Luisa Rossi

Elevation of serum copper levels in Alzheimer’s disease

Objective To determine whether serum trace metals and oxidative species are related to abnormal cognition in AD. Methods The authors studied serum peroxides, copper, iron, transferrin, and antioxidant capacity in 79 patients with AD (mean age 74.3 years; 25 men, 54 women) and in 76 cognitively normal individuals (mean age 70.1 years; 33 men, 43 women). The relation of these oxidative and trace metals to APOE &egr;4 allele frequency, neuropsychological performance, and cerebrovascular or atrophic burden, as estimated by brain MRI and ultrasonography of cerebral vessels, was evaluated. Results Copper level was higher (p < 0.001) in subjects with AD than control subjects (specificity = 95%, sensitivity = 60%) with a cutoff serum level of 16 &mgr;mol/L (1.02 mg/L). An increase of 1 &mgr;mol/L in serum copper accounted for 80% of the risk of having AD and correlated with poor neuropsychological performance and medial temporal lobe atrophy (p < 0.03). Antioxidant capacity decreased and correlated with medial temporal lobe atrophy (p < 0.009) and with APOE &egr;4 allele (p = 0.004). Conclusions Copper may play a role in neurodegenerative processes in AD, and serum copper measurement may prove to be a peripheral diagnostic marker for AD.

Quinone toxicity in hepatocytes without oxidative stress

The toxicity of quinones is believed to be mediated via redox cycling involving formation of semiquinone radicals which autoxidize to form active oxygen species. However, when the cytotoxicity of benzoquinones was compared using freshly isolated rat hepatocytes, benzoquinones which did not mediate oxidative stress were highly toxic. Thus, the benzoquinone analogs in decreasing order of cytotoxicity were 2-CH3-, 2-Br-, unsubstituted, 2,6-(CH3)2-, 2,5-(CH3)2-, and 2,3,5-(CH3)3-benzoquinone. Cellular thiols were rapidly depleted and glutathione (GSH) was converted to a quinone conjugate without oxidation to glutathione disulfide. No increase in cyanide-resistant respiration was observed and benzoquinone-induced cytotoxicity was not enhanced by inactivation of catalase or glutathione reductase. In contrast, duroquinone [2,3,5,6-(CH3)4-benzoquinone], which stimulated cyanide-resistant respiration and GSH oxidation, was only cytotoxic when catalase or glutathione reductase was inactivated. These results suggest that alkylation and/or oxidative stress may be important mechanisms in the cytotoxicity of benzoquinone derivatives.

Excess of serum copper not related to ceruloplasmin in Alzheimer disease

Objective: To assess the role of serum copper in relation to ceruloplasmin and other peripheral markers of inflammation in Alzheimer disease (AD). Methods: The authors studied serum levels of copper, ceruloplasmin, and transferrin, as well as total peroxides, antioxidants, and other peripheral markers of inflammation in 47 patients with AD, 24 patients with vascular dementia (VaD), and 44 healthy controls. Biochemical variables were related to the patients’ and controls’ clinical status. Results: The authors found that copper (p < 0.001), peroxides (p = 0.026), and ceruloplasmin (p = 0.052) were increased and TRAP was decreased (p = 0.006) in patients with AD, while no other markers of inflammation were altered. The calculation of the ratio between copper and ceruloplasmin suggested the presence in the serum of AD patients, but not of VaD or normal controls, of a large pool of non-ceruloplasmin-bound copper. Conclusions: Changes in the distribution of the serum copper components, consisting of an increase of a copper fraction not explained by ceruloplasmin, seem to be characteristic of Alzheimer disease and may be implicated in the pathogenesis of the disease.

Effects of Oxidative Stress Caused by Hyperoxia and Diquat. A Study in Isolated Hepatocytes

The effects of oxidative stress caused by hyperoxia or administration of the redox active compound diquat were studied in isolated hepatocytes, and the relative contribution of lipid peroxidation, glutathione (GSH) depletion, and NADPH oxidation to the cytotoxicity of active oxygen species was investigated. The redox cycling of diquat occurred primarily in the microsomal fraction since diquat was found not to penetrate into the mitochondria. Depletion of intracellular GSH by pretreatment of the animals with diethyl maleate promoted lipid peroxidation and sensitized the cells to oxidative stress. Diquat toxicity was also greatly enhanced when glutathione reductase was inhibited by pretreatment of the cells with 1,3-bis(2-chloroethyl)-1-nitrosourea. Despite extensive lipid peroxidation, loss of cell viability was not observed, with either hyperoxia or diquat, until the GSH level had fallen below approximately 6 nmol/10(6) cells. The iron chelator desferrioxamine provided complete protection against both diquat-induced lipid peroxidation and loss of cell viability. In contrast, the antioxidant alpha-tocopherol inhibited lipid peroxidation but provided only partial protection from toxicity. The hydroxyl radical scavenger alpha-keto-gamma-methiol butyric acid, finally, also provided partial protection against diquat toxicity but had no effect on lipid peroxidation. The results indicate that there is a critical GSH level above which cell death due to oxidative stress is not observed. As long as the glutathione peroxidase - glutathione reductase system is unaffected, even relatively low amounts of GSH can protect the cells by supporting glutathione peroxidase-mediated metabolism of H2O2 and lipid hydroperoxides.

Features of ceruloplasmin in the cerebrospinal fluid of Alzheimer’s disease patients

The level of the apo-form of the copper enzyme ceruloplasmin (CP) is an established peripheral marker in diseases associated with copper imbalance. In view of the proposal that disturbances of copper homeostasis may contribute to neurodegeneration associated with Alzheimer’s disease (AD), the present work investigates, by Western blot and non-reducing SDS-PAGE followed by activity staining, the features of CP protein, and the copper/CP relationship in cerebrospinal fluid (CSF) and serum of AD patients. Results show that only a fraction of total copper is associated with CP in the CSF, at variance with serum, both in affected and in healthy individuals. Furthermore, a conspicuous amount of apo-ceruloplasmin and a decrease of CP oxidase activity characterize the CSF of the affected individuals, and confirm that an impairment of copper metabolism occurs in their central nervous system. In the CSF of AD patients the decrease of active CP, associated with the increase in the pool of copper not sequestered by this protein, may play a role in the neurodegenerative process.

Neuronal vulnerability following inhibition of mitochondrial complex II: a possible ionic mechanism for Huntington's disease

An impaired complex II (succinate dehydrogenase, SD) striatal mitochondrial activity is one of the prominent metabolic alterations in Huntingtons disease (HD), and intoxication with 3-nitropropionic acid (3-NP), an inhibitor of mitochondrial complex II, mimics the motor abnormalities and the pathology of HD. We found that striatal spiny neurons responded to this toxin with an irreversible membrane depolarization/inward current, while cholinergic interneurons showed a hyperpolarization/outward current. Both these currents were sensitive to intracellular concentration of ATP. The 3-NP-induced depolarization was associated with an increased release of endogenous GABA, while acetylcholine levels were reduced. Moreover, 3-NP induced a higher depolarization in presymptomatic R6/2 HD transgenic mice compared to wild-type (WT) mice, showing an increased susceptibility to SD inhibition. Conversely, the hyperpolarization did not significantly differ from the one recorded in WT mice. The diverse membrane changes induced by SD inhibition may contribute to the cell-type-specific neuronal death in HD.

Decrease of cytochrome c oxidase protein in heart mitochondria of copper-deficient rats

Copper deficiency has been reported to be associated withdecreased cytochrome c oxidase activity, whichin turn may be responsible for theobserved mitochondrial impairment and cardiac failure. We isolatedmito-chondriafrom hearts of copper-deficient rats: cytochrome c oxidase activity was found to be lowerthan incopper-adequate mitochondria. The residual activity paralleled coppercontent of mitochondria and also corresponded with the heme amount associated with cytochromeaa3. In fact, lower absorption in thea-band region of cytochrome aa3 was foundfor copper-deficient rat heart mitochondria. Gel electrophoresisof protein extractedfrom mitochondrial membranes allowed measurements of protein content of thecomplexes ofoxidative phosphorylation, revealing a lower content of complex IV protein incopper-deficientrat heart mitochondria. The alterations caused by copper deficiency appear to bespecific forcytochrome c oxidase. Changes were not observed for F 0 F 1 ATP synthase activity,for heme contents ofcytochrome c and b, and for protein contents of complexes I, III and V.The present study demonstrates that the alteration of cytochrome c oxidase activityobserved in copper deficiency is due to a diminishedcontent of assembled protein and that shortnessof copper impairs heme insertion into cytochrome c oxidase.

Quinone toxicity in hepatocytes: Studies on mitochondrial Ca2+ release induced by benzoquinone derivatives

Hepatocyte cytotoxicity caused by substituted benzoquinones was associated with increased cytosolic Ca2+ concentration. p-Benzoquinone-induced hepatotoxicity was enhanced when the hepatocytes were loaded with Ca2+ by preincubation with ATP. A similar order of potency of the substituted benzoquinones in releasing Ca2+ from isolated mitochondria and inducing hepatocyte cytotoxicity was found; in decreasing order, this was 2-Br-, unsubstituted-, 2-CH3-, 2,6-(CH3O)2-, 2,6-(CH3)2-, 2,5-(CH3)2-, 2,3,5-(CH3)3-, and 2,3,5,6-(CH3)4-benzoquinones (duroquinone). The cellular products of quinone metabolism, hydroquinones and glutathione conjugates, did not cause mitochondrial Ca2+ release. Benzoquinone-induced mitochondrial Ca2+ release was preceded by GSH conjugate formation and NAD(P)H oxidation but followed by mitochondrial swelling. With duroquinone, a slow GSH and NADPH oxidation preceded Ca2+ release, but GSH oxidation did not occur with Se-deficient mitochondria lacking glutathione peroxidase activity. Cyanide-insensitive respiration was also observed with duroquinone but not with benzoquinone, suggesting that duroquinone undergoes redox cycling. GSH was depleted by both arylation and oxidation with 2,6-(CH3O)2-, 2,6-(CH3)2-, 2,5(CH3)2-, and 2,3,5-(CH3)3-benzoquinones. Benzoquinone concentrations that totally depleted GSH did not cause Ca2+ release until intramitochondrial NAD(P)H was oxidized. Ca2+ release was also prevented when NAD(P)H generation was stimulated by the presence of isocitrate or 3-hydroxybutyrate. This suggests that mitochondrial Ca2+ release is associated with NAD(P)H oxidation catalyzed by NADH dehydrogenase with benzoquinone or by the glutathione peroxidase-glutathione reductase system with duroquinone.

Saturated dicarboxylic acids as products of unsaturated fatty acid oxidation.

Upon chemical, radiation-induced or enzymatic oxidation, cis-polyunsaturated fatty acids, i.e., C18:2(n-6), C18:3(n-3), C20:2(n-6), C20:3(n-6), C20:3(n-3), C20:4(n-6), C20:5(n-3), C22:2(n-3), C22:4(n-6), C22:6(n-3), were found to generate saturated short and medium-chain length dicarboxylic acids, which can be regarded as a distinctive feature of the particular double bonds positions in the polyunsaturated fatty acid molecule. Two different dicarboxylic acids, which were unambiguously quantified by GC-MS, were produced from a single fatty acid: one deriving from the oxidative splitting at the level of the first double bond in the molecule, the other being two-carbon-atoms lower homologous. Formation of dicarboxylic acids occurred also from triacylglycerols and phospholipids containing cis-polyunsaturated fatty acids. In this case, following oxidation, the diacids remained covalently bound to the starting molecule and transesterification was necessary for identification. Being extremely stable and easily detectable compounds, dicarboxylic acids may be considered potential markers of oxidative attack to both free and esterified unsaturated fatty acids.

Red blood cell copper, zinc superoxide dismutase activity is higher in Alzheimer's disease and is decreased by D-penicillamine

Copper, zinc superoxide dismutase (Cu, Zn SOD) activity was measured in red blood cells (RBC) of 32 patients affected by Alzheimers disease (AD), eight other AD patients treated with the copper-chelating agent D-penicillamine, 13 first-degree relatives and 22 controls. All AD patients enrolled in our study showed a higher level of Cu, Zn SOD activity early in the disease. No correlation between apolipoprotein E genotype and SOD activity was found in AD patients. D-penicillamine treatment of AD patients for 24 weeks lowered the enzyme activity even below the control value. These results support the hypothesis that a higher level of Cu, Zn SOD activity in RBC can be an early diagnostic peripheral marker of this disease and a sensor to monitor treatments with copper-chelating drugs.