Maternal Smoking Does Not Influence Vitamin A and E Concentrations in Mature Breastmilk

Abstract

Objectives:

The aim of this study was to analyze the effects of maternal smoking on the total antioxidant status (TAS) and the concentrations of vitamins A and E in human breastmilk.

Methodology:

The study group (n=20) comprised postpartum women who declared smoking more than five cigarettes per day (confirmed by urinalysis of the cotinine concentration). The control group included 25 nonsmoking postpartum women. Breastmilk samples were collected between day 30 and day 32 after delivery. TAS was determined by Rice-Evans and Miller method, whereas the amount of vitamins A and E was measured by high-performance liquid chromatography.

Results:

No significant differences were observed between breastmilk samples from smoking and nonsmoking mothers in terms of TAS and vitamin A and E concentrations. Additionally, no significant correlations were found between urinary cotinine and TAS (R=0.35, p=0.144) or vitamin A (R=0.14, p=0.571) and vitamin E (R=0.31, p=0.228) concentrations in breastmilk samples from smoking mothers.

Conclusions:

Maternal smoking is not reflected by decreased TAS and vitamin A and E concentrations in mature milk.

Introduction

Human breastmilk is the optimal source of nutrients for the rapidly growing neonate. Furthermore, breastmilk supports antioxidant and immune barriers of an infant.^1,2 Many factors modulate the composition of breastmilk; among them are diet, lifestyle, and nutritional status of the lactating woman.^3–5 Maternal smoking, during pregnancy and breastfeeding, is an additional important factor affecting both qualitative and quantitative composition of breastmilk. Moreover, smoking influences the quality and duration of breastfeeding and directly modifies the health of the neonate. Mothers who smoke decide on exclusive breastfeeding less frequently and earlier quit providing breastmilk in favor of modified formulas. As a result, lower weight gains are observed during the initial 6 months of life in infants of smoking mothers.^5,6 Additionally, lower secretion of breastmilk is reported in women who smoke, which may be associated with a decreased concentration in baseline prolactin.⁷ Besides affecting the secretion of breastmilk, maternal smoking may also markedly affect its composition, specifically in regards to the amount and concentration of polyunsaturated fatty acids, in particular, docosahexaenoic acid.⁸

Cigarette smoke contains many reactive oxygen species that may shift the antioxidant–prooxidant balance in favor of oxidation processes. Increased concentrations of protein, lipid, and DNA oxidation products have been reported in smokers, along with higher levels of oxidative stress markers.^9–14 Additionally, the effectiveness of the antioxidant barrier is impaired in smokers because of decreased activities of antioxidant enzymes and lower blood concentrations of antioxidants.^10,13,14 Vitamins A and E are important antioxidants, preventing lipid peroxidation. Decreased serum levels of these vitamins are observed among smokers.^13,15

In view of the marked disparity of antioxidant–prooxidant balance observed among smokers, the influence of maternal smoking during lactation on the antioxidant properties of breastmilk becomes a vital issue. Therefore, the aim of this study was to analyze the effects of maternal smoking during lactation on the total antioxidant status (TAS) and the concentrations of vitamins A and E in human breastmilk.

Subjects and Methods

Participants

This study centered on two hospital units in Gdansk (Northern Poland): the Obstetrical Ward of the Voivodeship Specialist Hospital and the Obstetrical Ward at the Institute of Obstetrics and Gynecology, Medical University of Gdansk. The study group (Group I, n=20) comprised postpartum women who met the following inclusion criteria: normal spontaneous full-term delivery and neonates in good general status, with normal birth weight (2,500–4,000 g) and breastfed exclusively (receiving only breastmilk and no other solids or liquids with the exception of vitamins, medicines, or mineral supplements). All participants declared smoking more than five cigarettes per day during lactation. The exclusion criteria included passive smoking, acute and chronic disorders (including gestational diabetes), and pharmacotherapy other than vitamin supplementation. The control group (Group II, n=25) comprised postpartum women who met all of these aforementioned inclusion criteria other than smoking during lactation. All participants (Groups I and II) used vitamin preparations designed for pregnant and lactating women throughout the period of pregnancy and breastfeeding (including daily intake of 2 mg of β-carotene and 12 mg of vitamin E).

Ethics

All the procedures were approved by the Local Ethics Committee of the Medical University in Gdansk. The subjects gave their informed consent before the start of any procedure.

Samples

Breastmilk samples were collected between day 30 and day 32 after delivery. Both breasts were expressed completely with the assistance of an electric breast pump 2 hours after the first morning feeding. The milk was collected into sterile glass containers. After careful mixing, 10-mL samples were taken, placed into another sterile container, and immediately frozen at −80°C. The remaining milk was fed to the infants. On the same day, first morning void urine samples were obtained from all participating mothers directly into plastic containers and frozen at −80°C until analysis.

Total antioxidant status

Breastmilk samples were centrifuged for 10 minutes at 2,800 g. The supernatant was collected and further centrifuged under the same conditions. The final supernatant was diluted 20 times with distilled water and used for further analysis. The TAS of the breastmilk was determined by the method of Rice-Evans and Miller¹⁶ with an aid of a commercially available reagent kit (Randox Laboratories, Crumlin, Northern Ireland, UK). Incubation of 2,2′-azino-bis(3-ethylbenzthiazoline sulfonate) (ABTS) with peroxidase and hydrogen superoxide leads to a blue-green–colored cationic radical (ABTS⁰) formation. Color intensity of this radical is decreased whenever antioxidants are present within the analyzed sample. The decrease in color intensity was measured spectrophotometrically (Ultrospec^® III spectrophotometer, Pharmacia LKB, Uppsala, Sweden) at a wavelength of 600 nm. The results were presented in mmol/L.

Vitamins A and E content

The amount of vitamins A and E in human milk was determined with an aid of a vitamin A/E high-performance liquid chromatography (HPLC) kit (Immundiagnostik AG, Bensheim, Germany). The standard solution (STD), internal standard solution (INSTD), dilution solution (DIL), precipitation solution (PREC), and controls 1 and 2 (CTRL1, CTRL2) were provided. The standards, controls, and samples were prepared in 1.5-mL reaction tubes, according to the manual of the vitamin A/E HPLC kit manufacturer. The preparation of the samples and their HPLC assays were performed on the same day to ensure sample stability. The STD preparations were prepared by adding 250 μL of STD to 50 μL of INSTD, then 250 μL of DIL, and finally 250 μL of PREC. All was mixed well (vortex-mixing for 2 minutes). The reaction tubes were left for 30 minutes at 4°C and centrifuged afterward at 5,591 g for 10 minutes. The supernatants were collected, placed in HPLC vials, and subjected to analysis. The human milk samples were thawed immediately before analysis. The preparation procedure for the samples and controls included adding 250 μL of sample (or CTRL1 and CTRL2) to 50 μL of INSTD and 500 μL of PREC. Like with the standards, all were mixed properly (vortex-mixing for 2 minutes). Then the reaction tubes were incubated for 30 minutes at 4°C and ultimately centrifuged at 5,591 g for 10 minutes. The supernatants were placed in HPLC vials and subjected to analysis. The HPLC analysis was performed according to the manufacturer's vitamin A/E HPLC kit manual. The HPLC apparatus (Younglin Instruments, An-Yang City, Korea) was equipped with an autosampler and a thermostat column oven controlled at 30°C, a gradient pump, and an ultraviolet-visible detector. The ultraviolet detection was done using the following wavelengths: 325 nm (vitamin A) and 300 nm (vitamin E), switched after 7 minutes. The chromatographic separation was performed on a C18 reversed-phase MZ-Analytical column (Nucleosil-100 C18, 125×4.0 mm, i.d. 10 μm; MZ Analysentechnik, Mainz, Germany). The 16-minute run was accomplished in isocratic mode. The flow rate was 0.8 mL/minute. The mobile phase was provided with the vitamin A/E HPLC kit. The injection volume was 100 μL. The concentrations of vitamins A and E in the analyzed human milk samples were calculated with the use of the following equations, described in the vitamin A/E HPLC kit manual: \documentclass{aastex}\usepackage{amsbsy}\usepackage{amsfonts}\usepackage{amssymb}\usepackage{bm}\usepackage{mathrsfs}\usepackage{pifont}\usepackage{stmaryrd}\usepackage{textcomp}\usepackage{portland, xspace}\usepackage{amsmath, amsxtra}\pagestyle{empty}\DeclareMathSizes{10}{9}{7}{6}\begin{document} \begin{align*} \hbox{Vitamin A concentration} \ ({\rm in} \mu {\rm g} / {\rm L}) = ([PH_{\rm VitAsample} \times C_{\rm VitASTD}] / PH_{\rm INSTDsample}) \times (PH_{\rm INSTD-STD} / PH_{\rm VitASTD}) \times 1,000 \end{align*} \end{document} \documentclass{aastex}\usepackage{amsbsy}\usepackage{amsfonts}\usepackage{amssymb}\usepackage{bm}\usepackage{mathrsfs}\usepackage{pifont}\usepackage{stmaryrd}\usepackage{textcomp}\usepackage{portland, xspace}\usepackage{amsmath, amsxtra}\pagestyle{empty}\DeclareMathSizes{10}{9}{7}{6}\begin{document} \begin{align*} \hbox{Vitamin E concentration} \ ({\rm in} \ {\rm mg} / {\rm L}) = ([PH_{\rm VitEsample} \times C_{\rm VitESTD}] / PH_{\rm INSTDsample}) \times (PH_{\rm INSTD-STD} / PH_{\rm VitESTD}) \end{align*} \end{document}

where PH_VitAsample is the peak height of vitamin A in the sample, C_VitASTD is the vitamin A concentration in the STD, PH_INSTDsample is the INSTD peak height in the sample, PH_INSTD-STD is the INSTD peak height in the STD, PH_VitASTD is the peak height of vitamin A in the STD, PH_VitEsample is the peak height of vitamin E in the sample, C_VitESTD is the vitamin E concentration in the STD, and PH_VitESTD is the peak height of vitamin E in the STD.

Urinary cotinine

The urinary concentration of creatinine was measured with a standard method based on the reaction of Jaffé.¹⁷ Urinary cotinine was determined with an aid of cotinine (urine) enzyme-linked immunosorbent assay (EIA-1377 or EIA-1726, DRG^® International, Inc., Mountainside, NJ). Similar to previous studies,¹⁸ a cotinine concentration equal to 200 ng/mL and a cotinine-to-creatinine ratio equal to150 ng of cotinine/mg of creatinine were considered as cutoff values distinguishing active smoking.

Statistical analysis

Normal distribution of continuous variables was tested with the Kolmogorow–Smirnov test. Depending on distribution, the results were presented as arithmetic means and their SDs or as medians and ranges. Arithmetic means between Groups I and II were compared with Student's t test, whereas median values were compared with the Mann–Whitney test. Associations between continuous variables were tested with Spearman's coefficient of correlation (R). Calculations were performed using Statistica version 8 software (StatSoft^®, Krakow, Poland) and the R 2.6.1 statistical environment, with statistical significance defined as p≤0.05.

Results

Characteristics of study participants and their neonates are summarized in Table 1. In all women from Group I, declared smoking was confirmed by the urinalysis of cotinine concentration.

Table 1.

Characteristics of Study Participants

Parameter	Group I (smokers, n=20)	Group II (nonsmokers, n=25)	p
Age (years)	27.2±5.22	26.1±3.18	0.388
Parity (n)	2.11±1.55	2.01±1.79	0.898
Birth weight (g)	3,402±601.3	3,601±509.2	0.236
Birth length (cm)	54.2±2.43	55.2±2.67	0.201
Apgar score at 1 minute (points)	9.41±0.82	9.57±0.67	0.475
Cigarettes per day (n)	11.2±5.87	0	—
Urinary cotinine (ng/mL)	819±616	59±101	0.001
Urinary CCR (ng/mg)	1,001±779	40±73	0.001

Data are mean±SD values.

CCR, cotinine-to-creatinine ratio.

No significant differences were observed between breastmilk samples from smoking and nonsmoking mothers in terms of TAS and vitamin A and E concentrations (Table 2). Additionally, no significant correlations were found between cotinine-to-creatinine ratio and TAS (R=0.35, p=0.144) or vitamin A (R=0.14, p=0.571) and vitamin E (R=0.31, p=0.228) concentrations in breastmilk samples from smoking mothers.

Table 2.

Total Antioxidant Status and Vitamin A and E Concentrations in the Mature Milk of the Study Participants

Parameter	Group I (smokers, n=20)	Group II (nonsmokers, n=25)	p
TAS (μmol/L)	4.66 (1.27–11.24)	5.42 (1.84–12)	0.245
Vitamin A (μg/L)	78.32 (33.41–201.55)	104.90 (84.69–296.52)	0.749
Vitamin E (mg/L)	1.17 (0.09–10.18)	1.00 (0.24–21.38)	1.000

Data are median (ranges) values.

TAS, total antioxidant status.

Discussion

Smoking during pregnancy markedly disturbs the antioxidant–prooxidant balance of mother and fetus. As a result, decreased TAS is observed in the blood of mothers^9,19–21 along with decreased concentrations of various antioxidants^9,22 and enzymes with antioxidant activity.²¹ Decreased TAS, on one hand, and exposure to reactive oxygen species present in tobacco smoke, on the other, are reflected by an increased oxidative stress in pregnant smokers. This in turn manifests by higher concentrations of lipid peroxidation products.²⁰

Additionally, significant disorders of antioxidant–prooxidant balance are observed in newborns whose mothers smoked in pregnancy. Decreased antioxidant concentrations and lower TAS are observed in the umbilical blood from such newborns along with an increase in the concentrations of lipid peroxidation products.^9,20,23

In this study, we analyzed the effects of maternal smoking during lactation on the antioxidant properties and vitamin A and E contents in mature breastmilk. No significant smoking-related differences were observed in TAS, which is consistent with the results of previous study by Ermis et al.²¹ It is notable that our previous studies revealed a decrease in TAS in the colostrum of smoking mothers compared with nonsmokers.²⁴ In the perinatal period, the neonate is exposed to harmful effects of reactive oxygen species because of incomplete maturation of the antioxidant barrier.^25,26 Consequently, providing antioxidant-rich breastmilk during this initial period of life seems particularly important for future health and development.

Vitamin E is a necessary antioxidant that prevents lipid peroxidation and, in particular, the oxidation of long-chain polyunsaturated fatty acids present in cell membranes.²⁷ The results of previous studies of the vitamin E concentrations in smoking pregnant women are inconclusive. Some authors did not observe significant smoking-related differences in serum content of vitamin E,^28–32 whereas others found decreased concentrations of vitamin E in smokers in the third trimester of pregnancy.⁹ In this study, we observed no effects of maternal smoking during lactation on the vitamin E concentration in mature breastmilk. Published research on the effects of smoking on breastmilk concentrations of vitamin E is scant. Orhon et al.²⁹ observed lower concentrations of vitamin E in smoking mothers, but their study referred to the very early period (namely, day 7) of lactation. In turn, Ortega et al.³⁰ documented lower concentrations of vitamin E in mature milk (day 40 of lactation) of smoking women, whereas no smoking-related differences in this vitamin level were observed in transitory milk (days 13–14 of lactation). Variance in breastmilk levels of vitamin E in nonsmoking women who were considered as a reference group in these aforementioned studies might constitute one potential reason for observed discrepancies. This potential confounder could be eliminated by providing the normal reference values of vitamin E for breastmilk from various periods of lactation. Unfortunately, defining such reference values is hindered by the fact that breastmilk concentrations of vitamin E in nonsmoking women are positively correlated with dietary content of this vitamin.³⁰ In this study we did not analyze dietary content of vitamin E in lactating women from both groups, which might constitute a potential confounder. We assumed, however, that the intake of this vitamin was optimal and did not differ significantly between smoking and nonsmoking women because they all used vitamin preparations for pregnant/lactating women at recommended doses.

Numerous biological activities of vitamin A include, among others, antioxidant properties. Most previous studies have not revealed decreased serum vitamin concentrations in pregnant smokers.^29,31,32 Nonetheless, Chechlowska et al.⁹ found lower concentrations of vitamin A in all trimesters of pregnancy in smokers. In this study, we did not observe significant differences in vitamin A content of mature breastmilk from smoking and nonsmoking women. This finding is consistent with the results of Orhon et al.²⁹ for breastmilk vitamin A content analyzed on day 7 of lactation.

Despite expected marked disparity in antioxidant–prooxidant balance, in this study we did not observe significant smoking-related changes in the breastmilk TAS and concentrations of antioxidant vitamins. Nonetheless, previous studies revealed that smoking is reflected in decreased activity of one principal antioxidant enzyme, superoxide dismutase, in breastmilk of active and passive smokers.²¹ Moreover, enhanced lipid peroxidation was reported in neonates who were breastfed by smoking mothers.³³ These aforementioned findings suggest further research on the association between maternal smoking during lactation and antioxidant activity of breastmilk as well as the long-term health consequences to the child. Mechanisms protecting us against harmful effects of reactive oxygen species are complex and modulated by various factors (including diet, health, and nutritional status, among others). Besides purely scientific aspects, expanding our knowledge of the harmful effect of smoking has clinical implications. It may constitute a base for dietary recommendations or supplementation programs for women who have not quit smoking during pregnancy and lactation.

Conclusion

In conclusion, maternal smoking is not reflected by decreased TAS and vitamin A and E concentrations in mature milk.

Footnotes

Disclosure Statement

No competing financial interests exist.

References

Goldman

, Goldblum

, Hanson

. Anti-inflammatory systems in human milk. Adv Exp Med Biol, 1990; 262:69–76.

Buescher

, McIlheran

. Colostral antioxidants: Separation and characterization of two activities in human colostrum. J Pediatr Gastroenterol Nutr, 1992; 14:47–56.

Imhoff-Kunsch

, Stein

, Villalpando

et al. Docosahexaenoic acid supplementation from mid-pregnancy to parturition influence breast milk fatty acid concentration at 1 month postpartum in Mexican women. J Nutr, 2011; 141:321–326.

Nakamori

, Ninh

, Isomura

et al. Nutritional status of lactating mothers and their breast milk concentration of iron, zinc and copper in rural Vietnam. J Nutr Sci Vitaminol (Tokyo), 2009; 55:338–345.

Kristiansen

, Lande

, Øverby

et al. Factors associated with exclusive breast-feeding and breast-feeding in Norway. Public Health Nutr, 2010; 13:2087–2096.

Rebhan

, Kohlhuber

, Schwegler

et al. Infant feeding practices and associated factors through the first 9 months of life in Bavaria, Germany. J Pediatr Gastroenterol Nutr, 2009; 49:467–473.

Matheson

, Rivrud

. The effect of smoking on lactation and infantile colic. JAMA, 1989; 261:42–43.

Agostoni

, Marangoni

, Grandi

et al. Earlier smoking habits are associated with higher serum lipids and lower milk fat and polyunsaturated fatty acid content in the first 6 months of lactation. Eur J Clin Nutr, 2003; 57:1466–1472.

Chechlowska

, Ambroszkiewicz

, Gajewska

et al. The effect of tobacco smoking during pregnancy on plasma oxidant and antioxidant status in mothers and newborn. Eur J Obstet Gynecol Reprod Biol, 2011; 155:132–136.

10.

Codandabany

. Erythrocyte lipid peroxidation and antioxidants in cigarette smokers. Cell Biochem Funct, 2000; 18:99–102.

11.

Morrow

, Frei

, Longmire

et al. Increase in circulating products of lipid peroxidation (F2-isoprostanes) in smokers. Smoking as a cause of oxidative damage. N Engl J Med, 1995; 332:1198–1203.

12.

Marangon

, Devaraj

, Jialal

. Measurement of protein carbonyls in plasma of smokers and in oxidized LDL by an ELISA. Clin Chem, 1999; 45:577–578.

13.

Mezzetti

, Lapenna

, Pierdomenico

et al. Vitamins E, C and lipid peroxidation in plasma and arterial tissue of smokers and non-smokers. Atherosclerosis, 1995; 112:91–99.

14.

Yildiz

, Kayaoglu

, Aksoy

. The changes of superoxide dismutase, catalase and glutathione peroxidase activities in erythrocytes of active and passive smokers. Clin Chem Lab Med, 2002; 40:612–615.

15.

Pincemail

, Vanbelle

, Degrune

et al.

Lifestyle behaviours and plasma vitamin C and β-carotene levels from the ELAN population (Liege, Belgium)

J Nutr Metab, 2011; 2011:494370.

16.

Rice-Evans

, Miller

. Total antioxidant status in plasma and body fluids. Methods Enzymol, 1994; 234:279–293.

17.

Jaffé

. About the precipitation caused by pikrinic acid in normal urine and about a new reaction of creatinine. Physiol Chem, 1886; 10:391–400.

18.

Riboli

, Haley

, Tredaniel

et al. Misclassification of smoking status among women in relation to exposure to environmental tobacco smoke. Eur Respir J, 1995; 8:285–290.

19.

Argüelles

, Machado

, Ayala

et al. Correlation between circulating biomarkers of oxidative stress of maternal and umbilical cord blood at birth. Free Radic Res, 2006; 40:565–570.

20.

Aycicek

, Erel

, Kocyigit

. Decreased total antioxidant capacity and increased oxidative stress in passive smoker infants and their mothers. Pediatr Int, 2005; 47:635–639.

21.

Ermis

, Yildirim

, Ors

et al. Influence smoking on serum and milk malondialdehyde, superoxide dismutase, glutathione peroxidase, and antioxidant potential levels in mothers at the postpartum seventh day. Biol Trace Elem Res, 2005; 105:27–36.

22.

Ortega

, López-Sobaler

, Quintas

et al. The influence of smoking on vitamin C status during the third trimester of pregnancy and on vitamin C levels in maternal milk. J Am Coll Nutr, 1998; 17:379–384.

23.

Aycicek

, Ipek

. Maternal active or passive smoking causes oxidative stress in cord blood. Eur J Pediatr, 2008; 167:81–85.

24.

Szlagatys-Sidorkiewicz

, Zagierski

, Korzon

. Total antioxidative status in colostrum. The influence of maternal smoking [in Polish] Med Wieku Rozwoj, 2005; 9:621–624.

25.

Frosali

, Di Simplicio

, Perrone

et al. Glutathione recycling and antioxidant enzyme activities in erythrocytes of term and preterm newborns at birth. Biol Neonate, 2004; 85:188–194.

26.

Ripalda

, Rudolph

, Wong

. Developmental patterns of antioxidant defense mechanisms in human erythrocytes. Pediatr Res, 1989; 26:366–369.

27.

Atkinson

, Epand

. Tocopherols and tosotrienols in membranes: A critical review. Free Radic Biol Med, 2008; 44:739–764.

28.

Kiely

, Cogan

, Kearney

et al. Relationship between smoking, dietary intakes and plasma levels of vitamin E and beta-carotene in matched maternal-cord pairs. Int J Vitam Nutr Res, 1999; 69:262–267.

29.

Orhon

, Ulukol

, Kahya

et al. The influence of maternal smoking on maternal and newborn oxidant and antioxidant status. Eur J Pediatr, 2009; 168:975–981.

30.

Ortega

, Lopez-Sobaler

, Martinez

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.

31.

Silva

, Rondó

, Bergamaschi

et al. Smoking during pregnancy and plasma concentrations beta-carotene and alpha-tocopherol in the immediate postpartum period in Brazilian women. Int J Vitam Nutr Res, 2005; 75:235–241.

32.

Bolisetty

, Naidoo

, Lui

et al. Postnatal changes in maternal and neonatal plasma antioxidant vitamins and the influence of smoking. Arch Dis Child Fetal Neonatal Ed, 2002; 86:F36–F40.

33.

Schwarz

, Cox

, Sharma

et al. Prooxidant effects of maternal smoking and formula in newborn infants. J Pediatr Gastroenterol Nutr, 1997; 24:68–74.