M.T. Ravi Subbiah

Estradiol-17β as an antioxidant : Some distinct features when compared with common fat-soluble antioxidants

Estrogens are potent antioxidants both in vitro and in vivo. In this study the antioxidant affect of estradiol-17beta (estradiol) was compared with those of fat-soluble antioxidants (alpha-tocopherol and beta-carotene) in terms of both fatty acid (thiobarbituric acid-reactive substances and diene conjugation) and cholesterol oxidation (oxysterols). The addition of alpha-tocopherol (54 micromol/L) inhibited low-density lipoprotein (LDL) oxidation by 92.6% and high-density lipoprotein (HDL) oxidation by 76.5%. In similar experiments, estradiol (54 micromol/L) inhibited LDL oxidation by 77.5% but inhibited HDL oxidation by only 55.4%. Beta-carotene had no antioxidant effect. Lag times (diene conjugation method) for alpha-tocopherol and beta-carotene increased by 175% and 125%, respectively. Estradiol markedly reduced the maximum formation of diene conjugates as compared with results with alpha-tocopherol and beta-carotene, and it exhibited a linear curve (no change in lag time). In terms of cholesterol oxidation, estradiol was far more effective than alpha-tocopherol or beta-carotene in inhibiting oxysterol formation (microg/ml plasma) (control = 24.56 +/- 2.31, beta-carotene = 20.59 +/- 3.32, alpha-tocopherol = 20.19 +/- 1.58, estradiol = 14.38 +/- 0.70). This study shows that estradiol is as effective an antioxidant as alpha-tocopherol in terms of fatty acid peroxidation but is far more effective than alpha-tocopherol in terms of cholesterol peroxidation.

Mechanisms involved in the protective effect of estradiol-17β on lipid peroxidation and DNA damage

Previous studies from our laboratory have shown that estrogens can protect against lipoprotein peroxidation and DNA damage. In this study, the mechanism of estradiol-17β (E2) action was investigated by comparing E2 with selective scavengers of reactive oxygen species (ROS) in terms of inhibition of 1) human low-density lipoprotein (LDL) peroxidation (measured by the diene conjugation method) and 2) DNA damage (measured by the formation of strand breaks in supercoiled OX-174 RFI DNA). In addition, the direct effect of E2 on the generation of individual ROS was also measured. By use of ROS scavengers, it was determined that lipoprotein peroxidation was predominantly due to superoxide (39%), with some contributions from hydrogen peroxide (23%) and peroxy (38%) radicals. E2 was a more effective inhibitor of peroxidation than all the ROS scavengers combined. In DNA damage, scavengers of hydrogen peroxide, hydroxyl, and superoxide radical offered significant protection (49-65%). E2 alone offered a similar degree of protection, and no additional effect was evident when it was combined with ROS scavengers. E2caused a significant reduction (37%) in the production of superoxide radical by bovine heart endothelial cells in culture but had no effect on the formation of either hydrogen peroxide or hydroxyl radicals. These studies show that 1) the protection offered by E2 in terms of lipid peroxidation could be due to its ability to inhibit generation of superoxide radical and prevent further chain propagation, and 2) in DNA damage protection, E2 mainly appears to inhibit chain propagation.

Superior and distinct antioxidant effects of selected estrogen metabolites on lipid peroxidation

The effect of the estrogen metabolites, 4-hydroxyestrone and 17alpha-dihydroequilin (metabolites of estradiol-17beta and equilin, respectively), were examined for antioxidant effects on plasma and lipoprotein lipid peroxidation . Lipid peroxidation was evaluated by products of both fatty acid (thiobarbituric acid-reactive substances [TBARS]) and cholesterol (oxysterols) oxidation from lipoproteins or whole plasma. Although all estrogens significantly reduced lipid peroxidation, 4-hydroxyestrone was far more potent than either equilin or 17alpha-dihydroequilin in inhibiting TBARS formation in lipoproteins induced by Cu2+. Similar effects were also noted on TBARS formation in THP-l macrophages in culture. However, 17alpha-dihydroequilin (along with equilin) strongly inhibited oxysterol formation, whereas 4-hydroxyestrone was ineffective. These studies suggest that different estrogens might act preferentially on distinct lipid substrates in exhibiting antioxidant effects.

Exercised-induced increase in lipid peroxidation parameters in amenorrhelc female athletes

OBJECTIVE To determine plasma lipid peroxidation parameters in eumenorrheic and amenorrheic athletes and to evaluate differences in their response to exercise-induced oxidative stress. In female athletes, intense physical exercise has been shown to be associated with an increased occurrence of menstrual dysfunction with lower levels of E2. Recently, a protective role has been demonstrated for estrogens as free radical scavengers. DESIGN Comparison of eumenorrheic and amenorrheic athletes before and after an acute bout of exercise. SETTING Academic Research Environment. PATIENT(S) Seven eumenorrheic (normally menstruating) and seven amenorrheic (<3 menses/year) female athletes aged 18 to 35 years participating in regular training. MAIN OUTCOME MEASURE(S) Plasma and low-density lipoprotein oxidation parameters, plasma E2 and vitamin E levels, and creatine kinase activity. RESULT(S) Both the amenorrheic and eumenorrheic athletes demonstrated a significant decrease in the lag time of conjugated diene formation after exercise (P < 0.01), with greater magnitude of change occurring in the amenorrheic athletes (P < 0.05). In addition, postexercise samples from amenorrheic (but not eumenorrhic) athletes showed a significant (P < 0.01) increase in oxysterol formation as compared to baseline values. Amenorrheic athletes also demonstrated a significantly higher baseline creatine kinase activity and a nonsignificant (P = 0.04) trend of an increase in creatine kinase activity after exercise. CONCLUSION(S) The results of this study shows that amenorrheic female athletes demonstrate an increased potential for lipid peroxidation after exercise. This could be related to lower plasma E2 levels in this group, considering the strong free radical scavenging ability of estrogens identified recently.

Estrogens protect against hydrogen peroxide and arachidonic acid induced DNA damage

The ability of estrogens to protect against DNA damage induced by either hydrogen peroxide or arachidonic acid alone or in combination with Cu2+ was investigated. DNA strand breaks were determined by conversion of double stranded supercoiled OX-174 RFI DNA to double stranded open circular DNA and linear single stranded DNA. Estradiol-17 beta significantly decreased the formation of single and double strand breaks in DNA induced by H2O2 alone or with Cu2+. Equilin (an equine estrogen) was more effective than estradiol-17 beta at the doses tested. Arachidonic acid in the presence of Cu2+ caused the formation of high levels of linear DNA which was protected by estrogen with equilen being more effective. These studies suggest that estrogens through this protective effect on DNA damage might contribute to cardioprotection.

Proteins and peptides bound to long-circulating liposomes

Liposome formulations with prolonged circulation time have recently been developed as a potential sustained-release drug delivery system. Data shown in this report indicate that such formulations can also be used to prolong the circulation time of proteins and peptides by conjugating them to the surface of liposomes. Increase of the circulation halflife ranged from 2- to 150-fold depending on the protein/lipid ratio of the liposomal formulation, liposome size, and the lipid composition of liposomes. Since the proteins/peptides localize on the liposome surface, instead of being entrapped inside the liposomes, they are directly available for binding to its receptor molecules and express the biological activity. This strategy has been successfully applied to two proteins with known fast clearance rate, i.e. asialofetuin and ricin A-chain. The biological activities of both proteins are preserved when they are formulated in liposomes. Incorporation of a peptide, i.e. a-factor of the yeast Saccharomyces cerevisiae, into the liposome membrane also significantly enhanced the circulation time of the peptide.

Studies on the Nature of Anti-Platelet Aggregatory Factors in the Seeds of the Amazonian Herb Guarana (Paullinia cupana)

Guarana (Paullinia cupana) is a popular herb native to the Amazon Basin and used extensively in soft drinks in Brazil, other Latin American countries, and more recently in the United States. Extracts derived from the dried seeds of guarana possess strong anti-platelet aggregatory properties. In this study, an active fraction containing this activity was purified and analyzed by high-performance liquid chromatography/mass spectrometry (HPLC/MS) techniques. It was noted that this fraction contains catechins, epicatechins, and their dimers, with a small amount of caffeine. It is suggested that complexes containing caffeine and catechins (and their dimers) might be responsible for anti-platelet aggregatory activity in guarana seeds and might offer health benefits towards decreasing risk of thrombosis and cardiovascular disease.

Newly Recognized Lipid Carrier Proteins in Fetal Life

Summary An attempt is made in this review to summarize new findings that propose the direct involvement of two fetal proteins (i.e, fetuin and α-fetoprotein) in fatty acid transport and delivery to cells. The presence of these proteins at a high plasma concentration in fetal life and the critical need for fatty acids during development strongly support this hypothesis. Tissue and cell specificities involved in the uptake of these fetal proteins and the mechanism of fatty acid delivery awaits further research.

Hepatic 3-hydroxy-3-methylglutaryl coenzyme a reductase activity amd cholesterol 7α-hydroxylase activity in neonatal guinea pig

Optimal assay conditions for hepatic HMG-CoA reducatase activity and cholesterol 7 alpha-hydroxylase activity in the guinea pig were determined. These two enzyme activities were studied in the liver of newborn guinea pigs during the first three postnatal weeks. Hepatic HMG-CoA reductase activity of neonatal guinea pigs was similar to that of adult animals. However, cholesterol 7 alpha-hydroxylase activity of newborns was about one-third of that in adult guinea pigs. This finding suggests that the system for bile acid synthesis in the neonatal guinea pigs is underdeveloped.

Uptake and protection against oxidative stress by estrogen esters in THP-1 human macrophage cell lines.

Estrogen replacement therapy offers protection from coronary artery disease in postmenopausal women. However, there is serious concern that long-term unopposed estrogen use increases the risk of breast and endometrial cancer through estrogen-receptor-driven mechanisms. In this communication, we have explored an alternate route of estrogen delivery to macrophages using hydrophobic derivatives that associate with lipoproteins. Unlike free estradiol (E2), long-chain fatty acid esters of E2 associate extensively with low-density lipoprotein (LDL). In THP-1 cells, E2 esters accumulated to a significantly higher level when compared to E2 in the presence of LDL. In the presence of oxidized LDL even greater amounts of E2 esters accumulated in cells. In THP-1 cells, E2 esters were capable of preventing the azo-bis-induced increase in oxidative stress (hydrogen peroxide formation). These studies suggest that (a) hydrophobic esters of estrogens that associate with LDL can be delivered to macrophages and (b) these esters can effectively function as antioxidants protecting against oxidative stress.