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Original Articles: Nutrition

Alpha-Tocopherol Concentration in Colostrum and Serum of Women With Premature Labor

Medeiros, Jeane Franco Pires*; da Silva Ribeiro Rodrigues, Karla Danielly; Lima, Mayara Santa Rosa*; da Silva, Anna Larissa Cortês; de Queiroz, Jaluza Luana Carvalho; Dimenstein, Roberto*

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Journal of Pediatric Gastroenterology and Nutrition: February 2016 - Volume 62 - Issue 2 - p 348–352
doi: 10.1097/MPG.0000000000000969
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Abstract

In premature births, which occur before 37 completed weeks of pregnancy, the risk of perinatal morbidity and mortality of infant and child is much greater the lower the gestational age and birth weight are. There are risks in adapting to extrauterine life, because of the immaturity of organs and systems (1), and with premature infants with gestational age of 32 weeks or less, having a higher risk of complications (2).

Premature children, especially those with very low birth weight, have a high risk of nutritional deficiencies (3,4) and have low body reserves of fat-soluble micronutrients, including vitamin E (5). The most active form of vitamin E is alpha-tocopherol (6). In newborns, serum levels of this vitamin are reduced (7). Authors suggest that the lower the gestational age and birth weight are, the higher the vitamin E deficiency is (8–10). It is believed that this event occurs because of limited placental transfer, alpha-tocopherol accumulation, especially in the third trimester, limited absorption, excessive fetal metabolism of vitamin E, oxidative stress in labor, and the increased requirements in the pre-term child (11,12).

Vitamin E is a powerful antioxidant. Several nonantioxidant functions have, however, been attributed to it such as posttranslational and transcriptional level modulator and inhibitor of cell proliferation, platelet aggregation, and monocyte adhesion (13). A lack of vitamin E has been associated with hemolytic anemia and involvement in the development of the central nervous system, immune system, and lungs in children, particularly in preterm (14–16).

Every year, 15 million neonates are born preterm and with low birth weight, one-third of which die before reaching 1 year of age. For every 10 newborns weighing <1000 g, 9 do not survive the first month of life (17). In 2013, of the 6.3 million estimated deaths of children worldwide, nearly 1.1 million were because of premature births (18). For the survival of these patients, it has been highlighted that the quality of food offered is key. Presently, The United Nations recommends that breast milk should exclusively be offered to children up to 6 months of life (19,20) for presenting a unique combination of proteins, lipids, carbohydrates, minerals, vitamins, enzymes, and live cells (21). Thus, breast milk should be the primary source of alpha-tocopherol for the newborn, avoiding the possible damage of oxidative stress.

Milk from mothers of premature infants presents differences in the composition of protein-energy intake and immune constituents compared with that produced by mothers of full-term newborns (22). For the alpha-tocopherol levels in breast milk, Quiles et al (23) found higher values in women with term delivery. This can be explained by the tendency to increase the serum concentration of vitamin E with the progress of pregnancy, except in cases of fetal complications or high-risk situations for the mother (24). This increase in maternal serum concentration occurs because during the third quarter there is a reserve of this vitamin in the fetus (25).

Therefore, if premature babies have low body reserves of vitamin E, it is important to assess whether the degree of prematurity is related to alpha-tocopherol levels in serum and maternal milk, and how the vitamin E needs of the premature infant supplied through breast milk decreases the damage caused by oxidative stress, thereby contributing to improvement in neonatal care.

METHODS

The study was cross-sectional, conducted between March 2013 and May 2014. The sample size was calculated using the GPower program version 3.1, considering an average of 200 births per month, and a minimum sample of 54 subjects was estimated for a confidence level of 95%.

Sixty-five adult women ages 18 to 45 years participated in the study, with gestational age <37 weeks and showed no multiple pregnancy, without malformation of the newborn; they did not use supplements containing vitamin E during pregnancy and did not have associated diseases (heart disease, cancer, gastrointestinal tract and liver diseases, syphilis, HIV). The recruited women were informed about the research objectives, signed the form and informed consent in accordance with the research ethics committee (CAAE 21778213.0.3001.5292) of the Federal University of Rio Grande do Norte. They were divided into 2 groups: group 1 consisted of mothers with gestational age of <32 weeks (n = 18), featuring severe prematurity; and group 2 was composed by mothers with gestational age of 32 weeks and <37 weeks (n = 47), featuring moderate prematurity (17). Gestational age was calculated by the Capurro method.

The variables of maternal profile used were maternal age, education, occupation, parity (defined as the number of previous pregnancies), type of delivery, marital status, and pregestational nutritional status. The newborn variables were the use of breast milk as food at birth and birth weight.

After overnight fasting, blood samples (5 mL) and colostrum milk (2 mL) of nursing mothers were collected up to 12 hours after birth. Samples were placed in polypropylene tubes protected from light and transported to the laboratory under refrigeration. The blood collection was done by venipuncture and colostrum was obtained manually from the breasts, always at the end of the feeding to avoid fluctuations in the tocopherol content and fat. Foremilk was discarded.

The Ortega (26) method was adopted for extracting tocopherol from the serum. An equal amount of 95% ethanol was added to 1 mL of serum, the mixture was stirred, and then 2 mL of hexane was added in 3 extraction steps; the mixture was stirred and centrifuged at each step (10 minutes, 1073g). The supernatant hexane layer was extracted and placed in another tube totaling 6 mL.

For milk, the Romeo-Nadal (27) was adapted, in which an equal amount of 95% ethanol was added to 500 μL of colostrum. The tube containing the mixture was mechanically stirred for 1 minute. The extraction procedure was carried out in 2 steps, each with 2 mL of hexane, stirred for 1 minute and centrifuged (10 minutes, 1073g), resulting in a 4-mL total removal of the hexane phase. The extracts of both samples were evaporated in a water bath at 37°C.

The concentration of alpha-tocopherol in samples of serum and colostrum were determined by high-performance liquid chromatography. After evaporation, the extracts were dissolved in absolute ethanol (Vetec) and 20 μL were applied onto the Shimadzu LC-20AT high-performance liquid chromatography chromatograph apparatus coupled with a SPD-20A Shimadzu UV-VIS detector, LC Solution software with a LiChroCART 250–4 column, 100% methanol mobile phase flow rate of 1.0 mL/min, and with 292 nm detection for alpha-tocopherol.

The identification and quantification of alpha-tocopherol in the samples were established by comparing the peak area in the chromatogram obtained with the area of the respective standard of alpha-tocopherol (SIGMA). The standard concentration was confirmed by the specific extinction coefficient in absolute ethanol and 1%, 1 cm = 75.8, as used by Nierenberg and Nann (28).

Maternal serum levels of alpha-tocopherol inferior to 11.6 μmol/L were indicative of vitamin E deficiency, between 11.6 and 16.2 μmol/L were at-risk values, and above 16.2 μmol/L were considered acceptable (29).

Statistical analyses were performed using Statistica V.7 program, and the data were expressed as average and standard deviation. To test the differences between the means of numerical data, we used the Student t test for unpaired samples. The relation between alpha-tocopherol concentrations in serum and colostrum, and serum and birth weight was performed by Pearson correlation. Differences were considered significant if P < 0.05.

RESULTS

The women included in the study had a mean age of 26 years and most were multiparous. Prepregnancy nutritional status of participants was 51% being of normal weight, followed by 25% overweight, 17% obese, and 7% were underweight. Approximately 75% of mothers were married or in stable relationships (Table 1).

TABLE 1
TABLE 1:
Assessment of the mothers and newborn in the study

All of the newborns received breast milk; however, 6.2% (n = 4) also received other milk as a complement. The average weight of children in groups I was 1223.3 ± 373.0 g and group II was 2128.3 ± 521.8 g, with a significant difference (P < 0.001). The average gestational age in group I was 29.2 weeks ± 2.4 and the group II was 34.5 weeks ± 1.5.

The average concentration of alpha-tocopherol in the serum of women in group I was 22.2 ± 4.4 μmol/L, and group II had a mean concentration of 27.1 ± 8.6 μmol/L, with a significant difference (P = 0.02). The serum levels of alpha-tocopherol indicated nutritional risk of 5.6% (n = 1) in women with severe prematurity and 4.3% (n = 2) for those with moderate prematurity. The concentration of maternal serum alpha-tocopherol was not related to birth weight, regardless of the degree of prematurity (P > 0.05) (data not shown).

The average values of alpha-tocopherol found in colostrum milk in groups I and II were 34.5 ± 20.2 μmol/L and 35.1 ± 16.3 μmol/L, respectively. There was no significant difference in colostrum between groups (P = 0.90). There was also no association between maternal serum and alpha-tocopherol levels in colostrum, as the result of the correlation between serum tocopherol in colostrum and milk severe prematurity obtained (r = −0.17 and P = 0.50) and moderate prematurity (r = −0.03 and P = 0.84).

DISCUSSION

Care for the premature babies went through several changes, focusing on individualized family-centered care aiming at the quality of life of the babies. This care includes the mother's practice to offer breast milk as early as possible (30), because breast milk is considered the ideal food for growth and proper development for children (31).

The average value of alpha-tocopherol found in the serum of severe preterm infants was lower than that found in the group with moderate prematurity. Both groups showed satisfactory biochemical nutritional status of vitamin E; however, by individually analyzing these women, it was found that serum levels of 5.6% alpha-tocopherol of the women with severe prematurity and 4.3% with moderate prematurity indicated nutritional risk. This may suggest that vitamin E deficiency, although rare in humans, can develop in vulnerable groups such as infants.

The difference in the concentration of alpha-tocopherol found between the groups can be explained by the positive association of vitamin E with present gestational age (32), because the alpha-tocopherol accumulation in the fetus occurs in the last quarter of pregnancy (33). The low concentration of alpha-tocopherol in serum pregnancy and postpartum periods can cause harm to mother and child. Research suggests that vitamin E increases the release of prostacyclin, an arachidonic acid metabolite, which inhibits platelet aggregation, soothes uterine contractility and increases vasodilation. Thus, it is possible that the circulating alpha-tocopherol concentrations are associated with fetal growth by increasing blood flow and passage of nutrients to the fetus (5), as well as preventing oxidative damage to DNA, proteins, and lipids (25,34). Furthermore, it is possible that low levels of alpha-tocopherol in maternal plasma are associated with increased risk of prematurity (35).

A study in Germany with mothers who had premature births found levels of alpha-tocopherol in the serum of 41.1 μmol/L, a higher value than the 2 groups of our research (36). Baydas et al (37) found a concentration of 30.2 μmol/L, which is similar to our group with moderate prematurity. These differences may be justified by the difference in eating habits between countries.

Alpha-tocopherol values found in colostrum of infants with severe prematurity were lower than moderate prematurity, but did not present significant difference. As the placental transfer of vitamin E during pregnancy is limited, human milk is the only source of vitamin E for babies who are exclusively breast-fed. Studies of premature babies have shown that breast-feeding reduces the incidence of infection, thrombocytosis, hemolytic anemia, retrolental fibroplasia, intraventricular hemorrhage, pulmonary bronchial dysplasia, spinocerebellar degeneration, and improves neurodevelopmental results (14,27).

Reference values for concentrations of alpha-tocopherol in human milk do not exist. David and Tudehope (38) showed that the composition of the milk varies with the time of gestation and continues to undergo changes over time. Our values were similar to those found by Grilo et al (39) (30.7 μmol/L) and Quiles et al (23) (38.0 μmol/L), who also evaluated the levels of alpha-tocopherol in colostrum milk in mothers with preterm births (<37 weeks’ gestation) without subdividing the degree of prematurity.

The health and nutritional states of breast-feeding mothers and newborns are closely linked with breast milk, because the interrelation of vitamin concentration in breast milk and the baby's plasma has been demonstrated (40). Mothers with poor nutritional status and low dietary intake are likely to have lower levels of vitamin E in milk and, as a result, their children may become vulnerable to vitamin deficiency. This risk increases when the baby is premature, and therefore at risk for vitamin E deficiency as they have low reserves of fat-soluble vitamins and need a greater contribution for the maturation of tissue (41–43).

No correlation was found when comparing the concentration of alpha-tocopherol in maternal serum with colostrum milk in both groups, even for those with severe prematurity. The lack of association between these variables indicates a compensatory mechanism of transfer in order to adapt the vitamin content of breast milk to the needs of the preterm baby (12). Mardones and Rigotti (44) suggested alternative mechanisms for alpha-tocopherol transport to breast milk, where alpha-tocopherol reaches the milk through routes for low-density lipoprotein receptors and/or transported by SR-B1 receptors. This transport may also involve membrane receptors and intracellular alpha-tocopherol in mammary epithelium (45).

Preterm birth is the most common direct cause of neonatal mortality, and proper neonatal care, including newborn feeding, can substantially reduce mortality (21). In many developing countries, children weighing <2000 g and having <32 weeks of gestation have little chance of survival. In contrast, the survival rate of babies born at 32 weeks is similar to that of full-term infants (46).

CONCLUSIONS

Our study contributes to improvements in the care of preterm newborns, because a difference in alpha-tocopherol maternal serum concentration was found between severe and moderate prematurity, making newborns with less gestational age more likely to develop vitamin E deficiency. This emphasizes the importance of breast-feeding to minimize the damage caused by oxidative stress and improve survival rate. In addition, our data provide new information on the alpha-tocopherol content in the milk of mothers with premature delivery. Thus, it is possible to conclude that severe prematurity affects the levels of alpha-tocopherol in maternal serum. The level of prematurity, however, did not alter the concentration of vitamin E in colostrum.

Infants and Alcohol

As early as the 4th century BCE, history has recorded incidences of wine and beer being fed to newborns:

It is a common thing for convulsions to attack an infant…Mischievous regards this affection is wine, dark more than white and wine not diluted with water

Aristotle (384–322 BCE), On the History of Animals, Book 7

Wine was perceived as a healthier source of fluid than plain water and generally was “cut” with water. Well into the apogee of the eastern Roman Empire (900–1100), white wine was the preferential drink for toddlers through adolescence. Following the Great Pestilence, wine fell into disfavor over beer (perhaps as a result of crop failures during the Little Ice Age, and the resulting dearth of grapes), and beer both in mainland Europe and the British Isles became the preferential fluid ingredient of pap.

Infants are given with good result broth made of beer mixed with boiled bread and butter which is quite nourishing. Wine should not be given to infants, but in our parts beer is given to them with advantage.

Daniel Sennert (1572–1637), De Mulierum et Infantius Morbis

Gin also was given to children as a soporific, and its consumption by adults and children was a significant public health issue in England during the eighteenth century. In the 17th century, gin had been an expensive libation imported from the Netherlands. The British government encouraged domestic distillation through a series of legislation with large tax incentives. Gin then became locally produced and a very cheap source of alcohol; a penny's worth was sufficient to intoxicate the average adult. Consumption of gin in England rose from 527,000 gallons in 1685 to 11,000,000 gallons in 1750 … parents gave their infants and children as much as 60 ml of gin on a routine basis to sedate them. William Hogarth's (1697–1764) Gin Lane rendered visual expression of the social malady.

A.R. and P.A. Colón, A History of Children

Engraving from Gin Lane (1751). Courtesy of Wikimedia Commons.

Figure
Figure

—Contributed by Angel R. Colón, MD

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Keywords:

maternal; milk; prematurity; vitamin E

© 2016 by European Society for Pediatric Gastroenterology, Hepatology, and Nutrition and North American Society for Pediatric Gastroenterology,