HPLC analysis of tetrahydrobiopterin and its pteridine derivatives using sequential electrochemical and fluorimetric detection: application to tetrahydrobiopterin autoxidation and chemical oxidation.

Tetrahydrobiopterin (BH(4)) is an essential cofactor of endothelial nitric oxide (NO) synthase and when depleted, endothelial dysfunction results with decreased production of NO. BH(4) is also an anti-oxidant being a good "scavenger" of oxidative species. NADPH oxidase, xanthine oxidase, and mitochondrial enzymes producing reactive oxygen species (ROS) can induce elevated oxidant stress and cause BH(4) oxidation and subsequent decrease in NO production and bioavailability. In order to define the process of ROS-mediated BH(4) degradation, a sensitive method for monitoring pteridine redox-state changes is required. Considering that the conventional fluorescence method is an indirect method requiring conversion of all pteridines to oxidized forms, it would be beneficial to use a rapid quantitative assay for the individual detection of BH(4) and its related pteridine metabolites. To study, in detail, the BH(4) oxidative pathways, a rapid direct sensitive HPLC assay of BH(4) and its pteridine derivatives was adapted using sequential electrochemical and fluorimetric detection. We examined BH(4) autoxidation, hydrogen peroxide- and superoxide-driven oxidation, and Fenton reaction hydroxyl radical-driven BH(4) transformation. We demonstrate that the formation of the primary two-electron oxidation product, dihydrobiopterin (BH(2)), predominates with oxygen-induced BH(4) autoxidation and superoxide-catalyzed oxidation, while the irreversible metabolites, pterin and dihydroxanthopterin (XH(2)), are largely produced during hydroxyl radical-driven BH(4) oxidation.

[Diabetic peripheral neuropathy: reflections and drug-rehabilitative treatment]. / Neuropatia diabetica periferica: riflessioni e proposte farmacologiche e riabilitative.

About 60 to 70 percent of people with diabetes have some neuropathy. Diabetic neuropathy can be classified as peripheral, autonomic, proximal, focal and multifocal or mixed. Peripheral neuropathy, the most common type of diabetic neuropathy, causes pain and/or loss of feeling in the toes, feet, legs, hands, and arms; extreme sensitivity to touch, loss of balance and coordination; muscle weakness and loss of reflexes, especially at the ankle, leading to changes in the way a person walks. The aim of this study is to underline the importance of drug and rehabilitative approach in the therapy of peripheral neuropathy, that frequently influences both diabetes mellitus type 1 and diabetes mellitus type 2.

Detection and scavenging of hydroxyl radical via D-phenylalanine hydroxylation in human fluids.

Hydroxyl radical (.OH) is highly reactive, and therefore very short-lived. Finding new means to accurately detect .OH, and testing the ability of known .OH scavengers to neutralize them in human biological fluids would leverage our ability to more effectively counter oxidative (.OH) stress-mediated damage in human diseases. To achieve this, we pursued the evaluation of secondary products resulting from .OH attack, using a detection system based on Fenton reaction-mediated D-phenylalanine (D-Phe) hydroxylation. This reaction in turn generates o-tyrosine (o-tyr), m-tyrosine (m-tyr) and p-tyrosine (p-tyr). Here, these isomers were separated by HPLC, equipped with fluorescence detectors due to the natural fluorescence of these hydrotyrosines. By extension, we found that, adding radical scavengers competed with D-Phe on .OH attack, thus allowing to determine the .OH quenching capacity of a given compound expressed as inhibition ratio percent (IR%). Using a kinetic approach, we then tested the .OH scavenging capacity (OHSC) of well-known antioxidant molecules. In a test tube, N,N'-dimethylthiourea (DMTU) was the most efficient scavenger as compared to Trolox and N-Acethyl-L-cysteine, with NAC being the less effective. OHSC assay was then applied to biological fluid samples as seminal plasma, human serum from normal subjects and patients undergoing hemodialysis (HD), colostrum and human breast milk from mothers that received daily doses of 30g of chocolate (70% cocoa) during pregnancy. We found that a daily administration of dark chocolate during pregnancy almost doubled OHSC levels in breast milk (1.88 ± 0.12 times, p < 0.01). Furthermore, HD treatment determined a significant reduction of serum OHSC concentration (54.63 ± 2.82%, p < 0.001). Our results provide evidence that Fenton reaction-mediated D-Phe hydroxylation is a suitable method for routine and non-invasive evaluation of .OH detection and its scavenging in human biological fluids.

Hydroxylation of D-phenylalanine as a novel approach to detect hydroxyl radicals: application to cardiac pathophysiology.

OBJECTIVE: Research in the pathophysiology of ischemia/reperfusion or redox signaling is hindered by lack of simple methodology to measure short-lived oxygen radicals. In the presence of hydroxyl radical ((*)OH), d-phenylalanine (d-Phe) yields para-, meta- and ortho-tyrosine. We have previously demonstrated that d-Phe can accurately detect (*)OH formation in chemical, enzymatic and cellular systems by simple HPLC methodology [Anal Biochem 290:138;2001]. In the present study, we tested whether d-Phe hydroxylation can be used to detect (*)OH formation in intact organs. METHODS: Rat hearts were perfused with buffer containing 5 mM d-Phe and subjected to 30 min of total global ischemia at 37 degrees C followed by 45 min of reperfusion. Quantitative analysis of the three hydroxytyrosine isomers was achieved by HPLC-based electrochemical detection of cardiac venous effluent, with the analytical cells operating in the oxidative mode. The detection limit of this assay was <10 fmol. RESULTS: Under baseline conditions, hydroxytyrosine release from the heart was very low ( congruent with0.8 nmol/min/g). However, a prominent tyrosine burst occurred immediately upon post-ischemic reflow. In cardiac effluent collected 40 s into reperfusion, the hydroxytyrosine concentration was more than 40 times greater than at baseline; hydroxytyrosine concentration then progressively declined, to return to pre-ischemic values by 5 min of reperfusion. In parallel experiments, formation of hydroxytyrosines was markedly reduced in hearts reperfused in the presence of the (*)OH scavenger mannitol. Inclusion of 5 mm d-Phe in the perfusion medium altered neither basal cardiac function nor coronary vascular tone, but it enhanced recovery of myocardial function during post-ischemic reperfusion, consistent with direct reaction with (*)OH. CONCLUSION: Our results demonstrate that d-Phe is a sensitive method for detection of (*)OH generation in the heart. Since d-Phe is not a substrate for endogenous enzymes, it can be exploited as a reliable method to measure (*)OH formation under a variety of pathophysiological conditions.

A pilot trial of levetiracetam in eyelid myoclonia with absences (Jeavons syndrome).

OBJECTIVE: Eyelid myoclonia with absences (EMA) or Jeavons syndrome characterized by eyelid myoclonia (EM) (with or without absences), eye closure-induced EEG paroxysms, and photosensitivity. We conducted an open-label trial of levetiracetam in EMA. PATIENTS AND METHODS: Patients were recruited in different Italian Epilepsy Centres. Levetiracetam was administrated at starting dose of 10 mg/kg/day up to 50-60 mg/kg/day in two doses. Treatment period included a 5-6 week up-titration phase and a 12-week evaluation phase. The number of days with EM (i.e., days with seizures, DwS) and number of generalized tonic-clonic seizures (GTCS) were evaluated. Analysis of intent-to-treat population was performed using Fisher's and Wilcoxon tests. RESULTS: Thirty-five patients (23 F) with a mean age of 19 +/- 6 years were recruited. Twenty-seven had previously undergone one to five adequate trials of antiepileptic drugs. The median number of DwS/month was 12 +/- 8.2. Twenty-one patients experienced GTCS (median number/month: 1 +/- 0.2). Thirty-four subjects completed the trial. Levetiracetam was well tolerated (mean dose: 1985 mg/day). Responders were 28/35 (80%) patients, nine taking levetiracetam as monotherapy. Six patients were seizure-free, 15 had > or =75% and seven >50% seizure reduction. GTCS remitted in 14 out of 21 (66.6%) patients. The number/month of DwS (median: 12 vs 5; p = 0.0001) and of GTCS (median: 1 vs 0; p = 0.0001) decreased compared to baseline period. Disappearance or clear reduction in paroxysmal abnormalities at eye closure occurred in 20 of the responders and photoparoxysmal response in 19. Mean follow-up was 23.9 +/- 18.5 months. CONCLUSION: Levetiracetam is effective and well tolerated in EMA. Placebo-controlled studies should confirm these findings.

Epilepsy and toxocariasis: a case-control study in Italy.

PURPOSE: To assess the relationship between epilepsy and toxocariasis in adult subjects by means of a case-control study in Catania, Italy. METHODS: People with epilepsy (PWE) were randomly selected from the database of the center of epilepsy of our department. Epilepsy was diagnosed according to the definition proposed by the International League Against Epilepsy. One healthy control per each case was selected among subjects who went to the central laboratory for a hematological check. Control subjects underwent a complete neurological examination to exclude the presence of neurological disorders. PWE and controls were assessed serologically for antibodies against Toxocara canis (T. canis) by an immunoblotting assay. RESULTS: Two hundred thirty-one PWE (110 men and 121 women) and 201 controls (126 men and 75 women) were enrolled in the study. Of the 231 PWE, 152 presented partial seizures. Antibodies anti-T. canis were found in 38 PWE (16.4%) and in 13 controls (6.6%) giving a crude OR of 2.85 (95% CI 1.47-5.51). Adjusted OR estimated by logistic regression was 3.90 (95% CI 1.91-7.98). This association was mainly due to a significant association between Toxocara antibodies and partial epilepsy (adjusted OR 4.69; 95% CI 2.24-9.80), while a positive, but not significant, association was found with generalized seizures (adjusted OR 1.74; 95% CI 0.60-5.05). CONCLUSION: We found a significant association between T. canis seropositivity and epilepsy and a stronger association was found with partial epilepsy. Our finding suggests that toxocariasis may increase the risk of epilepsy.

Myocardial ischemia results in tetrahydrobiopterin (BH4) oxidation with impaired endothelial function ameliorated by BH4.

Coronary vasodilation is impaired in the postischemic heart with a loss of endothelial nitric oxide synthase (eNOS) activity, but the mechanisms underlying ischemia-induced eNOS dysfunction are not understood. For nitric oxide (NO) synthesis, eNOS requires the redox-sensitive cofactor tetrahydrobiopterin (BH(4)); however, the role of BH(4) in ischemia-induced endothelial dysfunction remains unknown. Therefore, isolated rat hearts were subjected to varying durations of ischemia, and the alterations in NOS-dependent vasodilation were measured and correlated with assays of eNOS activity and cardiac BH(4) concentrations. Ischemia time-dependently decreased cardiac BH(4) content with 85, 95, or 97% irreversible degradation after 30, 45, or 60 min of ischemia, respectively. Paralleling the decreases in BH(4), reductions of eNOS activity were seen of 58, 86, or 92%, and NOS-derived superoxide production was greatly increased. Addition of 10 microM BH(4) enhanced eNOS activity in nonischemic hearts and partially restored activity after ischemia. It also suppressed NOS-derived superoxide production. Impaired coronary flow during postischemic reperfusion was improved by BH(4) infusion. Thus, BH(4) depletion contributes to postischemic eNOS dysfunction, and BH(4) treatment is effective in partial restoration of endothelium-dependent coronary flow. Supplementation of BH(4) may therefore be an important therapeutic approach to reverse endothelial dysfunction in postischemic tissues.

Metalloproteinase inhibitor counters high-energy phosphate depletion and AMP deaminase activity enhancing ventricular diastolic compliance in subacute heart failure.

Cardiac matrix metalloproteinases (MMPs) stimulated by the sympathomimetic action of angiotensin II (AII) exacerbate chamber diastolic stiffening in models of subacute heart failure. Here we tested the hypothesis that MMP inhibition prevents such stiffening by favorably modulating high-energy phosphate (HEP) stores more than by effects on matrix remodeling. Dogs were administered AII i.v. for 1 week with tachypacing superimposed in the last two days (AII+P; n = 8). A second group (n = 9) underwent the same AII+P protocol but was preceded by oral treatment with an MMP inhibitor PD166793 [(S)-2-(4-bromo-biphenyl-4-sulfonylamino-3-methyl butyric acid] 1 week before and during the AII+P period. Pressure-volume analysis was performed in conscious animals, and myocardial tissue was subjected to in vitro and in situ zymography, collagen content, and HEP analysis (high-performance liquid chromatography). As reported previously, AII+P activated MMP9 and MMP2 and specifically exacerbated diastolic stiffening (+130% in chamber stiffness). PD166793 cotreatment prevented these changes, although myocardial collagen content, subtype, and cross-linking were unaltered. AII+P also reduced ATP, free energy of ATP hydrolysis (DeltaG(ATP)), and phosphocreatine while increasing free [ADP], AMP catabolites (nucleoside-total purines), and lactate. PD166793 reversed most of these changes, in part due to its inhibition of AMP deaminase. MMP activation may influence cardiac diastolic function by mechanisms beyond modulation of extracellular matrix. Interaction between MMP activation and HEP metabolism may play an important role in mediating diastolic dysfunction. Furthermore, these data highlight a potential major role for increased AMP deaminase activity in diastolic dysfunction.

Clinical and genetic findings in 26 Italian patients with Lafora disease.

PURPOSE: EPM2B mutations have been found in a variable proportion of patients with Lafora disease (LD). Genotype-phenotype correlations suggested that EPM2B patients show a slower course of the disease, with delayed age at death, compared with EPM2A patients. We herein report clinical and genetic findings of 26 Italian LD patients. METHODS: Disease progression was evaluated by means of a disability scale based on residual motor and cognitive functions and daily living and social abilities, at 4 years from the onset. Mutational analysis was performed by sequencing the coding regions of the EPM2A and EPM2B genes. RESULTS: Age at onset ranged from 8.5 to 18.5 years (mean, 13.7+/-2.6). The mean duration of follow-up was 7.1+/-3.9 years. Daily living activities and social interactions were preserved in five of 24 patients. The remaining patients showed moderate to extremely severe limitations of daily living and social abilities. Sixteen (72%) of 22 families showed mutations in the EPM2B gene, and five (22%), in the EPM2A gene. One family showed no mutations. A novel EPM2B mutation also was identified. CONCLUSIONS: In our series, EPM2B mutations occurred in 72% of families, thus indicating that EPM2B is the major gene for LD in the Italian population. Moreover, we found that six of 17 EPM2B patients preserved daily living activities and social interactions at 4 years from onset, suggesting a slow disease progression. Additional clinical and functional studies will clarify whether specific mutations may influence the course of the disease in LD patients.

Clinical heterogeneity in eyelid myoclonia, with absences, and epilepsy.

Eyelid myoclonia with absences should always be considered in the investigation of children with epilepsy.

Epilepsy and multiple sclerosis in Sicily: a population-based study.

PURPOSE: To evaluate the association between epilepsy and multiple sclerosis (MS), we analyzed the incidence of epilepsy in a population-based incidence cohort of MS in Catania, Sicily. METHODS: According to Poser's diagnostic criteria, 170 incident cases of MS have been identified from 1975 to 1994 in the city of Catania. All these subjects underwent a complete neurological examination to confirm the diagnosis of MS and to identify those patients with a history of seizures. Diagnosis of epilepsy was based on the criteria proposed by the International League Against Epilepsy (ILAE) in 1993, and seizures were classified according to the classification of the ILAE, 1981. RESULTS: From 1975 to 1994, 170 subjects with MS had the clinical onset of the disease. The mean annual incidence of MS was 2.3/100,000 (95% CI, 2.0-2.6). Of the 170 defined MS patients, four developed epilepsy after the onset and also diagnosis of MS, giving an incidence rate of epilepsy of 285/100,000 person years at risk (95% CI, 119-684) and 147.8/100,000 when age adjusted to the world standard population. The cumulative risk of developing epilepsy after the onset of MS, evaluated by using the life-table methods, was zero at 1 year and 1.76% at 5 years. Of these four patients, three were classified as having partial seizures with secondary generalization and one with tonic-clonic seizures. CONCLUSIONS: Our data are consistent with those reported in literature suggesting that the risk of developing epilepsy is threefold higher among MS patients than in the general population.

Autosomal dominant lateral temporal epilepsy: clinical spectrum, new epitempin mutations, and genetic heterogeneity in seven European families.

PURPOSE: [corrected] To describe the clinical and genetic findings of seven additional pedigrees with autosomal dominant lateral temporal epilepsy (ADLTE). METHODS: A personal and family history was obtained from each affected and unaffected member, along with a physical and neurologic examination. Routine and sleep EEGs, computed tomography (CT), or magnetic resonance imaging (MRI) were performed in almost all the patients. DNAs from family members were typed with several microsatellite markers localized on either side of LGI1 at 10q24 and screened for LGI1 mutations. RESULTS: The seven families included a total of 34 affected individuals (10 deceased). The age at onset ranged between 8 and 50 years (average, 22 years). Twenty-six patients had clear-cut focal (elementary, complex, or secondarily generalized) seizures, characterized by prominent auditory auras in 68% of the cases. Less frequent ictal symptoms were visual, psychic, or aphasic seizures, the latter occurring in isolation in one family. The attacks were rare and well controlled by antiepileptic drug treatment but recurred after drug discontinuation. Interictal EEGs were usually unrevealing. MRI or CT scans were negative. Analysis of LGI1/Epitempin exons failed to show mutations in three pedigrees. Linkage analysis strongly suggested exclusion of linkage in one of these families. We found two novel missense mutations, a T-->C substitution in exon 6 at position 598, and a T-->A transition in exon 8 at position 1295, the latter being detected in a family with aphasic seizures. CONCLUSIONS: Our data confirm the inclusion of aphasic seizures within the ADLTE clinical spectrum, suggest the existence of locus heterogeneity in ADLTE, and provide new familial cases with LGI1 missense mutations associated with the disease.