Vitamin E is naturally found in various foods and has 8 stereoisomers, each comprising a 2-methyl-6-chromanol ring; the isomers can be differentiated based on the methylation status of the α, β, γ, δ tocopherols and a phytyl side chain (1,2). Commercially, vitamin E is available both as natural RRR-α-tocopherol and as the synthetic form all-rac-α-tocopherol. Synthetic vitamin E is generated from the reaction of trimethyl hydroquinone with synthetic isophytol, resulting in a mixture of 8 optical isomers, half with chiral centers in the 2R form and the other half in 2S(3–6).
The most abundant form of vitamin E found in human tissues is alpha-tocopherol (α-TOH), its most common form being RRR-α-TOH in healthy adults. This is attributable to the presence of methyl groups in a 2R configuration in the chromanol ring, which results in additional hydrophobic interactions between the tocopherol and α-TOH transfer protein (α-TTP) (7). This occurs in the liver, where α-TTP has a higher affinity for RRR-α-TOH stereoisomers, followed by the 2R-RSR-, RRS-, and RSS-α-TOH forms. 2S forms have not been found in plasma or tissues, and no differences between the activity of natural and synthetic forms have been demonstrated (8–11).
Newborns are considered a high-risk group for vitamin E deficiency and are recommended an intake of 4 mg/day (4). Insufficient consumption of vitamin E at this phase of life can compromise the immune system and affect lung development (12). Premature newborns are more susceptible to vitamin E deficiency, and low levels of α-TOH in this group may lead to complications such as thrombocytosis, hemolytic anemia, retrolental fibroplasia, and intraventricular hemorrhage (13).
The main source of α-TOH for newborns is breast milk. α-TOH prevents possible damage from oxidative stress generated by the transition from a prenatal intrauterine environment relatively poor in oxygen to a postnatal extrauterine environment considerably richer in oxygen (14,15).
Studies have shown that serum levels of α-TOH increase significantly in neonates fed colostrum (16). Maternal supplementation with α-TOH can be an efficient way to boost vitamin E levels in neonates. Most studies evaluating α-TOH supplementation have, however, been conducted in animals, and little is known about the effect of this supplementation in humans, or the influence of supplementation with natural or synthetic forms of α-TOH on the levels of α-TOH in human breast milk (16–19). Thus, the aim of this study was to evaluate levels of vitamin E in human colostrum when lactating mothers were given supplements of either natural or synthetic forms of α-TOH.
A randomized clinical study was performed at the Januário Cicco public maternity clinic located in Natal, RN, Brazil, from June 2012 to December 2013. To calculate the minimum sample size required, a power analysis using the software GPower (version 3; Shimadzu Corporation, Kyoto, Japan) was conducted. The parameters considered were effect size d = 0.5, probability of error α = 0.05, test power β = 0.95, and a confidence interval of 95%. Using these parameters, the required minimum sample size was 22 subjects per group.
The study was a randomized double-blind clinical trial with 3 treatment arms: a control group (CG) without treatment, a group receiving an acetate capsule with natural RRR-α-TOH (GNAT), and a group receiving an acetate capsule with synthetic all-rac-α-TOH (GSINT). All of the participants and researchers were blinded to the treatment groups. Unblinding was performed only after the statistical analysis was completed.
Enrolled participants included healthy women ages 18 to 40 years without signs of infections, syphilis, HIV, gastrointestinal tract and liver diseases, heart disease, diabetes, hypertension, or cancer. Only women who had full-term, healthy deliveries, no history of miscarriage, and no vitamin supplement use during pregnancy were included in the study.
The study was approved by the ethics in research committee—ethics of the Universidade Federal do Rio Grande do Norte, according to protocol 234/11—CEP/UFRN CAAE 0260.0.051.294-11. The clinical trial was registered under number U1111-1144-1649. All of the women formalized their participation by signing the informed consent form.
Sample Collection and Preparation
Blood and milk colostrum were collected from participants after an overnight fast, approximately 12 hours postpartum. The supplemented groups (GSINT and GNAT) received a capsule containing 400 IU of RRR-α-TOH or 400 IU of all-rac-α-TOH following the first milk collection. Milk was collected 24-h after supplementation.
For serum analysis, 5 mL of blood was collected by venipuncture into light-protected polypropylene tubes and transported to a laboratory under refrigeration. The blood was centrifuged, and 1 mL of serum was transferred to a new tube and stored at −18°C.
Colostrum was obtained by manual expression at the end of breast-feeding to prevent fluctuations in fat and tocopherol contents. The foremilk was discarded, and 2 mL of colostrum was collected before and after supplementation. Following collection, colostrum was incubated at 37°C and homogenized, after which a 500-μL aliquot was removed and stored at −18°C.
The technique used to extract serum tocopherol was adapted from Ortega et al (20). Briefly, 1 mL of 95% ethanol was added to an equal volume of serum, and after stirring, 2 mL of hexane was stirred into this mixture. The final mixture was centrifuged for 10 minutes at 500 × g. This step was repeated thrice, amounting to 3 extraction steps, and yielding a total extraction volume of 6 mL.
A similar method to extract tocopherol from colostrum was adapted from Romeu-Nadal et al (21). Equal volumes of colostrum and 95% ethanol were combined and subjected to mechanical agitation for 1 minute, following which extraction was performed in 2 steps with 2 mL of hexane, resulting in a total extraction of 4 mL from the hexane phase. Serum (2 mL) and colostrum (2 mL) extracts were dried and dissolved in 500 μL of absolute ethanol for further analysis.
The concentrations of α-TOH were determined by HPLC (LC-20AT; Shimadzu). Chromatographic separation was performed with a reverse-phase column (CLC-ODS; LC Shim-pack; Shimadzu): 4.6 mm × 25 cm with UV detection (λmax = 292 nm). The elution was isocratic, with a mobile phase consisting of methanol/ultrapure water (Milli-Q; Millipore Corporation, Darmstadt, Germany) 97:3 (v:v) at 1.0 mL/min flow rate). The chromatograms were integrated using the LC-solution program.
The α-TOH was identified and quantified in the samples by comparing the peak area obtained in the chromatogram with the area of the respective standard of α-TOH (Sigma, St Louis, MO) at a retention time of 12.5 minutes. The standard concentration was confirmed by the α-TOH–specific extinction coefficient (ε 1%, 1 cm = 75.8) in absolute ethanol (22). Participants who presented values lower than 499.6 μg/dL (11.6 μmol/L) of α-TOH were considered α-TOH deficient (23).
Quantification of α-TOH in Capsules
Solutions of α-TOH acetate from capsules were prepared and compared with a standard solution of commercially available α-TOH acetate (Sigma). α-TOH in the capsules was identified and quantified by comparing the peak area obtained in the chromatogram (UV detection at λmax = 286 nm) of the test sample with the corresponding area obtained from a standard solution of α-TOH acetate, as well as by the α-TOH acetate-specific extinction coefficient (ε 1%, 1 cm = 40) in absolute ethanol (23). The concentration values of RRR-α-TOH and all-rac-α-TOH were accurate to 98% and 99% of the values reported on the capsules, respectively.
Validation of the Method
For the quantification of α-TOH, calibration curves were established as follows. A stock solution of the standard α-TOH (Sigma) was prepared, and 6 dilutions were made with concentrations ranging between 1.2 and 41.3 μg/mL. The method demonstrated excellent linearity, and the linearity coefficient of the curves always presented values higher than 0.9998. To determine the precision and accuracy, recovery and repeatability tests were performed and expressed as relative standard deviation (RSD), with 3 concentrations (4, 8, and 16 μg/mL) covering the range of variation within the linearity curve when α-TOH was always detected in serum and colostrum. The average recovery rate of α-TOH in serum was 100% and in colostrum, 98.80%. The results showed satisfactory reproducibility, with RSD values of 7.70% for serum and 4.22% for milk.
To determine the limit of detection (LD) and limits of quantification (LQ), colostrum and serum samples from known concentrations were diluted. A sample of each dilution was applied, and the LD of the observed peak was determined when there was no longer a distinction between noise and the analytical signal at a concentration of 0.48 μg/mL. The LQ was determined as the point at which the analytical signal was detected at a dilution equivalent to 0.97 μg/mL.
SPSS version 19 (IBM SPSS Statistics, Armonk, NY) was used for statistical analysis. The concentration data for serum and colostrum are presented as means and standard errors. A preliminary comparison of the average α-TOH concentration before supplementation satisfied the basic normality assumptions of analysis of variance (Kolmogorov-Smirnov test), as well as homoscedasticity (Levene test). The response to treatment was calculated as a percentage increase in the concentration of α-TOH postsupplementation, and the treatment response data were converted into percentage variation (% var). To determine which capsule elicited the best response to the supplementation, the Kruskal-Wallis test for independent samples and Tukey test with 2-way analysis of group averages were performed. The scatter graph and Pearson correlation coefficient were used to determine the correlation between α-TOH levels in serum and milk at 0 hour and those after supplementation.
Of 151 women initially recruited, 109 completed the study. The participants were randomized into 3 arms, namely, a CG receiving no treatment (CG, n = 36), a treatment group receiving natural vitamin E supplementation (GNAT, n = 40), and a second treatment group receiving synthetic vitamin E supplementation (GSINT, n = 33). Sixty-five percent of the women were married, with an average age of 24 ± 6 years (Table 1). The level of education varied: 36% of women had completed primary education, and only 0.9% had attended university. In 81.4% of the women, the monthly family income ranged between US$ 264.08 and 1056.34. Regarding obstetric variables, 53.1% women were multiparous, 58.1% had undergone cesarean section, and the average gestation period was 39.1 ± 1.4 weeks. The women in CG, GNAT, and GSINT had average serum α-TOH concentrations of 1016 ± 52, 1236 ± 51, and 1083 ± 61 μg/dL, respectively. There were no significant differences among the groups (P = 0.546). Of the participants, 6.6% had low α-vitamin E levels, with α-TOH serum concentrations ranging from 499.6 to 697.7 μg/dL. None of the participants had α-TOH deficiency. The analysis of variance of α-TOH concentrations in colostrum before supplementation revealed no difference in the mean (P = 0.253) or the variance (P = 0.767) among the 3 treatment arms. A comparison of α-TOH concentrations (Fig. 1) in colostrum at 0 and 24 hours demonstrated that women who received no treatment (CG) had similar concentrations at both periods. Nevertheless, the average concentration of α-TOH in the milk collected at 24 hours from women supplemented with natural (GNAT) and synthetic (GSINT) α-TOH increased by 57.6% and 39.0%, respectively.
The average %var before and after supplementation (Fig. 2) were statistically different in the 2 experimental groups. The GNAT group presented the highest percentage of variation as confirmed by a Kruskal–Wallis test for independent samples (P = 0.000). The average concentration for each group was also compared using Tukey test and revealed that the average of CG differed from those of GNAT (P = 0.000) and GSINT (P = 0.040), and that the average of GNAT differed from that of GSINT (P = 0.040). When comparing the %var of CG with that of the supplemented groups, the variation after supplementation with the natural capsule was 101% and 51.6% higher than CG and GSINT, respectively, when compared with the same group without supplementation.
The means and %var in response to supplementation showed a significant difference between the GNAT and GSINT groups (P = 0.04). The women receiving the natural vitamin E supplement had on average a 49.6% higher concentration in the milk of α-TOH than those receiving the synthetic capsule, indicating that the natural form results in a greater increase in α-TOH in colostrum.
Concentrations of α-TOH in serum were compared with those of colostrum (0 hour) for all groups, and no correlation between these variables (r = 0.073, P = 0.440) was found. No correlations were observed in the α-TOH serum biochemical analysis between the GSINT and GNAT groups, and concentrations of α-TOH in colostrum after supplementation.
The inclusion criteria established in the study allowed us to compare maternal characteristics such as age, parity, weight, and type of delivery among the groups. Maternal serum α-TOH levels less than 499.6 μg/dL suggest vitamin E deficiency; values between 499.6 and 697.7 μg/dL indicate some risk of deficiency, while values more than 697.7 μg/dL are considered healthy (23). The mean concentrations of α-TOH in the serum of the studied groups were as follows: CG (1016 ± 52 μg/dL), GNAT (1236 ± 51 μg/dL), and GSINT (1083 ± 61 μg/dL). These values are acceptable, based on reference values for serum α-TOH concentrations. Similar concentrations were found in other studies (24–26).
When individual breastfeeding women were assessed, we found that 6.6% had a low nutritional status for α-TOH. This value is lower than the 12% previously reported by Lira et al, which can be explained by the participation of adolescents in their study.
The concentration of α-TOH in colostrum at 0 hour is within the expected range when compared with studies conducted in Brazil: Dimenstein et al (24) reported 1363.2 ± 727.9 μg/dL; Garcia et al (27), 1206 ± 858.5 μg/dL; and Campos (28), 1313.9 ± 798.7 μg/dL. These values are compatible with the present findings. Studies performed in the United States and Turkey also showed similar concentrations, that is, 1335.2 ± 198.1 and 1326.6 ± 68.9 μg/dL, respectively (29,30).
Other studies have reported varying levels of α-TOH in colostrum. Schweigert et al (31) and Quiles et al (32) reported colostrum α-TOH values of 2200.9 and 2454.9 μg/dL, which are higher than the ones reported in this study, whereas Ahmed et al (33) reported values lower than 919.1 μg/dL. The higher concentrations found in studies conducted by Schweigert et al (31) and Quiles et al (32) may be owing to the Mediterranean diet being rich in almonds, olives, nuts, and fish, which are rich sources of α-TOH. The below-average colostrum values found in the study by Ahmed et al (33) may be attributed to the low socioeconomic status of the population, which may possess a very low diversified supply of the vitamin (31–33).
In the present study, women treated with both the natural and the synthetic forms of α-TOH demonstrated an increase in concentration of α-TOH in colostrum. To date, similar studies with postpartum maternal supplementation with α-TOH have not been performed. Comparisons with studies in other mammals such as mares, pigs, rats, and cows, however, showed a significant increase after supplementation (16–18,34). Results demonstrating an increased efficacy of the natural form of α-TOH to improve maternal concentrations of vitamin E over the synthetic form have been reported in animal studies, wherein investigators supplemented cows with natural and synthetic forms and showed results similar to those found in humans (17,35,36). Cows fed diets supplemented with all-rac-α-TOH preferentially use the 2R-α-TOH stereoisomer, and such discrimination may occur in humans.
Slots et al (16) observed differing proportions of 2R-stereoisomers of α-TOH in cow milk. They reported that RSS, RRS, and RSR-α-TOH accounted for 2.4%, 2.9%, and 2.1%, respectively, in milk, which is considered low. The natural form represents 92.6% of total α-TOH in milk. None of the 2S stereoisomers was detected in the milk before or after supplementation with the synthetic form (16). The 2S-stereoisomers are not retained in the plasma or in tissue, and the relative activity of the synthetic form compared with the natural form is 50% (4). Similar values were observed in this study, as the natural capsule increased α-TOH concentrations in milk 49.6% more than the synthetic capsule. This suggests that there may be a transport mechanism of α-TOH to the mammary gland in humans, who depend on the activity of α-TTP. The stereoisomer selection mechanism works continuously from the liver into the plasma after birth, and according to Lauridsen et al (37), the presence of an α-TTP–dependent mechanism in the mammary gland cannot be excluded because the expression of α-TTP can facilitate α-TOH secretion into milk (37). Another alternative mechanism for transporting α-TOH has been suggested, wherein α-TOH can reach the breast tissue by means of low-density lipoprotein receptors and/or be transported by SR-B1 receptors, possibly involving intracellular membrane receptors for α-TTP in the mammary epithelium (38,39).
The lack of correlation between α-TOH in maternal serum with colostrum (r = 0.073, P = 0.440) has been reported (24,27,30,34,40). Dimenstein et al (24) proposed that the absence of correlation between these variables preclude the existence of passive transfer mechanisms during the passage of vitamin E from the mammary gland to the milk, thereby indicating the existence of a transport mechanism in the breast that is independent of plasma concentration. This could be valid because in late pregnancy and at the beginning of lactation, there is a gradual decrease in serum levels of α-TOH, which may explain the considerable increase in α-TOH in colostrum (37,41,42). This phenomenon may be because of an increased activity of low-density lipoprotein receptors by the mammary gland around parturition. The concentration of α-TOH has been found to depend on the plasma lipid fraction, that is, the ability of plasma lipoproteins to incorporate α-TOH, which ultimately results in the absorption of α-TOH by the mammary gland (2). Therefore, α-TTP would have an important role in the absorption of vitamin E to the mammary gland because its function is to transfer α-TOH to the lipoproteins.
Supplementation with natural and synthetic forms promoted α-TOH increase in colostrum when compared with the group without supplementation. Interestingly, the natural form of α-TOH was more efficient in increasing the preexisting levels than the synthetic form.
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