Carotenoid oxidative degradation products inhibit Na+-K+-ATPase

This study investigates the biological significance of carotenoid oxidation products using inhibition of Na⁺-K⁺-ATPase activity as an index. β-Carotene was completely oxidized by hypochlorous acid and the oxidation products were analyzed by capillary gasliquid chromatography and high performance liquid chromatography. The Na⁺-K⁺-ATPase activity was assayed in the presence of these oxidized carotenoids and was rapidly and potently inhibited. This was demonstrated for a mixture of β-carotene oxidative breakdown products, β-Apo-10'-carotenal and retinal. Most of the β-carotene oxidation products were identified as aldehydic. The concentration of the oxidized carotenoid mixture that inhibited Na⁺-K⁺-ATPase activity by 50% (IC₅₀) was equivalent to 10μM non-degraded β-carotene, whereas the IC₅₀ for 4-hydroxy-2-nonenal, a major lipid peroxidation product, was 120 μM. Carotenoid oxidation products are more potent inhibitors of Na⁺-K⁺-ATPase than 4-hydroxy-2-nonenal. Enzyme activity was only partially restored with hydroxylamine and/or β-mercaptoethanol. Thus, in vitro binding of carotenoid oxidation products results in strong enzyme inhibition. These data indicate the potential toxicity of oxidative carotenoid metabolites and their activity on key enzyme regulators and signal modulators.     

Simultaneous detection of carotenoids and vitamin E in human plasma

A simplified method for analysis of the antioxidants carotenoids and vitamin E in human plasma is presented. The method is based on high-performance liquid chromatography with a single column, a flow-rate gradient, and detection at 450 and 290 nm with a diode array detector. It gives good separation of the vitamin E isomers and the major carotenoids in plasma, with a 25 min analysis time. It was found that hydrolysis of triglycerides and cholesterol esters is required to obtain good recovery of non-polar carotenoids such as lycopene, α-carotene and β-carotene. Two methods were used for hydrolysis of the non-polar lipids, saponification with ethanolic KOH and digestion with an enzyme mixture of lipase and cholesterol esterase. It was found that the enzymatic digestion gave the best recoveries, better than 94% for all of the antioxidants, and preserved several carotenoids. A plasma pool is used for day to day calibration of the method, which eliminates the need for stock solutions of carotenoids that are stable for only a month due to oxidative breakdown and their tendency to crystallize when stored at −20°C in organic solvents.     

Dose- and Wavelength-Dependent Oxidation of Crystallins by UV Light-Selective Recognition and Degradation by the 20S Proteasome

The lens of the human eye is a suitable model for age-related alterations at the molecular level. Age-related cataract formation is closely related to the accumulation of oxidatively altered proteins. In this study the influence of UV-A, UV-B, and UV-C irradiation on the proteolytic susceptibility of -, β_L-, and β_H-crystallins by the isolated 20S proteasome was investigated. The proteins were irradiated with 280, 300, and 350 nm monochromatic light. Changes of the physical properties of the crystallins were characterized by absorbance measurements at 280 nm, fluorescence spectra, and SDS-PAGE-electrophoresis. The proteolytic susceptibility of crystallins was maximal after irradiation at 280 nm and three- to fivefold lower at 300 nm. Irradiation at 350 nm was not able to initiate proteolysis, probably due to protein-aggregate formation of higher molecular weight, as shown by SDS-PAGE. The damage of crystallins by UV-C light might be a signal for its proteolytic degradation by the 20S proteasome, whereas UV-B and UV-A do not increase the proteolytic susceptibility to the same extent but promote the formation of crosslinked proteins. Therefore, irradiation with UV, which is not followed by an increase in the proteolytic susceptibility, is accompanied by the formation of crosslinked proteins. It was concluded, that also long UV-B and UV-A may be involved in age-related alterations of the human lens and cataract formation.     

Formation of 4-hydroxynonenal and further aldehydic mediators of inflammation during bromotrichlorornethane treatment of rat liver cells

Bromotrichloromethane (CBrCl(3)) treatment is a model for studies on molecular mechanisms of haloalkane toxicity with some advantages compared with CCl(4) treatment. The formation of 4-hydroxynonenal and similar aldehydic products of lipid peroxidation, which play a role as mediators of inflammatory processes, was clearly demonstrated in rat hepatocytes treated with CBrCl(3). It may be assumed that haloalkane toxicity is connected with the biological effects of those inflammation mediatory aldehydic compounds.