Dealkylation and loss of capacity for reactivation of cholinesterase inhibited by sarin.

Inhibition of rat brain acetylcholinesterase by (32)P-sarin in vivo results initially in (32)P-isopropylmethylphosphonylated enzyme. The percentage of inhibited enzyme that could not be reactivated by pyridinium aldoxime methochloride (aged enzyme) approximated the amount of radioactivity identified as (32)P-methylphosphonate. The (32)P-isopropyl methylphosphonate not released fromthe inhibited enzyme by the oxime accounted for 51 +/- 10 percent ( standard deviation) of the radioactivity fixed to brain tissue. It showed no correlation with aging and was probably bound to sites other than acetylcholinesterase.

Urinary excretion of beta-aminopropionitrile and cyanoacetic acid.

Human serum did not convert beta-aminopropionitrile (BAPN) to the deaminated, nonlathyrogenic metabolite, cyanoacetic acid (CAA). Instead, its enzymic activity for oxidizing benzylamine was inhibited by BAPN (I50 = 2 X 10(-3) M). BAPN was found in the urine within one hour of oral administration. Oral 250 mg BAPN at 6-hr intervals each day for 21 days resulted in urinary BAPN recoveries approximating 16% of the total dose. BAPN was not detected in urine in specimens collected later than 7 hr after cessation of BAPN dosage. Urinary CAA appeared more slowly than BAPN and increased gradually to approximately three times that of urinary BAPN. After BAPN was discontinued, there was prolonged urinary excretion of BAPN-derived CAA. These along with earlier findings in experimental animals suggest that unexcreted BAPN is sequestered in tissues where it is metabolized to CAA before slowly released.

Immunochemical properties of malondialdehyde-protein adducts.

Malondialdehyde (MDA), a product of lipid peroxidation, can bind to and modify proteins by adduct formation. To determine whether MDA adducts were immunogenic, MDA was added to rabbit serum albumin (RSA) in order to characterize MDA-proteins and to immunize rabbits. Bound MDA was proportional to the concentration of MDA added in the range of 2.5-20 mM as measured by thiobarbituric acid reactivity. MDA adducts of RSA migrated further toward the anode than native serum protein in zone and immunoelectrophoresis indicating increased negative charge. Rabbits immunized with MDA-RSA produced high titers of IgG antibodies to MDA-RSA as measured by enzyme-linked immunosorbent assay (ELISA). Hapten specificity of the antibody was demonstrated by antisera reactivity with MDA-RSA but not with unaltered RSA. Our findings support the possibility that MDA may serve as a hapten to form neoantigens which may represent a pathway by which lipid peroxidation could produce tissue damage via an immunologic mechanism.

Estimation of urinary diamines and polyamines by thin-layer chromatography.

The dansylated derivatives of ammonia, 1,3-diaminopropane, 1,4-diaminobutane, 1,5-diaminopentane, spermidine, histamine, and spermine were separated by one ascending development in chloroform-triethylamine (5:1) on a 250-mum silica gel 60 plate. Putrescine, cadaverine, spermidine, and spermine in human urine were quantitated by a direct scan of the fluorescent intensity of the spots corresponding to these compounds. Higher amounts of spermidine and spermine were found in the urines of cancer patients compared to the values of these substances in normal urine.

Acetaldehyde-albumin adduct formation: possible relevance to an immunologic mechanism in alcoholism.

Acetaldehyde (A), an ethanol metabolite, was incubated with rabbit serum albumin (RSA) and human serum albumin (HSA) to form the corresponding soluble haptenized proteins, A-RSA and A-HSA respectively. Both protein adducts showed conjugation with acetaldehyde evidenced by more rapid anodal migration compared to RSA and HSA. A-RSA immunized rabbits produced titers greater than 1:100,000 by enzyme-linked immunosorbent assay (ELISA). Rabbit serum antibodies to A-RSA diluted 1:10,000 showed high specificity for the hapten when reacted with acetaldehyde conjugates of RSA, HSA, bovine serum albumin, bovine gamma globulin and human gamma globulin. Our findings support the possibility that acetaldehyde may serve as a hapten to form neoantigens relevant to an immunologic mechanism which might be associated with alcoholic liver disease.