2-4-dinitrophenylhydrazine and Alzheimer-Disease

The development of diagnostic markers for earlier and more reliable diagnosis of Alzheimer disease (AD) is essential, particularly because therapeutic medication is available for AD. Cerebrospinal fluid (CSF) is a useful source of diagnostic information. Previously we found the increase of total tau protein in CSF in normal pressure hydrocephalus patients, and others reported the increase also in corticobasal degeneration and frontotemporal dementia. To differentiate the AD from other diseases, further approach was employed and oxidized protein in CSF was investigated. Heat stable fractions of CSF were analyzed on the content of carbonyl residues, which are derivatives of protein oxidization. The result suggests that protein oxidation is highly involved in AD and that this method might be useful to differentiate AD from other neurological disease.

Topics: Alzheimer Disease; Amyloid beta-Peptides; Biomarkers; Humans; Nerve Tissue Proteins; Oxidative Stress; Phenylhydrazines; tau Proteins

We review an array of newly developed in situ detection methods that can be used for the qualitative and semi-quantitative measurement of various indices related to oxidative stress. The importance of in situ methods over bulk analysis cannot be overstated when considering the structural and cellular complexity of tissue and the effects of diseases thereof. Indeed, in situ detection allows detection of specific cell types affected or specific localization such that a process affecting only a small fraction of the tissue or cells can be readily visualized. Consequently, a positive signal in situ indicates real levels that cannot be masked by unrelated or compensatory responses in adjacent cells, and corrections can be easily made for the modifications to long-lived proteins during physiological aging. In fact, the damage to extracellular matrix proteins of major vessels, provides a cumulative record of long-term oxidative insult. Yet the same properties that make vessels ideal markers for aging limits their sensitivity to detect disease-specific changes unless in situ techniques are used.

Topics: Alzheimer Disease; Biomarkers; DNA Damage; Glycation End Products, Advanced; Histocytochemistry; Humans; Immunohistochemistry; Metals; Oxidation-Reduction; Oxidative Stress; Phenylhydrazines; Tyrosine

Diverse oxidative pathways, such as direct oxidation of amino acids, glycoxidation, and lipoxidation could contribute to Alzheimer disease pathogenesis. A global survey for the amount of structurally characterized probes for these reactions is lacking and could overcome the lack of specificity derived from measurement of 2,4-dinitrophenylhydrazine reactive carbonyls. Consequently we analyzed (i) the presence and concentrations of glutamic and aminoadipic semialdehydes, N(epsilon)-(carboxymethyl)-lysine, N(epsilon)-(carboxyethyl)-lysine, and N(epsilon)-(malondialdehyde)-lysine by means of gas chromatography/mass spectrometry, (ii) the biological response through expression of the receptor for advanced glycation end products, (iii) the fatty acid composition in brain samples from Alzheimer disease patients and age-matched controls, and (iv) the targets of N(epsilon)-(malondialdehyde)-lysine formation in brain cortex by proteomic techniques. Alzheimer disease was associated with significant, although heterogeneous, increases in the concentrations of all evaluated markers. Alzheimer disease samples presented increases in expression of the receptor for advanced glycation end products with high molecular heterogeneity. Samples from Alzheimer disease patients also showed content of docosahexaenoic acid, which increased lipid peroxidizability. In accordance, N(epsilon)-(malondialdehyde)-lysine formation targeted important proteins for both glial and neuronal homeostasis such as neurofilament L, alpha-tubulin, glial fibrillary acidic protein, ubiquinol-cytochrome c reductase complex protein I, and the beta chain of ATP synthase. These data support an important role for lipid peroxidation-derived protein modifications in Alzheimer disease pathogenesis.

Topics: Aged; Aged, 80 and over; Alzheimer Disease; Blotting, Western; Brain; Carbon; Cerebral Cortex; Databases as Topic; Electrophoresis, Gel, Two-Dimensional; Electrophoresis, Polyacrylamide Gel; Fatty Acids; Fatty Acids, Unsaturated; Female; Glial Fibrillary Acidic Protein; Glycation End Products, Advanced; Glycolysis; Humans; Lipid Metabolism; Lipid Peroxidation; Lysine; Male; Malondialdehyde; NADH Dehydrogenase; Oxygen; Phenylhydrazines; Proteins; Silver Staining; Ubiquinone

A proteomic approach was employed to elucidate possible differential expression of native and oxidized glycoproteins using pooled plasma samples derived from ten patients with sporadic Alzheimer's disease (AD) and pooled plasma samples from nine normal elderly control (NEC) subjects. The plasma samples were fractionated by sequential affinity chromatography on heparin-agarose (HepA) and concanavalin A-agarose (ConA) columns followed by separation on one-dimensional and two-dimensional polyacrylamide gels. Carbonylation (oxidation) of proteins was monitored by in-strip derivatization with 2,4-dinitrophenylhydrazine (DNP) and anti-DNP immunoblotting. Nine spots representing glycoproteins which showed enrichment or high specific oxidation indices in AD HepA-ConA 2-D gels relative to NEC samples were analyzed by matrix-assisted laser desorption-time of flight-mass spectrometry and identified with high probability (p < 0.001) as isoforms of human transferrin (Tf), hemopexin (Hpx) and alpha-1-antitrypsin (alpha-1-AT). These glycoproteins were concentrated, respectively, 5-, 6.5- and 107-fold in HepA-ConA eluates derived from AD plasma relative to the NEC samples. Specific oxidation indices of the identified Tf and Hpx isoforms in AD plasma were respectively, 7.4 and 2.8 relative to NEC. Our findings provide further evidence for systemic derangements in heme/iron/redox homeostasis and activation of the acute phase response in sporadic AD. Moreover, the data implicate isoforms of Tf, Hpx and alpha-1-AT as potential biological markers of this condition.

Topics: alpha 1-Antitrypsin; Alzheimer Disease; Chromatography, Affinity; Concanavalin A; Electrophoresis, Gel, Two-Dimensional; Glycoproteins; Hemopexin; Heparin; Humans; Oxidation-Reduction; Phenylhydrazines; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; Transferrin

The levels of oxidatively modified proteins were examined in blood from Alzheimer's disease (AD) patients, non-AD controls, and AD relatives. Oxidative modification was measured by reacting the protein carbonyls with 2,4-dinitrophenyl hydrazine (DNPH). The total oxidized proteins were determined by HPLC, while specific protein oxidation was assessed from Western blots of electrophoretic gels using antibody to the DNP derivatives. Statistically significant elevations (P < 0.05) of total oxidized proteins were observed in both AD subjects and AD relatives when compared with non-AD controls. Moreover, a protein band (e.g., MW = 78-kDa) was uniquely oxidized in the plasma of AD subjects. Furthermore, this protein from AD subjects was more susceptible to in vitro oxidation. These data suggest that such oxidized proteins may be useful as biomarkers for the detection and evaluation of AD.

Topics: Aged; Aged, 80 and over; Alzheimer Disease; Blood Proteins; Humans; Middle Aged; Oxidation-Reduction; Phenylhydrazines

Topics: Aldehydes; Alzheimer Disease; Analysis of Variance; Biomarkers; DNA Damage; Glycation End Products, Advanced; Heme Oxygenase (Decyclizing); Heme Oxygenase-1; Histocytochemistry; Humans; Iron; Membrane Proteins; Neurodegenerative Diseases; Neurofibrillary Tangles; Neurons; Nitrates; Oxidation-Reduction; Oxidative Stress; Oxygen; Phenylhydrazines; Plaque, Amyloid; Tyrosine

Formation of carbonyls derived from lipids, proteins, carbohydrates, and nucleic acids is common during oxidative stress. For example, metal-catalyzed, "site-specific" oxidation of several amino acid side-chains produces aldehydes or ketones, and peroxidation of lipids generates reactive aldehydes such as malondialdehyde and hydroxynonenal. Here, using in situ 2,4-dinitrophenylhydrazine labeling linked to an antibody system, we describe a highly sensitive and specific cytochemical technique to specifically localize biomacromolecule-bound carbonyl reactivity. When this technique was applied to tissues from cases of Alzheimer disease, in which oxidative events including lipoperoxidative, glycoxidative, and other oxidative protein modifications have been reported, we detected free carbonyls not only in the disease-related intraneuronal lesions but also in other neurons. In marked contrast, free carbonyls were not found in neurons or glia in age-matched control cases. Importantly, this assay was highly specific for detecting disease-related oxidative damage because the site of oxidative damage can be assessed in the midst of concurrent age-related increases in free carbonyls in vascular basement membrane that would contaminate biochemical samples subjected to bulk analysis. These findings demonstrate that oxidative imbalance and stress are key elements in the pathogenesis of Alzheimer disease.

Topics: Adult; Aged; Aged, 80 and over; Alzheimer Disease; Extracellular Matrix; Hippocampus; Humans; Immunohistochemistry; Middle Aged; Neurons; Oxidative Stress; Phenylhydrazines; Sensitivity and Specificity

Topics: Adult; Aged; Aged, 80 and over; Alzheimer Disease; Case-Control Studies; Hippocampus; Humans; Immunoenzyme Techniques; Middle Aged; Oxidation-Reduction; Oxidative Stress; Phenylhydrazines

Apolipoprotein E, the most common apolipoprotein found in the brain, is linked to several pathologies like Alzheimer's disease. Apolipoprotein E directly binds to beta-amyloid with a strong affinity. Myeloperoxidase, a protein secreted by neutrophils and involved in the inflammatory process, is also present in the brain. In vitro myeloperoxidase oxidation of recombinant human apolipoprotein E leads to fragmentation of the protein with low concentrations of hydrogen peroxide and polymerization with higher concentrations. Comparison with bovine serum albumin shows a higher susceptibility of apolipoprotein E to myeloperoxidase oxidation, which may have importance in the Alzheimer's disease process.

Topics: Alzheimer Disease; Apolipoproteins E; Brain; Escherichia coli; Humans; Immunoblotting; Molecular Weight; Oxidation-Reduction; Peroxidase; Phenylhydrazines; Recombinant Proteins

Four biomarkers of neuronal protein oxidation [W/S ratio of MAL-6 spin-labeled synaptosomes, phenylhydrazine-reactive protein carbonyl content, glutamine synthetase (GS) activity, creatine kinase (CK) activity] in three brain regions [cerebellum, inferior parietal lobule (IPL), and hippocampus (HIP)] of Alzheimer's disease (AD)-demented and age-matched control subjects were assessed. These endpoints indicate that AD brain protein may be more oxidized than that of control subjects. The W/S ratios of AD hippocampal and inferior parietal synaptosomes are 30 and 46% lower, respectively, than corresponding values of tissue isolated from control brain; however, the difference between the W/S ratios of AD and control cerebellar synaptosomes is not significant. Protein carbonyl content is increased 42 and 37% in the Alzheimer's HIP and IPL regions, respectively, relative to AD cerebellum, whereas carbonyl content in control HIP and IPL is similar to that of control cerebellum. GS activity decreases an average of 27% in the AD brain; CK activity declines by 80%. The brain regional variation of these oxidation-sensitive biomarkers corresponds to established histopathological features of AD (senile plaque and neurofibrillary tangle densities) and is paralleled by an increase in immunoreactive microglia. These data indicate that senile plaque-dense regions of the AD brain may represent environments of elevated oxidative stress.

Topics: Aged; Alzheimer Disease; Biomarkers; Brain; Creatine Kinase; Female; Glutamate-Ammonia Ligase; Humans; Male; Membrane Proteins; Microglia; Nerve Tissue Proteins; Oxidation-Reduction; Phenylhydrazines; Spin Labels; Synaptosomes