A rapid, sensitive and selective method for the assay of glutathione in cultured cells has been developed using ion-pair reversed phase rapid high performance liquid chromatography. The use of a 4 micron particle, 5 cm long column (Superspher 100 RP 18 end-capped) allowed complete analysis of glutathione within 3 min. A postcolumn derivatization reaction with o-phthalaldehyde and fluorometric detection made the assay fully selective with regard to other endogenous thiols and sensitive (the detection limit was 0.5 ng of glutathione injected). The linearity range was between 0.1 and 2.0 microgram/mL with good repeatability (relative standard deviation less than 5% for the lowest concentration quantitated). Recoveries of GSH from cultured cell samples were above 98%. The rapid analysis enabled the processing of a large number of samples in a short time (up to 20 per hour). The method was applied to the measurement of the intracellular glutathione amount in V79 fibroblasts along cell growth in culture.

Animals↗

PubMedDate not supplied

Repression of cytochrome P450 by cytokines: IL-1 beta counteracts clofibric acid induction of CYP4A in cultured fetal rat hepatocytes.

Interleukin-1 is known to repress a number of hepatic drug-metabolizing enzymes in rats and humans. The effect of interleukin-1 beta on lauric acid 12-hydroxylase (CYP4A family) was studied in cultured fetal rat hepatocytes after clofibric acid induction. Dexamethasone was used as an agent promoting differentiation and long-term maintenance of active hepatocytes. Dexamethasone and clofibric acid in combination allowed maximal (13.5-fold) induction of CYP4A1. Lauric acid 12-hydroxylase activity was found to increase with time in culture. Interleukin-1 beta adversely affected P4504A clofibric acid-induced activity, totally eliminating the effect of induction at doses exceeding 5 ng/ml. This repression/inhibition was dose-dependent. The mechanism by which interleukin-1 beta prevents the development of cytochrome P4504A activity is unclear.

Animals↗

PubMedDate not supplied

Inducibility of rat brain drug-metabolizing enzymes.

Cytochrome P450 from rat brain mitochondrial and microsomal fractions was found to be inducible by 3-methylcholanthrene, both in quantity of enzyme and in activity towards 7-ethoxyresorufin, which is a model substrate for the cytochrome P450 isoform specifically induced by 3-methylcholanthrene. Conversely, a phenobarbital treatment resulted in an induction of the microsomal cytochrome P450 only. On the other hand, the microsomal 1-naphthol-UDP-glucuronosyl transferase and epoxide hydrolase seemed to be non-inducible by 3-methylcholanthrene or by phenobarbital. The toxicological implications of these data are discussed.

Animals↗

PubMedDate not supplied

Drug metabolizing enzymes in the brain and cerebral microvessels.

Several families of brain parenchyma and microvessel endothelial cell enzymes can metabolize substrates of exogenous origin. This xenobiotic metabolism includes functionalization and conjugation reactions and results in detoxication, but also possibly in the formation of pharmacologically active or neurotoxic products. The brain is partially protected from chemical insults by the physical barrier formed by the cerebral microvasculature of endothelial cells, which prevents the influx of hydrophilic molecules. These cells provide also, as a result of their drug-metabolizing enzyme activities, a metabolic barrier against penetrating lipophilic substances. The involvement of these enzymatic activities in neurotoxic events, probably responsible for neuronal dysfunctioning and/or death, neurodegenerative diseases and normal aging, is discussed.

Animals↗

PubMedDate not supplied

Increased protein glycation in cerebrospinal fluid of Alzheimer's disease.

Accumulation of advanced glycation end products occurs in the brain with ageing and was proposed to be involved in pathogenesis of Alzheimer's disease. We studied changes in the level of an early glycation product, an Amadori product, in cerebrospinal fluid (CSF) in ageing and in late-onset Alzheimer's disease. The work was carried out on 99 consecutive patients. The concentration of Amadori product in CSF correlated with CSF glucose concentration but was not changed with age (n = 70). In contrast, level of CSF Amadori product was 1.7-fold higher in Alzheimer's disease patients (n = 29) as compared with non-demented age-matched control group (n = 20; P < 0.0005), although CSF glucose concentration was similar in both groups (4.1 +/- 1.3 vs. 3.8 +/- 0.6 mmol/liter, resp.). An increased accumulation of Amadori products was found in all major proteins of CSF of Alzheimer's disease including albumin, apolipoprotein E and transthyretin. We propose that the increased early glycation of CSF proteins in the Alzheimer's patients may stimulate the formation and the consequent deposition of advanced glycation end products as well as oxidative stress in the brain.

Adult↗