Effect of medroxyprogesterone acetate on endothelium-dependent vasodilation in postmenopausal women receiving estrogen.

BACKGROUND: Estrogen increases endothelium-dependent vasodilation in postmenopausal women. However, use of progestins in combination with estrogen may counter beneficial effects of estrogen on endothelium. We investigated the effect of medroxyprogesterone acetate (MPA) on estrogen-induced increase in endothelium-dependent vasodilation in postmenopausal women. METHODS AND RESULTS: Postmenopausal women were treated daily with conjugated equine estrogen (CEE) 0.625 mg (n=14), CEE 0.625 mg and MPA 2.5 mg (n=15) or CEE 0.625 mg and MPA 5.0 mg (n=16) for 3 months. Plasma lipids and hormones were measured before and after treatment. Vasodilatory responses of the brachial artery were evaluated by measuring flow-mediated vasodilation (FMD) and nitroglycerin-induced vasodilation by use of high-resolution ultrasonography. Susceptibility of LDL to oxidation was analyzed by incubation with CuSO(4) while kinetics of conjugated diene formation was monitored. Plasma total and LDL cholesterol concentrations were decreased significantly in all groups. CEE increased FMD significantly, from 4.5+/-1.7% to 8.5+/-2.8% (P<0.001). Addition of MPA reversed this effect in a concentration-dependent manner (for MPA 2.5 mg, from 5.0+/-3.2% to 6.2+/-3.1%; for MPA 5.0 mg, from 4.9+/-3.4% to 3.6+/-3.7%; P=NS for each). No treatment significantly altered nitroglycerin-induced dilation. Lag time for conjugated diene formation was prolonged significantly in all groups, and the oxidation rate was significantly reduced. CONCLUSIONS: Concurrent MPA administration may offset favorable effects of estrogen on endothelial function in postmenopausal women. Because MPA did not diminish LDL-lowering and antioxidant effects of estrogen, MPA-induced inhibition of endothelium-dependent vasodilation may be independent of changes in oxidative susceptibility and plasma concentration of LDL.

Melatonin protects against oxidized low-density lipoprotein-induced inhibition of nitric oxide production in human umbilical artery.

We evaluated the antioxidative effect of melatonin on the oxidized low-density lipoprotein (LDL)-induced impairment of nitric oxide (NO) production in human umbilical artery, which may be the prime cause of endothelial dysfunction in pre-eclampsia. Umbilical artery sections with intact endothelium were obtained from healthy pregnant women who were delivered between 37 and 40 wk of gestation. The production of NO in the umbilical arteries was stimulated by adding L-arginine followed by incubation for 60 min. NO concentrations were estimated by measuring nitrite ions (NO(2) using high-performance liquid chromatography. LDL was oxidized by incubation with 5 microM CuSO(4) at 37 degrees C for 4 hr, followed by dialysis at 4 degrees C for 24 hr. Prior to the addition of L-arginine, the segments were treated with native or oxidized LDL (0, 50, 100, 200, 400 microg/mL), or were pre-treated with either mannitol (50 mM) or melatonin (20, 100, 500 microM) before adding oxidized LDL. Changes in L-arginine-induced NO(2)(-) production were expressed as a percentage of NO(2)(-) production at the end of pre-incubation. Treatment with oxidized LDL significantly reduced L-arginine-induced NO(2)(-) production (P<0.05), while NO(2)(-) production did not change by incubation with native LDL. Pre-treatment with melatonin significantly increased NO(2)(-) production that had been decreased by oxidized LDL (P<0.05). Similarly, pre-treatment with mannitol reversed the oxidized LDL-induced reduction in NO(2)(-) production (P<0.05). These results indicate that melatonin protects against oxidized LDL-induced inhibition of NO production in the endothelium of human umbilical arteries, most likely through its ability to scavenge hydroxyl radicals.

Melatonin protects against ischemia/reperfusion-induced oxidative damage to mitochondria in fetal rat brain.

We investigated the effects of melatonin on ischemia/reperfusion-induced oxidative damage to mitochondria in fetal rat brain. The utero-ovarian arteries were occluded bilaterally for 20 min in female Wistar rats on day 19 of pregnancy to induce fetal ischemia. Reperfusion was achieved by releasing the occlusion and restoring circulation for 30 min. A sham operation was performed in control rats. Melatonin (10 mg/kg) or vehicle was injected intraperitoneally 60 min prior to occlusion. We measured the respiratory control index (RCI) and the adenosine 5-diphosphate (ADP)/oxygen ratio as indicators of mitochondrial respiratory activity, as well as the concentration of thiobarbituric acid-reactive substances (TBARS) in the mitochondria of fetal brain. Ischemia/reperfusion significantly elevated the concentration of TBARS and significantly reduced the RCI as well as the ADP/oxygen ratio. Melatonin treatment reversed the ischemia/reperfusion-induced reductions in the RCI (2.29 +/- 0.06-2.64 +/- 0.09, P < 0.05) and in the ADP/oxygen ratio (1.48 +/- 0.03-1.57 +/- 0.02, P < 0.05), and also reduced the elevation in concentration of TBARS (11.00 +/- 0.34-7.57 +/- 0.74 nM/mg protein, P < 0.01), resulting in values similar to those in untreated, sham-ischemic animals. The results indicate that administration of melatonin to pregnant rats may prevent ischemia/reperfusion-induced oxidative mitochondrial damage in fetal rat brain.

Molecular characterization of a 2-Cys peroxiredoxin from the human malaria parasite Plasmodium falciparum.

We have identified the 2-Cys peroxiredoxin (PfPrx-1) from the human malaria parasite Plasmodium falciparum. The PfPrx-1 showed the highest identity at amino acid level to the type II Prx among the currently known six subfamilies of mammalian Prx. The sequence identity between the PfPrx-1 and the previously reported 1-Cys Prx of P. falciparum (PfPrx-2), which corresponded to mammalian type VI Prx, was 25%. This suggests that the parasite possesses two Prx subfamilies. The PfPrx-1 showed significant sequence similarities with those of 2-Cys peroxiredoxins of plants in the BLASTX search. This may reflect the consequences of a genetic transfer from an algal endosymbiont to the parasite nucleus during evolution. The recombinant PfPrx-1 protein (rPfPrx-1) was expressed as a histidine fusion protein in Escherichia coli and purified with Ni chromatography. The rPfPrx-1 existed as dimers under non-reducing conditions and dissociated into monomers in the presence of dithiothreitol. The PfPrx-1 protein also exists as a dimer in the parasites themselves. The reduction of the oxidized enzyme by the donation of electrons from E. coli thioredoxin (Trx)/Trx reductase system was demonstrated in its reaction with H(2)O(2), using the rPfPrx-1 protein. These results suggested that the PfPrx-1 can act as a terminal peroxidase of the parasite Trx system. An elevated expression of the PfPrx-1 protein seen in the trophozoite, the stage with active metabolism, suggests an association of the parasite Trx system with its intracellular redox control.

Effects of short-term melatonin administration on lipoprotein metabolism in normolipidemic postmenopausal women.

OBJECTIVE: To investigate the effects of short-term administration of melatonin on lipoprotein metabolism in normolipidemic postmenopausal women. METHODS: Fifteen such women received 6.0 mg melatonin daily for 2 weeks. Blood was sampled before and after treatment. We measured concentrations of total cholesterol and total triglyceride in the plasma, as well as the levels of cholesterol, triglyceride, and protein in the very low-density lipoprotein (VLDL), low-density lipoprotein (LDL), and high-density lipoprotein (HDL). Plasma apolipoprotein levels were determined by immunoturbidimetric assay. Activities of lipoprotein lipase, hepatic triglyceride lipase, and lecithin cholesterol acyltransferase were also determined by enzymatic analysis. RESULTS: Melatonin administration significantly increased the plasma levels of triglyceride by 27.2% (P < 0.05), of VLDL-cholesterol by 37.2% (P < 0.01), of VLDL-triglyceride by 62.2% (P < 0.001), and of VLDL-protein by 30.0% (P < 0.05). However, the plasma total cholesterol level and the concentration of lipid and protein in LDL and HDL were not significantly affected. Melatonin significantly increased the plasma levels of apolipoprotein C-II by 29.5% (P < 0.005), of C-III by 17.1% (P < 0.001), and of E by 7.6% (P < 0.05). The plasma levels of apolipoprotein A-I, A-II, and B were not altered. Melatonin significantly inhibited the activity of lipoprotein lipase by -14.1% (P < 0.05), but did not significantly affect the activities of hepatic triglyceride lipase or of lecithin cholesterol acyltransferase. CONCLUSIONS: Findings indicate that melatonin increases the plasma level of VLDL particles by inhibiting the activity of lipoprotein lipase, but may not affect the plasma levels of LDL and HDL particles in postmenopausal women with normolipidemia.

Lipolytic enzyme effect on small low-density lipoprotein particles in women treated with estrogen.

OBJECTIVE: To test whether hydrolysis of low-density lipoprotein (LDL) triglyceride by lipolytic enzymes decreases the size of LDL particles in women treated with estrogen replacement. METHODS: Fifteen postmenopausal women received 0.625 mg conjugated equine estrogens daily for 3 months. Plasma concentrations of total cholesterol, triglyceride, and high-density lipoprotein (HDL) cholesterol were measured before and after therapy. We also assayed levels of total, free, and esterified cholesterol, triglyceride, and protein in LDL. Plasma samples were incubated at 37C for 24 hours and LDL fractions were isolated by ultracentrifugation. After LDL samples were further incubated with or without lipoprotein lipase (500, 700, and 1000 ng/mL) at 37C for 24 hours, LDL triglyceride, LDL protein, and the diameter of LDL particles were measured. RESULTS: Estrogen decreased total cholesterol and increased triglyceride and HDL cholesterol in plasma. Estrogen treatment decreased the ratio of cholesteryl ester/protein, whereas the ratio of triglyceride/protein increased. Estrogen decreased LDL particle diameter. Incubation of plasma increased the ratio of LDL triglyceride/protein from 0.40 +/- 0.14 to 0.48 +/- 0.15 (P <.05) and decreased the ratio of LDL cholesteryl ester/protein from 1.17 +/- 0.25 to 1.09 +/- 0.22 (P <.05), but LDL particle diameter did not change. Incubation of LDL with lipoprotein lipase reduced the LDL triglyceride/protein ratio, and decreased the diameter of LDL particles from 25.61 +/- 0.87 nm to 24.89 +/- 0.88 nm (500 ng/mL, P <.05), 24.62 +/- 1.20 nm (700 ng/mL, P <.05), and 24.67 +/- 1.19 nm (1000 ng/mL, P <.05). CONCLUSION: In women treated with estrogen, hydrolysis of triglyceride in LDL particles might be accompanied by reduced particle size.

Estrogen-induced small low density lipoprotein particles may be atherogenic in postmenopausal women.

OBJECTIVES: The purpose of this study was to investigate the susceptibility of estrogen-induced small low density lipoprotein (LDL) particles to oxidation. BACKGROUND: Estrogen replacement therapy in postmenopausal women has an antioxidant effect that opposes oxidation of LDL particles. Estrogen-induced increases in plasma triglyceride concentrations, however, decrease LDL particle size, which may act counter to this antioxdant effect. It has not been evaluated whether estrogen-induced small LDL particles are atherogenic. METHODS: In 24 lean and healthy postmenopausal women treated with conjugated equine estrogen (0.625 mg daily) for three months, plasma lipid concentrations and diameter of LDL particles were measured before and after therapy. Susceptibility of LDL to oxidation was determined by measuring the concentration of thiobarbituric acid-reactive substances (TBARS) after incubation with CuSO4. RESULTS: Estrogen significantly decreased plasma concentrations of total cholesterol, LDL-cholesterol and apolipoprotein B, while increasing concentrations of triglyceride, high-density lipoprotein cholesterol and apolipoprotein A-I. Estrogen-induced changes in LDL particle diameter correlated negatively with changes in plasma triglyceride concentrations (r = -0.55, p < 0.005) and with changes in concentrations of LDL-derived TBARS (r = -0.49, p < 0.005). In subjects with substantial estrogen-induced plasma triglyceride increases, estrogen significantly reduced the diameter of LDL particles (p < 0.05) and significantly increased the concentration of LDL-derived TBARS (p < 0.05). In contrast, estrogen significantly reduced the concentration of LDL-derived TBARS (p < 0.05) and caused no significant change in LDL particle diameter in subjects whose plasma triglyceride concentration was unchanged with estrogen therapy. CONCLUSIONS: Because estrogen-induced plasma triglyceride increases may produce small LDL particles that are more susceptible to oxidation, antioxidant effects of estrogen might be offset in patients showing such a triglyceride increase.

Melatonin protects fetal rat brain against oxidative mitochondrial damage.

Our objective was to investigate the effects of melatonin on the free radical-induced oxidative damage to mitochondria in fetal rat brain. Female Wistar rats on day 19 of pregnancy were used. Melatonin (10 mg/kg) or vehicle (control) was injected intraperitoneally 60 min prior to laparotomy for removal of the fetuses. The mitochondrial fraction was isolated from the fetal rat brain of each group. Superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) activities were measured. As indicators of mitochondrial respiratory activity, we determined the respiratory control index (RCI) and the adenosine 5-diphosphate/oxygen (ADP/O) ratio in the presence and absence of 2.5 microM hypoxanthine and 0.02 units/mL xanthine oxidase. Mitochondrial lipid peroxidation was determined by measuring the concentration of thiobarbituric acid reactive substances in fetal brain mitochondria in the presence or absence of 2.5 microM hypoxanthine, 0.02 units/mL xanthine oxidase, and 50 microM FeSO4. The free radical-induced rates of inhibition of mitochondrial RCI and the ADP/O ratio were both significantly lower in the fetal rat brains treated with melatonin compared with those of the controls (RCI, 44.25 +/- 15.02% vs. 25.18 +/- 5.86%, P < 0.01; ADP/O ratio, 50.74 +/- 23.05% vs. 13.90 +/- 7.80%, P < 0.001). The mitochondrial lipid peroxidation induced by free radicals was significantly reduced in the melatonin-treated group compared with the controls (484.2 +/- 147.2%) vs. 337.6 +/- 61.0%, P < 0.01). Pretreatment with melatonin significantly increased the activity of GSH-Px (20.35 +/- 5.27 to 28.93 +/- 11.01 mU/min mg(-1) protein, P < 0.05) in fetal rat brain mitochondria, but the activity of SOD did not change significantly. Results indicate that the administration of melatonin to the pregnant rat may prevent the free radical-induced oxidative mitochondrial damage to fetal rat brain by a direct antioxidant effect and the activation of GSH-Px.

Non-linear pharmacokinetics of ofloxacin after a single intravenous bolus dose in pigs.

The pharmacokinetics of ofloxacin (OFLX) was investigated after intravenous administration of 3, 10 and 30 mg/kg of body weight in pigs. Plasma OFLX concentration-time course collected from the highest dosage showed a convex decline, indicating a capacity-limited process in drug elimination (Michaelis-Menten elimination). Dose-normalized area under curve (AUC/Dose) and mean resident time (MRT) were dose-dependent, indicating a classical pattern of non-linear elimination pharmacokinetics. Based on simultaneous curve fitting from three doses, non-linear pharmacokinetic parameters were as follows: 0.87 mg/h/kg for maximum velocity, 2.20 microg/mL in Michaelis-Menten constant and 2.06 L/kg for apparent volume of distribution. Based on a model-independent analysis, the apparent volume of distribution at steady-state (Vdss) was dose-independent whereas total body clearance (CLtot) was dose-dependent, mainly contributed by renal clearance (CLr) with the regression line of CLtot=1.14xCLr+0.09 (r=0.92). The intercept of the regression line indicates non-renal clearance (CLnr), corresponding to the value of observed CLnr without dose-dependency. Because of a higher CLr compared with glomerular filtration rate (GFR) in spite of drug reabsorption, the CLr must contain the renal active tubular secretion. With increasing dosage, the level of saturation of tubular secretion of OFLX decreased the CLr, resulting in the decrease in CLtot. The plasma protein binding to OFLX was dose-independent: mean free fraction (fp)=0.73, with probably no influential effect on OFLX disposition. In conclusion, the degree of saturation in the renal active tubular secretion of OFLX could be a major causal factor in the alteration of CLr in an increasing dosage of OFLX. Accordingly, the alteration of CLr could directly induce the non-linear pharmacokinetics of OFLX in pigs, an important consideration in clinical therapeutics.

Melatonin inhibits vasospastic action of oxidized low-density lipoprotein in human umbilical arteries.

We evaluated the antioxidant property of melatonin in countering the vasospastic effect of oxidized low-density lipoprotein (ox-LDL), which has been reported to be the most important risk factor for atherosclerosis and also may be linked to preeclampsia. Helical sections of umbilical arteries were obtained from human placentas at elective cesarean deliveries between 37 and 39 weeks of gestation. Changes in maximal tension induced by potassium chloride were measured in arterial sections with intact endothelium. ox-LDL (200 or 300 microg protein/mL) increased vascular tension by 15.6 +/- 2.3 or 31.9 +/- 4.0%, respectively. In contrast, native LDL only slightly increased vascular tension (2.7 +/- 1.0% for 200 microg protein/mL and 6.0 +/- 1.7% for 300 microg protein/mL). Pretreatment with L-N(G)-monomethyl-arginine (2 x 10(-4) M) significantly reduced the vasospastic effect of ox-LDL, as did pretreatment with mannitol (30 mM). Melatonin (10 microM) significantly reduced the vasospastic effect of ox-LDL. These findings suggest that ox-LDL potentiates vascular tension in the human umbilical artery, possibly by suppressing endothelial synthesis of nitric oxide. Melatonin significantly suppressed the vasospastic effect of ox-LDL, probably because it scavenges hydroxyl radical arising from ox-LDL.

Lipoprotein particles in preeclampsia: susceptibility to oxidative modification.

OBJECTIVE: To investigate the susceptibility to oxidation of low-density lipoprotein (LDL) and high-density lipoprotein (HDL) in women with preeclampsia. METHODS: Plasma levels of total cholesterol, total triglyceride, and concentrations of cholesterol, triglyceride, and protein in LDL and HDL were measured in 12 preeclamptic women and 12 normal pregnant women. Oxidation of LDL or HDL was assessed by incubation with copper ions and evaluated by monitoring the kinetics of conjugated diene formation. RESULTS: The plasma levels of total triglyceride and concentration of LDL protein were significantly higher in preeclamptic women than in normals. Levels of HDL lipid did not differ significantly. Analysis of kinetics of conjugated diene production showed a significantly shorter lag time for LDL (83.1 +/- 5.5 minutes versus 67.4 +/- 10.2 minutes, P <.001) and HDL (76.9 +/- 7.3 minutes versus 59.5 +/- 9.2 minutes, P <.001) and a significantly higher oxidation rate for LDL (3.6 +/- 0.4 nmol/minutes/mg LDL versus 4.4 +/- 1.0 nmol/minutes/mg LDL, P <.05) in preeclamptic women. CONCLUSION: Low-density lipoprotein and HDL particles were more susceptible to oxidative modification, and plasma concentration of LDL particles, but not of HDL particles, was increased in preeclampsia.

Effects of combination therapy with estrogen plus simvastatin on lipoprotein metabolism in postmenopausal women with type IIa hypercholesterolemia.

We investigated the effects of estrogen and simvastatin, administered both alone and in combination, on the plasma lipid levels and lipoprotein-related enzymes in 45 postmenopausal women with type IIa hypercholesterolemia. They received 0.625 mg conjugated equine estrogen (n=15), 5 mg simvastatin (n=15), or the combination (n=15) daily for 3 months. We measured the concentrations of cholesterol and triglyceride in the plasma, and in the very low-density lipoprotein (VLDL), intermediate-density lipoprotein (IDL), low-density lipoprotein (LDL)1 (1.019

Melatonin inhibits oxidative modification of low-density lipoprotein particles in normolipidemic post-menopausal women.

In this study, we investigated the short-term effect of melatonin on the susceptibility of low-density lipoprotein (LDL) to oxidation in normolipidemic post-menopausal women. Fifteen post-menopausal women received 6.0 mg melatonin daily for 2 wk. Blood samples were obtained before and after the treatment and the plasma levels of total cholesterol, total triglyceride, high-density lipoprotein (HDL)-cholesterol, LDL-cholesterol, LDL-triglyceride, and LDL-apolipoprotein B were determined. LDL oxidation was performed by incubation with copper ions and was analyzed by monitoring the kinetics of conjugated diene formation and measuring the concentration of thiobarbituric-acid-reactive substances (TBARS). LDL-apolipoprotein B derivatization was analyzed by measuring trinitrobenzene sulfonic acid (TNBS) reactivity. Melatonin treatment significantly increased the plasma triglyceride levels (P<0.05), but did not significantly alter the plasma levels of total cholesterol, HDL-cholesterol, or LDL-lipids. The kinetics analysis of conjugated diene production revealed that melatonin treatment significantly prolonged the lag time of conjugated diene formation (from 64.71+/-11.89 to 70.15+/-10.52 min, P<0.05). The oxidation rate and the amount of conjugated diene, however, did not change significantly. The TBARS concentration was significantly reduced by melatonin treatment (from 49.31+/-7.57 to 38.69+/-23.90 nM/mg LDL, P<0.05). Furthermore, melatonin treatment significantly reduced the copper-induced decrease of TNBS reactivity (from 79.43+/-6.19 to 86.50+/-9.07% at 1 hr and from 71.03+/-6.74 to 76.31+/-4.99% at 2 hr, P<0.05). These results indicate that melatonin treatment may reduce LDL susceptibility to oxidative modification in normolipidemic post-menopausal women.

Disease-induced alterations in plasma drug-binding proteins and their influence on drug binding percentages in dogs.

Disease-induced variations of plasma albumin (ALB) and alpha 1-acid glycoprotein (AAG) levels were investigated in dogs. Lower ALB (sometimes > 50% reduction) and higher AAG (sometimes > 10-fold increase) levels were observed in dogs with various diseases. Drug binding was determined at therapeutic concentrations using normal, low-ALB and high-AAG dog plasma. The binding percentages of the ALB-binding drugs decreased in low-ALB plasma, resulting in a large increase in unbound drug, particularly for naproxen (a 13-fold increase). The binding percentages of all AAG-binding drugs investigated in this study increased in high-AAG plasma, resulting in a large decrease in unbound drug, particularly for quinidine (99% decrease). The fluctuation in the unbound fraction of drugs could affect their efficacy or could cause side-effects. Veterinary clinicians should monitor the ALB and AAG levels in the plasma of patients and correct dosage regimens according to these levels, where field conditions permit this, in order to ensure the proper usage of drugs with high affinity for ALB or AAG.

Lipid transfer reactions and lipid composition of low-density lipoprotein particles in postmenopausal women receiving estrogen.

OBJECTIVE: To investigate the effects of estrogen on lipid transfer reactions and lipid composition of low-density lipoprotein (LDL) particles in postmenopausal women. METHODS: Twelve postmenopausal women were treated with conjugated equine estrogen, 0.625 mg daily, for 3 months. Plasma concentrations of total cholesterol, triglyceride, and high-density lipoprotein (HDL) cholesterol were measured before and after therapy. We also determined the amount of total, free, and esterified cholesterol, triglyceride, and apolipoprotein B in LDL. To evaluate lipid transfer reactions, plasma samples were incubated at 37C for 24 hours, and replacement of cholesteryl ester by triglyceride in LDL particles was analyzed. Cholesterol and triglyceride concentrations were measured enzymatically. Apolipoprotein B concentrations were determined by an immunoturbidimetric assay. RESULTS: Estrogen significantly reduced the plasma levels of total cholesterol and significantly increased those of triglyceride and HDL cholesterol. The ratio of cholesteryl ester to apolipoprotein B was reduced significantly, whereas the ratio of triglyceride to apolipoprotein B increased significantly after estrogen treatment. Both before and after estrogen treatment, incubation of plasma induced a significant increase in the ratio of LDL-triglyceride to apolipoprotein B with a concomitant decrease in the ratio of LDL-cholesteryl ester to apolipoprotein B. Incubation-induced changes in these ratios were significantly enhanced by estrogen therapy. The plasma concentration of triglyceride was correlated positively with incubation-induced changes in the ratio of LDL-triglyceride to apolipoprotein B (r = .83, P < .001) and correlated negatively with changes in the ratio of LDL-cholesteryl ester to apolipoprotein B (r = -.61, P < .01). CONCLUSION: Estrogen-induced increase in the plasma level of triglyceride may enhance lipid transfer reactions, resulting in triglyceride-rich and cholesteryl ester-poor LDL particles.

Effect of ischemia-reperfusion on xanthine oxidase activity in fetal rat brain capillaries.

OBJECTIVE: The purpose of this study was to investigate whether ischemia and subsequent reperfusion would affect xanthine oxidase activity in fetal rat brain capillaries. STUDY DESIGN: We used rats on day 19 of pregnancy. Fetal ischemia was induced by bilateral occlusion of the utero-ovarian artery for 20 minutes. Reperfusion was achieved by releasing the occlusion to restore the circulation for 30 minutes. Control rats underwent a sham operation. Fetal brain capillaries were isolated for measurement of concentrations of hypoxanthine, xanthine, and uric acid, as well as of concentrations of thiobarbituric acid-reactive substances. The brain capillaries were incubated with hypoxanthine for 1-5 hours at 25 degrees C. The activity of xanthine oxidase was estimated by measuring the amount of xanthine converted from hypoxanthine. RESULTS: Occlusion for 20 minutes markedly increased the concentration of hypoxanthine but had no effect on levels of xanthine, uric acid, and thiobarbituric acid-reactive substances. However, subsequent reperfusion led to significant increases in the levels of xanthine, uric acid, and thiobarbituric acid-reactive substances. Xanthine oxidase activity, as measured by the amount of xanthine produced, was significantly greater in the animals subjected to both ischemia and ischemia-reperfusion compared with the control group. CONCLUSION: Ischemic insult led to the accumulation of hypoxanthine and stimulated xanthine oxidase activity in fetal brain capillaries. Subsequent reperfusion enhanced the degradation of hypoxanthine to uric acid, which may induce cerebral lipid peroxidation.

Melatonin protects against ischemia and reperfusion-induced oxidative lipid and DNA damage in fetal rat brain.

To investigate whether melatonin reduces the susceptibility of the fetal rat brain to oxidative damage of lipids and DNA, we created a model of fetal ischemia/reperfusion using rats at day 19 of pregnancy. Fetal ischemia was induced by bilateral occlusion of the utero-ovarian artery for 20 min. Reperfusion was achieved by releasing the occlusion and restoring the circulation for 30 min. A sham operation was performed in control rats. Melatonin (10 mg/kg) or vehicle was injected intraperitoneally 60 min prior to the occlusion. We measured the concentration of thiobarbituric acid reactive substances (TBARS) in fetal brain homogenates, as well as levels of deoxyguanosine (dG) and 8-hydroxydeoxyguanosine (8-OHdG) in DNA extracted from those homogenates. Ischemia for 20 min did not significantly alter the levels of dG, 8-OHdG, and TBARS. Subsequent reperfusion, however, led to a significant reduction in the dG level (P < 0.05) and to significant increases in the levels of 8-OHdG (P < 0.05) and TBARS (P < 0.05), and in the 8-OHdG/dG ratio (P < 0.005). Melatonin administration prior to ischemia significantly reduced the ischemia/reperfusion-induced increases in the levels of 8-OHdG (14.33 +/- 6.52-5.15 +/- 3.28 pmol/mg of DNA, P < 0.001) and TBARS (11.61 +/- 3.85-4.73 +/- 3.80 nmol/mg of protein, P < 0.001) as well as in the 8-OHdG/dG ratio (7.19 +/- 2.49-1.61 +/- 0.98, P < 0.001). Furthermore, melatonin significantly increased the dG level (210.19 +/- 49.02-299.33 +/- 65.08 nmol/mg of DNA, P < 0.05). Results indicate that melatonin administration to the pregnant rat may prevent the ischemia/reperfusion-induced oxidative lipid and DNA damage in fetal rat brain.

Small low-density lipoprotein particles in women with natural or surgically induced menopause.

OBJECTIVE: To investigate the mechanism of the decrease in the size of low-density lipoprotein (LDL) particles in women with natural menopause and women with surgically induced menopause. METHODS: We measured plasma levels of total cholesterol; triglycerides; high-density lipoprotein (HDL) cholesterol; apolipoproteins A-I, A-II, and B; and sex hormones in 45 women; 15 women were premenopausal, 15 were naturally postmenopausal, and 15 were surgically menopausal. Lipoprotein lipase and hepatic triglyceride lipase activities were measured in postheparin plasma. Concentrations of total cholesterol and of apolipoprotein B in LDL also were measured. Low-density lipoprotein particle diameter was determined by gradient gel electrophoresis. RESULTS: Plasma levels of total cholesterol, triglycerides, apolipoprotein B, LDL-total cholesterol, LDL-apolipoprotein B, and the activity of postheparin plasma lipoprotein lipase were significantly higher and concentrations of estrone and estradiol were significantly lower in the naturally postmenopausal and surgically menopausal women than in the premenopausal women. Plasma levels of HDL cholesterol and apolipoproteins A-I and A-II and postheparin plasma hepatic triglyceride activity did not differ significantly between groups. The diameter of LDL particles was significantly reduced in the naturally (25.29 +/- 0.19 nm) and surgically (25.29 +/- 0.22 nm) menopausal women compared with the premenopausal women (25.88 +/- 0.22 nm). Plasma triglyceride levels were negatively correlated with LDL particle diameter in all three groups (premenopausal group: r = -0.64, P < .01; naturally postmenopausal group: r = -0.62, P < .01; and surgically menopausal group: r = -0.76, P < .001). The prevalence of LDL subclass pattern B was significantly increased in the naturally (67%, P < .05) and surgically (60%, P < .05) menopausal women. CONCLUSION: The plasma concentration of LDL particles was increased after menopause, whether natural or surgically induced. An increase in plasma triglyceride levels in women with low levels of endogenous estrogen appeared to cause the size of LDL particles to be reduced.

Effect of estrogen and simvastatin on low-density lipoprotein subclasses in hypercholesterolemic postmenopausal women.

OBJECTIVE: To identify the effects of estrogen and simvastatin, individually and in combination, on the levels of low-density lipoprotein (LDL) subclasses in postmenopausal women with type IIa hypercholesterolemia. METHODS: Fifty-five postmenopausal women with type IIa hypercholesterolemia were assigned randomly to 0.625 mg of conjugated equine estrogen (n = 20), 5 mg of simvastatin (n = 18), or both (n = 17) daily for 3 months. Cholesterol, triglyceride, and apolipoprotein B levels in the plasma and in the LDL1 (density 1.019-1.045 g/mL) and LDL2 (density 1.045-1.063 g/mL) fractions were measured before and after treatment. RESULTS: Estrogen treatment significantly reduced LDL1 cholesterol and LDL1 apolipoprotein B levels by 18.4% and 20.8%, respectively; simvastatin treatment by 21.9% and 29.2%, respectively; and combination therapy by 38.5% and 34.4%, respectively. In contrast to estrogen or simvastatin treatment, the combination therapy also significantly lowered the levels of LDL2 cholesterol by 19.5% and LDL2 apolipoprotein B by 30.5%. Posttreatment levels of total cholesterol, LDL1 cholesterol, and LDL1 apolipoprotein B were significantly lower after combination treatment than after estrogen treatment. Estrogen treatment, but not combination therapy, significantly increased total plasma triglyceride levels (103.1+/-26.0 mg/dL to 138.8+/-75.6 mg/dL, P < .01). Significantly more patients receiving combination therapy than those receiving estrogen had total and LDL cholesterol concentrations reduced to target levels. CONCLUSION: Combination therapy with estrogen and simvastatin favorably affected lipid metabolism by reducing large and small LDL particles and prevented the estrogen-induced increase in plasma triglyceride levels.