a history of gait disturbances since her teenage years. She was born after a normal delivery from a non consanguineous marriage. Her brother was affected by epilepsy (not available for biochemical and genetic tests). Initial motor milestones were normal. She normally frequented primary school with normal performances. Her motor problems became apparent from the age of 11 with frequent falls and subsequently toe walking. She developed balance problems and difficulties in coordination. She was unable to run after the age of 17, needing aid supports. She also developed joint deformities at hands, scoliosis, and pes cavus (Fig. 1). Neurologic examination showed a severe spastic paraparesis with abnormally brisk deep tendon reflexes and positive Babinski sign. Neuropsychological examination was normal. Nerve conduction velocities were decreased in both upper and lower limbs, consistent with a sensory motor polyneuropathy. Brain MRI showed signal changes (isosignal on T1 and high signal intensity on T2 and FLAIR images without gadolinium-enhancing) in bilateral posterior periventricular white matter and centrum semiovale, similarly to what

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.