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Abstract: Neuroglobin is a neuron-specific hexacoordinated globin capable of binding various ligands, including O2, NO, and CO, the biological function of which is still uncertain. Various studies seem to indicate that neuroglobin is a neuroprotective agent when overexpressed, acting as a potent inhibitor of oxidative and nitrosative stress. In this study, we evaluated the pathophysiological response of the neuroglobin gene and protein expression in the cerebral tissue of rats sustaining traumatic brain injury of differing severity, while simultaneously measuring the oxidant/antioxidant balance. Two levels of trauma (mild and severe) were induced in anesthetized animals using the weight-drop model of diffuse axonal injury. Rats were then sacrificed at 6, 12, 24, 48, and 120h after traumatic brain injury, and the gene and protein expression of neuroglobin and the concentrations of malondialdehyde (as a parameter representative of reactive oxygen species-mediated damage), nitrite + nitrate (indicative of NO metabolism), ascorbate, and glutathione (GSH) were determined in the brain tissue. Results indicated that mild traumatic brain injury, although causing a reversible increase in oxidative/nitrosative stress (increase in malondialdehyde and nitrite + nitrate) and an imbalance in antioxidants (decrease in ascorbate and GSH), did not induce any change in neuroglobin. Conversely, severe traumatic brain injury caused an over nine- and a fivefold increase in neuroglobin gene and protein expression, respectively, as well as a remarkable increase in oxidative/nitrosative stress and depletion of antioxidants. The results of this study, showing a lack of effect in mild traumatic brain injury as well as asynchronous time course changes in neuroglobin expression, oxidative/nitrosative stress, and antioxidants in severe traumatic brain injury, do not seem to support the role of neuroglobin as an endogenous neuroprotective antioxidant agent, at least under pathophysiological conditions. [Copyright &y& Elsevier]

Abstract: Objective: To verify the effect of and to date the unknown T677C mutation of the human N-acetylaspartoacylase (hASPA) gene on the function of the mutated enzyme. Design and methods: Wild type and I226T-mutated proteins were expressed and purified from a transformed Escherichia coli colony. Enzymatic activities were measured in the presence of varying substrate concentrations. Results: Whilst kinetic parameters of wild type hASPA were in line with data in literature, I226T-mutated hASPA showed no enzymatic activity. Conclusion: Data indicated that this new mutation might be responsible in homozygosis for the phenotype corresponding to Canavan disease. [Copyright &y& Elsevier]

Abstract: Objective: : To determine the type of mutation in a patient with clinical diagnosis of suspected APRT deficiency. Design and methods: : A 51-year-old male patient, with a clinical history of two prior episodes of renal colic with urinary stone excretion (reported as uric acid stones in the first episode and as calcium oxalate stones in the second), was admitted to the hospital with severe non-oliguric renal failure (1.06 mmol/L serum creatinine), severe hyponatremia (114 mmol/L Na+), metabolic acidosis (14 mmol/L HCO3 −) and uricemia in the normal range. Abnormalities at renal scan and persistency of severe renal failure required to start haemodialysis. Results of renal biopsy prompted us to undertake a biochemical and molecular biological evaluation of the patient for suspected adenine phosphoribosyltransferase (APRT) deficiency. Results: : HPLC analysis of serum and urine, for determining purine derivative profile, showed the pathological presence of adenine in both biological fluids (3.57 μmol/L and 7.11 μmol/mmol creatinine in serum and urine, respectively; not detectable in both fluids in healthy controls). APRT assay in a sample of patient hemolysate showed no detectable activity of the enzyme (25.56±9.55 U/L red blood cells in control healthy subjects). Molecular biological analysis of the amplified APRT gene revealed that the patient harboured in exon 3 a homozygous 254 bp deletion–8 bp insertion, previously described only once in a compound heterozygote. Analysis of the patient family showed that heterozygotes for this APRT gene mutation, in spite of a 69% lower APRT enzymatic activity than that of healthy subjects, had no detectable adenine concentrations in both serum and urine. Conclusions: : Results of the first patient harbouring the homozygous 254 bp deletion–8 bp insertion of the APRT gene strongly indicated that definitive diagnosis of APRT deficiency (often under or misdiagnosed) would require a combined clinical, biochemical and molecular biological evaluation. [Copyright &y& Elsevier]

Abstract: Objective: Canavan disease (OMIM 271900) is a severe autosomal recessive neurodegenerative disorder characterized by spongy degeneration of the brain and caused by mutations in the gene encoding for aspartoacylase (ASPA). The enzyme is responsible for the catalyses of the brain-specific compound N-acetylaspartate (NAA). Design and methods: We report the case of two Egyptian sibling patients suspected of Canavan disease (CD) showing clinical deterioration, white matter degeneration, megalencephaly and severe intellectual impairment. The patients underwent magnetic resonance imaging (MRI) and biochemical analysis of NAA in biological fluid samples (serum and urine). Subsequently, in order to determine the mutation responsible for CD in these two sibs, a molecular biological examination was performed. Results: MRI findings and quantification of high NAA excretion (1378.5 and 680.1μmolNAA/mmolcreatinine in urine of 4months and 4years old patients, respectively) confirmed the diagnosis of CD and prompted a search for the responsible mutation. The molecular biological analysis revealed homozygosity for the substitution T530C (Ile177Thr) in the exon 4 of the ASPA gene in both sibs. A total loss of enzymatic activity was also recorded. Conclusions: The substitution T530C (Ile177Thr) results in a novel missense mutation causing a CD phenotype with severe clinical characteristics. This mutation was not previously described in the literature. In these two sibs, urinary concentration of NAA appears to correlate inversely to symptom severity and CD progression. [Copyright &y& Elsevier]

In this study, the metabolic, enzymatic and gene changes causing cerebral glucose dysmetabolism following graded diffuse traumatic brain injury (TBI) were evaluated. TBI was induced in rats by dropping 450 g from 1 (mild TBI; mTBI) or 2 m height (severe TBI; sTBI). After 6, 12, 24, 48, and 120 h gene expressions and enzymatic activities of glycolysis and pentose phosphate pathway (PPP) enzymes, and levels of lactate, ATP, ADP, ATP/ADP (indexing mitochondrial phosphorylating capacity), NADP + , NADPH and GSH were determined in whole brain extracts (n = 9 rats at each time for both TBI levels). Sham-operated animals (n = 9) were used as controls. Results demonstrated that mTBI caused a late increase (48–120 h post injury) of glycolytic gene expression and enzymatic activities, concomitantly with mitochondrial functional recovery (ATP and ATP/ADP normalization). No changes in lactate and PPP genes and enzymes, were accompanied by transient decrease in GSH, NADP + , NADPH and NADPH/NADP + . Animals following sTBI showed early increase (6–24 h post injury) of glycolytic gene expression and enzymatic activities, occurring during mitochondrial malfunctioning (50% decrease in ATP and ATP/ADP). Higher lactate and lower GSH, NADP + , NADPH, NADPH/NADP + than controls were recorded at anytime post injury (p < 0.01). Both TBI levels caused metabolic and gene changes affecting glucose metabolism. Following mTBI, increased glucose flux through glycolysis is coupled to mitochondrial glucose oxidation. “True” hyperglycolysis occurs only after sTBI, where metabolic changes, caused by depressed mitochondrial phosphorylating capacity, act on genes causing net glycolytic flux increase uncoupled from mitochondrial glucose oxidation. [ABSTRACT FROM AUTHOR]

Abstract: Several in vitro and in vivo studies have suggested that carnosine (β-alanil-l-histidine) and homocarnosine (β-aminobutyril-l-histidine) can act as scavengers of reactive oxygen species. β-Cyclodextrin was functionalized with homocarnosine, obtaining the following new bioconjugate isomers: 6A-[(4-{[(1S)-1-carboxy-2-(1H-imidazol-4-yl)ethyl]amino}-4-oxobutyl)amino]-6A-deoxy-β-cyclodextrin and (2A S,3A R)-3A-[(4-{[(1S)-1-carboxy-2-(1H-imidazol-4-yl)ethyl]amino}-4-oxobutyl)amino]-3A-deoxy-β-cyclodextrin. Pulse radiolysis investigations show that the β-cyclodextrin homocarnosine bioconjugates are scavengers of OH radicals because of the formation of stable imidazole-centered radicals and the scavenger ability of glucose molecules of the macrocycle. The ability of these new β-cyclodextrin derivatives to inhibit the copper(II) driven LDL oxidation was determined in comparison with that displayed by the analogous carnosine derivatives. Both the β-cyclodextrin carnosine isomers show a higher protective effect than that of free dipeptide and homocarnosine derivatives, bringing into light the role of the β-CD cavity. The ability of these new β-cyclodextrin derivatives to inhibit the copper(II) driven LDL oxidation was determined in comparison with that displayed by the analogous carnosine derivatives. Both the β-cyclodextrin carnosine isomers show a higher protective effect than that of free dipeptide and homocarnosine derivatives, bringing into light the role of the β-CD cavity. [Copyright &y& Elsevier]

Abstract: Multiple sclerosis (MS) is a primary inflammatory demyelinating disease associated with a probably secondary progressive neurodegenerative component. Impaired mitochondrial functioning has been hypothesized to drive neurodegeneration and to cause increased anaerobic metabolism in MS. The aim of our multicentre study was to determine whether MS patients had values of circulating lactate different from those of controls. Patients (n=613) were recruited, assessed for disability and clinically classified (relapsing–remitting, secondary progressive, primary progressive) at the Catholic University of Rome, Italy (n=281), at the MS Centre Amsterdam, The Netherlands (n=158) and at the S. Camillo Forlanini Hospital, Rome, Italy (n=174). Serum lactate levels were quantified spectrophotometrically with the analyst being blinded to all clinical information. In patients with MS serum lactate was three times higher (3.04±1.26mmol/l) than that of healthy controls (1.09±0.25mmol/l, p<0.0001) and increased across clinical groups, with higher levels in cases with a progressive than with a relapsing–remitting disease course. In addition, there was a linear correlation between serum lactate levels and the expanded disability scale (EDSS) (R2 =0.419; p<0.001). These data support the hypothesis that mitochondrial dysfunction is an important feature in MS and of particular relevance to the neurodegenerative phase of the disease. Measurement of serum lactate in MS might be a relative inexpensive test for longitudinal monitoring of “virtual hypoxia” in MS and also a secondary outcome for treatment trials aimed to improve mitochondrial function in patients with MS. [Copyright &y& Elsevier]

Abstract: Lactate has been identified as an alternative fuel for the brain in situations of increased energy demand, as following a traumatic brain injury (TBI). This study investigates the effect of treatment with sodium lactate (NaLac) on the changes in brain energy state induced by a severe diffuse TBI. Rats were assigned to one of the eight groups (n=10 per group): 1—sham, normal saline; 2—TBI, normal saline; 3—TBI, hypertonic saline; 4—TBI, 100mM NaLac, 5—TBI, 500mM NaLac; 6—TBI, 1280mM NaLac; 7—TBI, 2000mM NaLac and 8-TBI-500mM NaLac+magnesium sulfate. Cerebrums were removed 6h after trauma. Metabolites representative of the energy state (ATP, ATP-catabolites), N-acetylaspartate (NAA), antioxidant defenses (ascorbic acid, glutathione), markers of oxidative stress (malondialdehyde, ADP-ribose) and nicotinic coenzymes (NAD+) were measured by HPLC. TBI induced a marked decrease in the cerebral levels of ATP, NAA, ascorbic acid, glutathione and NAD+ and a significant rise in the content of ATP-catabolites, malondialdehyde and ADP-ribose. These alterations were not ameliorated with NaLac infusion. We observed a significant reduction in cerebral NAD+, an essential co-enzyme for mitochondrial lactate-dehydrogenase that converts lactate into pyruvate and thus replenishes the tricarboxylic acid cycle. These results suggest that the metabolic pathway necessary to consume lactate may be compromised following a severe diffuse TBI in rats. [Copyright &y& Elsevier]

Abstract: Objectives: In this study, the concentrations of uric acid, purine profile and creatinine in samples of cerebrospinal fluid and serum of multiple sclerosis (MS) patients were measured by HPLC and compared with corresponding values recorded in patients without MS (cerebrospinal fluid) and healthy subjects (serum). Design and methods: All samples were deproteinized with ultrafiltration (which ensures minimal sample manipulation and efficient protein removal) and then assayed for the synchronous HPLC separation of uric acid, hypoxanthine, xanthine, inosine, adenosine, guanosine and creatinine. Results: The values of all compounds assayed were significantly higher in both biological fluids of MS patients with respect to values measured in controls. In particular, serum hypoxanthine, xanthine, uric acid and sum of oxypurines were, respectively, 3.17, 3.11, 1.23 and 1.27-fold higher in these patients than corresponding values recorded in controls (p <0.001). Conclusions: Differently from what previously reported, we here demonstrate that all purine compounds, including uric acid, are elevated in biological fluids of MS patients. Reinforced by the trend observed for creatinine, this corroborates the notion of sustained purine catabolism, possibly due to imbalance in ATP homeostasis, under these pathological conditions. These results cast doubt on the hypothesis that uric acid is depleted in MS because of increased oxidative stress, rather suggesting that this disease causes a generalized increase in purine catabolism. As observed in other pathological states, uric acid, purine compounds and creatinine, can be considered markers of metabolic energy imbalance rather than of reactive oxygen species, even in MS. [Copyright &y& Elsevier]

Abstract: Synthesis, antioxidant properties and resistance to carnosinase hydrolysis of histidine-containing dipeptides are reported in this study. Carnosine (β-alanyl-l-histidine), homocarnosine (γ-aminobutyryl-l-histidine) and anserine (β-alanyl-3-methyl-l-histidine) were covalently derivatized with β-cyclodextrin to form different OH- or NH-bound conjugates. Mass spectroscopic and 1H NMR data were used to determine the structure and the purity of the various β-cyclodextrin derivatives. The inhibitory effect towards oxidation of human LDL induced by Cu2+ ions, was estimated by measuring malondialdehyde formation as a function of increasing concentrations of these newly synthesized compounds (the β-cyclodextrin-anserine conjugated in 3 had the highest antioxidant effect). All derivatives had higher antioxidant effects than those of the corresponding free histidine-containing dipeptides. Resistance to rat brain carnosinase hydrolysis of the most active derivatives indicated that these compounds are good candidates for further studies in more complex cellular and animal models. Their possible applications for remedies in neurodegenerative disorders, such as Alzheimer''s and Parkinson''s diseases, are discussed. [Copyright &y& Elsevier]

Abstract: Objectives: : To set up a novel simple, sensitive, and reliable ion-pairing HPLC method for the synchronous separation of several purines, pyrimidines, N-acetylated amino acids, and dicarboxylic acids for the chemical diagnosis and screening of inborn errors of metabolism (IEM). Design and methods: : The separation was set up using a Hypersil C-18, 5-μm particle size, 250 × 4.6 mm column, and a step gradient using two buffers and tetrabutylammonium hydroxide as the pairing reagent. A highly sensitive diode array UV detector was set up at a wavelength between 200 and 300 nm that revealed purines and pyrimidines at 260 nm and other compounds at 206 nm. Results: : Compounds were determined in the plasma of 15 healthy adults, in the urine of 50 healthy subjects (1–3 years, 4–6 years, 8–10 years, 12–18 years, 25–35 years), and in 10 non-pathological amniotic fluid samples. To assess the validity of the chemical diagnosis of IEM, plasma and urine samples were analyzed in patients affected by Canavan disease (n = 10; mean age 4.6 ± 2.3). Low plasma levels of N-acetylaspartate (16.96 ± 19.57 μmol/L plasma; not detectable in healthy adults) and dramatically high urinary N-acetylaspartate concentrations (1872.03 ± 631.86 μmol/mmol creatinine; 450 times higher than that which was observed in age-matched controls) were recorded. Neither N-acetylglutamate nor N-acetylaspartylglutamate could be detected in the plasma or urine of controls or patients with Canavan disease. Conclusions: : The results demonstrate the suitability of the present ion-pairing HPLC separation with UV detection of cytosine, cytidine, creatinine, uracil, uridine, β-pseudouridine, adenine, 3-methyladenine, hypoxanthine, xanthine, xanthosine, inosine, guanosine, ascorbic acid, thymine, thymidine, uric acid, 1-methyluric acid, orotic acid, N-acetylaspartate, N-acetylglutamate, N-acetylaspartylglutamate, malonic acid, methylmalonic acid, GSH, and GSSG as a reliable method for the prenatal and neonatal chemical diagnosis and screening of IEM using biological fluids. [Copyright &y& Elsevier]