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Circulating antioxidant profile of pregnant women with asthma
Clinical Nutrition 31 (2012) 99e107
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Clinical Nutrition journal homepage: http://www.elsevier.com/locate/clnu
Original article
Circulating antioxidant profile of pregnant women with asthma Penelope C. McLernona, b, Lisa G. Woodc, Vanessa E. Murphyc, Nicolette A. Hodyla, Vicki L. Cliftona, b, * a
Robinson Institute, University of Adelaide, Adelaide, SA, Australia Mothers and Babies Research Centre, Hunter Medical Research Institute, University of Newcastle, NSW, Australia c Centre for Asthma and Respiratory Diseases, University of Newcastle and Hunter Medical Research Institute, Newcastle NSW, Australia b
a r t i c l e i n f o
s u m m a r y
Article history: Received 18 July 2011 Accepted 3 September 2011
Background & aims: One of the most prevalent complications of pregnancy is asthma which is associated with an increased incidence of intrauterine growth restriction. The mechanisms that affect fetal development in pregnancies complicated by asthma are not clearly defined. Antioxidants are particularly important during pregnancy due to their protective role against a state of high oxidative stress as gestation progresses. The current study was designed to characterise the circulating profile of tocopherols and carotenoids in pregnant women with asthma to determine whether asthma severity and dietary intake were associated with an altered antioxidant profile. Methods: Maternal dietary intake and plasma and erythrocyte concentrations of tocopherols and carotenoids were examined in women with (n ¼ 84) and without asthma (n ¼ 47) at 18, 30 and 36 weeks gestation. Tocopherol and carotenoid levels were related to fetal and birth outcomes. Results: Pregnant women with moderate/severe asthma were found to have increased plasma concentrations of total carotenoids (P < 0.05), lutein (P < 0.05 and a-tocopherol (P < 0.02) late in gestation compared to those women with mild asthma and healthy pregnant controls. Moderate/severe asthmatics had higher erythrocyte a-tocopherol quinone levels early in gestation relative to the controls (P < 0.02) but this marker of oxidative stress decreased as gestation progressed. Tocopherols and carotenoids were positively associated with birth weight centile (P < 0.05). Conclusion: These findings suggest that the maternal system adjusts antioxidant pathways in response to the presence of a high oxidative load induced by asthma during pregnancy in an attempt to ensure continued fetal growth in an adverse environment. Ó 2011 Elsevier Ltd and European Society for Clinical Nutrition and Metabolism. All rights reserved.
Keywords: Pregnancy Fetal growth Asthma Tocopherol Carotenoid Antioxidant
1. Introduction Dietary antioxidants such as tocopherols and carotenoids are likely to have a significant effect in modulating systemic oxidative stress. Tocopherols (vitamin E) are low molecular weight substances that act to break free radical chain reactions involved in lipid peroxidation.1 In this way, they are thought to provide a protective mechanism against oxidative damage to the body.2 The most active of the tocopherols is a-tocopherol which converts lipid peroxyl radicals and oxygen radicals found in lipid membranes to less reactive forms2 in order to maintain the integrity of membrane fatty acids.3 Carotenoids are tetraterpenoid organic pigments that are naturally occurring in the chloroplasts of plants. There are two
* Corresponding author. Robinson Institute, University of Adelaide, Lyell McEwin Hospital, Haydown Rd, Elizabethvale, SA 5112, Australia. Tel.: þ61 8 81332133; fax: þ61 8 8303 4099. E-mail address: [email protected] (V.L. Clifton).
classes, xanthophylls which include lutein and zeaxanthine and carotenes which include a-carotene, b-carotene and lycopene. Carotenoids (b-carotene, a-carotene, g-carotene, and b-cryptoxanthin) primarily act as antioxidants by scavenging free radicals. Normal pregnancy has been characterised as being a state of high oxidative stress.4 Oxidative stress is defined as an imbalance between the cellular generation of Reactive Oxygen Species (ROS) and the capacity of antioxidants to prevent oxidative damage. The increased maternal and fetal utilisation of energy, as well as an increased oxygen intake as gestation progresses promotes oxidative stress and places huge demands on the maternal system to balance the generation of ROS via the up regulation of antioxidant mechanisms. Pregnancies complicated by inflammation and oxidative stress such as pre-eclampsia5 have been reported to have higher levels of maternal circulating a-tocopherol.6 Gamma (g) tocopherol was found to be decreased in pre-eclamptic women relative to healthy pregnancies.7 Levels of lycopene were found to be reduced in pre-eclamptic women.8 These studies suggest that in a high oxidative stress environment antioxidant defences are
0261-5614/$ e see front matter Ó 2011 Elsevier Ltd and European Society for Clinical Nutrition and Metabolism. All rights reserved. doi:10.1016/j.clnu.2011.09.006
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compromised and in some cases there may be a compensatory increase in the circulation of some dietary antioxidants in an attempt to protect the maternal system from oxidative stress. However it is currently unknown whether antioxidant mechanisms are altered in pregnant asthmatic mothers. We have previously reported that a-tocopherol levels are lower in non-pregnant asthmatics than healthy controls9 and reduced circulating levels of antioxidants are associated with airway hyperresponsiveness.10 Other studies reported reduced levels of circulating carotenoids in serum,11 and whole blood12 of non-pregnant asthmatic subjects. These studies suggest that worsening asthma disturbs the oxidant-antioxidant balance. Pregnancies complicated by maternal asthma are associated with an increased incidence of intrauterine growth restriction 13, 14, with significant implications for both the short and long term health of the offspring15 Reduced fetal growth may be a consequence of high oxidative stress in pregnancies complicated by asthma. Cigarette use by pregnant women with asthma is a highly prevalent comorbidity of these pregnancies14 that could also significantly lower antioxidant defences and impact on fetal development. It has been previously shown that cigarette use can reduce circulating antioxidants in non-pregnant subjects.16 Since we have previously identified that tocopherols9 and carotenoids10 are altered in non-pregnant asthmatics, the current study was designed to assess if there are any differences in the circulating tocopherols and carotenoids of pregnant women with asthma compared to women without asthma and whether these differences were related to dietary intake, asthma treatment, asthma severity or cigarette use. It was hypothesised that maternal asthma during pregnancy would be associated with reduced circulating concentrations of tocopherols and carotenoids which would be further depleted by increased asthma severity and cigarette use. We also hypothesised that a reduction in circulating antioxidants would be associated with poor fetal outcomes. 2. Methods 2.1. Experimental subjects The study was approved by the Hunter New England Area Health Service and University of Newcastle Human Research Ethics Committees. Pregnant women were recruited at the John Hunter Hospital antenatal clinic during the first trimester (n ¼ 135; controls n ¼ 47, and asthmatics n ¼ 84) and provided written informed consent for participation. The protocol for this study has been described in detail previously.17,18. Using the smallest difference observed previously in our analyses of carotenoid levels in non-pregnant asthmatic and healthy control women,12 a power calculation was performed allowing a-priori for analysis of asthma severity and treatment effects. To identify a true difference in means of 15 ug/l with standard deviation 12, we needed to recruit a minimum of 32 control women and 64 women with asthma (32 with mild asthma and 32 with mod-severe asthma) for power equal to 80% and a type 1 error rate of 0.05. The inclusion criteria for women with asthma were women with a doctor diagnosis of asthma who were less than 18 weeks pregnant at the time of consent. The inclusion criteria for control subjects were women with no pre-existing health problems who were less than 18 weeks at the time of consent. Smokers and obese women were included in the study as these are common co-morbidities associated with pregnancies complicated by asthma. Those women with complications other than asthma such as pre-eclampsia, gestational diabetes, infection or preterm delivery were excluded from the analysis retrospectively (n ¼ 4). Birth weight and fetal sex were determined at birth.
Clinical asthma severity was rated as mild, moderate or severe using the integrated severity score described in the Australian Asthma Management Guidelines,19 which closely approximate the National Heart, Lungs and Blood Institute Guidelines.20 Proper inhaler use and compliance was assessed by the study research nurse.21 Cumulative, inhaled corticosteroids (ICS) dose was calculated for each trimester, and summarised as the mean daily dose of beclomethasone dipropionate or equivalent used during pregnancy, where 1 mg BDP was considered equal to 1 mg budesonide or 0.5 mg fluticasone propionate.22 For data analysis, the low, moderate and high ICS dose groups were combined. Some women (n ¼ 36) were using the combination of a long acting b2 agonist with ICS. Asthmatic women in all groups, including the no glucocorticoid group, used the inhaled short acting b2 agonist, salbutamol for symptom relief when required. Current smoking status was assessed by direct questioning at recruitment. All women participating in the study underwent ultrasound assessment at 18, 30 and 36 weeks of gestation. Fetal biparietal diameter (BPD), head circumference (HC), abdominal circumference (AC) and femur length (FL) were measured. Birth weight, length and head circumference were recorded at delivery. Customised birth weight centiles were calculated using www.gestation.net, which accounts for maternal height and weight, ethnicity, parity, fetal sex and gestational age. Length and head circumferences were converted to centiles using John Hunter Hospital growth charts. Gestational age was determined by date of the last menstrual period and confirmed at 18 week ultrasound. Gestational age was not determined at a 12 week ultrasound as these scans are conducted in private clinics and the data was unavailable to the study. 2.2. Blood collection Twenty mL of whole blood was collected into EDTA coated tubes from the median cubital vein at each visit (18, 30 and 36 weeks). Whole blood was centrifuged at 3000 rpm at 4 C for 10 min. Plasma and erythrocytes were decanted into 500 mL aliquots and stored at 80 C until required for analysis. Erythrocytes were used as a representative measure of antioxidant accumulation in tissues and provided a longer term measure of antioxidant status as well as an indication of the degree of protection of cell membranes from oxidation. 2.3. 24 hour food recall At each gestational visit, participants completed a 24 h (hr) food recall questionnaire which has previously been shown to be a useful tool for analysing mean dietary intakes.23 The questionnaire asked for detailed information on meals and snacks consumed 24 h prior to the clinic visit. For this study, the data was analysed from a subgroup of women (i.e. those who had each provided a blood sample and filled out the 24 h food recall questionnaire at each gestational time point), incorporating controls (n ¼ 32) and asthmatics (n ¼ 57). Data were entered into the Foodworks (Xyris, Brisbane) database, incorporating the AusFoods (Brands) and AusNut (All Foods; Food Standards Australia & New Zealand) were extracted. 3. Biochemical analyses 3.1. Analysis of tocopherols and carotenoids in plasma Antioxidant analysis was performed based on an established method using high performance liquid chromatography. All work was carried out in the dark to prevent photooxidative degeneration and samples were kept on ice. 400 mL (mL) of plasma was transferred into polypropylene culture tubes to which 1 mL (mL) of
P.C. McLernon et al. / Clinical Nutrition 31 (2012) 99e107 Table 1 Maternal Characteristics. Maternal characteristics
Control n ¼ 47
Mild n ¼ 31
Maternal age (yrs) Range Body mass index Range Gravidity Range Parity Range Inhaled corticosteroid use (%) Cigarette use (n)
28 24e32 28.9 25e31.9 2 1e3 0 0e1 0 7
27 24e29 25.7 22.6e32.1 3 1e4 1 0e2 32 8
Mod-severe n ¼ 53 28 23e32 26.4 23e30.9 2 1e3 1 0e2 83 19
P value 0.6 0.14 0.18 0.26 0.00* 0.06
Values reported as the median and range. *P<0.05 with significant differences observed between moderate severe asthmatic group relative to the control and mild asthmatic group.
101
(mild; n ¼ 31, moderate/severe; n ¼ 53) were analysed. Not all women contributed a blood sample or completed the questionnaires at each gestational time point (18, 30 and 36 weeks) and some women did not participate in all fetal ultrasound scans (n ¼ 37, controls: n ¼ 10 and asthmatics: n ¼ 21). Subjects who did not contribute data at all time points were included in the cross sectional analyses but excluded for the temporal analyses. Plasma and matched erythrocytes tocopherols from controls (n ¼ 47) and asthmatic (n ¼ 84); (mild; n ¼ 31, moderate/severe; n ¼ 53) women were examined. Three tocopherol isomers were analysed in both maternal plasma and erythrocytes which are d, g and a. In addition, a-tocopherol quinone was measured in erythrocytes, and the ratio of a-tocopherol quinone:a-tocopherol levels was calculated. 3.2. Statistical analysis
ethanol (Sigma, Australia) and 1 mL of ethyl acetate (containing internal standards: 2.5 mg per millilitre (mg/mL)) canthaxanthin [Carotenature, Switzerland] and 20 mg/mL of a-tocopherol acetate (Sigma, Australia) was added. Samples were vortexed for 30 s (sec), centrifuged at 3000 revs per minute (rpm) at 4 C for 10 min (min) and the subsequent supernatant was decanted to a fresh polypropylene tube. The pellet was then washed in 1 mL of ethyl acetate, vortexed for 30 s, centrifuged at 3000 rpm at 4 C for 6 min and the subsequent supernatant was added to previous supernatant. This process was repeated again, and then 1 mL of Hexane (Sigma, Australia) was used for the wash. Sample pellets were discarded. 1 mL of ultra pure water (Millipore, Australia) was added to the supernatant tubes and vortexed for 1 min, centrifuged at 3000 rpm at 4 C for 10 min and the subsequent supernatant was decanted to a clean glass high recovery culture tube and evaporated using high purity nitrogen gas (Linde, Australia). Dried samples were resuspended in 100 mL of dichloromethane:methanol (2:1 v/v [Sigma, Australia]) injection solvent, vortexed for 30 s and then centrifuged at 3000 rpm at 4 C for 5 min. Supernatant was extracted and stored at 80 C until analysed with an Agilent 1200 high performance liquid chromatograph (Agilent Technologies, USA) with a 100 mm (mm) long (2.1 mm ID) with 5 mm particle size Hypersil ODS C18 column (Thermo Electron Corporation, USA). The isocratic mobile phase was acetonitrile, with dichloromethane and methanol (0.05% ammonium acetate added for buffering) as organic modifiers (85:10:5 volume (v)/v/v). Mobile phase flow rate was set at 0.3 mL/min. Tocopherols and carotenoids were detected using a diode array detector (Agilent Technologies, USA) at 297 and 450 nm (nm) respectively. Plasma concentrations and 24 h recall dietary intake questionnaire data from controls (n ¼ 47) and asthmatic (n ¼ 84) women
All statistical analysis was conducted using the Statistical Package for the Social Sciences (SPSS/PASW v17). Most of the data was not normally distributed and therefore non parametric statistics were used. Friedman’s Analysis of Variance (ANOVA) Test was conducted to analyse temporal changes of tocopherol and carotenoids within groups, with Wilcoxon Signed Rank Tests used for post hoc comparisons. The Kruskal Wallis Test was used for multiple comparisons, and ManneWhitney U Tests were used for post hoc comparisons. All analyses were two tailed. Spearman’s bivariate correlation analysis was used to determine any significant relationships between maternal circulating antioxidant levels and fetal/birth growth parameters. Fisher’s Test for Exact Significance was used where possible to determine significance levels. Bonferroni corrections were applied to all post hoc analyses to adjust for multiple comparisons. An alpha level was set at P 0.05 for all analyses unless specified. 4. Results 4.1. Maternal and neonatal characteristics There were no significant differences between the mild, moderate/severe asthmatics and the control groups with respect to maternal age or body mass index (BMI) (P > 0.05, Fisher’s Exact Test, Table 1), gravidity or parity. A higher proportion of moderate/ severe asthmatics used inhaled corticosteroids to treat their asthma (83%) when compared to the mild asthmatics (32%). There were no significant differences in fetal growth parameters as measured by ultrasound (data not shown) or neonatal outcomes between the groups (p > 0.05 Fisher’s Exact Test, Table 2).
Table 2 Neonatal Characteristics. Neonatal characteristics
Control n ¼ 47
Mild n ¼ 31
Mod-severe n ¼ 53
P value
Gestational age (weeks) Range Birth weight (g) Range Birth weight centile Range Birth length (cm) Range Head circumference (cm) Range Placental weight (g) Range Male/female (n/n)
40.4 39.4e41.2 3402.5 3115e3715 35.6 19.3e51.4 51 50e53 35 34e35.5 630.4 602.8e671.7 18/25
40.3 39.6e41 3660 3220e3940 52.3 14.6e73.5 51 50e54 34.5 33.6e36 671.8 607.4e800.8 14/17
39.8 38.6e40.6 3340 3040e3647 52 22.9e63.1 52 50e53 34.5 33.5e35.5 645.1 592.8e781.6 24/29
0.06
Values reported as the median and inter-quartile range.
0.18 0.29 0.24 0.26 0.27
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4.2. Dietary intake
4.3. Plasma carotenoids
Dietary intake was examined in a subset of women from the control group (n ¼ 32) and asthmatic women (n ¼ 57) at 18, 30 and 36 weeks gestation. Asthmatic groups were subdivided based on severity (mild; n ¼ 25, moderate/severe; n ¼ 34). There were no significant differences between the groups as gestation progressed. At 36 weeks gestation, (Table 3) the mild asthmatic group (n ¼ 25) consumed higher quantities of energy, protein, fats, carbohydrates, starch, thiamin, riboflavin, niacin equivalents, magnesium and phosphorous than the moderate/severe asthmatics (n ¼ 34) or control population (n ¼ 32) (P < 0.01, Fisher’s Exact Test, Table 3). The moderate/severe asthmatic group had a reduced consumption of all the food groups relative to the control or mild asthmatic group (P < 0.01, Fisher’s Exact Test, Table 3) at 36 weeks.
When cross sectional data was analysed by asthma severity, there were no significant differences in maternal plasma (Table 4) total carotenoids, a-carotene, b-carotene, lutein, b-cryptoxanthin or lycopene (mg/L, data not shown) between the control (n ¼ 37), mild (n ¼ 26) or moderate/severe asthmatic (n ¼ 37) groups at 18, 30 or 36 weeks gestation (p > 0.05, Kruskal Wallis Tests). A subgroup of women from the total cohort provided blood samples at each gestational time point (control (n ¼ 21), mild asthmatics (n ¼ 16) and moderate/severe asthmatics (n ¼ 19)). A temporal analysis of maternal plasma total carotenoids (mg/L) and lutein revealed a significant increase of concentrations in the moderate/severe asthmatic group as gestation progressed
Table 3 Maternal total dietary intake at 36 weeks gestation. Dietary factors
Control n ¼ 32
Mild n ¼ 25
Energy (MJ) Range Protein (g) Range Total fat (g) Range Saturated fat (g) Range Polyunsaturated fat (g) Range n6-Polyunsaturated fat (g) Range n3-Polyunsaturated fat (g) Range Monounsaturated fat (g) Range Cholesterol (mg) Range Carbohydrates (g) Range Sugars (g) Range Starch (g) Range Dietary fibre (g) Range Thiamin (mg) Range Riboflavin (mg) Range Niacin equivalents (mg) Range Vitamin C (mg) Range Total folate (ug) Range Viamin A equivalents (ug) Range Retinol (ug) Range Beta carotene (ug) Range Potassium (mg) Range Magnesiums (mg) Range Calcium (mg) Range Phosphorous (mg) Range Iron (mg) Range
9.6 7.2e11.4 86.2 58.3e113 97.9 68.8e122.3 41.5 32.3e54.1 10.9 6.6e16.6 6.5 5.0e12.1 0.9 0.7e1.5 30.9 22e47.2 254.6 160.9e354.6 269.6 192.9e324 167.9 112.5e205.7 118 69.1e136.9 17.3 14.1e25.8 1.4 0.9e2.1 2.1 1.1e3 35.4 24.6e47 135.8 50.9e238.9 280.9 178.4e366.9 970.6 547e1392 432.4 292.2e692.8 2159 971e4432 3020.8 1997.4e3880.5 306.7 201.9e987.9 865.9 561.9e1491.2 1367.9 1024.2e1936.7 11.1 6.9e13.4
10.7* 9e14. 96.8* 80.2e141 108.2 87.5e148.9 43.6 35.9e66.7 14.6 9.2e19.9 7.2 3.8e12.5 0.8 0.6e1.8 40.9 29.9e50.1 331.7* 229.6e515 309.5* 241.8e407.4 156.4 124.9e209.9 138.8 107.2e182.4 21.5 17.6e26.6 1.8* 1.5e2.4 2.4 1.6e3.1 43.1* 34.5e59.2 128.3 41.5e289.8 304.7 233.2e403.5 1143.1 652e1336 498.3 437.7e691.9 2552 586e5413.1 3055.9 2625.4e3940.3 333.9 293.1e372.3 984.8 819.6e1465.9 1698.5* 1452.4e2022.4 13.4* 10.3e18.5
Moderate/severe n ¼ 34
P value
8.2** 6.9e9.8 76.5** 56e96.7 80.6* 56.7e100.6 33.3* 24.8e46.4 7.8* 5.5e14.8 6 4.4e10.2 0.8 0.5e1.23 29.3* 20.6e38.6 269.4 148.2e410.7 224.1 189.2e294.5 128.1 91.9e170 101.9* 84.6e144.8 17.8 12e26.1 1.4 0.9e1.8 1.6* 1.1e2.3 36.3 23.8e44.6 142.3 78.5e257.7 257.7 182.8e307 955.3 568e1311 363.5 274.9e633.2 1816.4 658e4999 3115.8 2255.9e3987.5 265.2* 220.9e322.8 825.5 485.8e1122.3 1244 1002.7e1662.4 11.6 7.9e13.9
0.004* 0.02* 0.01* 0.04* 0.02* 0.3 0.5 0.03* 0.02* 0.02* 0.1 0.04* 0.4 0.03* 0.05* 0.04* 0.8 0.2 0.8 0.1 0.9 0.9 0.04* 0.3 0.01* 0.04*
Values are median and inter-quartile range. The Kruskal-Wallis and ManneWhitney U test with Bonferroni correction were used. *P <0.05 was considered significant compared to control population. ** significant compared to mild asthmatics.
P.C. McLernon et al. / Clinical Nutrition 31 (2012) 99e107 Table 4 Plasma carotenoids at 18, 30 and 36 weeks gestation. n
Control 37
Mild 26
Mod/severe 37
P value
18 weeks gestation Total carotenoids Range b-Carotene Range a-carotene Range b-cryptoxanthine Range Lycopene Range Lutein Range
1.2 0.84e1.7 0.1 0.06e0.25 0.02 0.01e0.04 0.07 0.04e0.15 0.27 0.13e0.4 0.63 0.5e0.9
1.3 0.76e1.8 0.12 0.05e0.31 0.02 0.01e0.04 0.11 0.06e0.18 0.27 0.15e0.4 0.54 0.4e0.9
0.94 0.65e1.3 0.08 0.06e0.2 0.01 0.01e0.08 0.07 0.03e0.12 0.23 0.13e0.3 0.53 0.4e0.7
0.2
30 weeks gestation Total carotenoids Range beCarotene Range aecarotene Range becryptoxanthine Range Lycopene Range Lutein Range
1.44 0.97e2 0.09 0.07e0.3 0.02 0.01e0.06 0.11 0.06e0.23 0.25 0.16e0.38 0.75 0.6e1
1.37 0.94e1.9 0.12 0.05e0.3 0.02 0.01e0.04 0.11 0.07e0.16 0.26 0.2e0.4 0.65 0.5e1
1.1 0.83e1.4 0.09 0.06e0.3 0.02 0.01e0.04 0.09 0.05e0.15 0.18 0.1e0.3 0.69 0.5e0.9
0.2
36 weeks gestation Total carotenoids Range b-Carotene Range a-carotene Range b-cryptoxanthine Range Lycopene Range Lutein Range
1.36 1.05e1.8 0.14 0.07e0.23 0.02 0.01e0.05 0.1 0.05e0.2 0.3 0.2e0.4 0.84 0.6e1.0
1.56 8.2e2.1 0.2 0.06e0.3 0.03 00e0.05 0.11 0.06e0.14 0.3 0.2e0.6 0.7 0.5e1.0
1.3 1.04e1.6 0.1 0.07e0.2 0.02 0.01e0.04 0.09 0.05e0.13 0.25 0.13e0.3 0.75 0.6e1.1
0.8
0.5
Plasma carotenoids were not significantly different between women who used inhaled corticosteroids and those who did not in either the mild or the moderate/severe asthmatic groups. Smokers had significantly lower maternal circulating plasma levels of carotenoids compared to non-smokers (p 0.001 Bonferroni correction). 4.4. Relationships between birth weight centile and carotenoids
0.3 0.08 0.3 0.4
0.9 0.9 0.2 0.06 0.3
0.9 0.9 0.5 0.5 0.7
Concentrations in mg/L. Values are median with inter-quartile range. A Kruskal Wallis test was used to examine differences between control (n ¼ 37), mild asthma (n ¼ 26) and moderate severe asthma (n ¼ 37).
(p < 0.05, Fisher’s Exact Test; Figs. 1 and 2). Maternal circulating b-cryptoxanthin (mg/L), lycopene (mg/L), b-carotene (mg/L) or a-carotene (mg/L) did not change as gestation progressed (p > 0.05, Fisher’s Exact Test).
A
103
B
No relationships were found between maternal circulating plasma levels of carotenoids and birth weight centile (BWC) in the control group. In the moderate/severe asthmatics, positive correlations were found between BWC and maternal circulating plasma levels of lycopene at 18 weeks (R2 ¼ 0.19, r ¼ 0.432, p ¼ 0.008, n ¼ 37) and 30 weeks (R2 ¼ 0.14, r ¼ 0.385, p ¼ 0.05, n ¼ 27). 4.5. Tocopherols There were significantly higher concentrations of total plasma tocopherols and a-tocopherols in the moderate/severe asthmatics (p < 0.02, Bonferroni correction) at both 30 and 36 weeks gestation compared to the control group (Table 6). In erythrocytes, a-tocopherol levels at 30 weeks gestation were significantly higher in the mild asthmatic group relative to the controls (p ¼ 0.05, Kruskal Wallis ANOVA, Table 5). Erythrocyte atocopherol quinone levels were significantly higher in the moderate/severe asthmatic group at 18 weeks gestation relative to the controls (p < 0.02, Bonferroni correction, Table 5). There were no significant differences in the ratio of a-tocopherol quinone-atocopherol between the groups. Plasma d- and erythrocyte d- and g-tocopherols were close to the limit of detection, and therefore the data are not shown. When the data was analysed temporally, maternal circulating concentrations of total tocopherols significantly increased during pregnancy in all groups (Fig. 3, p < 0.05, Fisher’s Test). Maternal circulating plasma a-tocopherol levels significantly increased as gestation progressed in the mild (p < 0.001, Fisher’s Test) and moderate/severe asthmatic groups (p < 0.001, Fisher’s Test, Fig. 4). There were no significant differences found in maternal circulating plasma g-tocopherol levels between any of the groups. No significant differences were found temporally in maternal erythrocyte levels of tocopherols between any of the groups. Maternal plasma and erythrocyte levels of tocopherols were not significantly different between the women who used inhaled
C
Fig. 1. Temporal analysis of plasma total carotenoids (mg/L) in control pregnancies (panel A), mild (panel B) and moderate/severe asthmatic pregnancies (panel C) at 18, 30 and 36 weeks gestation. Data are presented as median and inter-quartile ranges with 95% confidence intervals. A Friedman Test was used to record differences over pregnancy. Wilcoxon’s Signed Rank Test was used for post hoc analysis with Bonferroni correction; *P < 0.02.
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Fig. 2. Temporal analysis of plasma lutein (mg/L) in control pregnancies (panel A), mild (panel B) and moderate/severe asthmatic pregnancies (panel C) at 18, 30 and 36 weeks gestation. Data are presented as median and inter-quartile ranges with 95% confidence intervals. A Friedman Test was used to record differences over pregnancy. Wilcoxon’s Signed Rank Test was used for post hoc analysis with Bonferroni correction; *P < 0.02.
corticosteroids and those who did not in either the mild or the moderate/severe asthmatic groups (p > 0.05, Bonferroni correction). There was also no effect of cigarette use on tocopherols in plasma or erythrocytes (p > 0.05, Bonferroni correction).
4.6. Relationships between birth outcomes and tocopherols In the presence of maternal asthma there was a positive correlation between erythrocyte a tocopherol at 36 weeks gestation and neonatal HC (r ¼ 0.447, p ¼ 0.002, n ¼ 44), and a negative correlation between erythrocyte a-tocopherol quinone:a tocopherol at 36 weeks gestation and neonatal HC (r ¼ 0.403, p ¼ 0.01, n ¼ 44). Maternal circulating plasma a-tocopherol levels at 36 weeks gestation and BWC were positively correlated (r ¼ 0.330, p ¼ 0.029, Table 5 Plasma tocopherols during gestation in relation to asthma severity. n
n ¼ 44). These correlations remained when split by asthma severity but were not observed in the control population. 5. Discussion This study has identified that pregnancies complicated by moderate/severe asthma are associated with an altered antioxidant profile with higher concentrations of some tocopherols and carotenoids in plasma as gestation progresses. The data suggests that the maternal system compensates by adjusting its antioxidant defences in response to high oxidative load of both pregnancy and asthma. These adjustments appear to be beneficial to fetal growth. In the current study it was observed that moderate severe asthmatics had higher erythrocyte a-tocopherol quinone levels early in gestation relative to the controls. Tocopherol quinone is the oxidized Table 6 Erythrocyte tocopherols during gestation in relation to asthma severity.
Control 37
Mild 26
Mod/severe 37
P value
18 weeks gestation Total tocopherols Range g-tocopherol Range a-tocopherol Range
19.1 17.2e21.6 0.06 0.4e0.7 18.7 16.8e20.8
20.7 14.9e25.5 0.6 0.4e0.8 20.2 14.5e25.2
22.3 17.8e26.9 0.6 0.4e0.8 21.8 17.3e26.1
0.06
30 weeks gestation Total tocopherols Range g-tocopherol Range a-tocopherol Range
21.5 19.7e25.8 0.65 0.5e0.9 20.8 19.2e25.2
24 19.5e31.3 0.68 0.6e0.9 23.4 19.1e30.3
26.4 # 21.9e31.9 0.68 0.5e0.9 25.6 # 21.5e31.4
0.02
36 weeks gestation Total tocopherols Range g-tocopherol Range a-tocopherol Range
21.9 19.2e25.6 0.7 0.6e0.8 21.5 18.6e24.9
24.4 20.4e31.9 0.64 0.6e0.8 23.8 19.7e31.2
29.3 #* 26.2e37.4 0.7 0.6e1.0 28.6 # 2.4e36.21
n
Control 35
Mild 23
Mod/severe P value 38
0.8 0.5e0.9 0.7 0.5e0.8 0.9 0.7e1.1
0.9 0.6e1.0 0.7 0.6e0.9 0.9 0.6e1.3
0.8 0.6e1.0 0.8* 0.6e0.9 1.1 0.8e1.3
0.9
Range a-tocopherol quinone Range a-tocopherol quinone/aetocopherol Range 30 weeks gestation a-tocopherol Range a-tocopherol quinone Range a-tocopherol quinone/aetocopherol Range
0.7 0.6e0.9 0.7 0.5e0.9 1 0.7e1.3
0.9* 0.8e1.1 0.7 0.6e1.0 0.8 0.6e1.0
0.7 0.5e1.0 0.8 0.6e1.0 1 0.6e1.3
0.05*
36 weeks gestation a-tocopherol Range a-tocopherol quinone Range a-tocopherol quinone/aetocopherol Range
0.9 0.8e0.96 0.7 0.6e0.9 0.9 0.6e1.1
0.8 0.7e1.3 0.7 0.6e0.8 0.7 0.5e0.9
0.7 0.5e1.0 0.7 0.5e1.1 0.9 0.7e1.4
18 weeks gestation
0.4 0.06
0.9 0.02
<0.01 0.3 <0.01
Concentrations in mg/L. Values are median with inter-quartile range. A Kruskal Wallis test was used to examine differences between controls, mild and moderate/ severe asthmatics. ManneWhitney U tests were used for post hoc analysis with Bonferroni corrections. Significant difference to Control # and mild * are indicated.
a-tocopherol
0.04* 0.3
0.6 0.1
0.3 0.7 0.09
Concentrations in mg/L. Values are median with inter-quartile range. A Kruskal Wallis test was used to examine differences between controls, mild and moderate/ severe asthmatics. ManneWhitney U tests were used for post hoc analysis with Bonferroni corrections. Significant difference to Control * are indicated.
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105
Fig. 3. Temporal analysis of plasma total tocopherol levels (mg/L) in control pregnancies (panel A), mild (panel B) and moderate/severe asthmatic pregnancies (panel C) at 18, 30 and 36 weeks gestation. Data are presented as median and inter-quartile ranges with 95% confidence intervals. A Friedman Test was used to record differences over pregnancy. Wilcoxon Signed Rank Tests were used for post hoc analysis using Bonferroni correction; *P < 0.02.
form of a-tocopherol. Our previous work has identified that nonpregnant asthmatics had higher concentrations of whole blood atocopherol quinone than healthy controls which was correlated with asthma control.10 These data would support the concept that asthma is a disease state associated with reduced antioxidant defences that may contribute to asthma severity. The current findings suggest that early gestational profile of asthmatics may be comparable to the non-pregnant state in terms of antioxidant activity. However the antioxidant profile appears to change as gestation progresses. Maternal asthma during pregnancy was associated with an increase in plasma concentrations of total carotenoids, lutein and a-tocopherol late in gestation. These antioxidants correlated positively with birthweight centile suggesting that increased concentrations of circulating antioxidants at 30 and 36 weeks gestation in the cohort of moderate/severe asthmatic women was protective against the growth restricting effects of maternal asthma during pregnancy. In pregnant sheep, maternal tocopherol supplementation during pregnancy reduced maternal circulating mediators of oxidative stress including 8-epi-prostglandin F224 and up regulated placental gene networks involved in angiogenesis including increased placental expression of endothelial nitric
oxide synthase, vascular endothelial growth factor and hypoxia inducible factor25 to promote fetal growth. The programming effects of prenatal malnutrition on renal function were reversed with maternal tocopherol supplementation during lactation in the rat.26 In humans maternal tocopherol concentrations during pregnancy were positively correlated with fetal crown-rump length, as a marker of fetal growth and associated with a reduced risk of development of childhood asthma.27 Conversely, supranutritional supplementation of pregnant rats with tocopherols resulted in permanent impairments in hippocampal development in offspring.28 Similarly a randomised placebo-controlled trial of high dose tocopherol and vitamin C supplementation in pregnant women with pre-eclampsia resulted in an increased incidence of low birth weight babies in the supplemented group relative to the placebo group.29 In this study, a combination of vitamin E and vitamin C was used due to the role of vitamin C in regenerating vitamin E and restoring it’s antioxidant function once it has been oxidised. These studies indicate that endogenous concentrations of tocopherols derived from dietary intake or low dose supplementation may be central to protecting the fetus from the oxidative effects of maternal asthma.
Fig. 4. Temporal analysis of plasma a-tocopherol levels (mg/L) in control pregnancies (panel A), mild (panel B) and moderate/severe asthmatic pregnancies (panel C) at 18, 30 and 36 weeks gestation. Data are presented as median and inter-quartile ranges with 95% confidence intervals. A Friedman Test was used to record differences over pregnancy. Wilcoxon Signed Rank Tests were used for post hoc analysis using Bonferroni correction; *P < 0.02.
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Higher concentrations of total plasma tocopherols and atocopherols, total plasma carotenoids and lutein in the moderate/ severe asthmatic group at both 30 and 36 weeks gestation were not related to differences in dietary intake. In fact moderate/severe asthmatics had reduced intake of most food groups relative to the mild asthmatic and control groups. Overall dietary intake in this particular population of pregnant women is comparable to the diet of other pregnant cohorts.30e32 Intake of total energy and macronutrients, including polyunsaturated fat, which is closely linked to a-tocopherol intake, was lower in the moderate/severe asthma group. Also, dietary intake for the antioxidant vitamin C was not increased in the moderate/severe asthma group. Hence, these data suggest that a systemic compensatory antioxidant response occurs in the presence of moderate/severe asthma as gestation progresses and fetal demand increases. This may involve the release of antioxidants from tissue stores, to boost antioxidant defences and ensure continued fetal growth and survival in a sub-optimal intrauterine environment. As expected the use of cigarettes during pregnancy significantly altered the antioxidant profile especially in relation to the plasma carotenoids which were reduced in the smokers relative to the nonsmokers. Previous studies have reported that smoking depletes antioxidant defences,16 contributes to worsening asthma during pregnancy18 and is associated with a significant reduction in fetal growth.33 Smoking during pregnancy is more prevalent in asthmatic women relative to non-asthmatic women14 and is a serious health issue contributing to poor outcomes observed in this population. Supplementation of smokers with vitamin E has recently been reported in the Women’s Health Study.34 It was found that vitamin E supplementation to women over the age of 45 in a placebo-controlled randomised trial significantly reduced the incidence of chronic lung disease which was not modified by the presence of cigarette use.34 Smoking has been shown to be associated with a reduced consumption of carotenoids, derived from dietary intake of fruit and vegetables.35 These data suggest improved dietary intake of fruit and vegetables along with a multivitamin supplement may be important for smokers during pregnancy. Furthermore, pregnant women should also be encouraged to quit or reduce cigarette use during pregnancy. The use of 24 h recall dietary analysis, as a measure of recent dietary intake was considered a limitation of the study and a 4-day food record would have provided a more accurate representation. However 24 h recalls were used in order to minimise participant burden and are a useful measure of group mean intakes.23 Another limitation was the use of non-fasting samples which could not be avoided as the request for an overnight fast was ethically inappropriate for a non-medical assessment of pregnant women. The current study is the first to characterise the circulating antioxidant profile of pregnant women with asthma. Moderate/ severe asthmatic women appear to have a compensatory increase in the concentration of circulating antioxidants which is positively associated with birth outcomes. These findings suggest the maternal system adjusts metabolic pathways in response to the presence of a high oxidative load induced by asthma in an attempt to ensure continued fetal growth. Future studies will examine the relationship between the maternal, placental and fetal antioxidant profiles in pregnancies complicated by asthma.
Statement of authorship and conflict of interest statement PM conducted the study and analysed the data. NH analysed the data. LW, VM and VC designed the study and wrote the manuscript. There are no identified conflicts of interest in the production of this work or manuscript.
Acknowledgements The work was funded by Department of Health and Aging Award and National Health and Medical Research Council (NHMRC). P. McLernon was funded by an Australian Postgraduate Award for the work outlined in this manuscript. V. Clifton is the recipient of a NHMRC Senior Fellowship (ID 510703). V. Murphy is the recipient of a NHMRC Research Training Fellowship (Part time, ID 455626). N. Hodyl is the recipient of a NHMRC Peter Doherty Fellowship (APP1016379). The authors would like to thank Prof Manohar Garg and staff from the John Hunter Hospital Antenatal Clinic and Delivery Suite. References 1. Greene LS. Asthma, oxidant stress, and diet. Nutrition 1999;15:899e907. 2. de Luis DA, Armentia A, Aller R, Asensio A, Sedano E, Izaola O, et al. Dietary intake in patients with asthma: a case control study. Nutrition 2005;21:320e4. 3. Traber MG, Atkinson J. Vitamin E, antioxidant and nothing more. Free Radic Biol Med 2007;43:4e15. 4. Toescu V, Nuttall SL, Martin U, Kendall MJ, Dunne F. Oxidative stress and normal pregnancy. Clin Endocrinol (Oxf) 2002;57:609e13. 5. Siddiqui IA, Jaleel A, Tamimi W, Al Kadri HM. Role of oxidative stress in the pathogenesis of preeclampsia. Arch Gynecol Obstet 2010;282:469e74. 6. Bakheit KH, Ghebremeskel K, Zaiger G, Elbashir MI, Adam I. Erythrocyte antioxidant enzymes and plasma antioxidant vitamins in Sudanese women with preeeclampsia. J Obstet Gynaecol 2010;30:147e50. 7. Ishihara O, Hayashi M, Osawa H, Kobayashi K, Takeda S, Vessby B, et al. Isoprostanes, prostaglandins and tocopherols in preeeclampsia, normal pregnancy and nonepregnancy. Free Radic Res 2004;38:913e8. 8. Sharma JB, Sharma A, Bahadur A, Vimala N, Satyam A, Mittal S. Oxidative stress markers and antioxidant levels in normal pregnancy and preeeclampsia. Int J Gynaecol Obstet 2006;94:23e7. 9. Wood LG, Garg ML, Blake RJ, Simpson JL, Gibson PG. Oxidized vitamin E and glutathione as markers of clinical status in asthma. Clin Nutr 2008;27:579e86. 10. Wood LG, Gibson PG. Reduced circulating antioxidant defences are associated with airway hypereresponsiveness, poor control and severe disease pattern in asthma. Br J Nutr 2010;103:735e41. 11. Ford ES, Mannino DM, Redd SC. Serum antioxidant concentrations among U.S. adults with selfereported asthma. J Asthma 2004;41:179e87. 12. Wood LG, Garg ML, Blake RJ, GarciaeCaraballo S, Gibson PG. Airway and circulating levels of carotenoids in asthma and healthy controls. J Am Coll Nutr 2005;24:448e55. 13. Schatz M, Zeiger RS. Treatment of asthma and allergic rhinitis during pregnancy. Ann Allergy 1990;65:427e9. 14. Clifton VL, Engel P, Smith R, Gibson P, Brinsmead M, Giles WB. Maternal and neonatal outcomes of pregnancies complicated by asthma in an Australian population. Aust New Zealand J Obstetircs Gynaecol 2009;49:619e26. 15. Godfrey KM, Barker DJ. Fetal nutrition and adult disease. Am J Clin Nutr 2000;71:1344Se52S. 16. Faure H, Preziosi P, Roussel AM, Bertrais S, Galan P, Hercberg S, et al. Factors influencing blood concentration of retinol, alphaetocopherol, vitamin C, and betaecarotene in the French participants of the SU.VI.MAX trial. Eur J Clin Nutr 2006;60:706e17. 17. Murphy VE, Gibson PG, Giles WB, Zakar T, Smith R, Bisits AM, et al. Maternal asthma is associated with reduced female fetal growth. Am J Respir Crit Care Med 2003;168:1317e23. 18. Murphy VE, Clifton VL, Gibson PG. The effect of cigarette smoking on asthma control during exacerbations in pregnant women. Thorax 2010;65:739e44. 19. Gibson PG, Wilson AJ. The use of continuous quality improvement methods to implement practice guidelines in asthma. J Qual Clin Pract 1996;16:87e102. 20. National Heart LaBI. New NHLBI guidelines for the diagnosis and management of asthma. Lippincott Health Promot Lett 1997;2:8e9. 21. Murphy VE, Gibson PG, Talbot PI, Kessell CG, Clifton VL. Asthma selfemanagement skills and the use of asthma education during pregnancy. Eur Respir J 2005;26:435e41. 22. Barnes PJ, Pedersen S. Efficacy and safety of inhaled corticosteroids in asthma. Report of a workshop held in Eze, France, October 1992. Am Rev Respir Dis 1993;148:S1e26. 23. Block G. A review of validations of dietary assessment methods. Am J Epidemiol 1988;115:492e505. 24. Kasimanickam RK, Kasimanickam VR. Effect of tocopherol supplementation on serum 8eepieprostaglandin F2 alpha and adiponectin concentrations, and mRNA expression of PPARgamma and related genes in ovine placenta and uterus. Theriogenology 2011. 25. Kasimanickam RK, Kasimanickam VR, Rodriguez JS, Pelzer KD, Sponenberg PD, Thatcher CD. Tocopherol induced angiogenesis in placental vascular network in late pregnant ewes. Reprod Biol Endocrinol 2010;8:86. 26. VieiraeFilho LD, Lara LS, Silva PA, Santos FT, Luzardo R, Oliveira FS, et al. Placental malnutrition changes the regulatory network of renal NaeATPase in
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adult rat progeny: reprogramming by maternal alphaetocopherol during lactation. Arch Biochem Biophys 2011;505:91e7. Turner SW, Campbell D, Smith N, Craig LC, McNeill G, Forbes SH, et al. Associations between fetal size, maternal {alpha}etocopherol and childhood asthma. Thorax 2010;65:391e7. Betti M, Ambrogini P, Minelli A, Floridi A, Lattanzi D, Ciuffoli S, et al. Maternal dietary loads of alphaetocopherol depress protein kinase C signaling and synaptic plasticity in rat postnatal developing hippocampus and promote permanent deficits in adult offspring. J Nutr Biochem 2011;22:60e70. Poston L, Briley AL, Seed PT, Kelly FJ, Shennan AH, Consortium VT. Vitamin C and vitamin E in pregnant women at risk for preeeclampsia (VIP trial): randomised placeboecontrolled trial. Lancet 2006;367:1145e54. Hure A, Young A, Smith R, Collins C. Diet and pregnancy status in Australian women. Public Health Nutr 2009;12:853e61.
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31. Prasad M, Lumia M, Erkkola M, Tapanainen H, KronbergeKippila C, Tuokkola J, et al. Diet composition of pregnant Finnish women: changes over time and across seasons. Public Health Nutr 2010;13:939e46. 32. Guelinckx I, Devlieger R, Mullie P, Vansant G. Effect of lifestyle intervention on dietary habits, physical activity, and gestational weight gain in obese pregnant women: a randomized controlled trial. Am J Clin Nutr 2010;91:373e80. 33. Clifton VL, Hodyl NA, Murphy VE, Giles WB, Baxter RC, Smith R. Effect of maternal asthma, inhaled glucocorticoids and cigarette use during pregnancy on the newborn insulinelike growth factor axis. Growth Horm IGF Res 2010;20:39e48. 34. Agler AH, Kurth T, Gaziano JM, Buring JE, Cassano PA. Randomised vitamin E supplementation and risk of chronic lung disease in the Women’s Health Study. Thorax 2011;66:320e5. 35. Padrao P, SilvaeMatos C, Damasceno A, Lunet N. Association between tobacco consumption and alcohol, vegetable and fruit intake across urban and rural areas in Mozambique. J Epidemiol Community Health 2011;65:445e53.
Contents lists available at SciVerse ScienceDirect
Clinical Nutrition journal homepage: http://www.elsevier.com/locate/clnu
Original article
Circulating antioxidant profile of pregnant women with asthma Penelope C. McLernona, b, Lisa G. Woodc, Vanessa E. Murphyc, Nicolette A. Hodyla, Vicki L. Cliftona, b, * a
Robinson Institute, University of Adelaide, Adelaide, SA, Australia Mothers and Babies Research Centre, Hunter Medical Research Institute, University of Newcastle, NSW, Australia c Centre for Asthma and Respiratory Diseases, University of Newcastle and Hunter Medical Research Institute, Newcastle NSW, Australia b
a r t i c l e i n f o
s u m m a r y
Article history: Received 18 July 2011 Accepted 3 September 2011
Background & aims: One of the most prevalent complications of pregnancy is asthma which is associated with an increased incidence of intrauterine growth restriction. The mechanisms that affect fetal development in pregnancies complicated by asthma are not clearly defined. Antioxidants are particularly important during pregnancy due to their protective role against a state of high oxidative stress as gestation progresses. The current study was designed to characterise the circulating profile of tocopherols and carotenoids in pregnant women with asthma to determine whether asthma severity and dietary intake were associated with an altered antioxidant profile. Methods: Maternal dietary intake and plasma and erythrocyte concentrations of tocopherols and carotenoids were examined in women with (n ¼ 84) and without asthma (n ¼ 47) at 18, 30 and 36 weeks gestation. Tocopherol and carotenoid levels were related to fetal and birth outcomes. Results: Pregnant women with moderate/severe asthma were found to have increased plasma concentrations of total carotenoids (P < 0.05), lutein (P < 0.05 and a-tocopherol (P < 0.02) late in gestation compared to those women with mild asthma and healthy pregnant controls. Moderate/severe asthmatics had higher erythrocyte a-tocopherol quinone levels early in gestation relative to the controls (P < 0.02) but this marker of oxidative stress decreased as gestation progressed. Tocopherols and carotenoids were positively associated with birth weight centile (P < 0.05). Conclusion: These findings suggest that the maternal system adjusts antioxidant pathways in response to the presence of a high oxidative load induced by asthma during pregnancy in an attempt to ensure continued fetal growth in an adverse environment. Ó 2011 Elsevier Ltd and European Society for Clinical Nutrition and Metabolism. All rights reserved.
Keywords: Pregnancy Fetal growth Asthma Tocopherol Carotenoid Antioxidant
1. Introduction Dietary antioxidants such as tocopherols and carotenoids are likely to have a significant effect in modulating systemic oxidative stress. Tocopherols (vitamin E) are low molecular weight substances that act to break free radical chain reactions involved in lipid peroxidation.1 In this way, they are thought to provide a protective mechanism against oxidative damage to the body.2 The most active of the tocopherols is a-tocopherol which converts lipid peroxyl radicals and oxygen radicals found in lipid membranes to less reactive forms2 in order to maintain the integrity of membrane fatty acids.3 Carotenoids are tetraterpenoid organic pigments that are naturally occurring in the chloroplasts of plants. There are two
* Corresponding author. Robinson Institute, University of Adelaide, Lyell McEwin Hospital, Haydown Rd, Elizabethvale, SA 5112, Australia. Tel.: þ61 8 81332133; fax: þ61 8 8303 4099. E-mail address: [email protected] (V.L. Clifton).
classes, xanthophylls which include lutein and zeaxanthine and carotenes which include a-carotene, b-carotene and lycopene. Carotenoids (b-carotene, a-carotene, g-carotene, and b-cryptoxanthin) primarily act as antioxidants by scavenging free radicals. Normal pregnancy has been characterised as being a state of high oxidative stress.4 Oxidative stress is defined as an imbalance between the cellular generation of Reactive Oxygen Species (ROS) and the capacity of antioxidants to prevent oxidative damage. The increased maternal and fetal utilisation of energy, as well as an increased oxygen intake as gestation progresses promotes oxidative stress and places huge demands on the maternal system to balance the generation of ROS via the up regulation of antioxidant mechanisms. Pregnancies complicated by inflammation and oxidative stress such as pre-eclampsia5 have been reported to have higher levels of maternal circulating a-tocopherol.6 Gamma (g) tocopherol was found to be decreased in pre-eclamptic women relative to healthy pregnancies.7 Levels of lycopene were found to be reduced in pre-eclamptic women.8 These studies suggest that in a high oxidative stress environment antioxidant defences are
0261-5614/$ e see front matter Ó 2011 Elsevier Ltd and European Society for Clinical Nutrition and Metabolism. All rights reserved. doi:10.1016/j.clnu.2011.09.006
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compromised and in some cases there may be a compensatory increase in the circulation of some dietary antioxidants in an attempt to protect the maternal system from oxidative stress. However it is currently unknown whether antioxidant mechanisms are altered in pregnant asthmatic mothers. We have previously reported that a-tocopherol levels are lower in non-pregnant asthmatics than healthy controls9 and reduced circulating levels of antioxidants are associated with airway hyperresponsiveness.10 Other studies reported reduced levels of circulating carotenoids in serum,11 and whole blood12 of non-pregnant asthmatic subjects. These studies suggest that worsening asthma disturbs the oxidant-antioxidant balance. Pregnancies complicated by maternal asthma are associated with an increased incidence of intrauterine growth restriction 13, 14, with significant implications for both the short and long term health of the offspring15 Reduced fetal growth may be a consequence of high oxidative stress in pregnancies complicated by asthma. Cigarette use by pregnant women with asthma is a highly prevalent comorbidity of these pregnancies14 that could also significantly lower antioxidant defences and impact on fetal development. It has been previously shown that cigarette use can reduce circulating antioxidants in non-pregnant subjects.16 Since we have previously identified that tocopherols9 and carotenoids10 are altered in non-pregnant asthmatics, the current study was designed to assess if there are any differences in the circulating tocopherols and carotenoids of pregnant women with asthma compared to women without asthma and whether these differences were related to dietary intake, asthma treatment, asthma severity or cigarette use. It was hypothesised that maternal asthma during pregnancy would be associated with reduced circulating concentrations of tocopherols and carotenoids which would be further depleted by increased asthma severity and cigarette use. We also hypothesised that a reduction in circulating antioxidants would be associated with poor fetal outcomes. 2. Methods 2.1. Experimental subjects The study was approved by the Hunter New England Area Health Service and University of Newcastle Human Research Ethics Committees. Pregnant women were recruited at the John Hunter Hospital antenatal clinic during the first trimester (n ¼ 135; controls n ¼ 47, and asthmatics n ¼ 84) and provided written informed consent for participation. The protocol for this study has been described in detail previously.17,18. Using the smallest difference observed previously in our analyses of carotenoid levels in non-pregnant asthmatic and healthy control women,12 a power calculation was performed allowing a-priori for analysis of asthma severity and treatment effects. To identify a true difference in means of 15 ug/l with standard deviation 12, we needed to recruit a minimum of 32 control women and 64 women with asthma (32 with mild asthma and 32 with mod-severe asthma) for power equal to 80% and a type 1 error rate of 0.05. The inclusion criteria for women with asthma were women with a doctor diagnosis of asthma who were less than 18 weeks pregnant at the time of consent. The inclusion criteria for control subjects were women with no pre-existing health problems who were less than 18 weeks at the time of consent. Smokers and obese women were included in the study as these are common co-morbidities associated with pregnancies complicated by asthma. Those women with complications other than asthma such as pre-eclampsia, gestational diabetes, infection or preterm delivery were excluded from the analysis retrospectively (n ¼ 4). Birth weight and fetal sex were determined at birth.
Clinical asthma severity was rated as mild, moderate or severe using the integrated severity score described in the Australian Asthma Management Guidelines,19 which closely approximate the National Heart, Lungs and Blood Institute Guidelines.20 Proper inhaler use and compliance was assessed by the study research nurse.21 Cumulative, inhaled corticosteroids (ICS) dose was calculated for each trimester, and summarised as the mean daily dose of beclomethasone dipropionate or equivalent used during pregnancy, where 1 mg BDP was considered equal to 1 mg budesonide or 0.5 mg fluticasone propionate.22 For data analysis, the low, moderate and high ICS dose groups were combined. Some women (n ¼ 36) were using the combination of a long acting b2 agonist with ICS. Asthmatic women in all groups, including the no glucocorticoid group, used the inhaled short acting b2 agonist, salbutamol for symptom relief when required. Current smoking status was assessed by direct questioning at recruitment. All women participating in the study underwent ultrasound assessment at 18, 30 and 36 weeks of gestation. Fetal biparietal diameter (BPD), head circumference (HC), abdominal circumference (AC) and femur length (FL) were measured. Birth weight, length and head circumference were recorded at delivery. Customised birth weight centiles were calculated using www.gestation.net, which accounts for maternal height and weight, ethnicity, parity, fetal sex and gestational age. Length and head circumferences were converted to centiles using John Hunter Hospital growth charts. Gestational age was determined by date of the last menstrual period and confirmed at 18 week ultrasound. Gestational age was not determined at a 12 week ultrasound as these scans are conducted in private clinics and the data was unavailable to the study. 2.2. Blood collection Twenty mL of whole blood was collected into EDTA coated tubes from the median cubital vein at each visit (18, 30 and 36 weeks). Whole blood was centrifuged at 3000 rpm at 4 C for 10 min. Plasma and erythrocytes were decanted into 500 mL aliquots and stored at 80 C until required for analysis. Erythrocytes were used as a representative measure of antioxidant accumulation in tissues and provided a longer term measure of antioxidant status as well as an indication of the degree of protection of cell membranes from oxidation. 2.3. 24 hour food recall At each gestational visit, participants completed a 24 h (hr) food recall questionnaire which has previously been shown to be a useful tool for analysing mean dietary intakes.23 The questionnaire asked for detailed information on meals and snacks consumed 24 h prior to the clinic visit. For this study, the data was analysed from a subgroup of women (i.e. those who had each provided a blood sample and filled out the 24 h food recall questionnaire at each gestational time point), incorporating controls (n ¼ 32) and asthmatics (n ¼ 57). Data were entered into the Foodworks (Xyris, Brisbane) database, incorporating the AusFoods (Brands) and AusNut (All Foods; Food Standards Australia & New Zealand) were extracted. 3. Biochemical analyses 3.1. Analysis of tocopherols and carotenoids in plasma Antioxidant analysis was performed based on an established method using high performance liquid chromatography. All work was carried out in the dark to prevent photooxidative degeneration and samples were kept on ice. 400 mL (mL) of plasma was transferred into polypropylene culture tubes to which 1 mL (mL) of
P.C. McLernon et al. / Clinical Nutrition 31 (2012) 99e107 Table 1 Maternal Characteristics. Maternal characteristics
Control n ¼ 47
Mild n ¼ 31
Maternal age (yrs) Range Body mass index Range Gravidity Range Parity Range Inhaled corticosteroid use (%) Cigarette use (n)
28 24e32 28.9 25e31.9 2 1e3 0 0e1 0 7
27 24e29 25.7 22.6e32.1 3 1e4 1 0e2 32 8
Mod-severe n ¼ 53 28 23e32 26.4 23e30.9 2 1e3 1 0e2 83 19
P value 0.6 0.14 0.18 0.26 0.00* 0.06
Values reported as the median and range. *P<0.05 with significant differences observed between moderate severe asthmatic group relative to the control and mild asthmatic group.
101
(mild; n ¼ 31, moderate/severe; n ¼ 53) were analysed. Not all women contributed a blood sample or completed the questionnaires at each gestational time point (18, 30 and 36 weeks) and some women did not participate in all fetal ultrasound scans (n ¼ 37, controls: n ¼ 10 and asthmatics: n ¼ 21). Subjects who did not contribute data at all time points were included in the cross sectional analyses but excluded for the temporal analyses. Plasma and matched erythrocytes tocopherols from controls (n ¼ 47) and asthmatic (n ¼ 84); (mild; n ¼ 31, moderate/severe; n ¼ 53) women were examined. Three tocopherol isomers were analysed in both maternal plasma and erythrocytes which are d, g and a. In addition, a-tocopherol quinone was measured in erythrocytes, and the ratio of a-tocopherol quinone:a-tocopherol levels was calculated. 3.2. Statistical analysis
ethanol (Sigma, Australia) and 1 mL of ethyl acetate (containing internal standards: 2.5 mg per millilitre (mg/mL)) canthaxanthin [Carotenature, Switzerland] and 20 mg/mL of a-tocopherol acetate (Sigma, Australia) was added. Samples were vortexed for 30 s (sec), centrifuged at 3000 revs per minute (rpm) at 4 C for 10 min (min) and the subsequent supernatant was decanted to a fresh polypropylene tube. The pellet was then washed in 1 mL of ethyl acetate, vortexed for 30 s, centrifuged at 3000 rpm at 4 C for 6 min and the subsequent supernatant was added to previous supernatant. This process was repeated again, and then 1 mL of Hexane (Sigma, Australia) was used for the wash. Sample pellets were discarded. 1 mL of ultra pure water (Millipore, Australia) was added to the supernatant tubes and vortexed for 1 min, centrifuged at 3000 rpm at 4 C for 10 min and the subsequent supernatant was decanted to a clean glass high recovery culture tube and evaporated using high purity nitrogen gas (Linde, Australia). Dried samples were resuspended in 100 mL of dichloromethane:methanol (2:1 v/v [Sigma, Australia]) injection solvent, vortexed for 30 s and then centrifuged at 3000 rpm at 4 C for 5 min. Supernatant was extracted and stored at 80 C until analysed with an Agilent 1200 high performance liquid chromatograph (Agilent Technologies, USA) with a 100 mm (mm) long (2.1 mm ID) with 5 mm particle size Hypersil ODS C18 column (Thermo Electron Corporation, USA). The isocratic mobile phase was acetonitrile, with dichloromethane and methanol (0.05% ammonium acetate added for buffering) as organic modifiers (85:10:5 volume (v)/v/v). Mobile phase flow rate was set at 0.3 mL/min. Tocopherols and carotenoids were detected using a diode array detector (Agilent Technologies, USA) at 297 and 450 nm (nm) respectively. Plasma concentrations and 24 h recall dietary intake questionnaire data from controls (n ¼ 47) and asthmatic (n ¼ 84) women
All statistical analysis was conducted using the Statistical Package for the Social Sciences (SPSS/PASW v17). Most of the data was not normally distributed and therefore non parametric statistics were used. Friedman’s Analysis of Variance (ANOVA) Test was conducted to analyse temporal changes of tocopherol and carotenoids within groups, with Wilcoxon Signed Rank Tests used for post hoc comparisons. The Kruskal Wallis Test was used for multiple comparisons, and ManneWhitney U Tests were used for post hoc comparisons. All analyses were two tailed. Spearman’s bivariate correlation analysis was used to determine any significant relationships between maternal circulating antioxidant levels and fetal/birth growth parameters. Fisher’s Test for Exact Significance was used where possible to determine significance levels. Bonferroni corrections were applied to all post hoc analyses to adjust for multiple comparisons. An alpha level was set at P 0.05 for all analyses unless specified. 4. Results 4.1. Maternal and neonatal characteristics There were no significant differences between the mild, moderate/severe asthmatics and the control groups with respect to maternal age or body mass index (BMI) (P > 0.05, Fisher’s Exact Test, Table 1), gravidity or parity. A higher proportion of moderate/ severe asthmatics used inhaled corticosteroids to treat their asthma (83%) when compared to the mild asthmatics (32%). There were no significant differences in fetal growth parameters as measured by ultrasound (data not shown) or neonatal outcomes between the groups (p > 0.05 Fisher’s Exact Test, Table 2).
Table 2 Neonatal Characteristics. Neonatal characteristics
Control n ¼ 47
Mild n ¼ 31
Mod-severe n ¼ 53
P value
Gestational age (weeks) Range Birth weight (g) Range Birth weight centile Range Birth length (cm) Range Head circumference (cm) Range Placental weight (g) Range Male/female (n/n)
40.4 39.4e41.2 3402.5 3115e3715 35.6 19.3e51.4 51 50e53 35 34e35.5 630.4 602.8e671.7 18/25
40.3 39.6e41 3660 3220e3940 52.3 14.6e73.5 51 50e54 34.5 33.6e36 671.8 607.4e800.8 14/17
39.8 38.6e40.6 3340 3040e3647 52 22.9e63.1 52 50e53 34.5 33.5e35.5 645.1 592.8e781.6 24/29
0.06
Values reported as the median and inter-quartile range.
0.18 0.29 0.24 0.26 0.27
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4.2. Dietary intake
4.3. Plasma carotenoids
Dietary intake was examined in a subset of women from the control group (n ¼ 32) and asthmatic women (n ¼ 57) at 18, 30 and 36 weeks gestation. Asthmatic groups were subdivided based on severity (mild; n ¼ 25, moderate/severe; n ¼ 34). There were no significant differences between the groups as gestation progressed. At 36 weeks gestation, (Table 3) the mild asthmatic group (n ¼ 25) consumed higher quantities of energy, protein, fats, carbohydrates, starch, thiamin, riboflavin, niacin equivalents, magnesium and phosphorous than the moderate/severe asthmatics (n ¼ 34) or control population (n ¼ 32) (P < 0.01, Fisher’s Exact Test, Table 3). The moderate/severe asthmatic group had a reduced consumption of all the food groups relative to the control or mild asthmatic group (P < 0.01, Fisher’s Exact Test, Table 3) at 36 weeks.
When cross sectional data was analysed by asthma severity, there were no significant differences in maternal plasma (Table 4) total carotenoids, a-carotene, b-carotene, lutein, b-cryptoxanthin or lycopene (mg/L, data not shown) between the control (n ¼ 37), mild (n ¼ 26) or moderate/severe asthmatic (n ¼ 37) groups at 18, 30 or 36 weeks gestation (p > 0.05, Kruskal Wallis Tests). A subgroup of women from the total cohort provided blood samples at each gestational time point (control (n ¼ 21), mild asthmatics (n ¼ 16) and moderate/severe asthmatics (n ¼ 19)). A temporal analysis of maternal plasma total carotenoids (mg/L) and lutein revealed a significant increase of concentrations in the moderate/severe asthmatic group as gestation progressed
Table 3 Maternal total dietary intake at 36 weeks gestation. Dietary factors
Control n ¼ 32
Mild n ¼ 25
Energy (MJ) Range Protein (g) Range Total fat (g) Range Saturated fat (g) Range Polyunsaturated fat (g) Range n6-Polyunsaturated fat (g) Range n3-Polyunsaturated fat (g) Range Monounsaturated fat (g) Range Cholesterol (mg) Range Carbohydrates (g) Range Sugars (g) Range Starch (g) Range Dietary fibre (g) Range Thiamin (mg) Range Riboflavin (mg) Range Niacin equivalents (mg) Range Vitamin C (mg) Range Total folate (ug) Range Viamin A equivalents (ug) Range Retinol (ug) Range Beta carotene (ug) Range Potassium (mg) Range Magnesiums (mg) Range Calcium (mg) Range Phosphorous (mg) Range Iron (mg) Range
9.6 7.2e11.4 86.2 58.3e113 97.9 68.8e122.3 41.5 32.3e54.1 10.9 6.6e16.6 6.5 5.0e12.1 0.9 0.7e1.5 30.9 22e47.2 254.6 160.9e354.6 269.6 192.9e324 167.9 112.5e205.7 118 69.1e136.9 17.3 14.1e25.8 1.4 0.9e2.1 2.1 1.1e3 35.4 24.6e47 135.8 50.9e238.9 280.9 178.4e366.9 970.6 547e1392 432.4 292.2e692.8 2159 971e4432 3020.8 1997.4e3880.5 306.7 201.9e987.9 865.9 561.9e1491.2 1367.9 1024.2e1936.7 11.1 6.9e13.4
10.7* 9e14. 96.8* 80.2e141 108.2 87.5e148.9 43.6 35.9e66.7 14.6 9.2e19.9 7.2 3.8e12.5 0.8 0.6e1.8 40.9 29.9e50.1 331.7* 229.6e515 309.5* 241.8e407.4 156.4 124.9e209.9 138.8 107.2e182.4 21.5 17.6e26.6 1.8* 1.5e2.4 2.4 1.6e3.1 43.1* 34.5e59.2 128.3 41.5e289.8 304.7 233.2e403.5 1143.1 652e1336 498.3 437.7e691.9 2552 586e5413.1 3055.9 2625.4e3940.3 333.9 293.1e372.3 984.8 819.6e1465.9 1698.5* 1452.4e2022.4 13.4* 10.3e18.5
Moderate/severe n ¼ 34
P value
8.2** 6.9e9.8 76.5** 56e96.7 80.6* 56.7e100.6 33.3* 24.8e46.4 7.8* 5.5e14.8 6 4.4e10.2 0.8 0.5e1.23 29.3* 20.6e38.6 269.4 148.2e410.7 224.1 189.2e294.5 128.1 91.9e170 101.9* 84.6e144.8 17.8 12e26.1 1.4 0.9e1.8 1.6* 1.1e2.3 36.3 23.8e44.6 142.3 78.5e257.7 257.7 182.8e307 955.3 568e1311 363.5 274.9e633.2 1816.4 658e4999 3115.8 2255.9e3987.5 265.2* 220.9e322.8 825.5 485.8e1122.3 1244 1002.7e1662.4 11.6 7.9e13.9
0.004* 0.02* 0.01* 0.04* 0.02* 0.3 0.5 0.03* 0.02* 0.02* 0.1 0.04* 0.4 0.03* 0.05* 0.04* 0.8 0.2 0.8 0.1 0.9 0.9 0.04* 0.3 0.01* 0.04*
Values are median and inter-quartile range. The Kruskal-Wallis and ManneWhitney U test with Bonferroni correction were used. *P <0.05 was considered significant compared to control population. ** significant compared to mild asthmatics.
P.C. McLernon et al. / Clinical Nutrition 31 (2012) 99e107 Table 4 Plasma carotenoids at 18, 30 and 36 weeks gestation. n
Control 37
Mild 26
Mod/severe 37
P value
18 weeks gestation Total carotenoids Range b-Carotene Range a-carotene Range b-cryptoxanthine Range Lycopene Range Lutein Range
1.2 0.84e1.7 0.1 0.06e0.25 0.02 0.01e0.04 0.07 0.04e0.15 0.27 0.13e0.4 0.63 0.5e0.9
1.3 0.76e1.8 0.12 0.05e0.31 0.02 0.01e0.04 0.11 0.06e0.18 0.27 0.15e0.4 0.54 0.4e0.9
0.94 0.65e1.3 0.08 0.06e0.2 0.01 0.01e0.08 0.07 0.03e0.12 0.23 0.13e0.3 0.53 0.4e0.7
0.2
30 weeks gestation Total carotenoids Range beCarotene Range aecarotene Range becryptoxanthine Range Lycopene Range Lutein Range
1.44 0.97e2 0.09 0.07e0.3 0.02 0.01e0.06 0.11 0.06e0.23 0.25 0.16e0.38 0.75 0.6e1
1.37 0.94e1.9 0.12 0.05e0.3 0.02 0.01e0.04 0.11 0.07e0.16 0.26 0.2e0.4 0.65 0.5e1
1.1 0.83e1.4 0.09 0.06e0.3 0.02 0.01e0.04 0.09 0.05e0.15 0.18 0.1e0.3 0.69 0.5e0.9
0.2
36 weeks gestation Total carotenoids Range b-Carotene Range a-carotene Range b-cryptoxanthine Range Lycopene Range Lutein Range
1.36 1.05e1.8 0.14 0.07e0.23 0.02 0.01e0.05 0.1 0.05e0.2 0.3 0.2e0.4 0.84 0.6e1.0
1.56 8.2e2.1 0.2 0.06e0.3 0.03 00e0.05 0.11 0.06e0.14 0.3 0.2e0.6 0.7 0.5e1.0
1.3 1.04e1.6 0.1 0.07e0.2 0.02 0.01e0.04 0.09 0.05e0.13 0.25 0.13e0.3 0.75 0.6e1.1
0.8
0.5
Plasma carotenoids were not significantly different between women who used inhaled corticosteroids and those who did not in either the mild or the moderate/severe asthmatic groups. Smokers had significantly lower maternal circulating plasma levels of carotenoids compared to non-smokers (p 0.001 Bonferroni correction). 4.4. Relationships between birth weight centile and carotenoids
0.3 0.08 0.3 0.4
0.9 0.9 0.2 0.06 0.3
0.9 0.9 0.5 0.5 0.7
Concentrations in mg/L. Values are median with inter-quartile range. A Kruskal Wallis test was used to examine differences between control (n ¼ 37), mild asthma (n ¼ 26) and moderate severe asthma (n ¼ 37).
(p < 0.05, Fisher’s Exact Test; Figs. 1 and 2). Maternal circulating b-cryptoxanthin (mg/L), lycopene (mg/L), b-carotene (mg/L) or a-carotene (mg/L) did not change as gestation progressed (p > 0.05, Fisher’s Exact Test).
A
103
B
No relationships were found between maternal circulating plasma levels of carotenoids and birth weight centile (BWC) in the control group. In the moderate/severe asthmatics, positive correlations were found between BWC and maternal circulating plasma levels of lycopene at 18 weeks (R2 ¼ 0.19, r ¼ 0.432, p ¼ 0.008, n ¼ 37) and 30 weeks (R2 ¼ 0.14, r ¼ 0.385, p ¼ 0.05, n ¼ 27). 4.5. Tocopherols There were significantly higher concentrations of total plasma tocopherols and a-tocopherols in the moderate/severe asthmatics (p < 0.02, Bonferroni correction) at both 30 and 36 weeks gestation compared to the control group (Table 6). In erythrocytes, a-tocopherol levels at 30 weeks gestation were significantly higher in the mild asthmatic group relative to the controls (p ¼ 0.05, Kruskal Wallis ANOVA, Table 5). Erythrocyte atocopherol quinone levels were significantly higher in the moderate/severe asthmatic group at 18 weeks gestation relative to the controls (p < 0.02, Bonferroni correction, Table 5). There were no significant differences in the ratio of a-tocopherol quinone-atocopherol between the groups. Plasma d- and erythrocyte d- and g-tocopherols were close to the limit of detection, and therefore the data are not shown. When the data was analysed temporally, maternal circulating concentrations of total tocopherols significantly increased during pregnancy in all groups (Fig. 3, p < 0.05, Fisher’s Test). Maternal circulating plasma a-tocopherol levels significantly increased as gestation progressed in the mild (p < 0.001, Fisher’s Test) and moderate/severe asthmatic groups (p < 0.001, Fisher’s Test, Fig. 4). There were no significant differences found in maternal circulating plasma g-tocopherol levels between any of the groups. No significant differences were found temporally in maternal erythrocyte levels of tocopherols between any of the groups. Maternal plasma and erythrocyte levels of tocopherols were not significantly different between the women who used inhaled
C
Fig. 1. Temporal analysis of plasma total carotenoids (mg/L) in control pregnancies (panel A), mild (panel B) and moderate/severe asthmatic pregnancies (panel C) at 18, 30 and 36 weeks gestation. Data are presented as median and inter-quartile ranges with 95% confidence intervals. A Friedman Test was used to record differences over pregnancy. Wilcoxon’s Signed Rank Test was used for post hoc analysis with Bonferroni correction; *P < 0.02.
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Fig. 2. Temporal analysis of plasma lutein (mg/L) in control pregnancies (panel A), mild (panel B) and moderate/severe asthmatic pregnancies (panel C) at 18, 30 and 36 weeks gestation. Data are presented as median and inter-quartile ranges with 95% confidence intervals. A Friedman Test was used to record differences over pregnancy. Wilcoxon’s Signed Rank Test was used for post hoc analysis with Bonferroni correction; *P < 0.02.
corticosteroids and those who did not in either the mild or the moderate/severe asthmatic groups (p > 0.05, Bonferroni correction). There was also no effect of cigarette use on tocopherols in plasma or erythrocytes (p > 0.05, Bonferroni correction).
4.6. Relationships between birth outcomes and tocopherols In the presence of maternal asthma there was a positive correlation between erythrocyte a tocopherol at 36 weeks gestation and neonatal HC (r ¼ 0.447, p ¼ 0.002, n ¼ 44), and a negative correlation between erythrocyte a-tocopherol quinone:a tocopherol at 36 weeks gestation and neonatal HC (r ¼ 0.403, p ¼ 0.01, n ¼ 44). Maternal circulating plasma a-tocopherol levels at 36 weeks gestation and BWC were positively correlated (r ¼ 0.330, p ¼ 0.029, Table 5 Plasma tocopherols during gestation in relation to asthma severity. n
n ¼ 44). These correlations remained when split by asthma severity but were not observed in the control population. 5. Discussion This study has identified that pregnancies complicated by moderate/severe asthma are associated with an altered antioxidant profile with higher concentrations of some tocopherols and carotenoids in plasma as gestation progresses. The data suggests that the maternal system compensates by adjusting its antioxidant defences in response to high oxidative load of both pregnancy and asthma. These adjustments appear to be beneficial to fetal growth. In the current study it was observed that moderate severe asthmatics had higher erythrocyte a-tocopherol quinone levels early in gestation relative to the controls. Tocopherol quinone is the oxidized Table 6 Erythrocyte tocopherols during gestation in relation to asthma severity.
Control 37
Mild 26
Mod/severe 37
P value
18 weeks gestation Total tocopherols Range g-tocopherol Range a-tocopherol Range
19.1 17.2e21.6 0.06 0.4e0.7 18.7 16.8e20.8
20.7 14.9e25.5 0.6 0.4e0.8 20.2 14.5e25.2
22.3 17.8e26.9 0.6 0.4e0.8 21.8 17.3e26.1
0.06
30 weeks gestation Total tocopherols Range g-tocopherol Range a-tocopherol Range
21.5 19.7e25.8 0.65 0.5e0.9 20.8 19.2e25.2
24 19.5e31.3 0.68 0.6e0.9 23.4 19.1e30.3
26.4 # 21.9e31.9 0.68 0.5e0.9 25.6 # 21.5e31.4
0.02
36 weeks gestation Total tocopherols Range g-tocopherol Range a-tocopherol Range
21.9 19.2e25.6 0.7 0.6e0.8 21.5 18.6e24.9
24.4 20.4e31.9 0.64 0.6e0.8 23.8 19.7e31.2
29.3 #* 26.2e37.4 0.7 0.6e1.0 28.6 # 2.4e36.21
n
Control 35
Mild 23
Mod/severe P value 38
0.8 0.5e0.9 0.7 0.5e0.8 0.9 0.7e1.1
0.9 0.6e1.0 0.7 0.6e0.9 0.9 0.6e1.3
0.8 0.6e1.0 0.8* 0.6e0.9 1.1 0.8e1.3
0.9
Range a-tocopherol quinone Range a-tocopherol quinone/aetocopherol Range 30 weeks gestation a-tocopherol Range a-tocopherol quinone Range a-tocopherol quinone/aetocopherol Range
0.7 0.6e0.9 0.7 0.5e0.9 1 0.7e1.3
0.9* 0.8e1.1 0.7 0.6e1.0 0.8 0.6e1.0
0.7 0.5e1.0 0.8 0.6e1.0 1 0.6e1.3
0.05*
36 weeks gestation a-tocopherol Range a-tocopherol quinone Range a-tocopherol quinone/aetocopherol Range
0.9 0.8e0.96 0.7 0.6e0.9 0.9 0.6e1.1
0.8 0.7e1.3 0.7 0.6e0.8 0.7 0.5e0.9
0.7 0.5e1.0 0.7 0.5e1.1 0.9 0.7e1.4
18 weeks gestation
0.4 0.06
0.9 0.02
<0.01 0.3 <0.01
Concentrations in mg/L. Values are median with inter-quartile range. A Kruskal Wallis test was used to examine differences between controls, mild and moderate/ severe asthmatics. ManneWhitney U tests were used for post hoc analysis with Bonferroni corrections. Significant difference to Control # and mild * are indicated.
a-tocopherol
0.04* 0.3
0.6 0.1
0.3 0.7 0.09
Concentrations in mg/L. Values are median with inter-quartile range. A Kruskal Wallis test was used to examine differences between controls, mild and moderate/ severe asthmatics. ManneWhitney U tests were used for post hoc analysis with Bonferroni corrections. Significant difference to Control * are indicated.
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105
Fig. 3. Temporal analysis of plasma total tocopherol levels (mg/L) in control pregnancies (panel A), mild (panel B) and moderate/severe asthmatic pregnancies (panel C) at 18, 30 and 36 weeks gestation. Data are presented as median and inter-quartile ranges with 95% confidence intervals. A Friedman Test was used to record differences over pregnancy. Wilcoxon Signed Rank Tests were used for post hoc analysis using Bonferroni correction; *P < 0.02.
form of a-tocopherol. Our previous work has identified that nonpregnant asthmatics had higher concentrations of whole blood atocopherol quinone than healthy controls which was correlated with asthma control.10 These data would support the concept that asthma is a disease state associated with reduced antioxidant defences that may contribute to asthma severity. The current findings suggest that early gestational profile of asthmatics may be comparable to the non-pregnant state in terms of antioxidant activity. However the antioxidant profile appears to change as gestation progresses. Maternal asthma during pregnancy was associated with an increase in plasma concentrations of total carotenoids, lutein and a-tocopherol late in gestation. These antioxidants correlated positively with birthweight centile suggesting that increased concentrations of circulating antioxidants at 30 and 36 weeks gestation in the cohort of moderate/severe asthmatic women was protective against the growth restricting effects of maternal asthma during pregnancy. In pregnant sheep, maternal tocopherol supplementation during pregnancy reduced maternal circulating mediators of oxidative stress including 8-epi-prostglandin F224 and up regulated placental gene networks involved in angiogenesis including increased placental expression of endothelial nitric
oxide synthase, vascular endothelial growth factor and hypoxia inducible factor25 to promote fetal growth. The programming effects of prenatal malnutrition on renal function were reversed with maternal tocopherol supplementation during lactation in the rat.26 In humans maternal tocopherol concentrations during pregnancy were positively correlated with fetal crown-rump length, as a marker of fetal growth and associated with a reduced risk of development of childhood asthma.27 Conversely, supranutritional supplementation of pregnant rats with tocopherols resulted in permanent impairments in hippocampal development in offspring.28 Similarly a randomised placebo-controlled trial of high dose tocopherol and vitamin C supplementation in pregnant women with pre-eclampsia resulted in an increased incidence of low birth weight babies in the supplemented group relative to the placebo group.29 In this study, a combination of vitamin E and vitamin C was used due to the role of vitamin C in regenerating vitamin E and restoring it’s antioxidant function once it has been oxidised. These studies indicate that endogenous concentrations of tocopherols derived from dietary intake or low dose supplementation may be central to protecting the fetus from the oxidative effects of maternal asthma.
Fig. 4. Temporal analysis of plasma a-tocopherol levels (mg/L) in control pregnancies (panel A), mild (panel B) and moderate/severe asthmatic pregnancies (panel C) at 18, 30 and 36 weeks gestation. Data are presented as median and inter-quartile ranges with 95% confidence intervals. A Friedman Test was used to record differences over pregnancy. Wilcoxon Signed Rank Tests were used for post hoc analysis using Bonferroni correction; *P < 0.02.
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Higher concentrations of total plasma tocopherols and atocopherols, total plasma carotenoids and lutein in the moderate/ severe asthmatic group at both 30 and 36 weeks gestation were not related to differences in dietary intake. In fact moderate/severe asthmatics had reduced intake of most food groups relative to the mild asthmatic and control groups. Overall dietary intake in this particular population of pregnant women is comparable to the diet of other pregnant cohorts.30e32 Intake of total energy and macronutrients, including polyunsaturated fat, which is closely linked to a-tocopherol intake, was lower in the moderate/severe asthma group. Also, dietary intake for the antioxidant vitamin C was not increased in the moderate/severe asthma group. Hence, these data suggest that a systemic compensatory antioxidant response occurs in the presence of moderate/severe asthma as gestation progresses and fetal demand increases. This may involve the release of antioxidants from tissue stores, to boost antioxidant defences and ensure continued fetal growth and survival in a sub-optimal intrauterine environment. As expected the use of cigarettes during pregnancy significantly altered the antioxidant profile especially in relation to the plasma carotenoids which were reduced in the smokers relative to the nonsmokers. Previous studies have reported that smoking depletes antioxidant defences,16 contributes to worsening asthma during pregnancy18 and is associated with a significant reduction in fetal growth.33 Smoking during pregnancy is more prevalent in asthmatic women relative to non-asthmatic women14 and is a serious health issue contributing to poor outcomes observed in this population. Supplementation of smokers with vitamin E has recently been reported in the Women’s Health Study.34 It was found that vitamin E supplementation to women over the age of 45 in a placebo-controlled randomised trial significantly reduced the incidence of chronic lung disease which was not modified by the presence of cigarette use.34 Smoking has been shown to be associated with a reduced consumption of carotenoids, derived from dietary intake of fruit and vegetables.35 These data suggest improved dietary intake of fruit and vegetables along with a multivitamin supplement may be important for smokers during pregnancy. Furthermore, pregnant women should also be encouraged to quit or reduce cigarette use during pregnancy. The use of 24 h recall dietary analysis, as a measure of recent dietary intake was considered a limitation of the study and a 4-day food record would have provided a more accurate representation. However 24 h recalls were used in order to minimise participant burden and are a useful measure of group mean intakes.23 Another limitation was the use of non-fasting samples which could not be avoided as the request for an overnight fast was ethically inappropriate for a non-medical assessment of pregnant women. The current study is the first to characterise the circulating antioxidant profile of pregnant women with asthma. Moderate/ severe asthmatic women appear to have a compensatory increase in the concentration of circulating antioxidants which is positively associated with birth outcomes. These findings suggest the maternal system adjusts metabolic pathways in response to the presence of a high oxidative load induced by asthma in an attempt to ensure continued fetal growth. Future studies will examine the relationship between the maternal, placental and fetal antioxidant profiles in pregnancies complicated by asthma.
Statement of authorship and conflict of interest statement PM conducted the study and analysed the data. NH analysed the data. LW, VM and VC designed the study and wrote the manuscript. There are no identified conflicts of interest in the production of this work or manuscript.
Acknowledgements The work was funded by Department of Health and Aging Award and National Health and Medical Research Council (NHMRC). P. McLernon was funded by an Australian Postgraduate Award for the work outlined in this manuscript. V. Clifton is the recipient of a NHMRC Senior Fellowship (ID 510703). V. Murphy is the recipient of a NHMRC Research Training Fellowship (Part time, ID 455626). N. Hodyl is the recipient of a NHMRC Peter Doherty Fellowship (APP1016379). The authors would like to thank Prof Manohar Garg and staff from the John Hunter Hospital Antenatal Clinic and Delivery Suite. References 1. Greene LS. Asthma, oxidant stress, and diet. Nutrition 1999;15:899e907. 2. de Luis DA, Armentia A, Aller R, Asensio A, Sedano E, Izaola O, et al. Dietary intake in patients with asthma: a case control study. Nutrition 2005;21:320e4. 3. Traber MG, Atkinson J. Vitamin E, antioxidant and nothing more. Free Radic Biol Med 2007;43:4e15. 4. Toescu V, Nuttall SL, Martin U, Kendall MJ, Dunne F. Oxidative stress and normal pregnancy. Clin Endocrinol (Oxf) 2002;57:609e13. 5. Siddiqui IA, Jaleel A, Tamimi W, Al Kadri HM. Role of oxidative stress in the pathogenesis of preeclampsia. Arch Gynecol Obstet 2010;282:469e74. 6. Bakheit KH, Ghebremeskel K, Zaiger G, Elbashir MI, Adam I. Erythrocyte antioxidant enzymes and plasma antioxidant vitamins in Sudanese women with preeeclampsia. J Obstet Gynaecol 2010;30:147e50. 7. Ishihara O, Hayashi M, Osawa H, Kobayashi K, Takeda S, Vessby B, et al. Isoprostanes, prostaglandins and tocopherols in preeeclampsia, normal pregnancy and nonepregnancy. Free Radic Res 2004;38:913e8. 8. Sharma JB, Sharma A, Bahadur A, Vimala N, Satyam A, Mittal S. Oxidative stress markers and antioxidant levels in normal pregnancy and preeeclampsia. Int J Gynaecol Obstet 2006;94:23e7. 9. Wood LG, Garg ML, Blake RJ, Simpson JL, Gibson PG. Oxidized vitamin E and glutathione as markers of clinical status in asthma. Clin Nutr 2008;27:579e86. 10. Wood LG, Gibson PG. Reduced circulating antioxidant defences are associated with airway hypereresponsiveness, poor control and severe disease pattern in asthma. Br J Nutr 2010;103:735e41. 11. Ford ES, Mannino DM, Redd SC. Serum antioxidant concentrations among U.S. adults with selfereported asthma. J Asthma 2004;41:179e87. 12. Wood LG, Garg ML, Blake RJ, GarciaeCaraballo S, Gibson PG. Airway and circulating levels of carotenoids in asthma and healthy controls. J Am Coll Nutr 2005;24:448e55. 13. Schatz M, Zeiger RS. Treatment of asthma and allergic rhinitis during pregnancy. Ann Allergy 1990;65:427e9. 14. Clifton VL, Engel P, Smith R, Gibson P, Brinsmead M, Giles WB. Maternal and neonatal outcomes of pregnancies complicated by asthma in an Australian population. Aust New Zealand J Obstetircs Gynaecol 2009;49:619e26. 15. Godfrey KM, Barker DJ. Fetal nutrition and adult disease. Am J Clin Nutr 2000;71:1344Se52S. 16. Faure H, Preziosi P, Roussel AM, Bertrais S, Galan P, Hercberg S, et al. Factors influencing blood concentration of retinol, alphaetocopherol, vitamin C, and betaecarotene in the French participants of the SU.VI.MAX trial. Eur J Clin Nutr 2006;60:706e17. 17. Murphy VE, Gibson PG, Giles WB, Zakar T, Smith R, Bisits AM, et al. Maternal asthma is associated with reduced female fetal growth. Am J Respir Crit Care Med 2003;168:1317e23. 18. Murphy VE, Clifton VL, Gibson PG. The effect of cigarette smoking on asthma control during exacerbations in pregnant women. Thorax 2010;65:739e44. 19. Gibson PG, Wilson AJ. The use of continuous quality improvement methods to implement practice guidelines in asthma. J Qual Clin Pract 1996;16:87e102. 20. National Heart LaBI. New NHLBI guidelines for the diagnosis and management of asthma. Lippincott Health Promot Lett 1997;2:8e9. 21. Murphy VE, Gibson PG, Talbot PI, Kessell CG, Clifton VL. Asthma selfemanagement skills and the use of asthma education during pregnancy. Eur Respir J 2005;26:435e41. 22. Barnes PJ, Pedersen S. Efficacy and safety of inhaled corticosteroids in asthma. Report of a workshop held in Eze, France, October 1992. Am Rev Respir Dis 1993;148:S1e26. 23. Block G. A review of validations of dietary assessment methods. Am J Epidemiol 1988;115:492e505. 24. Kasimanickam RK, Kasimanickam VR. Effect of tocopherol supplementation on serum 8eepieprostaglandin F2 alpha and adiponectin concentrations, and mRNA expression of PPARgamma and related genes in ovine placenta and uterus. Theriogenology 2011. 25. Kasimanickam RK, Kasimanickam VR, Rodriguez JS, Pelzer KD, Sponenberg PD, Thatcher CD. Tocopherol induced angiogenesis in placental vascular network in late pregnant ewes. Reprod Biol Endocrinol 2010;8:86. 26. VieiraeFilho LD, Lara LS, Silva PA, Santos FT, Luzardo R, Oliveira FS, et al. Placental malnutrition changes the regulatory network of renal NaeATPase in
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