BREASTFEEDING MEDICINE Volume 10, Number 4, 2015 ª Mary Ann Liebert, Inc. DOI: 10.1089/bfm.2014.0143
Effect of Vitamin C and E Supplementation on Total Antioxidant Content of Human Breastmilk and Infant Urine Asghar Zarban,1,2 Mahsa Mostafavi Toroghi,2 Marziye Asli,2 Masumeh Jafari,2 Morteza Vejdan,2 and Gholamreza Sharifzadeh 3
Abstract
Objectives: After delivery and birth, mothers and neonates are exposed to oxidative stress. The present study examined the effect of supplementation of the diet of breastfeeding mothers with vitamin C and E to improve the antioxidant content of breastmilk and evidence of antioxidant activity in infant urine. Subjects and Methods: The subjects were 60 healthy lactating breastfeeding mothers and their infants 1–6 months of age. They were randomly allocated to a control group (n = 30) consuming a free diet or an experimental group (n = 30) consuming a free diet supplemented each day with effervescent tablets of vitamin C (500 mg) and chewable tablets of vitamin E (100 IU). After 30 days, the total antioxidant content of the mothers’ breastmilk and evidence of antioxidant activity in the infants’ urine were measured by the ferric reducing/antioxidant power assay. The free radical scavenging activity of the urine samples was measured by the a,a-diphenyl-b-picrylhydrazyl method. Differences pre- and postintervention were compared within and between the groups. Results: Significantly higher levels of antioxidants in the breastmilk (610 – 295.5 to 716 – 237.5 lmol/L) and infant urine (43.2 – 21.8 to 75.0 – 49.2 lmol/mg creatinine) were observed in the experimental group over the control group ( p < 0.05). A significant increase in evidence of free radical scavenging in infant urine was observed in the experimental group after 30 days of supplementation by mothers ( p < 0.05). Conclusions: Consumption of vitamin C and E supplements appears to have a positive effect on total antioxidant content of breastmilk and evidence of antioxidant activity in infant urine.
Introduction
H
uman breastmilk contains essential components (vitamins, minerals, essential amino acids, and fatty acids) for infant health and is considered to be the ideal nutrient source for neonates and infants for the first 6 months of life.1,2 The composition of human milk can be affected by maternal nutritional factors.3 After birth, infants are exposed to high concentration of oxygen, which is potentially toxic because of the production of reactive oxygen species and free radicals.2,4 When the rate of free radical production exceeds the rate of its removal by cellular defense mechanisms, a condition known as oxidative stress occurs. This is a contributing factor to the pathogenesis of neonatal diseases such as bronchopulmonary dysplasia and necrotizing enterocolitis.4–6
Human breastmilk contains defense mechanisms that can suppress oxidative stress.7 The antioxidant capacity of human milk derives from numerous bioactive components having varying capacities for antioxidant activity. Enzymatic antioxidant ingredients, such as catalase, superoxide dismutase, and glutathione peroxidase, and nonenzymatic ingredients, such as vitamins E and C, retinol, b-carotene, lactoferrin, and glutathione, present in human breastmilk play a protective role against reactive oxygen species.2,7,8 It appears that the antioxidant content of breastmilk is affected by maternal antioxidant status and can influence the antioxidant status of breastfed infants.9 The present study evaluated the effect of consumption of vitamin C and E supplements by the mother on the antioxidant content of breastmilk and evidence of antioxidant activity in the urine of breastfed infants.
1 Biochemistry Department, 2Antioxidants & Metabolism Research Center, and 3Social Medicine Department, Birjand University of Medical Sciences, Birjand, Islamic Republic of Iran. This study is registered with the Iranian Registry of Clinical Trials with clinical trial registration number IRCT2012072410379N1.
214
ANTIOXIDANT CONTENT OF HUMAN BREASTMILK
215
Subjects and Methods
This study was approved by the Ethics Committee of the Birjand University of Medical Sciences, Birjand, Islamic Republic of Iran. It was a randomized clinical trial with a control group. In total, 60 healthy lactating women and their infants having similar socioeconomic backgrounds were selected from a health center in Birjand. The women were 20– 50 years of age, and the infants were 1–6 months of age. Mothers were fully informed about the aim of the investigation and consented to the participation of the infant in the study. The primary requisite for inclusion was the mother’s intent to exclusively breastfeed from birth. Exclusion criteria were consumption of infant formula by the infants, illness requiring intensive care admission, major congenital anomaly, diabetes, infectious disease, or genetic disease of either the mother or infant. The mothers and their infants were randomly and alternately divided into two groups; the mothers were the criterion for randomization, and the infants were placed in the same group as their mothers after randomization. The placement of the first mother and infant into an experimental or control group was done by asking the mother to randomly select one of two cards on which were written the terms ‘‘control’’ and ‘‘experimental.’’ The remaining mothers with their infants were then alternately assigned to the two groups. The age, weight, and height of all subjects were measured at the onset of the study. Samples of milk from the right breast were collected manually between 9:00 and 11:00 a.m. when the infants had not breastfed for at least 1 hour. Urine samples were collected from the infants using a standard urine bag. All samples were transferred to the laboratory under sterile and cold conditions and stored at - 70C for further analysis. The mothers in the experimental and control groups were directed to consume a free diet. Both groups received suggested dietary guidelines for nutritional dietary habits. They were requested not to consume other supplements and not to change their diets during the study. The mothers in the experimental group asked to consume one effervescent tablet of vitamin C (500 mg) and one chewable tablet of vitamin E (100 IU) each day for 30 days. The control group did not receive supplements. Milk and urine samples were again collected from the mothers and infants, respectively, at the end of the 30-day treatment program. Analytical method
All samples collected before and after intervention were analyzed together. The total antioxidant capacities of milk and urine were measured using the ferric reducing/antioxidant power (FRAP) assay developed by Benzie and Strain10 as a direct method for measuring total antioxidant power of biological fluids with slight modification. To increase the accuracy of analysis, each breastmilk sample was tested three times, and the results were averaged. The free radical scavenging activity of the urine samples was measured using the a,a-diphenyl-b-picrylhydrazyl (DPPH) assay proposed by Brand-Williams et al.11 with slight modification. The creatinine content of the urine was measured using the Jaffe method with a standard kit (Pars-Azmoon, Tehran, Iran). The ratio of parameters related to the antioxidant capacity of creatinine in the urine was used to control for the effect of urine concentration. The result for the FRAP assay was
Table 1. Comparison of the Mothers’ and Infants’ Demographic Variables for Case and Control Groups
Mothers Age (years) BMI (kg/m2) Parturition NVD Cesarean section Infants Age (days) Weight (kg) Sex Male Female
Control group (n = 30)
Case group (n = 30)
29.1 – 8.4 23.6 – 4.5
27.4 – 7.4 23.6 – 4.9
23 (76.7) 7 (23.3)
24 (80.0) 6 (20.0)
99.8 – 31.2 5.6 – 1.1
85.5 – 36.6 5.5 – 1.2
14 (46.7) 16 (53.3)
14 (46.7) 16 (53.3)
p value 0.44 0.9 0.75
0.13 0.7 1
Data are presented as mean – standard deviation values or frequency (percentage), as indicated. BMI, body mass index; NVD, normal vaginal delivery.
presented as lmol of creatinine/mg, and that for the DPPH method was presented as %/mg creatinine. Statistical analysis
The data was expressed in terms of frequency (percentage) and mean – standard deviation value to test for a normal distribution using the Kolmogorov–Smirnov test. The pairedsamples t test was used to compare the means before and after intervention (consumption of vitamin C and E supplements). An independent-samples t test was used to compare mean change (difference between values before and after intervention) between the experimental and control groups. Nonparametric tests (Mann–Whitney U test and Wilcoxon) were used for those variables that were not normally distributed. The chi-squared test was used to determine differences between groups for gender of infants and method of parturition. All differences were considered to be statistically significant at p < 0.05. All statistical analyses were done using SPSS version 16 software (SPSS, Inc., Chicago, IL). Results
No significant differences were observed between groups for type of parturition (normal vaginal delivery and cesarean section), mean age, and body mass index of the mothers and for gender, mean age, and weight of the infants (Table 1). Table 2 shows the results of the FRAP assay for breastmilk and the FRAP/creatinine and DPPH/creatinine assays of the infant urine before and after intervention for the experimental and control groups. A significant increase in all values was seen for the experimental group after intervention. The mean change was also significantly different for the FRAP of breastmilk and the FRAP/creatinine and DPPH/creatiinine of infant urine (Table 2). Discussion
This investigation assessed the effect of vitamin C and E supplementation on the antioxidant content of breastmilk of
216
ZARBAN ET AL.
Table 2. Comparison of the Mean of the Antioxidant Indices and the Mean Change, Before and After the Intervention, in Case and Control Groups Control group (n = 30) Variable Breastmilk FRAP (lmol/L) Infant’s urine DPPH/Cr (%/mg Cr) FRAP/Cr (lmol/mg Cr)
Case group (n = 30) Before
After
p valuea
p valueb
0.388
610 – 295.5
716 – 237.5
0.017c
0.02c
0.2 0.8
4.85 – 1.9 43.2 – 21.8
8.2 – 4.5 75.0 – 49.2
0.005c 0.025c
0.002c 0.03c
Before
After
p value
690 – 285.7
653 – 220.6
6.5 – 5.7 54.8 – 23.4
5.03 – 1/7 52.7 – 33
a
Data are mean – standard deviation values. a Comparison of the mean of antioxidant indices before and after the intervention. b Comparison of the mean change (the difference between the values before and after the intervention in each group) between the case and control groups. c Significant at p < 0.05. CR, creatinine; DPPH, a,a-diphenyl-b-picrylhydrazyl; FRAP, ferric reducing/antioxidant power.
lactating mothers. A significantly higher level of antioxidants was recorded for the breastmilk in the experimental group over that from the control group. It appears that this increase is in response to the consumption of vitamin C and E supplements by the mothers in the intervention group because there was no such increase in the control group (Table 2). Previous studies have determined several antioxidant nutrients and correlated their values in a mother’s diet. A relationship between antioxidant vitamins and provitamin levels in human breastmilk and diet during pregnancy has also been reported.12 The total antioxidant content of breastmilk decreases during the course of lactation and can be the result of depletion of antioxidants stored in the mothers. The large variation in total antioxidant content reported suggests the need for mothers with low values to take steps to improve their nutrition, especially the consumption of natural antioxidants during lactation.2 Micronutrients such as vitamins C and E are essential to the health of the antioxidant defense system that protects against damaging reactive species that arise as by-products of metabolism.13 Because breastmilk is the only provider of antioxidants for breastfed infants, the mother’s diet is essential to development of a shield against oxidative stress for infants exclusively fed breastmilk. It has been reported that the antioxidant content of transitional and mature milk may be related to the mother’s dietary intake of vitamins A, C, and E during the third trimester of pregnancy.12–16 Byerley et al.17 reported no significant influence on ascorbic acid output by human breastmilk of lactating women after supplementation with 1,000 mg/day; however, this study was a short-term intervention with only five participants. Daneel-Otterbech et al.18 found that the ascorbic acid content of human breastmilk can increase two- to threefold with increased intake of ascorbic acid by lactating women with breastmilk showing low ascorbic acid content. The results of that investigation highlighted the importance of encouraging regular consumption of ascorbic acid–rich foods like fresh orange juice by lactating mothers to ensure adequate human milk ascorbic acid output. Hoppu et al.19 found that maternal intake of vitamin C in the diet, but not from supplements, influenced the concentration of vitamin C in breastmilk. Codon˜er-Franch et al.20 reported that supplementing the diet of breastfeeding mothers
with nonalcoholic beer, a product rich in antioxidants, increased the antioxidant content of breastmilk. The results of the present study were in accordance with the results of Nikniaz et al.,21 who found a significant increase of total antioxidant content of breastmilk of mothers in the treatment group receiving symbiotic supplementation, but a decrease for the placebo group. Previous studies have primarily examined the effect of supplements and vitaminrich foods on the contents of breastmilk. The present investigation also measured a significant increase in the antioxidant power in the urine of infants after ingestion of supplements by mothers as measured by the FRAP and DPPH assays (Table 2). These findings confirm the previous highly significant correlation between the antioxidant content of milk and vitamin supplementation of mothers. Codon˜er-Franch et al.20 found no difference in oxidant markers in infant urine after consumption of nonalcoholic beer by breastfeeding mothers. The data from the present study show a high antioxidant status of the mothers was transferred via the breastmilk to the infants as evidenced by the excessive load discharged in the urine of the infants. A previous study indicated that a healthy balanced diet may outweigh single dietary supplements, but the results of the present study suggest that supplementation of high doses of vitamins C and E can increase the total antioxidant content of breastmilk. This is especially important where there are large differences between maternal diet and the nutritional content of the breastmilk, given the obvious importance of breastmilk as the sole antioxidant source for breastfed infants.2,18,19 Further study is required to evaluate antioxidant indices for infant plasma to confirm these results. Conclusions
Daily intake of 500 mg of vitamin C and 100 mg of vitamin E as supplements by lactating mothers with breastfed infants for 30 days significantly increased the antioxidant capacity of the breastmilk. It also significantly increased the antioxidant content of infant urine, which indicates the transfer of antioxidants from the breastmilk to the infants and the removal of the excessive load in the urine of the infants. The results of this study suggest that the use of antioxidants early in life can decrease oxidative stress and help prevent broad-spectrum diseases by balancing between free radicals and antioxidants.
ANTIOXIDANT CONTENT OF HUMAN BREASTMILK
217
Acknowledgments
The authors wish to thank the Deputy of Research and Technology of Birjand University of Medical Sciences for his support of this project. The contributions of the mothers and their newborns are also gratefully acknowledged.
13. 14.
Disclosure Statement
No competing financial interests exist.
15.
References
1. Tijerina-Sa´enz A, Innis SM, Kitts DD. Antioxidant capacity of human milk and its association with vitamins A and E and fatty acid composition. Acta Paediatr 2009;98:1793–1798. 2. Zarban A, Taheri F, Chahkandi T, et al. Antioxidant and radical scavenging activity of human colostrums, transitional and mature milk. J Clin Biochem Nutr 2009;45:150–154. 3. Picciano MF. Human milk: Nutritional aspects of a dynamic food. Biol Neonate 1998;74:84–93. 4. Saugstad OD. Bronchopulmonary dysplasia—Oxidative stress and antioxidants. Semin Neonatol 2003;8:39–49. 5. Knuppel RA, Hassan MI, McDermott JM, et al. Oxidative stress and antioxidants: Preterm birth and preterm infants. In: Morrison JC, ed. Preterm Birth—Mother and Child. In Tech, Rijeka, Croatia, 2012, pp. 126–127. 6. Thompson AM, Bizzarro MJ. Necrotizing enterocolitis in newborns: Pathogenesis, prevention and management. Drugs 2008;68:1227–1238. 7. Shoji H, Shinohara K, Oguchi S, et al. Suppressive effects of breast milk on oxidative DNA damage in very low birthweight infants. Arch Dis Child Fetal Neonatal Ed 2004; 89:136–138. 8. Kasapovic J, Pejic S, Mladenovic M, et al. Superoxide dismutase activity in colostrum, transitional and mature human milk. Turk J Pediatr 2005;47:343–347. 9. Newburg DS. Bioactive components of human milk: Evolution, efficiency, and protection. Adv Exp Med Biol 2001; 501:3–10. 10. Benzie IF, Strain JJ. The ferric reducing ability of plasma (FRAP) as a measure of ‘‘antioxidant power’’: The FRAP assay. Anal Biochem 1996;239:70–76. 11. Brand-Williams W, Cuvelier ME, Berset C. Use of a free radical method to evaluate antioxidant activity. Lebensm Wiss Technol 1995;28:25–30. 12. Ortega RM, Andres P, Martinez RM, et al. Vitamin A status during the third trimester of pregnancy in Spanish
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women: Influence on concentrations of vitamin A in breast milk. Am J Clin Nutr 1997;66:564–568. Gate L, Pual J, Ba GN, et al. Oxidative stress induced in pathologies: The role of antioxidants. Biomed Pharmacother 1999;53:169–180. Ortega RM, Lopez-Sobaler AM, Martinez RM, et al. Influence of smoking on vitamin E status during the third trimester of pregnancy and on breast-milk tocopherol concentrations in Spanish women. Am J Clin Nutr 1998;68:662–667. Ortega RM, Quintas ME, Andres P, et al. Ascorbic acid levels in maternal milk: Differences with respect to ascorbic acid status during the third trimester of pregnancy. Br J Nutr 1998;79:431–437. Alberti-Fidanza A, Burini G, Perriello G. Total antioxidant capacity of colostrum, transitional and mature human milk. J Matern Fetal Neonatal Med 2002;11:275–279. Byerley LO, Kirksey A. Effects of different level of vitamin C intake on the vitamin C concentration in human milk and the vitamin C intakes of breast-fed infants. Am J Clin Nutr 1985;41:665–671. Daneel-Otterbech S, Davidsson L, Hurrell R. Ascorbic acid supplement and regular consumption of fresh orange juice increase the ascorbic acid content of human milk: Studies in European and African lactating women. Am J Clin Nutr 2005;81:1088–1093. Hoppu U, Rinne M, Salo-Vaananen P, et al. Vitamin C in breast milk may reduce the risk of atopy in the infant. Eur J Clin Nutr 2005;59:123–128. Codon˜er-Franch P, Herna´ndez-Aguilar MT, Navarro-Ruiz A, et al. Diet supplementation during early lactation with non-alcoholic beer increases the antioxidant properties of breastmilk and decreases the oxidative damage in breastfeeding mothers. Breastfeed Med 2013;8:164–169. Nikniaz L, Mahdavi R, Ostadrahimi A, et al. Effects of synbiotic supplementation on total antioxidant capacity of human breastmilk. Breastfeed Med 2013;8:217–222.
Address correspondence to: Asghar Zarban, PhD Biochemistry Department Birjand University of Medical Sciences Ghaffari Street Birjand, Iran E-mail: [email protected] [email protected]
Effect of Vitamin C and E Supplementation on Total Antioxidant Content of Human Breastmilk and Infant Urine Asghar Zarban,1,2 Mahsa Mostafavi Toroghi,2 Marziye Asli,2 Masumeh Jafari,2 Morteza Vejdan,2 and Gholamreza Sharifzadeh 3
Abstract
Objectives: After delivery and birth, mothers and neonates are exposed to oxidative stress. The present study examined the effect of supplementation of the diet of breastfeeding mothers with vitamin C and E to improve the antioxidant content of breastmilk and evidence of antioxidant activity in infant urine. Subjects and Methods: The subjects were 60 healthy lactating breastfeeding mothers and their infants 1–6 months of age. They were randomly allocated to a control group (n = 30) consuming a free diet or an experimental group (n = 30) consuming a free diet supplemented each day with effervescent tablets of vitamin C (500 mg) and chewable tablets of vitamin E (100 IU). After 30 days, the total antioxidant content of the mothers’ breastmilk and evidence of antioxidant activity in the infants’ urine were measured by the ferric reducing/antioxidant power assay. The free radical scavenging activity of the urine samples was measured by the a,a-diphenyl-b-picrylhydrazyl method. Differences pre- and postintervention were compared within and between the groups. Results: Significantly higher levels of antioxidants in the breastmilk (610 – 295.5 to 716 – 237.5 lmol/L) and infant urine (43.2 – 21.8 to 75.0 – 49.2 lmol/mg creatinine) were observed in the experimental group over the control group ( p < 0.05). A significant increase in evidence of free radical scavenging in infant urine was observed in the experimental group after 30 days of supplementation by mothers ( p < 0.05). Conclusions: Consumption of vitamin C and E supplements appears to have a positive effect on total antioxidant content of breastmilk and evidence of antioxidant activity in infant urine.
Introduction
H
uman breastmilk contains essential components (vitamins, minerals, essential amino acids, and fatty acids) for infant health and is considered to be the ideal nutrient source for neonates and infants for the first 6 months of life.1,2 The composition of human milk can be affected by maternal nutritional factors.3 After birth, infants are exposed to high concentration of oxygen, which is potentially toxic because of the production of reactive oxygen species and free radicals.2,4 When the rate of free radical production exceeds the rate of its removal by cellular defense mechanisms, a condition known as oxidative stress occurs. This is a contributing factor to the pathogenesis of neonatal diseases such as bronchopulmonary dysplasia and necrotizing enterocolitis.4–6
Human breastmilk contains defense mechanisms that can suppress oxidative stress.7 The antioxidant capacity of human milk derives from numerous bioactive components having varying capacities for antioxidant activity. Enzymatic antioxidant ingredients, such as catalase, superoxide dismutase, and glutathione peroxidase, and nonenzymatic ingredients, such as vitamins E and C, retinol, b-carotene, lactoferrin, and glutathione, present in human breastmilk play a protective role against reactive oxygen species.2,7,8 It appears that the antioxidant content of breastmilk is affected by maternal antioxidant status and can influence the antioxidant status of breastfed infants.9 The present study evaluated the effect of consumption of vitamin C and E supplements by the mother on the antioxidant content of breastmilk and evidence of antioxidant activity in the urine of breastfed infants.
1 Biochemistry Department, 2Antioxidants & Metabolism Research Center, and 3Social Medicine Department, Birjand University of Medical Sciences, Birjand, Islamic Republic of Iran. This study is registered with the Iranian Registry of Clinical Trials with clinical trial registration number IRCT2012072410379N1.
214
ANTIOXIDANT CONTENT OF HUMAN BREASTMILK
215
Subjects and Methods
This study was approved by the Ethics Committee of the Birjand University of Medical Sciences, Birjand, Islamic Republic of Iran. It was a randomized clinical trial with a control group. In total, 60 healthy lactating women and their infants having similar socioeconomic backgrounds were selected from a health center in Birjand. The women were 20– 50 years of age, and the infants were 1–6 months of age. Mothers were fully informed about the aim of the investigation and consented to the participation of the infant in the study. The primary requisite for inclusion was the mother’s intent to exclusively breastfeed from birth. Exclusion criteria were consumption of infant formula by the infants, illness requiring intensive care admission, major congenital anomaly, diabetes, infectious disease, or genetic disease of either the mother or infant. The mothers and their infants were randomly and alternately divided into two groups; the mothers were the criterion for randomization, and the infants were placed in the same group as their mothers after randomization. The placement of the first mother and infant into an experimental or control group was done by asking the mother to randomly select one of two cards on which were written the terms ‘‘control’’ and ‘‘experimental.’’ The remaining mothers with their infants were then alternately assigned to the two groups. The age, weight, and height of all subjects were measured at the onset of the study. Samples of milk from the right breast were collected manually between 9:00 and 11:00 a.m. when the infants had not breastfed for at least 1 hour. Urine samples were collected from the infants using a standard urine bag. All samples were transferred to the laboratory under sterile and cold conditions and stored at - 70C for further analysis. The mothers in the experimental and control groups were directed to consume a free diet. Both groups received suggested dietary guidelines for nutritional dietary habits. They were requested not to consume other supplements and not to change their diets during the study. The mothers in the experimental group asked to consume one effervescent tablet of vitamin C (500 mg) and one chewable tablet of vitamin E (100 IU) each day for 30 days. The control group did not receive supplements. Milk and urine samples were again collected from the mothers and infants, respectively, at the end of the 30-day treatment program. Analytical method
All samples collected before and after intervention were analyzed together. The total antioxidant capacities of milk and urine were measured using the ferric reducing/antioxidant power (FRAP) assay developed by Benzie and Strain10 as a direct method for measuring total antioxidant power of biological fluids with slight modification. To increase the accuracy of analysis, each breastmilk sample was tested three times, and the results were averaged. The free radical scavenging activity of the urine samples was measured using the a,a-diphenyl-b-picrylhydrazyl (DPPH) assay proposed by Brand-Williams et al.11 with slight modification. The creatinine content of the urine was measured using the Jaffe method with a standard kit (Pars-Azmoon, Tehran, Iran). The ratio of parameters related to the antioxidant capacity of creatinine in the urine was used to control for the effect of urine concentration. The result for the FRAP assay was
Table 1. Comparison of the Mothers’ and Infants’ Demographic Variables for Case and Control Groups
Mothers Age (years) BMI (kg/m2) Parturition NVD Cesarean section Infants Age (days) Weight (kg) Sex Male Female
Control group (n = 30)
Case group (n = 30)
29.1 – 8.4 23.6 – 4.5
27.4 – 7.4 23.6 – 4.9
23 (76.7) 7 (23.3)
24 (80.0) 6 (20.0)
99.8 – 31.2 5.6 – 1.1
85.5 – 36.6 5.5 – 1.2
14 (46.7) 16 (53.3)
14 (46.7) 16 (53.3)
p value 0.44 0.9 0.75
0.13 0.7 1
Data are presented as mean – standard deviation values or frequency (percentage), as indicated. BMI, body mass index; NVD, normal vaginal delivery.
presented as lmol of creatinine/mg, and that for the DPPH method was presented as %/mg creatinine. Statistical analysis
The data was expressed in terms of frequency (percentage) and mean – standard deviation value to test for a normal distribution using the Kolmogorov–Smirnov test. The pairedsamples t test was used to compare the means before and after intervention (consumption of vitamin C and E supplements). An independent-samples t test was used to compare mean change (difference between values before and after intervention) between the experimental and control groups. Nonparametric tests (Mann–Whitney U test and Wilcoxon) were used for those variables that were not normally distributed. The chi-squared test was used to determine differences between groups for gender of infants and method of parturition. All differences were considered to be statistically significant at p < 0.05. All statistical analyses were done using SPSS version 16 software (SPSS, Inc., Chicago, IL). Results
No significant differences were observed between groups for type of parturition (normal vaginal delivery and cesarean section), mean age, and body mass index of the mothers and for gender, mean age, and weight of the infants (Table 1). Table 2 shows the results of the FRAP assay for breastmilk and the FRAP/creatinine and DPPH/creatinine assays of the infant urine before and after intervention for the experimental and control groups. A significant increase in all values was seen for the experimental group after intervention. The mean change was also significantly different for the FRAP of breastmilk and the FRAP/creatinine and DPPH/creatiinine of infant urine (Table 2). Discussion
This investigation assessed the effect of vitamin C and E supplementation on the antioxidant content of breastmilk of
216
ZARBAN ET AL.
Table 2. Comparison of the Mean of the Antioxidant Indices and the Mean Change, Before and After the Intervention, in Case and Control Groups Control group (n = 30) Variable Breastmilk FRAP (lmol/L) Infant’s urine DPPH/Cr (%/mg Cr) FRAP/Cr (lmol/mg Cr)
Case group (n = 30) Before
After
p valuea
p valueb
0.388
610 – 295.5
716 – 237.5
0.017c
0.02c
0.2 0.8
4.85 – 1.9 43.2 – 21.8
8.2 – 4.5 75.0 – 49.2
0.005c 0.025c
0.002c 0.03c
Before
After
p value
690 – 285.7
653 – 220.6
6.5 – 5.7 54.8 – 23.4
5.03 – 1/7 52.7 – 33
a
Data are mean – standard deviation values. a Comparison of the mean of antioxidant indices before and after the intervention. b Comparison of the mean change (the difference between the values before and after the intervention in each group) between the case and control groups. c Significant at p < 0.05. CR, creatinine; DPPH, a,a-diphenyl-b-picrylhydrazyl; FRAP, ferric reducing/antioxidant power.
lactating mothers. A significantly higher level of antioxidants was recorded for the breastmilk in the experimental group over that from the control group. It appears that this increase is in response to the consumption of vitamin C and E supplements by the mothers in the intervention group because there was no such increase in the control group (Table 2). Previous studies have determined several antioxidant nutrients and correlated their values in a mother’s diet. A relationship between antioxidant vitamins and provitamin levels in human breastmilk and diet during pregnancy has also been reported.12 The total antioxidant content of breastmilk decreases during the course of lactation and can be the result of depletion of antioxidants stored in the mothers. The large variation in total antioxidant content reported suggests the need for mothers with low values to take steps to improve their nutrition, especially the consumption of natural antioxidants during lactation.2 Micronutrients such as vitamins C and E are essential to the health of the antioxidant defense system that protects against damaging reactive species that arise as by-products of metabolism.13 Because breastmilk is the only provider of antioxidants for breastfed infants, the mother’s diet is essential to development of a shield against oxidative stress for infants exclusively fed breastmilk. It has been reported that the antioxidant content of transitional and mature milk may be related to the mother’s dietary intake of vitamins A, C, and E during the third trimester of pregnancy.12–16 Byerley et al.17 reported no significant influence on ascorbic acid output by human breastmilk of lactating women after supplementation with 1,000 mg/day; however, this study was a short-term intervention with only five participants. Daneel-Otterbech et al.18 found that the ascorbic acid content of human breastmilk can increase two- to threefold with increased intake of ascorbic acid by lactating women with breastmilk showing low ascorbic acid content. The results of that investigation highlighted the importance of encouraging regular consumption of ascorbic acid–rich foods like fresh orange juice by lactating mothers to ensure adequate human milk ascorbic acid output. Hoppu et al.19 found that maternal intake of vitamin C in the diet, but not from supplements, influenced the concentration of vitamin C in breastmilk. Codon˜er-Franch et al.20 reported that supplementing the diet of breastfeeding mothers
with nonalcoholic beer, a product rich in antioxidants, increased the antioxidant content of breastmilk. The results of the present study were in accordance with the results of Nikniaz et al.,21 who found a significant increase of total antioxidant content of breastmilk of mothers in the treatment group receiving symbiotic supplementation, but a decrease for the placebo group. Previous studies have primarily examined the effect of supplements and vitaminrich foods on the contents of breastmilk. The present investigation also measured a significant increase in the antioxidant power in the urine of infants after ingestion of supplements by mothers as measured by the FRAP and DPPH assays (Table 2). These findings confirm the previous highly significant correlation between the antioxidant content of milk and vitamin supplementation of mothers. Codon˜er-Franch et al.20 found no difference in oxidant markers in infant urine after consumption of nonalcoholic beer by breastfeeding mothers. The data from the present study show a high antioxidant status of the mothers was transferred via the breastmilk to the infants as evidenced by the excessive load discharged in the urine of the infants. A previous study indicated that a healthy balanced diet may outweigh single dietary supplements, but the results of the present study suggest that supplementation of high doses of vitamins C and E can increase the total antioxidant content of breastmilk. This is especially important where there are large differences between maternal diet and the nutritional content of the breastmilk, given the obvious importance of breastmilk as the sole antioxidant source for breastfed infants.2,18,19 Further study is required to evaluate antioxidant indices for infant plasma to confirm these results. Conclusions
Daily intake of 500 mg of vitamin C and 100 mg of vitamin E as supplements by lactating mothers with breastfed infants for 30 days significantly increased the antioxidant capacity of the breastmilk. It also significantly increased the antioxidant content of infant urine, which indicates the transfer of antioxidants from the breastmilk to the infants and the removal of the excessive load in the urine of the infants. The results of this study suggest that the use of antioxidants early in life can decrease oxidative stress and help prevent broad-spectrum diseases by balancing between free radicals and antioxidants.
ANTIOXIDANT CONTENT OF HUMAN BREASTMILK
217
Acknowledgments
The authors wish to thank the Deputy of Research and Technology of Birjand University of Medical Sciences for his support of this project. The contributions of the mothers and their newborns are also gratefully acknowledged.
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Disclosure Statement
No competing financial interests exist.
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Address correspondence to: Asghar Zarban, PhD Biochemistry Department Birjand University of Medical Sciences Ghaffari Street Birjand, Iran E-mail: [email protected] [email protected]
