Efecto de la suplementación materna con dha durante la gestación y la lactancia sobre el estrés oxidativo y el remodelado óseo materno y neonatal

Resumen

INTRODUCTION Oxidative stress appears in both the mother and fetus in the early stages of pregnancy because of the role it plays in the development of the placenta. Hypoxia is essential to control oxygen homeostasis, and it is considered essential in early embryonic development, as well as in placental development through the regulation of angiogenesis in human placenta (De Marco & Caniggia, 2002) (Mutinati et al., 2014). However, oxidative stress has also been associated with the pathophysiology of diseases that may develop during pregnancy such as abortions, eclampsia, restricted intrauterine growth retardation (IUGR), and membrane premature rupture (Burton & Jauniaux 2011; Mutinati et al. 2014). Therefore, the antioxidant defense system is essential to maintain the balance between pro-oxidants and antioxidants (oxidative balance). Thus we analyzed the enzymes of the primary antioxidant defense (superoxide dismutase, catalase and glutathione peroxidase), as well as fat-soluble vitamins, which are major components of the non-enzymatic antioxidant system (¿-tocopherol, Coenzyme Q10, ß-carotene and retinol). On the other hand, during pregnancy and lactation key physiological adaptations occur in women, among others we can highlight the changes happening in bone metabolism, such as compensatory mechanisms to ensure the proper mineral development in the fetus and the suitable protection on mother¿s skeleton (Glerean 2000; Vidal et al., 2008). Thereby, in early pregnancy osteocalcin levels are comparable to non-pregnant women; during the second trimester those levels decrease and then in the third trimester of pregnancy osteocalcin levels increase. Osteocalcin remains high during the breastfeeding, reflecting the high bone turnover that occurs in the mother, which helps to provide the necessary amount of calcium for skeletal maturation in fetus and the newborn (Glerean, 2000). After pregnancy during the breastfeeding period, calcium demand increases so that calcium can be concentrated in breast milk. To compensate for the greater demand of calcium, maternal adaptations include both mechanisms, bone resorption and renal calcium reabsorption. These compensatory physiological mechanisms allow, in most cases, to cope with the requirements needed for the formation and mineralization of the fetal skeleton and infant nutrition (Yoon et al. 2000). The fetal and neonatal skeleton requires a suitable mineral content in order to develop and mineralize appropriately. Both PTH and PTHrP, are essential to maintain high serum levels of calcium and phosphorus in the womb, to carry out an optimal peak bone mineralization. Similarly, endochondral bone development during fetal development requires PTH and PTHrP, but not calcitriol, calcitonin or (probably) sex steroids. It is in the neonatal period when the intestinal absorption of calcium and therefore, skeletal development and mineralization become dependent on vitamin D/calcitriol (Kovacs, 2013). During lactation, most studies have shown an increase in bone remodeling. This can occur partly due to reduced plasma estradiol (P-E2), as well as due to an increase in plasma prolactin and PTHrP during lactation (Møller et al., 2013; Kovacs, 2013). During childhood, especially during the first years of life, is the time when the phenomena of bone turnover reached a peak intensity. This is manifested by the high levels of serum osteocalcin and propeptide (Kovacs 2011). Regarding to the link between bone remodeling and oxidative stress, according to evidence there is a correlation between bone metabolism and redox regulation, indicating that reactive oxygen species (ROS) may play an important role because they inhibit forming osteoblasts and therefore bone formation. In addition ROS are involved in homeostasis and cartilage degradation. ROS, specifically H2O2, play a crucial role in osteoclast function and differentiation. ROS increase the number of osteoclasts and thus bone resorption by stimulating RANKL and TNF-¿ expression through the ERK (extracellular signal regulated kinase) and nuclear factor kappaß (NF-kß) activation, TNF-¿ caused cell damage, and it also inhibits SOD (Bai et al. 2005). The RANK-L binds to its receptor, RANK, promoting osteoclast differentiation. Furthermore, OPG prevents the binding between RANKL and RANK. Alterations in the RANK-RANKL-OPG system due to an increased activity of RANK-L, appear to be related to the pathogenesis of several bone diseases including osteoporosis and inflammatory bone disease. Increased osteoclast activity can lead to increased formation of superoxide anion (O2 -) generation and/or inhibit the activity of SOD and GPx, with consequent bone destruction (Sheweita & Khoshhal, 2007). As mentioned before, the RANKL plays a key role in controlling osteoclastogenesis. During this process the inflamed synovial tissue produces a variety of cytokines and hormones which may also influence the activity of osteoclasts. Among these factors we can find: interleukins IL-1¿, IL-1ß, IL-6, IL-17, TNF-¿, the Macrophage colony-stimulating factor (M-CSF), and Parathyroid hormone-related protein (PTHrP) (Anandarajah & Schwarz 2006; Barbour et al. 2012). The role of these cytokines in bone resorption and inflammation, shows that there might be a correlation between the immune system and bone resorption. According to some studies, T-cells activated after the inflammation process that occurs in rheumatoid arthritis, promote the formation of RANK-L. In fact, it is known that the active T-cells play a central role in the pathogenesis of rheumatoid arthritis, contributing to bone resorption mediated by osteoclasts through the expression of RANKL (Gravallese et al. 2000; Clowes et al. 2005; Barbour et al. 2012). This study evaluates the effect of maternal supplementation of docosahexaenoic acid (DHA), an omega-3 long chain polyunsaturated fatty acid (LC-PUFA), which has proven to be a major structural component in cell membrane, it helps to maintain the adequate fluidity and functionality. It is especially abundant in neuronal tissue and the retina, therefore it is essential for the development of brain and retina in the fetus (Walczewska et al. 2011; De Giuseppe et al. 2014). During prenatal stage, DHA is provided to the fetus by maternal-fetal transfer across the placenta; after birth, the main source through which the infant gets the DHA is the breast milk. As DHA synthesis is very limited in the newborn, it is essential to ensure the presence and the adequate concentration of this fatty acid in the mother, so that she can provide the suitable amount for the newborn (Helland et al. 2008; Poniedzia¿ek-Czajkowska et al. 2014). Omega-3 PUFAs are involved in antioxidant and anti-inflammatory (resolvins mediated) processes. Several placental disorders such as intrauterine growth restriction, preterm labor and premature rupture of membranes (PROM), are associated with inflammatory processes and oxidative stress in the placenta, in this way DHA supplementation has shown beneficial effects on these anomalies (Pietrantoni et al. 2014). However, DHA is a highly polyunsaturated fatty acid and therefore very susceptible to oxidative stress. For this reason, there is some controversy with several studies showing antioxidant protection of omega-3 LC-PUFA (Mas et al. 2010; Garcia-Rodriguez et al. 2012), while some others report an increase in oxidative damage after this supplementation (Shoji et al. 2009; Boulis et al. 2014). This link between DHA and oxidative stress is of great importance, since it must be assessed the beneficial effects of this this fatty acid as an antioxidant, vs. the possible effect as an prooxidant which promotes oxidative damage. Pregnancy appears to be accompanied by a high metabolic demand, an increased requirement of oxygen and thus an increased production of free radicals. We observed a higher oxidative stress in pregnant women than in non-pregnant ones, in the same way, in the neonate at birth we observed en elevated oxidative damage (Ochoa et al. 2003 and 2007; Díaz-Castro et al., 2015). The birth also involves a strong oxidative stress for both, the mother and the newborn. In case of healthy full term newborn, this oxidative stress disappears during the first hours of life. In the premature infants due to several reasons (immaturity of the organs, immature antioxidant system, etc.) oxidative stress is even greater than the observed in full term neonates (Ochoa et al. 2007). Although the effect of DHA supplementation has been extensively studied on various aspects of pregnancy, childbirth and infant development, the potential effect of this supplementation on oxidative stress observed in newborns and the evolution has not been studied so far. On the other hand, omega-3 fatty acids might have beneficial effects on bone metabolism regulation. Thus, optimal amounts of n-3 PUFA appear to inhibit bone resorption as they promote bone formation (Griel et al. 2007; Kajarabille et al. 2013). The mechanism of action of omega-3 fatty acids in the bone is quite complex and its effects can be multifactorial, involving several signaling pathways, cytokines and growth factors. There are several mechanisms by which the omega-3 may regulate bone metabolism, including a decrease in the release of prostaglandin PGE2, as well as in the most important factor in osteoclast differentiation, the RANKL. In addition, omega-3 fatty acids can modulate several inflammatory cytokines, increase the production of IGF-1 (insulin-like growth factor-1) and improve the accretion of calcium in bones (Griel et al. 2007; Kruger et al. 2010). PGE2 are potent modulators of bone remodeling involved in bone resorption, since they stimulate osteoclastogenesis via the RANK-L. Both processes bone formation and resorption are related to PGE2 and its effects on the bone can be dose-dependent. Thus, low levels PGE2 stimulates bone formation through osteoblasts, while high levels suppress osteoblast differentiation and promote bone resorption mediated by osteoclasts via RANK-L (Korotkova et al. 2004; Kruger et al. 2010; Lukas et al. 2011). It has been shown that inflammatory signals can modulate the OPG/RANK-L system decreasing OPG and increasing RANK levels, thereby producing bone resorption. According to biography, DHA might be beneficial for bone due to its anti-inflammatory actions among others (Martin-Bautista et al. 2010). BACKGROUND AND AIMS DHA supplementation during pregnancy and lactation has shown positive effects on different aspects related to the newborn development, such as the association between the maternal DHA intake and the proper cognitive and visual development, underlying the need to ensure a suitable supply to the fetus and infant through an adequate maternal nutrition. However, there are certain aspects of the DHA supplementation related with the development and health of the fetus that have not been studied to date and they could be decisive factors in the early stage of life. Among these aspects we can find the correlation between DHA supplementation during pregnancy and lactation with respect to bone metabolism and oxidative stress in both the mother and the neonate, where maternal supplementation could play an important role. The controversy mentioned above together with the lack of information in the physiological stages of pregnancy, lactation and postnatal development is what has motivated the development of this work Therefore, the aim of this study is to evaluate the effect of maternal supplementation with a dairy product enriched with fish oil (320 mg DHA + 72 mg EPA) during pregnancy (last trimester) and breastfeeding on oxidative stress and bone metabolism in both the mother and the full term newborn. METHODS This study involved a group of mothers and their healthy term neonates enrolled in a registered, double-blind, controlled, lasting form the sixth month gestation to fourth month of newborn¿s life. 110 volunteers were recruited into the study from ¿Hospital Materno-Infantil¿ (Granada, Spain) and ¿Hospital Universitario Materno-Infantil¿ (Canary Islands, Spain). The study was approved by the Bioethical Committee on Research Involving Human Subjects at both Hospitals. Written informed consent was obtained from each participant after a complete explanation of the study details. The study is registered at www.clinicaltrials.gov (Clinical Trial Identifier NCT01947426). Women were randomly assigned to one of the following intervention groups: Supplemented group: Consumption of 400 ml/day of fish oil enriched dairy drink (320 mg DHA + 80 mg EPA); Control Group: Consumption of 400 ml/day of the control dairy drink. Detailed information on the composition of the dairy drink using during the intervention is given in Table 1. We supervised the mother¿s diet during the intervention period. Maternal dietary intake was assessment using a 110-item food frequency questionnaire that included specific questions about consumption of sources of DHA such as freshwater fish, seafood, canned tuna and sardines, salmon, trout, and cod liver oil (Parra-Cabrera et al. 2011) (García-Rodríguez et al. 2012) (Annex I). Together with these questionnaires, nutritional recommendations adapted to the conditions of the mothers (gestation - lactation) were given and especially those related to the suitable consumption of fish (2/3 portions per week as daily sources of EPA+ DHA), something really important from an ethical point of view. Samples of mother¿s blood (5 mL) were obtained at the enrollment (28th week of pregnancy) (SM0)), at delivery (SM1), at 2.5 months of lactation (SM2) and at the end of the dietetic intervention (four month postpartum) (SM3). After delivery blood samples were collected from the umbilical vein and arteries (SHOV and SHOA, respectively) and at 2.5 months of life a sample of blood form all the neonates was obtained (SH1). The samples were centrifuged to separate plasma from red blood cell pellets. Erythrocyte cytosolic and membrane fractions were prepared by differential centrifugation according to the method of Hanahan and Ekholm (Hanahan et al., 1974). Samples were stored at -80 °C until analysis. Oxidative damage in plasma and erythrocyte membrane Plasma hydroperoxides content was assessed using a commercial kit (Oxystat BI-5007, Biomedica Gruppe, Vienna, Austria). Erythrocyte membranes was assessed using another commercial kit Pierce Quantitative Peroxide Assay Kit, aqueous-compatible formulation (Ref: 23280) (Thermo Scientific, Rockford, USA). Plasma fat soluble antioxidants and total antioxidant capacity in plasma Fat soluble antioxidants in plasma were also determined: Vitamin A (Retinol), Vitamin E (a-Tocoferol), ß-Carotene, Coenzyme Q9 and Coenzyme Q10. Samples were mixed with ethanol in polypropylene tubes and left on ice for 10 minutes, then hexane was added, and we left them on ice for 5 minutes. Samples were centrifuged at 2200g for 10 minutes at 4 °C and finally dried in a speed-vacum). A mixture of ethanol: isopropanol (90:10, v/v) was added to the sample and then analyzed by Ultra performance liquid chromatography-tandem mass spectrometry, UPLC-MS / MS system. The equipment used was an ACQUITY UPLC H-Class detector coupled to a triple quadrupole Xevo TQ-S (Waters Corporation, Milford, USA). Total plasma antioxidant capacity in plasma samples was measured using a kit (TAS Randox® kit, Randox Laboratories Ltd., Crumlin, UK). Fat soluble antioxidants in erythrocyte membranes and antioxidants enzyme activity Fat soluble antioxidants in erythrocyte membrane (Vitamin E and Coenzyme Q10) were also determined following the same procedure described above for fat soluble antioxidants in plasma. Determinations of antioxidant enzymes, Glutathione Peroxidase (GPx), Superoxide dismutase (SOD) and Catalase (CAT) in erythrocyte cytosol were measured as previously described by Díaz-Castro et al. (2014). Bone metabolism biomarkers analysis Luminex xMAP technology based MILLIPLEX MAP kits were used. Human Bone Panel 1A Millipore (USA) (Cat No. HBN1A-51k) was used for ACTH, PTH, osteocalcin, osteopontin, OPG, leptin, insulin, TNF-¿ and IL-6 determination, and MILLIPLEX MAP RANKL Single Plex (Cat No. HBN51k1RANKL) was used for RANKL determination. Mineral content in erythrocyte cytosol The concentration of minerals (Ca, Mg, Fe, Cu and Zn) in the cytosolic fractions was determined by atomic absorption spectrophotometry (PerkinElmer AAnalyst 1100B spectrometer with WinLab32 for AA software, Massachusetts, USA). The samples had previously been mineralized by the wet method in a sand bath (J.R. Selecta, Barcelona, Spain), according to the procedure described previously by Diaz-Castro et al., (2008). Phosphorous (P) concentration was analyzed using a commercial kit: Phosphorous-UV SPINREACT (Barcelona, Spain) (Cat. No. MI1001155). STATISTICAL ANALYSES The sample size was calculated by detecting a difference in the DHA levels in mothers of 2.26, with a standard deviation of 1.43 and using a potency of 80% and 0.05 of significance level. This calculation indicates the need for 45 mothers, amount increased by 20%, to counteract possible losses and thus obtaining a final sample size of 55 mothers per group. The results are shown as mean ± standard error of the Mean (SEM). Kolmogorov-Smirnoff test was performed following normality criteria. Categorical variables were compared using chi-square test. Differences between groups were analyzed with Student-T test for independent samples. Student-T test was also used to compare differences between mothers (SM0, SM1, SM2 and SM3) and newborns (SH0V, SH0A and SH1). To test the differences between parameters determined in different periods of time, in both mothers and newborn, a General Linear Model for repeated measures or ANOVA was used, with a posterior adjustment with Bonferroni for testing the differences between different periods of time. For data analysis we used the SPSS version 20.0 (SPSS Statistics for Windows, 20.0.0. SPSS INC. Chicago, IL, USA). RESULTS AND DISCUSSION As mentioned above, a noteworthy aspect to consider during pregnancy, labor and postnatal life is the evoked oxidative stress, affecting both the mother and neonate (Ochoa et al., 2003,2007; (Di Nunzio & Bordoni, 2011). Kankofer (2011) reported a maximum peroxides output in mother plasma at delivery and during pregnancy, results which are in agreement with those found in the current study. We have found that plasma hydroperoxides were also elevated in neonates, being similar to those found in the mother during lactation. Omega-3 LC-PUFA supplementation showed a clear beneficial effect on hydroperoxides levels in the newborn, by decreasing them in umbilical cord artery, as well as in the postnatal life of the newborns at 2.5 months. Similarly, we have observed the effect of DHA supplementation in mothers at delivery and postpartum (2.5 and 4 months), and we found that peroxide levels in erythrocyte membrane were also significantly reduced. The decrease in plasma and erythrocyte membrane peroxides content induced by omega-3 LC-PUFA supplementation may be due to the effect of the LC-PUFA on some key factors responsible of free radicals generation and the effect on the antioxidant system in the mother and the newborn. One of the possible protective mechanisms of the omega-3 LC-PUFA to oxidative stress would underlie in the generating mechanisms of free radicals, including the inflammatory signaling. DHA has an anti-inflammatory effect that appears to be correlated with its inhibiting action on the arachidonic acid (AA) (Pietrantoni et al., 2014). AA is released via selective Ca2+ dependent cytosolic phospholipase A2 (IVA cPLA2), however this mechanism also implies the conversion in eicosanoids, generating free radicals (Vericel et al., 2015). In this sense, we can postulate that a possible antioxidant role of DHA can be attributed to its inhibiting action on the AA/eicosanoids production and to the anti-inflammatory and protective properties of the protectins and resolvins (Pietrantoni et al., 2014). In this sense, Gonzalez-Periz et al. (2006), demonstrated that DHA prevents DNA damage and oxidative stress in liver cells, effect that was associated with a decrease in the hepatic synthesis of n-6-derived eicosanoids, as well as an increase in the generation of protective DHA-derived lipid mediators. Another aspect to be taken into account is the effect on the antioxidant defense. We have analyzed the plasma antioxidant capacity. Our results showed that the highest antioxidant values were found in mothers during pregnancy and lactation, whereas these levels diminished at delivery. In both groups, the antioxidant capacity is almost double in mothers compared to their neonates. Thereby, the main differences between control and supplemented groups were found in mothers at parturition and in the newborn at 2.5 months of life, although with different behavior. The lower value of total antioxidant capacity found in the mothers supplemented with omega-3 LC-PUFA at the moment of childbirth, together with the lower peroxides level, especially in erythrocyte membrane could indicate a lower need of antioxidants in the supplemented group, probably due to a lower production of free radicals1 (Yavin 2006; Shoji et al., 2009; Smithers et al., 2011). In this sense, we could also consider a lower transfer of antioxidants to the neonate, explaining the lower content in plasma hydroperoxides found in umbilical cord artery in the fish oil-supplemented group. Newborns are particularly susceptible to oxidative stress for several reasons33. Our results show that omega-3 LC-PUFA supplementation avoids the decrease of antioxidant capacity at 2.5 months of life in newborns and, in addition, this supplementation also reduces peroxide levels in plasma compared with the control (non-supplemented) group. Regarding to the fat soluble antioxidants in plasma and erythrocyte membrane, in mothers we did not find any significant differences between the control and the fish oil-supplemented group during the last trimester of pregnancy, finding which is in agreement with the data from an study carried out with mothers who were supplemented with salmon during pregnancy (García-Rodríguez et al., 2012). In this study, the authors did not observed significant differences for ¿-tocopherol, ß-carotene and CoQ10 in the supplemented group compared to control. However, retinol levels in plasma were different from those found in our study, since they observed significantly higher retinol concentrations in mothers who were supplemented with two portions of salmon per week during gestation, fact that could be attributed to the great retinol concentration in salmon. Nevertheless, we found significant differences in plasma and membrane samples at 2.5 months postpartum as well as in umbilical cord artery, suggesting that fish oil-supplementation has positive effects on the neonate, enhancing the antioxidant capacity and reducing the evoked oxidative stress. After parturition we observed higher of retinol, CoQ9 and ¿-tocopherol in plasma in the supplemented group at 2.5 months postpartum and ß-carotene and CoQ10 at 4th month postpartum. In erythrocyte membrane, higher values of CoQ10 were found at delivery and ¿-tocoferol at 2.5 months postpartum. These data suggest a higher fat soluble antioxidant defense in the supplemented mothers, especially after lactation. The result observed for ¿ -tocoferol in plasma at 2.5 months postpartum is interesting. Florian et al. (2004), reported a significant decrease in ¿-tocopherol levels in plasma at 2.5 months postpartum, which is in agreement with our results, although in the supplemented group this decrease was not observed. With regard to the newborns, in general, a higher content in retinol, CoQ10 and ¿- tocopherol and erythrocyte membrane was observed in the samples obtained in umbilical cord of the supplemented group. This information indicates, once again, the higher antioxidant defense at the moment of the childbirth in the neonate and this is the moment when the maximum oxidative stress occurs (Saugstad, 2005; Ochoa et al., 2007). In the same way, at 2.5 months of postnatal life, a higher CoQ10 content is observed in the supplemented group. All these findings can be correlated with the results found in plasma peroxides, because fish oil-supplemented group showed a decrease of peroxides in umbilical cord vein and artery, as well as at 2.5 months of newborn¿s life. To avoid induced oxidative damage in cells, the antioxidant defense system provides protection and the capacity of this defense system is determined by a dynamic interaction between individual components, which include fat soluble vitamins and several antioxidant enzymes (superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GPx)). Regarding to our data for antioxidant enzymes, in the supplemented group as well as in the control group, both mothers and their neonates showed similar activity . With regard to SOD we found the highest activity at 4 month postpartum in mothers and in umbilical cord artery in neonates. In general, the omega 3 LC-PUFA supplementation leads to an increase in the activity of this antioxidant enzyme, though it is only significant at the moment of the delivery in the mother and at 2.5 months of postnatal life in the newborn. Garcia-Rodriguez et al.23 did not report any differences in the activity of this enzyme due to the maternal supplementation with two portions of salmon during gestation, though this study was only focused in the period before the childbirth and not during the labor, neither in the newborn child. On the other hand, in a study carried out by Garrel et al. (2012), they observed a significant increase in SOD activity in brain of rats that were fed with a diet rich in DHA, during postnatal development. With regard to CAT , in the fish oil-supplemented group, we observed higher values at 2.5 months postpartum in mothers, as well as at 2.5 months of newborn¿s life. We did not found significant differences for CAT during pregnancy neither the birth, which is in agreement with the results found by Garrel et al.(2012) and García-Rodríguez et al. (2012). Regarding to GPx, we found the highest values in both groups after delivery, particularly at 4th month postpartum and the lowest values were observed in the newborns at 2.5 months postpartum. Though a higher activity of this enzyme was observed in the supplemented group in umbilical cord artery and in the newborn at 2.5 months of life, it was just a trend and not statistically significant. García-Rodríguez et al. (2012) found a higher activity GPx in DHA-supplemented mothers with salmon, though this increased activity was associated with the increase in the levels of selenium, due to the high content of this mineral in salmon and it is known the effect of selenium as a cofactor in the activity of this enzyme. In general, it is noteworthy the effect of the mother supplementation with omega-3 LC-PUFA in the newborn child at 2.5 months of life on the enzymatic activity of the complex SOD-CAT, a very interesting finding, due to the importance in the development of the newborn child, because as it has been previously reported, a decrease in the activity of these enzymes promotes the oxidative damage in proteins and lipids (Slater et al., 1987; Saugstad 2005). Regarding to the effect of omega-3 LC-PUFA on bone metabolism as well as the effect on mineral content in erythrocyte cytosol, we found a significant increase in PTH during lactation in mothers who were supplemented. PTH is a hypercalcemic hormone, which stimulates osteoclasts, this could be negative for the mother, but on the other hand beneficial to the newborn, as the processes of resorption increases, calcium recovery increases in the kidneys, as well as, intestinal absorption of calcium, which increases calcium content in breast milk. This is consistent with one of the physiological mechanisms described at this stage, where bone resorption increases in order to meet the needs of calcium in the breast milk for the newborn (Glerean 2000; Yoon et al. 2000; Vidal et al. 2008). Regarding to ACTH, we found maximum values in the supplemented group in mothers at delivery and especially in the fetus in samples of umbilical cord vein. As it is known physiological levels of cortisol do not have deleterious effects on bone formation, but they appear to be necessary for cell differentiation and probably also for the bones (Riancho J.A. 2003), moreover it has been shown that ACTH may stimulate osteoblast proliferation through specific receptors on these cells, which would enhance bone formation (Zhong et al. 2005; Isales et al. 2010). Furthermore, the increase of ACTH, also leads to increased levels of glucocorticoids such as cortisol, which are responsible for the stress response, so that this could improve not only the ability of the newborn to face the challenge of birth and their adaptation to extrauterine environment, promoting the mobilization of energy substrates, the maintenance of blood glucose level and even lung maturity, but also it could help the mother to cope with the stress during labour (Márquez et al., 2005; Raga et al., 2006). Our results also showed significantly higher OPN values in neonates, which indicates the high bone turnover in the newborn. We observed the effect of supplementation only during childbirth, showing higher values in women who were supplemented with omega-3 LC-PUFA. This could be related to the increased bone resorption in mother in order to concentrate more calcium in breast milk, as it has been described above. On the other hand, this cytokine has also a role as an inflammatory signal, and parturition leads to a significant inflammatory process which can promote an increase in OPN values (Terzidou et al. 201; Chen et al. 2012; Straburzy¿ska-Lupa et al. 2013). Regarding to OC our data showed the greatest values in the newborn, pointing out the high bone turnover, especially bone formation (Manjón 2003). In the case of mothers, we observed a progressive increase, reaching the maximum values at lactation, showing the high bone turnover in the latter period, an increased bone resorption to get more calcium in milk for the newborn as mentioned above. Our data are in agreement with other studies that have also shown higher levels of osteocalcin during lactation (Yoon et al. 2000; Barba 2011). We observed the effect of the supplementation at delivery in mothers and at 2,5 months of newborn¿s life , where we found an increase in OC, which promotes neonate¿s development and bone formation. Lecke et al. (2011) reported that leptin values decreased in relation to the days of life in newborns, which matches our results, where we observed a very low leptin levels in neonates at 2.5 months. Furthermore, we observed an increase of leptin in umbilical cord blood in newborns whose mothers received the supplementation. According to biography, leptin has a double mechanism on bone metabolism, anyways based on in vivo studies it appears that the peripheral effect which stimulates bone formation is the one that predominates, and which could be beneficial for the neonate (Capellán 2000; Quesada Gomez JM 2006; Williams et al. 2011). So, if we consider that leptin has a potential positive effect on bone remodeling, omega-3 LC-PUFA supplementation could also promote a positive effect in this first stage of life, but perhaps the most interesting effect is it¿s role on energy balance and which would be enhanced by omega-3 fatty acids. With regard to insulin, we observed an increase in venous umbilical cord blood in neonates whose mothers received supplementation. In contrast, another study evaluating the influence of a DHA supplementation during pregnancy and lactation, found lower concentration of insulin in umbilical cord blood of newborns whose mothers consumed DHA, compared to placebo (Courville et al. 2011). However, with the data obtained in this study, we cannot draw a conclusion about the effect of omega-3 fatty acids on insulin and bone remodeling. On the other hand, we found higher levels of RANKL in children at 2, 5 months and during breastfeeding. In mothers highest values were found during lactation compared to pregnancy and childbirth, this could be explained by the high degree of bone remodeling necessary which is needed to concentrate high calcium amounts in breast milk for the neonate. In newborn, this data suggest the high bone turnover during this stage. High levels of RANKL are related to an increased bone resorption, as it leads to osteoclast differentiation and maturation, as well as an inhibition of apoptosis in osteoclasts (Fernández-Tresguerres et al. 2006). One of the proposed mechanisms by which omega-3 fatty acids, and especially DHA, can regulate bone metabolism is the influence on RANKL, inducing a decrease in this parameter which promotes bone resorption (Griel et al. 2007; Kruger et al. 2010). In this way, we found a significant decrease of this parameter in cord blood vein in mothers who were supplemented with omega-3 LC-PUFA, which may lead to a decrease in bone resorption processes. Regarding to OPG, we found the maximum values in mothers during labour, which might be related to high levels of estrogen, since it is known that this hormone may modify the levels of OPG (Murakami et al. 1998; Takai et al. 1998). This result are related to those shown by Uemura et al. (2002), in which no changes in OPG were detected during pregnancy, but they observed an increase in plasma during at delivery. On the other hand, another research found a gradual increase in OPG during pregnancy together with a decrease in RANKL (Taylor et al., 2003). The source of OPG during pregnancy remains unknown, but its fast decrease at postpartum, the low levels in neonate and the presence of OPG in placental tissue, suggests the placenta may be the source (Sarli et al., 2005). Our omega-3 LC-PUFA supplementation results in increased OPG levels in newborn at birth, showing significantly higher values in arterial cord blood, which could be beneficial for the newborn (Miller 2003; Neyro et al., 2011). We also analyzed the IL-6, which together with TNF-¿, both are cytokines that play a major role in bone remodeling. Evidence suggests that pro-inflammatory biomarkers act on mesenchymal stem cells and osteoclast precursors enhancing bone resorption (Barbour et al. 2012; Abdel Meguid et al. 2013; Korczowska et al., 2013). We found maximum values of IL-6 in mothers during labor, as well as in newborn¿s umbilical cord vein, where we also observed significant differences between groups, with higher IL-6 concentrations for both cases in the control group. Dietary intake of omega-3 LC-PUFA is known to be associated with the decrease pro-inflammatory factors as interleukins (Kajarabille et al. 2013). Omega-3 supplementation ranging from 3 to 6 g/day showed a modest, but rather consistent beneficial effect of these fatty acids in joint disease. In the same way, the syntheses of pro-inflammatory factors as interleukins and TNF-¿ in cartilage tissue were suppressed by dietary supplementation with fish oil containing both EPA and DHA (Shapiro et al. 1996; Watkins et al. 2001). Our omega-3 LC-PUFA supplementation results in decreased IL-6 levels in neonates at birth, showing significantly lower values in venous cord blood, which according to the mentioned above, could be beneficial for bone formation in newborn. However, this cytokine has a role as an inflammatory signal and parturition leads to a significant inflammatory process. Among the inflammatory chemokines and cytokines expressed by trophoblast cells, IL-6 is a classic multifunctional proinflammatory cytokine which is produced by the activated vascular endothelial cell and placenta (Yin et al. 2014; Natalie J. Hannan 2014; Demirturk et al., 2014). TNF-¿ is closely related to the IL-6, it promotes bone resorption and suppresses bone formation by increasing osteoblast apoptosis, and reduced differentiation and proliferation of osteoblasts and their progenitors (Kawai et al. 2012; Panuccio et al. 2012; Straburzy¿ska-Lupa et al. 2013). We found significant differences between both groups in mother at delivery, where supplemented group shows lower values, then omega-3 fatty acids may contribute to decrease the inflammatory process at labor (Marchioni & Lichtenstein 2013), as well as supplementation could also diminish the inflammatory process in the newborn at birth . Regarding to the newborn, we observed lower values of TNFa at 2,5 months after birth, in neonates whose mothers were supplemented, which means that omega-3 fatty acids could help to bone formation, decreasing bone resorption caused by TNFa. (Coates et al., 2011; Munro & Garg 2013; Kajarabille et al. 2013). On the other hand, and regarding to mineral content analysed in erythrocyte cytosol, Calcium (Ca) is one of the minerals which together with bone homeostasis suffers significant changes during pregnancy and lactation due to the increased fetal requirements of calcium, particularly during the third trimester when rapid mineralization of the fetal skeleton occurs, with the subsequent fragility fractures during late pregnancy or during the postpartum period in women (Iwamoto et al. 2012). We found that supplemented mothers at postpartum had higher Ca concentration, thus omega-3 LC-PUFA might help to cope with the great demand of Ca during lactation, in order to concentrate high amounts of Ca in breast milk (Yoon et al. 2000). According to bibliography omega-3 PUFA, and particularly DHA, improved calcium absorption, changing the composition of the intestinal membranes and reduced intestinal calcium loss (Bonnet & Ferrari 2011). With regard to the neonates, we observed significant differences at birth in umbilical cord vein and artery, where supplemented group also showed greater levels of Ca. This is consistent with studies that point out that omega-3 PUFA not only may modulate proinflammatory cytokines and increase the production of IGF1, but also improve the accretion of calcium in the bone (Griel et al. 2007; Kruger et al. 2010). Furthermore, Kruger & Schollum (2005), found that DHA concentrations in the membrane of erythrocyte were associated with bone density and calcium absorption in bone in a cohort of growing rats fed a diet supplemented with tuna oil, and in a randomized study with 40 patients with osteoporosis, subjects taking an omega-3 supplement, also had better absorption of calcium compared with placebo (Rahman et al. 2007). Together with Ca, phosphorus (P) is another essential mineral in developmental stage, during which infants exhibit the fastest mineral accretion and thus have the highest requirements during their lifetime (Christmann et al. 2014) . Regarding to our data, we found significant differences between both groups in mother at delivery and postpartum, where mothers who had supplementation showed higher concentration of P . In the newborn we observed significantly higher values of P in umbilical cord artery, for those whose mothers were supplemented. Therefore, omega-3 supplementation may allow not only on the recovery of P after labor on mothers, but also assures the required amount of P in breast milk which together with Ca, are the main minerals for bone mineralization in the neonate (Miller 2003). Newborns are particularly sensitive to imbalances in iron metabolism (Cornock et al., 2013). Although to date, no study has examined the correlation between Fe and omega-3 fatty acids during pregnancy, our results showed that mothers who were supplemented with omega-3 LC-PUFA during pregnancy and lactation had higher concentrations of Fe comparing to the control. Fe is an essential element for the developing fetus (Li et al. 2008), therefore omega-3 supplementation helps to increase the amount of Fe in order to assure a suitable development in fetus, in the same way, it helps to avoid Fe decrease during labor in mothers. Magnesium (Mg) and copper (Cu) are essential major elements, together with Ca and iron Fe, for the health of the pregnant women and the fetus. It¿s concentrations are altered during pregnancy with the requirements of growing fetus and changes in the mother's physiology (Zhang et al. 2013). For both minerals, our data showed significant differences between groups in mothers at postpartum (at 2,5 months) and in umbilical cord vein in the newborn, where supplemented group had greater Mg and Cu concentrations . Thus, omega-3 fatty acids might help in mineral recovery after labor and in the same way, it assures the optimum mineral content for the newborn. According to literature, zinc requirement during the third trimester of pregnancy is approximately twice as high as that in non-pregnant women (Mistry et al. 2014). Plasma zinc concentrations decline as pregnancy progresses and then paradoxically increase towards delivery (Izquierdo Alvarez et al., 2007). Zinc supplementation during pregnancy has been reported to significantly increase birth weight and head circumference, highlighting the importance of adequate zinc supply during pregnancy (Goldenberg et al. 1995; Mistry et al. 2014). In our study we observed significant differences in newborns in umbilical cord vein as well as at 2,5 months after birth, for both cases supplemented groups had greater Zn content compared to control group. These results suggest that omega-3 LC-PUFA might increase antioxidant function though increase of antioxidant enzymes (Cu/Zn SOD), therefore this antioxidant protection would benefit bone formation. CONCLUSIONS First conclusion During gestation and due to the increased metabolic rate, an increased production of free radicals occurs. We found the highest concentrations of plasma peroxides in the mother during the gestation, as well as during labor. Delivery also implies a strong oxidative stress for both the mother who suffers pain and stress, and for the newborn, who is in the transition between the uterine hypoxic/anaerobic environment to an oxygen saturated atmosphere, leading to an increased peroxide production. In erythrocyte membrane high levels of peroxides are also shown during the postpartum/breastfeeding period in mothers as well as, at 2,5 months of the newborn¿s life. Second conclusion DHA supplementation during last trimester of pregnancy and lactation showed a moderated beneficial effect on oxidative stress in mothers during labor and breastfeeding period. Supplementation decreases oxidative stress in mother, supplementation prevents oxidative damage by increasing fat soluble vitamins in plasma and erythrocyte membrane, as well an increase in activity of antioxidant enzymes such as superoxide dismutase and catalase in erythrocyte cytosol. DHA leads to a moderately higher level in plasma retinol, as well as an increased concentration in ¿-tocopherol and Coenzyme Q10 especially during the breastfeeding. Third conclusion DHA supplementation during the last trimester of pregnancy and lactation showed a beneficial effect on oxidative stress in the newborn at birth as well as during the first months of life. Supplementation decreased oxidative stress in the neonate, by increasing total antioxidant capacity in plasma and antioxidant enzymes activities such as superoxide dismutase and catalase in erythrocyte cytosol. Moreover, supplementation showed a beneficial effects preventing oxidative damage by increasing fat soluble vitamins in plasma and erythrocyte membrane. DHA leads to higher level in plasma retinol, as well as a higher concentration in ¿-tocopherol and Coenzyme Q10 in plasma and erythrocyte membrane of neonates. This increase shown in newborns was more evident compared to that observed in mothers. Fourth conclusion In mothers during labor bone formation seems to predominate, with high levels in ACTH, osteocalcin and OPG, along with lower levels of RANKL. In contrast, during lactation bone resorption predominates, with higher concentrations in osteopontin and RANKL and low levels of ACTH and OPG. Regarding to newborns, in general, an increased bone remodeling activity was observed, compared to the activity found in mothers. Thus, we observed PTH levels greatly reduced in umbilical cord blood, together with highest levels of RANKL in neonates at 2,5 months of life, and higher ACTH values in both, umbilical cord samples as well as at 2,5 months of newborn¿s life. Furthermore, we found highly decreased leptin levels in neonates at 2,5 months of life compared to those observed in umbilical cord. Fifth conclusion With regard to the effects of DHA supplementation in mothers, we could highlight the following ones: supplemented mothers showed higher OPN values during delivery as well as greater PTH values during lactation, which leads to an increased calcium mobilization, probably towards breast milk. Moreover, DHA decreases IL-6 levels, diminishing the inflammatory process in mothers at delivery. Supplementation also increased levels of osteocalcin (OC) in mothers during labor, which might help to restore the bone mass lost during pregnancy. Sixth conclusion DHA supplementation in newborns led to specific results on bone biomarkers, which overall they appear to be beneficial for the neonate. From the standpoint of bone formation, supplementation increased osteocalcin, and decreased PTH levels at 2,5 months of life, besides it increased ACTH levels and decreased RANKL levels in umbilical cord vein; OPG levels were higher in cord artery and leptin levels were also increased in umbilical cord vein and artery. On the other hand, from the point of view of energy metabolism, DHA supplementation increased leptin and ACTH levels at birth, together with a decrease in the inflammatory signaling, decreasing IL-6 and TNF-¿ at birth and at 2.5 months of newborn¿s life. Seventh conclusion DHA supplementation during the last trimester of pregnancy and lactation showed a beneficial effect on the mineral content in erythrocyte cytosol, increasing calcium, phosphorus and iron content. These findings are especially important from the point of view of bone metabolism and erythropoiesis, as well as to ensure an adequate development of the newborn during the early stages of life. Supplementation also increased copper and zinc concentration, which might benefit antioxidant function by increasing copper and zinc dependent antioxidant enzymes such as Cu/Zn SOD, this antioxidant protection would benefit bone formation, since it reduces oxidative damage. Overall conclusion DHA supplementation during the last trimester of pregnancy and lactation could be postulated as a nutritional strategy to avoid and ameliorate oxidative stress in mother and neonates. Supplementation decreased oxidative damage, leading to higher levels in plasma retinol, as well as increased concentration in tocopherol and Coenzyme Q10, in both plasma and erythrocyte membrane samples in neonate and in mothers, especially during lactation. Supplementation also increased the activity of antioxidant enzymes such as superoxide dismutase and catalase in erythrocyte cytosol. Moreover, DHA supplementation enhanced OPN and OC values at delivery, as it decreases IL-6 levels, and it enhanced PTH levels during lactation in mothers. In newborns, supplementation increased OC levels and decreased PTH levels at 2,5 months of life, besides it increased ACTH levels and decreased RANKL levels in umbilical cord vein; OPG levels were higher in cord artery and leptin levels were also increased in umbilical cord vein and artery. Therefore, DHA supplementation during the last trimester of pregnancy and lactation could also be postulated as a nutritional strategy to restore the bone mass lost during pregnancy in mothers and increase bone formation in neonate.