LC- and CZE-ICP-MS approaches for the in vivo analysis of the anticancer drug candidate sodium trans-[tetrachloridobis(1H-indazole)ruthenate(III)] (KP1339) in mouse plasma.

Ruthenium-indazole complexes are promising anticancer agents undergoing clinical trials. KP1339 is administered intravenously (i.v.), where serum proteins are the first available biological binding partners. In order to gain a better insight into the mode of action, mice were treated with different doses of KP1339 i.v. and sacrificed at different time points. The blood plasma was isolated from blood samples and analyzed by capillary zone electrophoresis (CZE) and size exclusion/anion exchange chromatography (SEC-IC) both combined on-line to inductively coupled plasma-mass spectrometry (ICP-MS). The performance of the analytical methodology was compared and the interaction of KP1339 with mouse plasma proteins characterized in vivo. Interestingly, the samples of the mice treated with 50 mg kg(-1) and terminated after 24 h showed a ca. 4-fold lowered albumin content and increased ruthenation of albumin aggregates as compared to the untreated control group and the 40 mg kg(-1) group. The majority of Ru was bound to albumin and the stoichiometry of the KP1339 protein binding was determined through the molar Ru/S ratio. In general, good agreement of the data obtained with both techniques was achieved and the SEC-IC method was found to be more sensitive as compared to the CZE-ICP-MS approach, whereas the latter benefits from the shorter analysis time and lower sample consumption.

Age- and stage-dependent glyoxalase I expression and its activity in normal and Alzheimer's disease brains.

The reaction of lysine and arginine residues of proteins with 1,2-dicarbonyl compounds result in the formation of advanced glycation end products (AGEs). Accumulation of AGEs is a characteristic feature of the aging brain and contributes to the development of neurodegenerative diseases such as Alzheimer's disease (AD). Therefore, it is of particular interest to study the cellular defense mechanisms against AGE formation and particularly the detoxification of their precursors. AGE precursor compounds such as methylglyoxal and glyoxal were cellulary detoxified by the glyoxalase system, consisting of glyoxalases I and II. Glyoxalase I levels are diminished in old aged brains but elevated in AD brains. However, it is still unknown how glyoxalase I level of AD brains changes in a disease and in an age-dependent manner. Therefore, we investigated the AD stage- and the age-dependent levels of glyoxalase I in the Brodmann area 22 of AD brains (n=25) and healthy controls (n=10). Our results obtained from RT-PCR reveal reducing glyoxalase I RNA levels with advancing stage of AD and with increasing age. Western Blot analysis indicates that in comparison to healthy controls, glyoxalase I protein amounts are 1.5-fold increased in early AD subjects and continuously decrease in middle and late stages of AD. The glyoxalase I protein amounts of AD patients also decrease with age. Results obtained from glyoxalase I activity measurement show 1.05-1.2-fold diminished levels in AD brains compared to healthy controls and no significant decrease neither with the stage of AD nor with age. The immunohistochemical investigations demonstrate an elevated number of glyoxalase I stained neurons in brains of early and middle but not in late AD subjects compared to age-matched healthy controls. In addition, the stage-dependent immunohistochemical investigation demonstrates that with reduced glyoxalase I staining AGE deposits prevail, specifically in late stage of AD. In conclusion, the decrease of glyoxalase I expression with increasing AD stage might be one reason for methylglyoxal-induced neuronal impairment, apoptosis, and AGE formation in plaques and tangles.

Age-dependent changes of glyoxalase I expression in human brain.

Increased modification and crosslinking of proteins by advanced glycation end products (AGEs) is a characteristic feature of aging, and contributes to the formation of many of the lesions of neurodegenerative diseases including neurofibrillary tangles and amyloid plaques in Alzheimer's disease. Therefore, defense mechanisms against AGE formation or detoxification of their precursors such as the glyoxalase system are of particular interest in aging research. Thus, we investigated the age-dependent protein expression, the activity as well as the RNA level of glyoxalase I in Brodmann area 22 (auditory association area of superior temporal gyrus) of the human cerebral cortex. Our immunohistochemical results demonstrate the localization of glyoxalase I in neurons, predominantly pyramidal cells, as well as in astroglia, located predominantly in the subpial region. The number of glyoxalase I expressing neurons and astroglia increases with age, with a peak at approximately 55 years, and progressively decreases thereafter. These results were confirmed by biochemical investigations in total brain tissue, where the RNA, the protein level as well as the activity of glyoxalase I enzyme were analyzed in different age groups. In conclusion, the increase in glyoxalase I expression up to the age of 55 may be a compensatory mechanism against high oxoaldyde levels and the accumulation of AGEs. However, the decline of glyoxalase expression and activity in old age, possibly caused by impairment in transcription or/and translation, may subsequently lead to increased levels of reactive carbonyl compounds, followed by protein crosslinking, inflammation, oxidative stress and neuronal degeneration.

Methylglyoxal, glyoxal, and their detoxification in Alzheimer's disease.

The accumulation of advanced glycation end products (AGEs) in brains with Alzheimer's disease (AD) has been implicated in the formation of insoluble deposits such as amyloid plaques and neurofibrillary tangles. AGEs are also known to activate glia, resulting in inflammation and neuronal dysfunction. As reactive intermediates of AGE formation, neurotoxic reactive dicarbonyl compounds such as glyoxal and methylglyoxal have been identified. One of the most effective detoxification systems for methylglyoxal and glyoxal is the glutathione-dependent glyoxalase system, consisting of glyoxalase I and glyoxalase II. In this study, we have determined the methylglyoxal and glyoxal levels in the cerebrospinal fluid of AD patients compared to healthy controls. Methylglyoxal levels in AD patients were twofold higher than in controls, but this difference was not significant due to the large intergroup variations and the small sample size. However, the concentrations of both compounds were five to seven times higher in CSF than in plasma. We also investigated the glyoxalase I level in AD and healthy control brains. The number of glyoxalase I- positive neurons were increased in AD brains compared to controls. Our findings suggest that glyoxalase I is upregulated in AD in a compensatory manner to maintain physiological methylglyoxal and glyoxal levels.