HTHQ (1-O-hexyl-2,3,5-trimethylhydroquinone), an anti-lipid-peroxidative compound: its chemical and biochemical characterizations.

Recently, it has become apparent that reactive oxygen species (ROS) play many important roles in biological systems. For example, relationships between many diseases, such as cancer, cardiac infarction and arteriosclerosis, and ROS have been found. It is also well known that anti-oxidative agents scavenge ROS in biological systems, which in turn prevents ROS-related diseases. In our previous efforts to develop effective anti-oxidative compounds, we found that 1-O-hexyl-2,3,5-trimethylhydroquinone (HTHQ), which is a hydroquinone monoalkyl ether, is a potent anti-oxidative agent. Here, the scavenging activities of HTHQ against ROS, such as superoxide anion radicals, hydroxyl radicals, t-butyl peroxyl radicals and singlet oxygens, were examined by the ESR (electron spin resonance)-spin trapping method. Among ROS, HTHQ scavenged t-butyl peroxyl radicals most effectively (IC50=0.31+/-0.04 mM), showing approximately twice the activity of a well-known lipophilic anti-oxidant, D,L-alpha-tocopherol (IC50=0.67+/-0.06 mM), as measured by IC50 values defined as the 50% inhibition concentration of the generated ROS. In addition, a relatively stable ESR spectrum of free radicals due to HTHQ was observed during the reaction of HTHQ and t-butyl peroxyl radicals, indicating a direct reaction of HTHQ and t-butyl peroxyl radicals. The free radicals due to HTHQ were more stable than those derived from D,L-alpha-tocopherol under the same conditions examined. On the basis of these results, we evaluated anti-lipid-peroxidative activity of HTHQ in three systems involving micelles, liposomes and rat liver microsomes. HTHQ exhibited a similar anti-oxidative activity to that of D,L-alpha-tocopherol against lipid peroxidation in linolate micelles initiated by addition of Fe2+. On the other hand, HTHQ exhibited approximately 4.8-fold higher anti-lipid-peroxidation activity than that of D,L-alpha-tocopherol against the peroxidation in phosphatidylcholine liposomes initiated by addition of Fe2+. Furthermore, HTHQ scavenged the lipid peroxides at a rate approximately 150 times higher than that of D,L-alpha-tocopherol against Fe3+ -ADP-induced lipid peroxidation in rat liver microsomes, indicating that the anti-lipid-peroxidation activity of HTHQ might be substantially elevated in biological systems in comparison with that of D,L-alpha-tocopherol. Based on these results, we suggest that HTHQ reacts directly with peroxyl radicals, such as t-butyl peroxyl radicals and peroxides of linolate micelles, liposomes and microsomes, by scavenging them to form stable free radicals. The resulting free radicals are presumed to be reduced by several reducing mechanisms in biological systems similarly to those of D,L-alpha-tocopherol, and then the lipid-peroxidation reactions will be terminated. In conclusion, HTHQ was found to be a potent anti-lipid-peroxidative compound and its antioxidation activity to be extremely elevated in biological systems, such as that of liver microsomes via the generation of stable free radicals. We propose that HTHQ is a potent anti-oxidative agent for use in future treatments for lipid-peroxide relevant diseases.

A novel fluorescent probe diphenyl-1-pyrenylphosphine to follow lipid peroxidation in cell membranes.

Diphenyl-1-pyrenylphosphine (DPPP) was tested whether it could be used as a fluorescent probe to monitor lipid peroxidation in cell membranes. DPPP reacted with organic hydroperoxides and hydrogen peroxide stoichiometrically to give DPPP oxide (DPPP = O). DPPP incorporated into phosphatidylcholine liposomal membranes and polymorphonuclear leukocytes (PMNs) reacted with methyl linoleate hydroperoxide rapidly but not with hydrogen peroxide nor with tert-butyl hydroperoxide. This novel method revealed that lipid peroxidation proceeded within membranes of PMNs stimulated with phorbol 12-myristate 13-acetate, which is known to produce several kinds of free radicals. It was found that DPPP is a suitable fluorescent probe to monitor lipid peroxidation within cell membranes specifically.

Sulphydryl groups and their relation to the antioxidant enzymes of chelonian red blood cells.

Thiol groups of hemoglobin and blood glutathione are higher in Geochelone carbonaria than in Geochelone denticulata. Exposure of stripped hemolysate of both tortoises to terc-butyl hydroperoxide, resulted in a higher ferroheme oxidation of G. denticulata hemoglobin. In this example glutathione reductase and glutathione peroxidase, were not active due to the absence of GSH and NADPH, suggesting that the thiol groups of G. carbonaria hemoglobin act as antioxidant, similar to GSH. In the total hemolysate, however, where the antioxidant enzymes are active, both species showed similar levels of hemoglobin oxidation, suggesting that the protective effect of thiol groups of hemoglobin are less effective for heme protection. The activity of glutathione reductase and glutathione peroxidase was higher in erythrocytes of G. denticulata and the activity of catalase and superoxide dismutase was higher in erythrocytes of G. carbonaria.

Chloroperoxidase-catalyzed enantioselective oxidations in hydrophobic organic media.

Chloroperoxidase from Caldariomyces fumago, a peroxidase that performs P450-like chemistry, was immobilized via covalent attachment into polyurethane foam as well as conjugated with a surfactant or polymer via colyophilization. The resulting preparations catalyzed enantio- and regioselective oxidations in hydrophobic organic media with tert-butyl hydroperoxide as the oxidant. Dried PUR-foam immobilized CPO mediated the selective oxidation of indole to 2-oxindole (regioselectivity: 99%) in water-saturated isooctane or 1-octanol. Thioanisole was converted into the corresponding (R)-sulfoxide (ee > 99%) in isooctane medium. The complexes of CPO with sodium octadecylsulphate or ethyl cellulose mediated the oxidation of thioanisole in water-immiscible organic media with variable enantioselectivity due to radical side-reactions. In the presence of alpha-tocopherol, acting as radical scavenger, the (R)-sulfoxide was formed with ee > 90%. The effect of the water activity on the catalytic activity of the complexes was investigated. The CPO complexes likewise mediated the regioselective oxidation of indole into 2-oxindole in water-saturated isooctane or 1-octanol and its kinetics were investigated. The reaction suffered from substrate inhibition when carried out in isooctane.