Sensitive fluorimetric quantitation of pyridinium and pentosidine crosslinks in biological samples in a single high-performance liquid chromatographic run.

A high-performance liquid chromatographic assay was developed for pyridinium crosslinks and pentosidine in mature collagen of a wide variety of connective tissue hydrolysates by a simple two-step isocratic assay using a reversed-phase column. The crosslinks (including the internal standard pyridoxine) were optimally detected by their native fluorescence by switching wavelengths of the detector during the assay. The method resulted in highly sensitive and accurate measurements, without need for precleaning of the samples: crosslink levels in 200 microm thin slices of the various zones of articular cartilage were easily quantified. The detection limit was as low as 0.4 pmol for the pyridinolines and 0.05 pmol for pentosidine. The intra-assay and inter-assay coefficients of variation were as low as 2% (pyridinolines) and 5% (pentosidine); calibration curves for all compounds were linear over a concentration range larger than two orders of magnitude. With our chromatographic system, the diglycosylated form of hydroxylysylpyridinoline in unhydrolyzed urine was separated as well.

8OHdG levels in brain do not indicate oxidative DNA damage in Alzheimer's disease.

Accumulation of oxidative DNA damage has been proposed to underlie aging and neurodegenerative diseases such as Alzheimer's Disease (AD). The DNA adduct 8-hydroxy-2'-deoxyguanosine (8OHdG) is considered a good indicator of oxidative DNA damage. To investigate whether this type of DNA damage is involved in AD etiology, 8OHdG levels were determined in postmortem human brain tissue of controls and AD patients (in frontal, occipital, and temporal cortex and in hippocampal tissue). Parametric studies in rat revealed no influences of postmortem delay, repeated freezing/thawing or storage time. In human brain, approximately two 8OHdG molecules were present per 10(5) 2'-deoxyguanosines. In AD patients and controls, 8OHdG-levels were not related to age, sex, or brain region. Also, no differences were found between controls and AD patients. It was concluded that 8OHdG in nuclear DNA, although present throughout the brain in fairly high amounts, does not accumulate with age, nor does it appear to be involved in AD. More detailed studies are required to extend this conclusion to other types of oxidative damage.

Cortisol production rate and the urinary excretion of 17-hydroxycorticosteroids, free cortisol, and 6 beta-hydroxycortisol in healthy elderly men and women.

BACKGROUND: Although many workers have tested adrenal function in the elderly, few have studied the effect of aging on cortisol production rate or urinary free cortisol or 6 beta-hydroxycortisol excretion, and none have published comparisons of these variables between old people of defined health status and young people. METHODS: We have measured cortisol production rate and the urinary excretion of free cortisol, 6 beta-hydroxycortisol, 17-hydroxycorticosteroids (Porter-Silber chromogens) and creatinine in elderly men and women screened by the SENIEUR protocol and in young men; 17-hydroxycorticosteroid and 6 beta-hydroxycortisol excretion were also measured in young women. The period of measurement was 24 h or, usually, 48 h. RESULTS: Only 6 beta-hydroxycortisol excretion was affected by aging; it was lower in the elderly men and women than in their younger counterparts. Urinary free cortisol excretion was lower in the elderly women than in the elderly men. There were no significant differences between groups in cortisol production rate or 17-hydroxycorticosteroid excretion. Excretion and (over the first 24 h) clearance of creatinine were lower in the old women than in the old men. The cortisol-related variables tended to be positively correlated with each other and with the relevant creatinine-related variables in the elderly subjects; over the first but not the second 24 h, most of the correlations were significant in the men and women combined. CONCLUSIONS: With the exception of 6 beta-hydroxycortisol, the data agree with measurements of plasma cortisol and the results of adrenal function tests in showing little change in hypothalamic-pituitary-adrenal function with aging in healthy people.

Sex and strain dependency of age-related changes in protein synthesis of isolated rat hepatocytes.

In previous studies, a decrease in protein synthesis by hepatocytes isolated from female WAG/Rij rats was observed in the first year of life, while an increase was seen in advanced age. In the present study, no change in protein synthesis in early age was found for hepatocytes isolated from male WAG/Rij and female BN/Bi rats, suggesting that the decline in protein synthesis is sex and strain dependent. In contrast, the increase in protein synthesis in advanced age could be demonstrated with hepatocytes from male WAG/Rij and female BN/Bi rats and therefore seems to be independent of strain and sex. The increase in protein synthesis in advanced age could be attributed to increased excretion of protein in the urine.