Cytochemical demonstration of oxidative damage in Alzheimer disease by immunochemical enhancement of the carbonyl reaction with 2,4-dinitrophenylhydrazine.
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.
Smith
M A
MA
Institute of Pathology, Case Western Reserve University, Cleveland, Ohio 44106, USA.
Sayre
L M
LM
Anderson
V E
VE
Harris
P L
PL
Beal
M F
MF
Kowall
N
N
Perry
G
G
D000328:Adult; D000368:Aged; D000369:Aged, 80 and over; D000544:Alzheimer Disease / Q000378:metabolism*; D005109:Extracellular Matrix / Q000737:chemistry; D006624:Hippocampus / Q000737:chemistry; D006801:Humans; D007150:Immunohistochemistry / Q000379:methods*; D008875:Middle Aged; D009474:Neurons / Q000737:chemistry; D018384:Oxidative Stress; D010659:Phenylhydrazines / Q000032:analysis / Q000737:chemistry*; D012680:Sensitivity and Specificity
D010659:Phenylhydrazines; C004787:2,4-dinitrophenylhydrazine