Bartolomé Bonet

In vitro effects of a flavonoid-rich extract on LDL oxidation

Flavonoids are phenolic compounds of vegetable origin with antioxidant effects. The present study aimed to determine their properties as LDL antioxidants. LDL were incubated with increasing concentrations of flavonoids (0-16 micrograms/ml) and LDL oxidation was started by adding CuCl2 (2 microM) to the media. When flavonoids were present in the media, vitamin E consumption, the lag phase of conjugated diene formation, LDL electrophoretic mobility in agarose gels and the appearance of thiobarbituric acid reacting substances (TBARS) were delayed in a concentration-dependent manner. To determine whether flavonoids could terminate LDL oxidation once initiated, two sets of experiments were performed. In the first, LDL oxidation was initiated as described above. At 2 or 4 h of incubation, flavonoids were added (4 micrograms/ml) and their effect compared to samples where butylated hydroxytoluene or EDTA were added. At 5 h, in the LDL samples where flavonoids were added, the electrophoretic mobility and TBARS production were the same as those present in LDL samples incubated for the whole period in the absence of flavonoids. However, when either butylate hydroxytoluene or EDTA was added, as would be expected, the LDL oxidation process was completely arrested as shown by a reduction in the appearance of TBARS and a lower LDL electrophoretic mobility. In the second experiment, LDL oxidation was initiated as described above and at 0, 10 and 20 min, flavonoids were added (4 micrograms/ml). When vitamin E was still present in the LDL solution, the flavonoids were able to both increase the lag phase in the formation of conjugated dienes and to delay the consumption of vitamin E. The present results show that in vitro, flavonoids prevent LDL oxidation in a concentration-dependent manner, delaying the consumption of vitamin E, but they cannot terminate or delay LDL oxidation once vitamin E in LDL is consumed.

ANTIOXIDANT AND PROOXIDANT EFFECTS OF ASCORBIC ACID, DEHYDROASCORBIC ACID AND FLAVONOIDS ON LDL SUBMITTED TO DIFFERENT DEGREES OF OXIDATION

UNLABELLED Although a high intake of antioxidants may decrease the risk of developing cardiovascular diseases, under certain circumstances they may promote free radical generation and lipid peroxidation. The objectives of the present study were to determine the antioxidant effects of ascorbic acid (AA), dehydroascorbic acid (DHA) and flavonoids on LDL submitted to different degrees of oxidation. LDL was submitted to oxidation with CuCl2 (2.4 microM). Before or at different times after the propagation of the oxidation process, 28 microM (5 micrograms/ml) of either AA or DHA or 5 micrograms/mL flavonoids extract were added. Alpha-tocopherol, conjugated dienes, thiobarbituric acid reacting substances (TBARS) and LDL electrophoretic mobility were determined as indices of LDL oxidation. The presence of any of the three antioxidants from the onset of the incubation delayed the oxidation process. However, the addition of both DHA and flavonoids to the oxidation process when it was already initiated and alpha-tocopherol consumed, accelerated the oxidation. In contrast, AA delayed the oxidation process even when added after alpha-tocopherol was consumed. Nevertheless, it also accelerated LDL oxidation when added during the propagation phase of the oxidation process. IN CONCLUSION although AA, DHA and flavonoids delay LDL oxidation when added before the initiation of the process, they accelerate the process if added to minimally oxidized LDL.

Intermediary Metabolism in Pregnancy: First Theme of the Freinkel Era

During the first half of gestation in the rat, maternal net body weight increases rapidly, whereas in the second half of gestation, the mass of maternal structures declines, coincident with the rate of maternal fat accumulation. Enhanced maternal food intake, extrahepatic tissue lipoprotein lipase (LPL) activity, and adipose tissue lipogenesis are responsible for the progressive accumulation of maternal fat. However, during late gestation, decreased fat synthesis in maternal adipose tissue, enhanced lipolytic activity, and decreased LPL activity deplete maternal fat depots. These changes, plus enhanced endogenous production of triglyceride-rich lipoproteins, are also responsible for maternal hypertriglyceridemia. This condition benefits the offspring in two ways: 1) enhanced LPL activity in maternal liver when fasting increases triglyceride consumption for ketone body synthesis, giving the basis for accelerated starvation; and 2) induction of LPL activity in the mammary gland before parturition diverts maternal circulating triglycerides to milk synthesis in preparation for lactation. The magnitude of the maternal-fetal glucose transfer was higher than that of any of the other substrates studied, including alanine, and despite actions to spare glucose, this transfer causes maternal hypoglycemia, which is especially intense in the fasting condition. This increases sympathoadrenal activity in the mother, which may contribute to her active gluconeogenesis. Glycerol was a more efficient glucose precursor than alanine and pyruvate, and whereas glycerol placental transfer is very small, it is proposed that the fetus benefits from this product of adipose tissue lipolysis when it is previously converted into glucose. In thyroidectomized pregnant rats treated with thyroxine for different periods, restraining maternal accumulation of fat depots during the early part of gestation compromises the normal metabolic adaptations during late gestation, including the capacity for accelerated starvation, which negatively affects fetal development.

Simultaneous determination of vitamins A and E in rat tissues by high-performance liquid chromatography

A high-performance liquid chromatographic (HPLC) method to determine vitamins A, Ap and E simultaneously was developed with direct extraction of vitamins from rat tissues with n-hexane and probe sonicating. The dry residue was redissolved in chloroform-methanol. Vitamins A and Ap were detected by UV-Vis and vitamin E by fluorescence. Vitamin K, used as internal standard, was detected both the UV-Vis and by fluorescence. Standards and samples were checked for linearity giving correlation coefficients that were higher than 0.99 in the concentration range of 3.1-9.4 for vitamin A, 8.2-24.7 for vitamin Ap and 0.6-1.7 nmol/g in the case of liver extracts and 0.5-3.0 nmol/g in the case of placenta. The inta-assay precision (R.S.D) varied between 1.48 and 7.25, whereas inter-assay precision was between 4.99 and 7.03. Recoveries ranged between 94 +/- 8 and 107 +/- 5%. Results from the application of this method to different rat tissues having wide range of vitamin content are presented.

Teratogenic Actions of Thermally-stressed Culinary Oils in Rats

Lipid oxidation products (LOPs), generated in culinary oils during episodes of thermal stressing can give rise to cellular damage. The aims of this study were to determine whether orally-administered, LOP-containing thermally-stressed safflower oil exerts teratogenic actions in rats, and whether this effect could be prevented by co-administration of f -tocopherol ( f -TOH). Safflower oil was heated for a period of 20 min according to standard frying practices and stored at m 20°C under N 2 . Four experimental groups of pregnant Wistar rats were employed; two received 0.30 ml of pre-heated oil (HO), one of which was also supplemented with 150 mg of f -TOH (HOE), and two served as controls, one treated with the non-heated oil (O) and the other without any treatment (C). The oil was administered daily by gavage from day 1 of pregnancy to day 11.5, when the animals were killed and the embryos examined. LOPs and f -TOH were determined both in the heated and non-heated oils. The percentage of embryo malformations and reabsorptions were determined in the above four experimental groups. Heating the oil substantially increased its concentration of LOPs and decreased its f -TOH content. The percentage of embryo malformations in the HO group was 21.73%, compared with 5.6 and 7% in the O and C groups, respectively. Supplementation of the pre-heated oil with f -TOH was found to decrease the percentage of malformations to 7%. The results obtained from these investigations indicate that LOPs detectable at millimolar levels in the heated cooking oils administered (e.g. saturated and f, g -unsaturated aldehydes, and/or their conjugated hydroperoxydiene precursors) exert potent teratogenic actions in experimental animals which are at least partially circumventable by co-administration of the chain-breaking antioxidant f -TOH. Plausible mechanisms for these processes and their health relevance to humans regarding diet and methods of frying/cooking are discussed.

Increased low-density lipoprotein susceptibility to oxidation in pregnancies and fetal growth restriction.

Objective: Atherosis and placental infarction have been observed in pregnancies complicated by fetal growth restriction (FGR). Low-density lipoprotein (LDL) oxidation plays a central role in the pathogenesis of atherosclerosis; therefore, it could be involved in the placental alterations observed in FGR. The aims of the present study were to estimate LDL susceptibility to oxidation in pregnancies complicated by FGR and to evaluate their relationship with fetal growth and placental hormone secretion. Methods: A cohort prospective study was carried out in 50 women with uncomplicated pregnancies and 55 women with FGR. Blood was drawn at 15, 24, and 32 weeks of gestation. Low-density lipoprotein oxidation was initiated by the addition of CuCl2 and formation of conjugated dienes was monitored. Cholesterol, triglycerides, vitamin E, estradiol, progesterone, and placental lactogen were determined. Results: Women with FGR showed a lag phase (minutes from addition of CuCl2) similar to the control group in the first trimester of pregnancy (85.3 ± 3.3 versus 81.3 ± 5.6). But in the second and third trimester, they showed a lower lag phase than the control group: 69.6 ± 3.6 versus 84.4 ± 3.5 (P < .05) and 69.9 ± 3.4 versus 95.6 ± 3.4 (P < .001). During the third trimester, pregnancies complicated with FGR showed lower levels of estradiol, progesterone, and human placental lactogen than those in the control group. In the third trimester, a positive correlation was found between the lag phase and the birth weight (P = .001) and with the plasma levels of estradiol (P = .002). Conclusion: Fetal growth restriction is associated with an increased LDL susceptibility to oxidation, a process that could damage the placenta, leading to alterations in placental endocrine function and fetal weight. Pregnancies complicated by fetal growth restriction show an increased LDL susceptibility to oxidation, a process that may lead to placental dysfunction and growth delay. Level of Evidence: II-2

Alpha-Tocopherol Concentration in Fetal and Maternal Tissues of Pregnant Rats Supplemented with Alpha-Tocopherol

We wanted to determine whether alpha-tocopherol supplementation to pregnant rats could increase the concentration of alpha-tocopherol in maternal and fetal plasma and tissues. Pregnant rats were treated with alpha-tocopherol on days 18 and 19 gestation and studied at day 20. A control group was studied in parallel. Treatment of pregnant rats with alpha-tocopherol increased its concentration in maternal and fetal plasma, in all maternal plasma lipoprotein fractions, in maternal and fetal liver and in the placenta. The fetal and maternal concentration of alpha-tocopherol were positively correlated.

Dual effect of glucose on LDL oxidation: dependence on vitamin E

The aim of the present study was to determine the direct effect of glucose on LDL oxidation, a key step in the development of atherosclerosis. Purified human LDL were incubated with glucose (500 mg/dl) and LDL oxidation was started by adding CuCl(2) to the media. Glucose delayed the vitamin E consumption, but accelerated the formation of conjugated dienes and increased both the formation of thiobarbituric acid reacting substances (TBARS) and LDL electrophoretic mobility. When LDL were incubated with increasing concentrations of glucose and submitted to oxidation, the formation of conjugated dienes, TBARS, and the electrophoretic mobility increased in a concentration-dependent manner. When LDL was enriched with vitamin E, it showed a delay in the formation of conjugated dienes, even in the presence of glucose. To determine whether glucose had any effect on LDL oxidation, once the process was started and vitamin E consumed, LDL were submitted to oxidation and, at different times thereafter, glucose was added into the media. Under these conditions glucose also accelerated the LDL oxidation. In summary, present results show that in LDL submitted to oxidation, glucose delays the early phases of the oxidation, slowing the vitamin E consumption, but it accelerates the rate of LDL oxidation once LDL vitamin E has been consumed; the effect being concentration-dependent.

Relationship between Alpha-Tocopherol Content in the Different Lipoprotein Fractions in Term Pregnant Women and in Umbilical Cord Blood

Aims: To determine the concentration of vitamin E in normal maternal and umbilical cord blood pairs, and to study the relationship between vitamin E content in maternal lipoprotein fractions and umbilical cord blood. Methods: Fifty healthy pregnant women were recruited randomly at term and blood samples were drawn from the mothers at delivery and cord blood was obtained immediately postpartum. Vitamin E was determined by HPLC in plasma, in the different lipoprotein fractions and in the placenta. Plasma levels of triglycerides and cholesterol were also measured. Results: The concentration of vitamin E in umbilical cord plasma was 250 µg/dl, lower than in maternal plasma (1,460 µg/dl) (p < 0.001). A positive correlation was found between the vitamin E concentration in maternal plasma, LDL and VLDL and in the umbilical cord plasma. In contrast, no correlation was found between maternal HDL concentration and umbilical cord blood. Conclusion: These results show that the concentration of vitamin E in umbilical cord blood is lower than in maternal plasma. LDL and VLDL seem to be the main source of vitamin E for the fetus.

Consequences of preeclampsia for the newborn: role of oxidative stress

Preeclampsia is a prevalent disorder that remains one of the more frequent sources of morbidity and mortality for pregnant women and their children. There is evidence that fetuses and newborns of preeclamptic women (NPW) are specifically affected by the maternal condition, independent of the uteroplacental flow restriction. Preeclampsia is thought to be an endothelial disorder in which pathophysiological oxidative stress plays an important role. Experimental and clinical studies suggest that maternal endothelial dysfunction and oxidative stress correlate with the severity of complications for NPW. In this regard, the fetus seems not to be a passive spectator suffering from the maternal condition, as fetal vasculature somehow shares the particular endothelial dysfunction; this has been demonstrated in umbilical vessels and endothelial cells from NPW, and in fetal systemic vessels in animal models of preeclampsia. In addition, oxidative stress is increased in fetal circulation in preeclampsia. Maternal symp...