Search

The effects of N-acetyl-L-cysteine (L-NAC), N,N-diacetyl-L-cystine (oxidized form of L-NAC) and N-acetyl-D-cysteine on the intracellular glutathione (GSH) level and their toxicity were investigated in the human melanoma cell culture IGR1. L-NAC applied in 3 mM concentration for 24 hr decreased; when applied for 48 hr it did not alter the intracellular GSH level. Treatment with 1 mM L-NAC for 24 hr had no effect on cellular glutathione, whereas the same concentration applied for 48 hr resulted in an increase in the level of GSH. Both concentrations also induced cell injury as determined by protein assay and trypan blue staining. N,N-diacetyl-L-cystine (0.5 and 1.5 mM, 24 hr) induced a decrease in cellular glutathione content without any apparent cell toxicity. D-NAC (1 and 3 mM, 24 hr) did not influence the GSH level of the melanoma cells; however, it had toxic effects resulting in cell loss.

Thiols are of great importance for the regulation of many cellular functions including metabolism, transport and cell protection. In this study the usefulness of L-cysteine methyl and octyl esters, of N,S-diacetyl-L-cysteine methyl ester and glutathione isopropyl ester as cellular cysteine and GSH delivery systems was investigated in the human IGR 1 melanoma cell line. The L-cysteine methyl and octyl esters proved to be highly toxic to the cells. Treatment of the cultures with 1 mM N,S-diacetyl-L-cysteine methyl ester or 3 mM glutathione isopropyl ester for 24 h resulted in marked elevation of the cellular glutathione level without apparent or with slight cell loss, respectively. Thus the administration of the latter two compounds seems to be suitable for inducing GSH elevation in the cultured melanoma cells.

A method for the determination of free fatty acids in minute serum samples has been developed. The acids are esterified by extractive alkylation, using tetrabutylammonium as a counter ion and pentafluorobenzyl bromide as alkylating reagent. The derivatization of palmitic acid required a reaction time of 25 min. The excess of pentafluorobenzyl bromide is removed by coupling it with a phenoalkylamine and extraction of the product into an acidic aqueous phase. Quantitation is carried out by gas chromatography with electron capture detection. The precision achieved in the determination of 6.2 mug of palmitic acid in 50 mul of mouse serum was 6.1% (S.D.).

The effects of buthionine sulphoximine (BSO) treatment on cellular glutathione (GSH) content and on the cytotoxic action of menadione were investigated in cultured IGRI human melanoma cells. Addition of BSO (10(-8)-0.5 X 10(-3) M) to the cultures resulted in a dose- and time-dependent depletion of cellular GSH. BSO (10(-5) and 10(-6) M) did not influence cell multiplication up to 48 h, as determined by trypan blue staining. Menadione (3 X 10(-5) M) treatment decreased the cellular GSH concentration and also reduced cell number after a 24 h exposure. Its cytotoxicity was increased by BSO (10(-5), 10(-6) M), though the potentiating effect was moderate.

A gas chromatographic method for the determination of phenobarbital in saliva has been developed. Phenobarbital is converted into its bispentafluorobenzyl derivative by extractive alkylation at pH 9 with 0.1 M tetrabutylammonium ion as extracting agent and 0.1 M pentafluorobenzyl bromide as alkylating reagent in methylene chloride. A reaction time of 20 min is required. Quantitation is effected by electorn-capture detection in a gas chromatograph equipped with a pre-column venting system for removal of methylene chloride and pentafluorobenzyl bromide. This procedure allows the direct introduction of the reaction mixture into the gas chromatograph. A 60-ng amount of phenobarbital in 100 mul of human saliva can be determined with a precision of 1.9% (S.D.) and a recovery of 93%.