A prospective study of diurnal cortisol and cognitive function in community-dwelling elderly people.

BACKGROUND: Elevated cortisol levels due to hypothalamic-pituitary-adrenal (HPA) axis stress response have been associated with cognitive impairment. However, the causal relationship between stress and subsequent cognitive impairment remains unclear, notably because of the small number of gender-stratified prospective studies. METHOD: Salivary cortisol secretion was evaluated in 197 non-depressed community-dwelling elderly people at three time points on the day of hospital attendance for a clinical examination and again on the following day at home, in a distinct environmental context. Cognitive performance was evaluated at baseline and at 2- and 4-year follow-up. RESULTS: Cross-sectional logistic analyses adjusted for age and education indicated that men with high morning cortisol at the hospital had higher risk of low cognitive performance in verbal fluency [odds ratio (OR) 3.0, p=0.05] and visuospatial performance (OR 5.1, p=0.03). Impairment in verbal fluency was observed in women with moderate high morning cortisol (OR 3.6, p=0.05) or moderate slow diurnal rhythm (OR 3.7, p=0.04). In longitudinal analyses, slow diurnal rhythm (flatter slope) was associated with decline over 4 years in visuospatial performance (OR 7.7, p=0.03) and visual memory (OR 4.1, p=0.03) in men, and in verbal fluency (OR 6.0, p=0.01) in women. High morning cortisol was associated with decline in visual memory in women (OR 5.1, p=0.06). CONCLUSIONS: HPA axis dysregulation seems to be associated with low cognitive performance in the elderly. Slower cortisol elimination rates could predict cognitive decline affecting principally non-verbal functioning in men and verbal functioning in women. The effects are independent of environmental context, apolipoprotein E (ApoE) genotype or psychopathology. Interventions blocking this pathway may provide new therapeutic options to prevent cognitive decline.

Prevalence of DSM-IV psychiatric disorder in the French elderly population.

BACKGROUND: France has high rates of psychotropic drug consumption and suicide in the elderly population, but it has not yet been possible to determine whether this is due to exceptionally high morbidity rates. AIMS: To describe the first longitudinal population study of psychiatric disorder undertaken in France, and to estimate current and lifetime prevalences and age of onset of psychiatric disorder. METHOD: A study group of 1873 non-institutionalised persons aged 65 years and over was randomly recruited from the Montpellier district electoral rolls. The Mini International Neuropsychiatric Interview was used to assess current and lifetime symptoms. Cases identified by the application of DSM-IV criteria were re-examined by a clinical panel. RESULTS: Forty-six per cent of the study population had experienced a mental disorder in their lifetime, and 3.7% had made a suicide attempt. Lifetime prevalence of major depression was 26.5% and 30% for anxiety disorders. Current prevalence rates were 14.2% for anxiety disorders, 10.7% for phobia, 3% for major depression and 1.7% for psychosis. CONCLUSIONS: Results show very high rates of lifetime but not current major depression. Rates of current phobia and suicidal ideation in the very elderly are also high compared with other studies. The rates reported are likely to be underestimates.

Electrotransfer of microsomal cytochrome P450 after isoelectric focusing (IEF blots): resolution of human 2A and 3A isozymes.

In this article a procedure is described which allows us to achieve electroblots of membrane-associated proteins after isoelectric focusing. Proteins are resolved in vertical acrylamide slab gels containing ampholines, urea, and nonionic detergents (dodecyl maltoside and Tergitol NP10). After migration the focusing gel is reinforced by a carrier gel (10% acrylamide) polymerized on only one face of the slab. Electroelution of the proteins toward a hydrophobic membrane (Immobilon P) is obtained by adding denaturing agents to the carrier gel (sodium dodecyl sulfate and mercaptoethanol) and by increasing the methanol concentration (60%) in the transfer buffer. This method is applied to analyzing P450 2A and 3A cytochromes from human liver microsomes.

Purification of two cytochrome P450 isozymes related to CYP2A and CYP3A gene families from monkey (baboon, Papio papio) liver microsomes. Cross reactivity with human forms.

Two cytochrome P450 isozymes, FA and FI, were isolated and characterized from liver microsomes of phenobarbital-induced baboons (Papio papio). The cytochrome FA possesses the same N-terminal amino acid sequence as P450 MK2 from crab-eating monkeys (Macaca irus) and closely resembles the human P450 3A isozymes. This cytochrome was able to oxidize nifedipine and hydroxylate testosterone at the 6 beta position. The second baboon cytochrome (FI) is closely related to the P450 2A subfamily and has the same N-terminal sequence as human P450 2A7. Like human P450 2A forms, it is highly active as a coumarin 7-hydroxylase. Antibodies against P450 FA and FI cross-react with two human liver proteins of 51 kDa and 49 kDa, respectively. The concentration of the first protein in the human samples, was five-times greater than the second. However, the latter showed marked interindividual variation. In primary cultures of human hepatocytes, rifampicin is a strong inducer of the 51-kDa protein and a moderate inducer of the 49-kDa protein, while phenobarbital has the opposite effect on the two proteins.

Isolation and characterization of a cytochrome P450 of the IIA subfamily from human liver microsomes.

Antibodies raised against cytochrome P450, which is overexpressed in mouse hepatic tumors, (P450tu) crossreact with two human liver microsomal proteins (49 kDa and 52 kDa). We have quantified these proteins in 60 human liver samples and found great interindividual variability in both of them. The concentration of the 49-kDa protein varies up to 144 fold in the various samples and represents typically 10% of the total mincrosomal P450 content. Its immunologically determined concentration correlates well (R = 0.78) with the microsomal coumarin-7-hydroylase (COH) activity. This activity is strongly and completely inhibited by anti-P450tu antibody (IC50 = 0.13 mg IgG/mg microsomal protein). The crossreacting 49-kDa protein shows an unusually high substrate specificity towards coumarin; it presents all human COH and part of 7-ethoxycoumarin O-deethylase (ECOD). Besides these two activities, we did not find any activity with other typical P450 substrates. In primary cultures of human hepatocytes, it is inducible by phenobarbital and dexamethasone, but not by pyrazole and beta-naphthoflavone. We isolated this protein from human liver microsomes and purified it to homogeneity by a combination of aminooctyl-amino-Sepharose chromatography and immunoaffinity chromatography. The protein was identified as a cytochrome P450 of the IIA subfamily. Its N-terminal amino-acid sequence was identical with the first 20 residues deduced from the nucleotide sequence of P450IIA6.

Identification of the rabbit and human cytochromes P-450IIIA as the major enzymes involved in the N-demethylation of diltiazem.

Oxidative metabolism of diltiazem (DTZ), a calcium channel blocker, was investigated in rabbit and human liver microsomes as well as in primary cultures of human hepatocytes. DTZ N-demethylation, the major metabolic pathway in man, was strongly increased by treatment of animals, patients, and hepatocyte cultures with rifampicin and other inducers of the P-450IIIA subfamily. In a reconstituted system with purified forms of P-450 and NADPH cytochrome P-450 reductase, P-450IIIA7 exhibited the highest DTZ N-demethylase activity. In both rabbit and human liver microsomes, this activity was highly correlated with erythromycin demethylase, a characteristic substrate of P-450IIIA, or with an immunoquantitated level of P-450IIIA, and was specifically inhibited by anti-P-450IIIA7 polyclonal and monoclonal antibodies. Cyclosporin A, another specific substrate of P-450IIIA in rabbit and human, competitively inhibited DTZ N-demethylase in both species. In primary cultures of human hepatocytes treated with various inducers, including rifampicin, dexamethasone, phenobarbital, phenylbutazone or beta-naphthoflavone, the rate of release of N-demethyl-DTZ in the extracellular medium was highly correlated with the intracellular level of P-450IIIA, which appeared to be strongly induced by rifampicin and phenobarbital and to a lesser extent by dexamethasone and phenylbutazone. In aggregate, these results are consistent with the view that in both rabbit and human, cytochromes P-450 from the P-450IIIA subfamily are the major enzymes involved in the N-demethylation of DTZ. Accordingly, drugs which may be specific substrates or inducers of this P-450 are likely to influence both the side effects and the efficacy of this molecule.

Triacetyloleandomycin as inducer of cytochrome P-450 LM3c from rabbit liver microsomes.

A cytochrome P-450 LM3 isozyme has been isolated and purified to electrophoretic homogeneity from liver microsomes of New Zealand white rabbits treated with TAO. On the basis of N-terminal sequence analysis and Ouchterlony double diffusion experiments, this isozyme appeared to be closely related to P-450 LM3c isolated from control animals and was designated LM3c (TAO). Anti LM3c (TAO) IgG totally inhibited both erythromycin demethylase and P-450-TAO metabolite complex formation, two monooxygenase activities specifically stimulated by TAO in liver microsomes from male and female rabbits. Moreover, immunoquantitation experiments showed that the level of LM3c (TAO) was increased 10-15 times above control values in liver microsomes from TAO treated male and female rabbits. We conclude that an isozyme identical or closely related to LM3c is the major form of P-450 induced by TAO in rabbit liver microsomes.

Cytochrome P-450 isozyme LM3b from rabbit liver microsomes. Induction by triacetyloleandomycin purification and characterization.

Oral administration of triacetyloleandomycin (TAO), 1 mmol/kg/day for 7 days to mature male New Zealand White rabbit results in a significant increase in the content of liver microsomal cytochrome P-450. This increase is accompanied by the occurrence on sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the microsomes of a strong band in the zone of electrophoretic mobility associated with the LM3 isozymes and the stimulation of a number of monooxygenase activities of these microsomes including aminopyrine, chlorcyclizine, TAO, and erythromycin demethylation as well as 2-OH-estradiol and 6 beta OH-testosterone hydroxylation. Cytochrome P-450 LM3 (TAO) from these liver microsomes, purified to electrophoretic homogeneity, had Mr = 52,000 as determined by calibrated sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Comparison with isozymes LM3a, LM3b, and LM3c isolated from control animals, by a number of criteria including spectral data, amino acid content, NH2-terminal sequence analysis, peptide mapping, immunological properties, and monooxygenase activities of reconstituted system, indicated that isozymes LM3 (TAO) and LM3b are very similar, if not identical, proteins. We conclude that TAO must be considered as a new type of inducer of microsomal cytochrome P-450 from rabbit liver.

Polyamines as modulators of drug oxidation reactions catalyzed by cytochrome P-450 from liver microsomes.

The effect of polyamines on the activity of the mixed-function oxidase (MFO) system from human, rat and rabbit liver microsomes was investigated in detail. It was shown that polyamine (spermine) stimulates NADPH-dependent activity of the MFO system several-fold whatever the substrate (foreign drug or natural), not only with microsomes but also with the reconstituted system consisting of highly purified cytochrome P-450 (LM2 isozyme), cytochrome P-450 NADPH reductase and dilauroylphosphorylcholine. Stimulation (extent and concentration dependence) appeared to be dependent on a number of parameters such as ionic strength, pH, animal species and treatment, nature of the substrate, and was stereospecific (different effect on 6 beta-and 16 alpha-testosterone hydroxylation). Further, the spermine effect was evaluated on some elementary steps of the cytochrome P-450 reaction cycle, like substrate binding, P-450 reduction and second electron transfer. Finally, it was shown that the organic peroxide dependent activity was not stimulated by spermine with microsomes nor with the purified P-450 LM2 isozyme. On the basis of this study, it was concluded that the locus of polyamine action is cytochrome P-450 and that stimulation could result either from increased stability of the oxyferrous intermediate of P-450 or from an increased rate of second electron transfer from reductase to P-450.

Inulinase activity of Debaromyces cantarellii.

Debaromyces cantarellii Capriotti contains an inulinase activity which is inducible by growth on inulin but not on other beta-fructosides. The induction is inhibited by glucose and fructose. The system is situated in the cell wall and can be best extracted with a 20 mM phosphate buffer at pH 8.5. The inulinase activity shows pH optima at 4 and 6, suggesting the presence of two enzymes, the latter being more tightly bound to the cell wall. Both enzymes degrade inulin from the nonreducin end. The cells also contain a constitutive beta-fructofuranosidase with a specificity partly overlapping with that of the inulinase(s).