Stopped-flow investigation of antioxidant activity of tocopherols. Finding of new tocopherol derivatives having higher antioxidant activity than alpha-tocopherol

Second-order rate constants, kappa s, for H-atom abstraction by phenoxyl radicals from five tocopherol (vitamin E) derivatives have been measured spectrophotometrically at 25.0 degrees C by the stopped-flow method, as a model reaction of tocopherols with unstable free radicals (LOO., LO., and HO.) in biological systems. Three new tocopherol derivatives with a five-membered heterocyclic ring were found to be 1.9-2.1 times more active than the alpha-tocopherol which has the highest antioxidant activity among natural tocopherols. The proton hyperfine splittings for the five tocopheroxyl radicals derived from these tocopherols by the reaction with phenoxyl were also determined by ESR measurements.     

Stopped-flow investigation of the reaction between vitamin E radical and vitamin C in solution

Kinetic study of the reaction between vitamin E radical and vitamin C has been performed. The rates of reaction of vitamin C (ascorbic acid 1, 6-0-stearyl ascorbic acid 2, and 2,6-O-dipalmitoyl ascorbic acid 3) with vitamin E radical (5,7-diisopropyl-tocopheroxyl) in benzene-ethanol (2:1, v/v) solution have been determined spectrophotometrically, using stopped-flow technique. The second-order rate constants obtained are 549 +/- 30 M-1s-1 for 1, 626 +/- 53 M-1s-1 for 2, and 4.84 +/- 1.41 M-1s-1 for 3 at 25.0 degrees C. The result shows that the ascorbic acid ester 2 having a long-alkyl-chain at 6-position is 1.14 times as reactive as the ascorbic acid 1, whereas the ascorbic acid ester 3 substituted at 2-position is only 0.01 times as reactive as the ascorbic acid 1.     

In vitro pro- and antioxidant properties of estrogens

The pro- and antioxidant properties of estrogens are subject of debate. The apparent discrepancy is largely caused by the chemical heterogeneity in the estrogen family and by their concentration and the environment in which they are found. To gain some insight into this debate, we determined whether estradiol (E(2)), estrone (E(1)), the 2-, 4- and 16alpha-hydroxyestrogens and also the 2- and 4-methoxyestrogens are: (1) good electron-donors; (2) capable of O(2) consumption and DNA strand break induction; (3) capable of inhibiting lipid peroxidation in vitro. E(2), E(1) and 16alpha-hydroxyestrone (16alpha-OHE(1)) were not pro-oxidants and were rather weak antioxidants, while the 2- and 4-hydroxyestrogens demonstrated both properties inducing DNA strand breaks damage as well as inhibiting lipid peroxidation. The 4-hydroxyestrogens consumed O(2) and induced DNA strand breaks to a level approximately 2.5-fold higher than the 2-hydroxyestrogens, but these hydroxyestrogens exhibited similar antioxidant capacity, as measured by inhibition of lipid peroxidation. The 4-methoxyestrogens cannot induce oxidative damage to DNA but can inhibit lipid peroxidation, although being less potent than the 2-methoxyestrogens and the 2- and 4-hydroxyestrogens. The 2-methoxyestrogens were both potent electron donors and inhibitors of lipid peroxidation. Although 2-methoxyestrogens cannot generate superoxide in vitro, they may also be considered pro-oxidants in vivo.     

The antioxidant activity of ubiquinol-3 in homogeneous solution and in liposomes.

With a view to determining the antioxidant effectiveness of ubiquinol, the autoxidation of egg phosphatidylcholine initiated by an azocompound was studied both in homogeneous solution and in liposomes, either in the presence or in the absence of ubiquinol-3. The results show that ubiquinol behaves as a chain-breaking antioxidant by trapping lipid peroxyl radicals, its inhibition rate constant being about one half of that of alpha-tocopherol in both systems under investigation. In organic solvents the stoichiometric factor was found approx. 2 and in liposomes approx. 0.5, i.e. one fourth of that of alpha-tocopherol. We suggest that the lower value found in model membranes is due to autoxidation of the quinol itself by a radical chain reaction taking place at the polar interface. Ubiquinol-3 exhibits a sparing effect toward alpha-tocopherol, both in liposomes and in tert-butanol. It is suggested, on a thermodynamic basis, that the regeneration of vitamin E from the corresponding radical is more likely to occur by reaction with the ubisemiquinone rather than with the ubiquinol. Although these results, obtained in in vitro systems, can not be directly extrapolated to an in vivo system, they may be useful to clarify the antioxidant role of ubiquinol in biomembranes.     

Stopped-flow and steady-state study of the diphenolase activity of mushroom tyrosinase

The reaction of mushroom (Agaricus bisporus) tyrosinase with dioxygen in the presence of several o-diphenolic substrates has been studied by steady-state and transient-phase kinetics in order to elucidate the rate-limiting step and to provide new insights into the mechanism of oxidation of these substrates. A kinetic analysis has allowed for the first time the determination of individual rate constants for several of the partial reactions that comprise the catalytic cycle. Mushroom tyrosinase rapidly reacts with dioxygen with a second-order rate constant k(+8) = 2.3 x 10(7) M(-)(1) s(-)(1), which is similar to that reported for hemocyanins [(1.3 x 10(6))-(5.7 x 10(7)) M(-)(1) s(-)(1)]. Deoxytyrosinase binds dioxygen reversibly at the binuclear Cu(I) site with a dissociation constant K(D)(O)()2 = 46.6 microM, which is similar to the value (K(D)(O)()2 = 90 microM) reported for the binding of dioxygen to Octopus vulgaris deoxyhemocyanin [Salvato et al. (1998) Biochemistry 37, 14065-14077]. Transient and steady-state kinetics showed that o-diphenols such as 4-tert-butylcatechol react significantly faster with mettyrosinase (k(+2) = 9.02 x 10(6) M(-)(1) s(-)(1)) than with oxytyrosinase (k(+6) = 5.4 x 10(5) M(-)(1) s(-)(1)). This difference is interpreted in terms of differential steric and polar effects that modulate the access of o-diphenols to the active site for these two forms of the enzyme. The values of k(cat) for several o-diphenols are also consistent with steric and polar factors controlling the mobility, orientation, and thence the reactivity of substrates at the active site of tyrosinase.     

Stopped-flow cryoenzymological investigation of the pre-steady-state kinetics of hydrolysis of Leu-Gly-NHNH-Dns by leucine aminopeptidase

Stopped-flow fluorescence experiments have been carried out to study the steady-state kinetics of hydrolysis of Leu-Gly-NHNH-Dns [Dns = 5-(dimethylamino)naphthalene-1-sulfonyl] by porcine kidney cytosol leucine aminopeptidase (LAP) in 50% v/v methanol/buffer solution at ambient temperature and the pre-steady-state kinetics of this reaction in the -35 to 0 degrees C temperature range. Experiments have been carried out on LAP species containing Mg(II), Mn(II), Cu(II), Ni(II), Zn(II), and no metal ion at the regulatory metal binding site. At ambient temperatures, the stopped-flow fluorescence changes observed on hydrolysis of the substrate have been used to measure the steady-state kinetic parameters kcat and KM. The results show that 50% v/v methanol lowers the values of kcat from 2- to 12-fold compared to the reactions in the absence of methanol for all of the metallo-LAP, but that the values of KM are essentially unaffected. The pre-steady-state reactions carried out under nonturnover conditions at -35 degrees C reveal a new relaxation for LAP species with Ni(II), Cu(II), and Zn(II) in the regulatory site. The value of kobsd for this relaxation reaches a plateau at high substrate concentrations, and the magnitude of its fluorescence change at a fixed concentration of substrate is proportional to the enzyme concentration. Thus, this relaxation corresponds to the production and decay of a new enzyme-substrate intermediate not observed at higher temperatures whose fluorescence differs from that of the succeeding intermediate that is normally seen above -26 degrees C.     

Stopped-flow kinetic study of the regeneration reaction of tocopheroxyl radical by reduced ubiquinone-10 in solution

Kinetic study of the reaction between tocopheroxyl (vitamin E radical) and reduced ubiquinone, n = 10) has been performed. The rates of reaction of ubiquinol with α-tocopheroxyl 1 and seven kinds of alkyl substituted tocopheroxyl radicals 2–8 in solution have been determined spectrophotometrically, using a stopped-flow technique. The result shows that the rate constants decrease as the total electron-donating capacity of the alkyl substituents on the aromatic ring of tocopheroxyls increases. For the tocopheroxyls with two alkyl substituents at ortho positions (C-5 and C-7), the second-order rate constants, k₁, obtained vary i n the order of 10², and decrease predominantly, as the size of two ortho-alkyl groups (methyl, ethyl, isopropyl and tert-buty) in tocopheroxyl increases. On the other hand, the reaction between tocopheroxyl and ubiquinone-10 (oxidized ubiquinone) has not been observed. The result indicates that ubiquinol-10 regenerates tocopherol by donating a hydrogen atom of the 1-OH and/or 4-OH group to the tocopheroxyl radical. For instance, the k₁ values obtained for α-tocopheroxyl are 3.74 · 10⁵ M^?1 · s^?1 and 2.15 · ⁵ M^?1 · s^?1 in benzene and ethanol solution at 25°C, respectively. The above reaction rates, k₁, obtained were compared with those of vitamin C with α-tocopheroxyl reported by Packer et al. (k₂ = 1.55 · 10⁶ M^?1 · s^?1) and Scarpa et al. (K₂ = 2 · 10⁵ 10⁵ M^?1 · s^?1), which is well known as a usual regeneration reaction of tocopheroxyl in biomembrane systems. The result suggests that ubiquinol-10 also regenerates the tocopheroxyl to tocopherol and prevents lipid peroxidation in various tissues and mitochondria.     

Role of estrogens on striatal dopaminergic activity

Studies were undertaken to evaluate the effects of estradiol and prolactin on striatal dopamine receptor activity. Dopamine receptors were quantified in partially purified striatal membranes by equilibrium binding using [3H]spiroperidol. When we investigated whether the D-2 dopamine receptor activity changes during the estrous cycle, the results suggest an increase in dopamine receptor density in diestrous, without modifications in the affinity. The finding that in ovariectomized rats the dopamine receptor binding parameters remained unchanged, suggests that gonadal steroids are not essential in the mechanism of action of this receptor. Results of activity of D-2 dopamine receptors showing that hyperprolactinemia fails to increase the number of these receptors do not support the hypothesis that circulating prolactin regulates the activity of these striatal dopamine receptors. Administration of estradiol benzoate (250 micrograms/kg per day) to hyperprolactinemic rats, by s.c. injection, significantly decreased both the density and the affinity of the striatal dopamine receptors. The present data indicate that, although prolactin does not seem to modify the activity of striatal dopamine receptors, it could modulate the estrogen-induced hypersensitivity of these receptors.     

Stopped-flow solution scattering using synchrotron radiation: Apparatus,data collection and data analysis

We have constructed an experimental system, under remote control, for stopped-flow X-ray scattering using synchrotron radiation. It has been used, in conjunction with an annular detector and its associated electronics, to obtain good scattering curves, with time-slices as short as 200 ms, in a new study of the dissociation of the enzyme complex aspartate transcarbamylase. The data have been analysed by new statistical methods, and they agree well with the results from parallel chemical quench experiments. For studying dissociation reactions, stopped-flow X-ray scattering is a quite practical method, which need not use very much more material than conventional stopped-flow experiments.     

Modulation of D-3-hydroxybutyrate dehydrogenase activity by estrogens

Liver D-3-hydroxybutyrate dehydrogenase (OHBD) is subjected to estrogen modulation. Estrogen action was demonstrated by (a) the lesser activity of liver OHBD in female rats, as compared with their male counterparts; (b) the increase of OHBD activity after ovariectomy of sexually mature rats; (c) the decrease of OHBD activity after treatment of gonadectomized or normal rats with 17 beta-estradiol or with artificial estrogens; (d) the decrease of OHBD activity in female rats during sexual development; (e) the effects of tamoxifen on the enzyme activity. The kinetics of OHBD reaction using liver mitochondria from estrogen-treated rats showed a 50% decrease of Vmax, as compared with the control value, in contrast to the other parameters which did not vary. These results, taken together with the effect of estrogens on liver mitochondrial phospholipids, point to a decreased content of OHBD in liver mitochondria from estrogen-treated rats. In contrast to OHBD, succinate dehydrogenase and cytochrome oxidase activities, mitochondrial protein synthesis and L-malate + L-glutamate oxidation by coupled liver mitochondria either increased or were not affected by estrogens. Kidney and heart OHBD were affected by ovariectomy and estrogens like the liver enzyme, though to a lesser degree.     

Mucolytics and antioxidant activity

We investigated effects of the mucolytics ambroxol and N-acetylcysteine on airways reactivity evoked by histamine in guinea pigs exposed to toluene vapors. We did not find significant changes in reactivity of tracheal smooth muscle from animals treated with mucolytics compared to the control group. However, the administration of ambroxol and N-acetylcysteine caused a significant decrease in lung tissue reactivity. The effect of ambroxol was more pronounced after intraperitoneal injection than after inhalation, while N-acetylcysteine was only effective after inhalation. The protective effects of mucolytics in the lung tissue may be due to their antioxidant activity together with other mechanisms.     

Spectrophotometric determination of antioxidant activity

Summary

The spectrophotometric technique for total antioxidant activity (TAA)^1,2 measures the relative abilities of antioxidants to scavenge the 2,2′-azino-bis(3-ethylbenz-thiazoline-6-sulfonic acid) (ABTS) radical cation (ABTS^?+) in comparison with the antioxidant potency of standard amounts of Trolox, the water-soluble vitamin E analogue. This method is based on the progressive consumption of antioxidant activity by ABTS^?+ as it is generated in the reaction cuvette and can be automated with a spectrophotometric analyzer. Several different analytical strategies are possible using the same reagents, enabling the assay system to be used to determine the antioxidant activity of plasma, saliva, lipoprotein fractions, foods and beverages. To determine the activity of pure antioxidant substances, a hydrogen peroxide concentration of 75 μM is used, together with a 6 min measuring time. For biological samples with endogenous peroxidase activity the hydrogen peroxide concentration is increased fivefold and the measuring time shortened to 3.25 min. Assays with improved sensitivity are described for low-density lipoprotein (LDL) preparations and saliva. Use of a spectrophotometric endpoint makes the assay simple to carry out without special laboratory equipment. Measurement at 734 nm avoids a range of potential interfering factors, such as sample turbidity and non-specific absorbance by sample constituents. Current applications of the ABTS antioxidant assay are described and discussed.     

The antioxidant activity of cyclohistidylproline

A cyclohistidyl-proline, cyclopeptide possessing a hormonal and neurotrophic activity is shown to be an inhibitor of the (Fe + ascorbic acid)-induced peroxidation of membrane lipids, its effect being dependent on its concentration. Inhibition of the malondialdehyde formation by cyclohistidil-proline is accompanied by protection of the membrane bound Ca-pump. In the test of the free radical cumole oxidation antioxidative effect of cyclohistidyl-proline is 4 times higher than that of the hydrophilic antioxidant carnosine. After peritoneal injection of cyclohistidil-proline (15 mg/kg of body weight) to rats the stationary level of thiobarbituric acid reactive products in rat brain or serum is pronouncedly decreased, this effect being in progress up to 6 h after injection. Antioxidative action of cyclohistidyl-proline suggests to be on the basis of a variety of its biological effects.