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Fatty acid profile of pregnant women with asthma
e-SPEN Journal 7 (2012) e78ee85
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Original article
Fatty acid profile of pregnant women with asthma Penelope McLernon a, b, Lisa Wood c, Vanessa E. Murphy c, Nicolette A. Hodyl a, Vicki L. Clifton a, b, * a
Robinson Institute, University of Adelaide, Lyell McEwin Hospital, Haydown Rd, Elizabethvale, Adelaide, SA 5112, 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 25 October 2011 Accepted 14 January 2012
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. Dietary fatty acids (FA) especially polyunsaturated fatty acids (PUFA) are particularly important during pregnancy due to their role in fetal growth and development. Dietary PUFAs also have a role in clinical outcomes for non-pregnant asthmatics. The current study was designed to characterize the fatty acid profile in pregnant women with asthma to determine whether asthma severity or reduced fetal growth were associated with an altered FA profile. Methods: Maternal dietary intake and plasma fatty acid profile were examined in women with and without asthma at 18, 30 and 36 weeks gestation. Maternal fatty acids levels were related to measures of fetal growth using Doppler ultrasound and birth outcomes. Results: The data found that pregnant women with moderate/severe asthma had increased circulating plasma fatty acid levels at 36 weeks gestation but reduced dietary intake of fats compared to those women with mild asthma and healthy pregnant controls. In addition, women with moderate/severe asthma had increased circulating n-3PUFA levels at 36 weeks gestation which was associated with reduced fetal and neonatal head circumference. Conclusion: These observations suggest moderate/severe asthma may disrupt lipid metabolism, transport or cellular uptake during pregnancy which subsequently contributes to reduced fetal growth. Ó 2012 European Society for Clinical Nutrition and Metabolism. Published by Elsevier Ltd. All rights reserved.
Keywords: Pregnancy Fetal growth Asthma Fatty acids
1. Introduction Dietary fatty acids (FA) are key contributors to daily energy requirements and play a central role in regulating physiological functions including the innate immune system, glucose metabolism and endothelial function. Fatty acids are classified according to the degree of saturation, as saturated fatty acids (SFA), monounsaturated fatty acids (MUFA) and polyunsaturated fatty acids (PUFA). PUFA can be further divided into n-3PUFA and n-6PUFA. The long chain n-3PUFA include eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), which have been shown to have antiinflammatory properties, due to their ability to modify production of eicosanoids, decrease production of pro-inflammatory cytokines
* Corresponding author. Robinson Institute, University of Adelaide, Lyell McEwin Hospital, Haydown Rd, Elizabethvale, Adelaide, SA 5112, Australia. Tel.: þ61 8 81332133; fax: þ61 8 8303 4099. E-mail address: [email protected] (V.L. Clifton).
and alter immune cell function.1e6 It has been hypothesized that increased prevalence of asthma and atopy in recent years may be linked to an increased intake of n-6PUFA relative to n-3PUFA.7 PUFAs are particularly important during pregnancy due to their role in fetal growth and development especially fetal brain development.8 It has been suggested that the anti-inflammatory properties of n-3PUFA may protect against the inflammation and oxidative damage that occurs in pregnancy.9,10 Indeed, it has been shown that insufficient n-3PUFA intake during pregnancy may contribute to suboptimal fetal neurodevelopment, and later life disease.11,12 One of the most prevalent complications of pregnancy is asthma and we are interested in the impact of this complication on fetal development.13e15 Pregnancies complicated by maternal asthma are associated with an increased incidence of intrauterine growth restriction,14,16 with significant implications for both the short and long term health of the offspring.17 The use of inhaled glucocorticoids for asthma treatment during pregnancy appears protective against the growth reducing effects of the disease.13 The maternal
2212-8263/$36.00 Ó 2012 European Society for Clinical Nutrition and Metabolism. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.clnme.2012.01.004
P. McLernon et al. / e-SPEN Journal 7 (2012) e78ee85
dietary contributions to reduced fetal growth in pregnancies complicated by asthma are not known. In particular, the maternal fatty acid profile may be associated with altered fetal development. Recently we have identified that circulating antioxidants in pregnant women with asthma are significantly increased as gestation progresses relative to a healthy population of pregnant women18 suggesting a compensatory 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. The current study was designed to assess if there are any differences in the FA profile of the same population of pregnant women with asthma compared to women without asthma18 and whether these differences are related to dietary intake, asthma treatment, asthma severity or cigarette use. 2. Methods 2.1. Experimental subjects The study was approved by the Hunter New England Research Ethics and University of Newcastle Human Research Ethics Committees. Pregnant women were recruited in the first trimester (n ¼ 131, controls n ¼ 47, and asthmatics n ¼ 84) and provided written informed consent. The protocol for this study has been described previously.13,19 Using the smallest difference observed previously in our analyses of tocopherol levels in pregnant asthmatic and healthy control women,18 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 mg/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 comorbidities associated with pregnancies complicated by asthma. Complications other than asthma such as pre-eclampsia, gestational diabetes, infection or preterm delivery were excluded from the analysis (n ¼ 4). Birth weight and fetal sex were determined at birth. The characteristics of this population were also published in a paper examining circulating antioxidants in this group of women.18 2.2. Materials and methods Clinical asthma severity was rated as mild, moderate or severe using the integrated severity score described in the Australian Asthma Management Guidelines,20 which closely approximate the National Heart, Lungs and Blood Institute Guidelines.21 Proper inhaler use and compliance was assessed by the study research nurse.22 Cumulative, inhaled corticosteroid (ICS) dose was calculated for each trimester, and summarized as the mean daily dose of beclomethasone dipropionate (BDP) or equivalent used during pregnancy, where 1 mg BDP was considered equal to 1 mg budesonide or 0.5 mg fluticasone propionate.23 For data analysis, the low, moderate and high inhaled corticosteroid (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 b2 agonist, salbutamol for symptom relief when required. Current smoking status was assessed by direct questioning at recruitment. Some of the control subjects were smokers (Table 1).
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Table 1 Maternal characteristics. Maternal characteristics
Maternal age (yrs) Range Body Mass Index Range Gravidity Range Parity Range Inhaled corticosteroid use (%) Cigarette use (n)
Control
Mild
Mod-severe
n ¼ 47
n ¼ 31
n ¼ 53
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
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.
All women participating in the study underwent ultrasound assessment at 18, 30 and 36 weeks of gestation. Fetal biparietal diameter, head circumference, abdominal circumference and femur length were measured. Birth weight, length and head circumference were recorded at delivery. Customized 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 hospital-specific growth charts. Ponderal index was calculated as birth weight (kg)/length3 (m). 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. 2.3. Blood collection Twenty mL of whole blood were collected into EDTA coated tubes from the median cubital vein at each visit (18, 30 and 36 weeks). Women were advised not to eat at least 2 h prior to blood sampling. An overnight fast from pregnant women was not appropriate for a non-medical assessment. Whole blood was centrifuged at 3000 rpm at 4 C for 10 min. Plasma was stored in 500 mL aliquots at 80 C until required for analysis. 2.4. 24 h 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 analyzing mean dietary intakes.24 The questionnaire asked for detailed information on meals and snacks consumed 24 h prior to the clinic visit. For this study, the data was analyzed 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. Dietary data was also published in the study of circulating antioxidants in pregnant women with asthma.18 3. Biochemical analyses 3.1. Maternal circulating levels of fatty acids Total fatty acids were analyzed by gas chromatography using established methodology.25,26 2 mL of methanol:toluene (4:1 v/v)
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containing C19:0 (4 mg/mL) was added to 100 mL of plasma. Fatty acids were methylated by adding 200 mL acetyl chloride dropwise whilst vortexing, then heated for 60 min at 100 C. After cooling, the reaction was stopped by adding 5 mL 6% potassium chloride (K2CO3). Samples were then centrifuged at 3000 rpm at 5 C for 10 min. Supernatant was analyzed by gas chromatograph for fatty acid methyl esters (FAME), using a HewlettePackard 6890 Gas Chromatograph equipped with a 30 m (0.25 mm ID) carbon-silica capillary column coated with 50% cyanoproylphenyl and 50% dimethylpolysiloxane (0.25 mm film thickness), a flame ionization detector, autosampler and autodetector. The initial oven temperature was set at 30 C, programmed to rise to a maximum of 240 C at 3 C/min. A split ratio of 10:1 with an injection volume of 3 mL was used. The carrier gas (hydrogen) was expelled at an average velocity of 7 cm/s. Chemstation version A.04.02 was used to quantify fatty acid methyl esters based on retention time compared to lipid standards. Plasma concentrations and 24 h recall dietary intake questionnaire data from controls (n ¼ 47) and asthmatic (n ¼ 84) women (mild; n ¼ 31, moderate/severe; n ¼ 53) were analyzed. 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. 3.2. Statistical analysis 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 analyze temporal changes of fatty acids 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 fatty acid 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) during pregnancy (P > 0.05, Fisher’s Exact Test, Table 1). A higher proportion of moderate/ severe asthmatics used inhaled corticosteroids to treat their asthma (83%) when compared to the mild asthmatics (32.3%). 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). There were no babies less than the 10th birth weight centile in this cohort. 4.2. Maternal dietary intake 4.2.1. Cross sectional analysis at 36 weeks gestation The mild asthmatic group (n ¼ 25) consumed higher quantities of energy, protein, fats, carbohydrates, starch, thiamin, riboflavin,
Table 2 Neonatal characteristics. Neonatal characteristics
Gestational age (weeks) Range Birth weight (g) Range Birth weight Centile Range Birth length (cm) Range Ponderal Index Range Head circumference (cm) Range Placental weight (g) Range male/female (n/n)
Control
Mild
Mod-severe
n ¼ 47
n ¼ 31
n ¼ 53
P value
40.4 39.4e41.2 3402.5 3115e3715 35.6 19.3e51.4 51 50e53 25.3 21.7e34.9 35 34e35.5 630.4 602.8e671.7 18/25
40.3 39.6e41 3660 3220e3940 52.3 14.6e73.5 51 50e54 24.5 19.6e31.8 34.5 33.6e36 671.8 607.4e800.8 14/17
39.8 0.06 38.6e40.6 3340 0.18 3040e3647 52 0.29 22.9e63.1 52 0.24 50e53 25.3 0.81 18.4e37.2 34.5 0.26 33.5e35.5 645.1 0.27 592.8e781.6 24/29
Values reported as the median and range. *P < 0.05.
niacin equivalents, magnesium and phosphorous than the moderate/severe asthmatics (n ¼ 34) or control population (n ¼ 32) at 36 weeks gestation (P < 0.01, Fisher’s Exact Test, Table 3). The moderate-severe asthmatic group had reduced consumption of all the fats relative to the control or mild asthmatic group (P < 0.01, Fisher’s Exact Test, Table 3) at 36 weeks. 4.2.2. Temporal analysis of dietary intake at 18, 30 and 36 weeks gestation Dietary intake data from controls (n ¼ 32) and asthmatic women (n ¼ 57) was analyzed at 18, 30 and 36 weeks gestation and asthmatic groups were compared based on severity (mild; n ¼ 25, moderate/severe; n ¼ 34). A temporal analysis revealed no significant changes from 18 to 36 weeks gestation in maternal dietary intake of total fatty acids, total saturated fatty acids, total monounsaturated fatty acids, total n-6 polyunsaturated fatty acids, total n-3 polyunsaturated fatty acids in the control, mild or the moderate/severe asthmatics. There were no significant changes over pregnancy in maternal dietary intake of the essential fatty acids (EFAs; linoleic [LA, 18:2n-6] and a-linolenic [aLNA, 18:3n-3]), nor the long chain polyunsaturated fatty acids (LCPUFA; Arachidonic [AA, 20:4n-6], eicosapentaenoic [EPA, 20:5n-3] or docosahexaenoic [DHA, 22:6n-3]) acids (P > 0.05, Friedman’s Test). The only temporal differences in the fatty acid dietary intake were observed in the moderate-severe asthmatics who had a significantly reduced percentage intake of total n-6PUFA at 36 weeks gestation which was unrelated to any individual n6 PUFA, though there was a trend toward reduced linoleic acid intake in the moderate-severe asthmatic group (18:2n-6, median controls 7.2 g/L vs moderate-severe 4.7 g/L; P ¼ 0.053). The intake of lignoceric acid (24:0) was also reduced in the moderate-severe asthmatic group relative to the control at 30 and 36 weeks gestation (data not shown). 4.3. Maternal plasma fatty acids 4.3.1. Cross sectional analysis at 36 weeks gestation Plasma total fatty acids were assessed in moderate/severe asthmatic (n ¼ 39), mild asthmatic (n ¼ 26) and control (n ¼ 38) subjects at 18, 30 and 36 weeks gestation (Table 4). Most fatty acids were not significantly different between the groups until 36 weeks gestation. Circulating SFA, MUFA and n-3PUFA were significantly increased in moderate-severe asthmatics relative to the control and mild asthmatic groups (Table 4). The ratio of n-6:n-3PUFA decreased in the moderate-severe asthmatics at 36 weeks gestation.
P. McLernon et al. / e-SPEN Journal 7 (2012) e78ee85
4.3.2. Temporal analysis at 18, 30 and 36 weeks gestation A subgroup of women from the total cohort provided blood samples at each gestational visit (control (n ¼ 21), mild asthmatics (n ¼ 16), moderate/severe asthmatics (n ¼ 22)). Data were analyzed to identify changes in circulating levels of fatty acids throughout gestation in relation to asthma severity and compared to 6 month post partum measurements. Total plasma fatty acids (Fig. 1), SFA (data not shown), MUFA (data not shown) and n-6PUFA (Fig. 2) were significantly increased with advancing pregnancy in all Table 3 Maternal total dietary intake at 36 weeks gestation. Dietary factors
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 fiber (g) Range Thiamin (mg) Range Riboflavin (mg) Range Niacin equivalents (mg) Range Vitamin C (mg) Range Total Folate (mg) Range Viamin A Equivalents (mg) Range Retinol (mg) Range Beta carotene (mg) Range Potassium (mg) Range Magnesiums (mg) Range Calcium (mg) Range Phosphorous (mg) Range Iron (mg) Range
Control
Mild
Mod-severe
n ¼ 32
n ¼ 25
n ¼ 34
9.6 7.2e11.4 86.2 58.3e113 97.9 68.8e122.3 41.5 32.3e54.1 10.9
10.7* 9e14. 96.8* 80.2e141 108.2 87.5e148.9 43.6 35.9e66.7 14.6
8.2** 6.9e9.8 76.5** 56e96.7 80.6* 56.7e100.6 33.3* 24.8e46.4 7.8*
0.02*
6.6e16.6 6.5
9.2e19.9 7.2
5.5e14.8 6
0.3
5.0e12.1 0.9
3.8e12.5 0.8
4.4e10.2 0.8
0.5
0.7e1.5 30.9
0.6e1.8 40.9
0.5e1.23 29.3*
0.03*
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
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*
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
24.6e47 135.8 50.9e238.9 280.9 178.4e366.9 970.6
34.5e59.2 128.3 41.5e289.8 304.7 233.2e403.5 1143.1
23.8e44.6 142.3 78.5e257.7 257.7 182.8e307 955.3
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
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
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
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groups (P < 0.01, Bonferroni correction). EPA and DHA n-3 fatty acids did not significantly change during pregnancy in any group (Fig. 3, p > 0.05, Fisher’s Exact test). The ratio of n-6:n-3PUFA was found to increase significantly over the course of pregnancy in the control and mild asthmatic groups Fig. 4, P < 0.01, Bonferroni correction). There was a significant increase in maternal circulating levels of the essential fatty acids: LA and aLNA over the duration of pregnancy in each of the groups (P < 0.05, Fisher’s Exact Test, data not shown). Maternal plasma levels of AA; (20:4n-6), did not change as gestation progressed in the control or asthmatic groups (P > 0.05, Fisher’s Exact Test, data not shown).
P value
0.004* 0.02* 0.01* 0.04*
0.02* 0.02* 0.1 0.04* 0.4 0.03* 0.05* 0.04*
4.3.3. Maternal smoking and ICS use Maternal circulating levels of fatty acids at 18, 30 and 36 weeks gestation 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 (p > 0.05, Bonferroni correction). Maternal circulating levels of fatty acids at 18, 30 and 36 weeks gestation were not significantly different between smokers and non smokers (p > 0.05, Bonferroni correction). 4.4. Relationships between maternal circulating fatty acids and fetal growth parameters in pregnancies complicated by asthma There was a positive correlation between fetal head circumference (HC) at 36 weeks gestation and the n-6:n-3PUFA ratio at 30 weeks gestation (r ¼ 0.481, p ¼ 0.032, n ¼ 20) and at 36 weeks gestation (r ¼ 0.716, p ¼ 0.001, n ¼ 17) in all groups. In the moderate/severe asthmatics, there was a negative correlation between fetal HC at 36 weeks gestation and the essential omega 3 a-linolenic acid (a-LNA, 18:3n-3) at 30 weeks gestation (r ¼ 0.405, p ¼ 0.032, n ¼ 28) and at 36 weeks gestation (r ¼ 0.386, p ¼ 0.043, n ¼ 28). In the moderate/severe asthmatic group, HC at birth was negatively correlated with DHA at 30 weeks gestation (r ¼ 0.415, p ¼ 0.025, n ¼ 29), LA at 18 (r ¼ 0.360, p ¼ 0.028, n ¼ 37) and 30 weeks gestation (r ¼ 0.384, p ¼ 0.040, n ¼ 29), AA at 30 weeks (r ¼ 0.436, p ¼ 0.018, n ¼ 29), total n-6PUFA at 18 weeks (r ¼ 0.331, p ¼ 0.046, n ¼ 37) and 30 weeks gestation (r ¼ 0.373, p ¼ 0.046, n ¼ 29), total n3 PUFA at 30 weeks (r ¼ 0.394, p ¼ 0.034, n ¼ 29), total MUFA at 30 weeks (r ¼ 0.448, p ¼ 0.015, n ¼ 29), total fatty acids at 30 weeks (r ¼ 0.416, p ¼ 0.025, n ¼ 29) and the ratio of n-6:n-3PUFA at 30 weeks (r ¼ 0.397, p ¼ 0.033, n ¼ 29).
0.8 0.2 0.8
Table 4 Cross sectional analysis of maternal plasma fatty acid concentrations at 36 weeks gestation. Fatty acids
0.1 0.9 0.9 0.04* 0.3 0.01* 0.04*
Values are median and inter-quartile range. The KW-ANOVA and ManneWhitney U test with Bonferroni correction were used. *P < 0.05 was considered significant compared to control population.
Saturated Fats (mg/L) Range MUFA (mg/L) Range n6 PUFA (mg/L) range n3 PUFA (mg/L) Range n6en3 ratio Range Total fatty acids (mg/L) Range
Control
Mild
Mod-severe
n ¼ 26
n ¼ 31
n ¼ 53
793.8 621e1831 665.2 548e1563 678.5 572e2109 94.7 64e351 6.9 6e8.5 2234.5 1789e5988
832.6 619e1425 647.7 503e1307 682.3 571e1825 84.3 71e268 7.7 6.5e9.4 2207.5 1808e4923
1688.9* 706e1955 1476.9* 649e1911 1918.9 625e2151 371.2* 93e439 5.4* 4.6e7.5 5635.3 1992e6452
P value
0.05* 0.03* 0.19 0.01* 0.001* 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. No significant differences were observed between controls and mild asthmatics. MUFA: monounsaturated fats; PUFA: polyunsaturated fats.
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A
B
CONTROL 8000
total fatty acids (mg/L)
MODERATE/SEVERE
8000
*
Maternal circulating plasma
C
MILD
8000
*
* *
*
6000
6000
6000
§ 4000
4000
4000
2000
2000
2000
0
0
0
18
30
36
Gestational Week
Post
18
30
36
Gestational Week
Partum
n=21
n=16
18
Post
30
36
Post
Gestational Week
Partum
Partum
n=22
Fig. 1. Maternal plasma total fatty acids (mg/L) in control pregnancies (panel A), mild (panel B) and moderate/severe asthmatic pregnancies (panel C) at 18, 30 and 36 weeks gestation, with post partum levels depicted for reference only. Data are presented as median and inter-quartile range. *P < 0.05.
None of the correlations observed in the moderate/severe asthmatics were observed in the control group or mild asthmatic group with respect to fatty acids and fetal or neonatal head growth in early, mid or late gestation.
Intake of energy (7.1e8.9 MJ/day) and protein (58e60 g/day) were above recommendations for pregnant women. Overall the total fats contributed to 37% of the total energy consumed by the women in all groups which above the recommended levels of 35% total energy and 15% of total energy was derived from saturated fats in all groups which is also above the recommendation that <10% of saturated fats should contribute to total energy. Fat intake of the women in the current study are only slightly higher than pregnant women in Finland29 and obese pregnant women in Belgium30 suggesting that the diet of these women does not differ significantly from other populations. Moderate-severe asthmatics had significantly higher concentrations of plasma FA compared to mild asthmatics and healthy controls, even though dietary intake was lower. Our findings suggest that altered maternal physiology in the presence of severe asthma greatly influences the circulating fatty acid profile. Normal pregnancy is well recognized as a hyperlipidemic state that may be induced by the increased concentrations of estrogens during pregnancy31 however the presence of moderate-severe asthma during pregnancy appears to further exacerbate hyperlipidemia. There may be numerous mechanisms contributing to hyperlipidemia in moderate/severe asthmatics. Lipolysis can be regulated by cortisol32 and possibly influenced by the use of inhaled corticosteroids (ICS) for the treatment of asthma. Glucocorticoids have been shown to increase lipase activity in adipocytes which results in increased release of circulating free fatty acids.32 Our research
5. Discussion This study demonstrates that in the final trimester of pregnancy, pregnant women with moderate/severe asthma have increased circulating plasma fatty acid levels. This is despite the fact that they have reduced dietary intake compared to those with mild asthma and healthy controls. In addition, for women with moderate/severe asthma, increased circulating n-3PUFA levels were associated with the smallest sized neonates although none of the babies in this cohort were growth restricted. Temporal analysis indicates that in moderate/severe asthma and healthy controls, levels of circulating fatty acids increase over the duration of the pregnancy, which was also independent of changes in dietary intake. These observations suggest that lipid metabolism is altered during pregnancy, particularly in those pregnancies complicated by asthma. Dietary intake in this particular population of pregnant women is comparable to data obtained in previous studies of Australian pregnant women.27 Most women had below the recommended intake of iron, calcium, magnesium, folate, retinol and dietary fiber. Riboflavin, niacin, vitamin C and vitamin A equivalents, phosphorous and potassium intake were all above recommended intakes.28
B
CONTROL
MILD
* *
2500
fatty acids (mg/L)
Maternal circulating plasma
total omega 6 polyunsaturated
A
C
*
2500
* *
2500
2000
2000
2000
MODERATE/SEVERE
§ 1500
1500
1500
1000
1000
1000
500
500
500
0
18
30
36
Gestational Week n= 21
Post Partum
0
18
30
36
Gestational Week n=16
Post Partum
0
18
30
36
Gestational Week
Post Partum
n=22
Fig. 2. Maternal plasma total omega 6 fatty acids (mg/L) in control pregnancies (panel A), mild (panel B) and moderate/severe asthmatic pregnancies (panel C) at 18, 30 and 36 weeks gestation, with post partum levels depicted for reference only. Data are presented as median and inter-quartile range. *P < 0.05.
P. McLernon et al. / e-SPEN Journal 7 (2012) e78ee85
Maternal circulating plasma
CONTROL
eicosapentaenoic acid (EPA [20:5n3] mg/L)
A
MILD
A
80
80
#
§ 40
40
40
20
20
20
0
0
0 30
36
18
Post
36
Post
18
30
400
MODERATE/SEVERE
B
400
C
#
300
300
300
Partum
n=22
MILD
A
Post
36
Gestational Week
Partum
n=16
CONTROL 400
30
Gestational Week
Partum
n=21
Maternal circulating plasma
C
60
60
60
Gestational Week
docosahexaenoic acid (DHA [22:6n3] mg/L)
MODERATE/SEVERE
B
80
18
B
e83
§ 200
200
200
100
100
100
0
18
30
0
36
Gestational Week
18
30
36
Gestational Week
n=21
Post
0
18
30
36
Post
Gestational Week
Partum
n=16
Partum
n=22
Fig. 3. Maternal plasma eicosapentaenoic acid (EPA [20:5n-3] mg/L, Fig. 3A) and docosahexaenoic acid (DHA [22:6n-3] mg/L, Fig. 3B) in control pregnancies (panel A), mild (panel B) and moderate/severe asthmatic pregnancies (panel C) at 18, 30 and 36 weeks gestation, with post partum levels depicted for reference only. Data are presented as median and interquartile range. A Friedman Test identified no significant differences in the levels of any n3 PUFAs during gestation.
has previously identified that ICS use during pregnancy for the treatment of asthma exerts systemic effects on glucocorticoid regulated pathways in the maternal system.33 In the current study 83% of moderate-severe asthmatics used ICS for the treatment of their asthma. The increased concentrations of circulating FA observed in the moderate-severe asthmatic women may be a consequence of ICS-induced lipolysis. In addition, the presence of oxidative stress in the moderate/ severe asthmatics34 may have altered lipolysis. Oxidative stress
A
*
fatty acid ratio
total n6-n3 polyunsaturated
14
Maternal circulating plasma
B
CONTROL
induces metabolic disturbances, including altered lipid metabolism. Lipid biosynthesis in the liver is regulated by a family of transcription factors: the sterol regulatory element binding proteins (SREBPs)35 which may be altered by oxidative stress.36,37 This raises the possibility that the increased levels of circulating fatty acids in the later stages of pregnancy in the moderate/severe asthmatics may occur due to increased lipolysis, induced by increased inflammation and oxidative stress that is induced by the combination of asthma and pregnancy.34
C
MILD *
14 12
12
10
10
10
8
8
8
6
6
6
4
4
4
2
2
2
18
30
36
Gestational Week n= 21
Post Partum
0
18
30
36
Gestational Week n=16
#
14
12
0
MODERATE/SEVERE
Post Partum
0
§
18
30
36
Gestational Week
Post Partum
n=22
Fig. 4. Maternal plasma n6-n3 fatty acid ratio in control pregnancies (panel A), mild (panel B) and moderate/severe asthmatic pregnancies (panel C) at 18, 30 and 36 weeks gestation, with post partum levels depicted for reference only. Data are presented as median and inter-quartile range. *P < 0.05.
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n-3PUFAs were amongst the classes of fatty acids that were elevated at 36 weeks gestation in the moderate-severe asthma group, despite PUFA intake being lower in this group. Unexpectedly, n-3PUFA were negatively associated with reduced head growth in utero and at birth. Although none of the babies in this study were growth restricted it was interesting to note that the smallest neonates were associated with the highest maternal fatty acid concentrations. n-3PUFAs are generally considered beneficial for fetal growth, though the data remains controversial.38e41 Furthermore n-3PUFA may increase susceptibility to oxidative stress, due to the presence of multiple double bonds, which are vulnerable to attack by free radicals. We42 and others,43 have shown that increased intracellular content of n-3PUFA results in increased susceptibility to lipid peroxidation. The current data suggest placental transfer of n-3PUFAs may be significantly compromised in asthmatic pregnancies and that supplementation with n-3PUFA in abnormal pregnancies may not improve fetal outcome or growth. It is not known whether placental transport of PUFAs is altered in asthmatic pregnancies or if the fetal circulation is deficient in fatty acids. This is an area that requires further investigation. It was expected from epidemiological studies of asthma during pregnancy that at least 10% of neonates would be small for gestational age or less than the 10th birth weight centile14 in this study. The absence of any growth restricted neonates in this cohort may be a result of the study protocol22 which involved women attending a respiratory nurse -led asthma clinic regularly during their pregnancy for monitoring of their asthma management. Women with well controlled asthma are known to have normal birth outcomes44 and we have previously reported that the use of inhaled glucocorticoids during pregnancy was associated with normal sized neonates in relation to both birth weight and head circumference.13 ICS do not cross the placenta33 and therefore do not exert a direct effect on fetal growth suggesting it is the inflammatory effects of the disease that has the greatest impact on fetal growth via alterations in placental function.15,45 A limitation of this study was the use of 24 h recall dietary analysis, as a measure of recent dietary intake. While alternative methods such as 4-day food records give a more accurate representation, 24 h recalls were used in order to minimize participant burden. Furthermore, this method has been shown to provide an accurate measure of group mean intakes.24 Another limitation is the use of non-fasting samples, which was ethically appropriate for a non-medical assessment of pregnant women. The fact that significant differences in groups were observed despite the potential confounders, further strengthens the importance of the observations. In summary, our data has revealed that despite lower dietary intake in moderate/severe asthmatic women, circulating fatty acid levels increase as pregnancy progresses and associated with negative fetal head growth. These observations suggest that maternal lipid metabolism and its transport to the fetus are altered in pregnant women with asthma and this may contribute to poor fetal outcomes. Conflict of interest There are no identified conflicts of interest in the production of this work or manuscript. Statement of authorship PM conducted the study and analyzed the data. NH analyzed the data. LW, VM and VC designed the study and wrote the manuscript.
Acknowledgments 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. Martin RE. Docosahexaenoic acid decreases phospholipase A2 activity in the neurites/nerve growth cones of PC12 cells. J Neurosci Res 1998;54:805e13. 2. Simopoulos AP. Omega-3 fatty acids in inflammation and autoimmune diseases. J Am Coll Nutr 2002;21:495e505. 3. Tamura Y, Terano J, Saito A, Hirai A, Shiina T. Enhancement of PGI2 formation by EPA in rat vascular smooth muscle cells. In: Ong ASH, Niki E, Packer L, editors. Nutrition, lipids, health and disease. Illinois: Champaign; 1995. p. 169. 4. Kang JX, Man SF, Brown NE, Labrecque PA, Garg ML, Clandinin MT. Essential fatty acid metabolism in cultured human airway epithelial cells. Biochim Biophys Acta 1992;1128:267e74. 5. Harbige LS. Fatty acids, the immune response, and autoimmunity: a question of n-6 essentiality and the balance between n-6 and n-3. Lipids 2003;38:323e41. 6. Calder PC. Polyunsaturated fatty acids, inflammation, and immunity. Lipids 2001;36:1007e24. 7. Black PN, Sharpe S. Dietary fat and asthma: is there a connection? Eur Respir J 1997;10:6e12. 8. Carlson SE. Docosahexaenoic acid and arachidonic acid in infant development. Semin Neonatol 2001;6:437e49. 9. Barden AE, Mori TA, Dunstan JA, Taylor AL, Thornton CA, Croft KD, et al. Fish oil supplementation in pregnancy lowers F2-isoprostanes in neonates at high risk of atopy. Free Radic Res 2004;38:233e9. 10. Dunstan JA, Mori TA, Barden A, Beilin LJ, Taylor AL, Holt PG, et al. Fish oil supplementation in pregnancy modifies neonatal allergen-specific immune responses and clinical outcomes in infants at high risk of atopy: a randomized, controlled trial. J Allergy Clin Immunol 2003;112:1178e84. 11. Schiefermeier M, Yavin E. n-3 deficient and docosahexaenoic acid-enriched diets during critical periods of the developing prenatal rat brain. J Lipid Res 2002;43:124e31. 12. Helland IB, Smith L, Saarem K, Saugstad OD, Drevon CA. Maternal supplementation with very-long-chain n-3 fatty acids during pregnancy and lactation augments children’s IQ at 4 years of age. Pediatrics 2003;111:e39e44. 13. 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. 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. Scott NM, Hodyl NA, Murphy VE, Osei-Kumah A, Wyper H, Hodgson DM, et al. Placental cytokine expression covaries with maternal asthma severity and fetal sex. J Immunol 2009;182:1411e20. 16. Schatz M, Zeiger RS. Treatment of asthma and allergic rhinitis during pregnancy. Ann Allergy 1990;65:427e9. 17. Godfrey KM, Barker DJ. Fetal nutrition and adult disease. Am J Clin Nutr 2000;71. 1344Se52S. 18. McLernon PC, Wood LG, Murphy VE, Hodyl NA, Clifton VL. Circulating antioxidant profile of pregnant women with asthma. Clin Nutr 2011. 19. Murphy VE, Clifton VL, Gibson PG. The effect of cigarette smoking on asthma control during exacerbations in pregnant women. Thorax 2010;65:739e44. 20. Gibson PG, Wilson AJ. The use of continuous quality improvement methods to implement practice guidelines in asthma. J Qual Clin Pract 1996;16:87e102. 21. National Heart LaBI. New NHLBI guidelines for the diagnosis and management of asthma. Lippincott Health Promot Lett 1997;2:8e9. 22. Murphy VE, Gibson PG, Talbot PI, Kessell CG, Clifton VL. Asthma selfmanagement skills and the use of asthma education during pregnancy. Eur Respir J 2005;26:435e41. 23. Barnes PJ, Pedersen S. Efficacy and safety of inhaled corticosteroids in asthma. Report of a workshop held in Eze, France. Am Rev Respir Dis October 1992;1993(148):S1e26. 24. Block G. A review of validations of dietary assessment methods. Am J Epidemiol 1988;115:492e505. 25. Lepage G, Roy CC. Direct transesterification of all classes of lipids in a one-step reaction. J Lipid Res 1986;27:114e20. 26. Wood LG, Fitzgerald DA, Gibson PG, Cooper DM, Garg ML. Increased plasma fatty acid concentration following respiratory exacerbations in cystic fibrosis is associated with elevated oxidative stress. Am J Clin Nutr 2002;75:668e75.
P. McLernon et al. / e-SPEN Journal 7 (2012) e78ee85 27. Hure A, Young A, Smith R, Collins C. Diet and pregnancy status in Australian women. Public Health Nutr 2009;12:853e61. 28. Council NHaMR. Nutrient reference values for Australia and New Zealand. Canberra: Commonwealth of Australia; 2006. 29. Prasad M, Lumia M, Erkkola M, Tapanainen H, Kronberg-Kippila C, Tuokkola J, et al. Diet composition of pregnant Finnish women: changes over time and across seasons. Public Health Nutr 2010;13:939e46. 30. 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. 31. Herrera E, Lopez-Soldado I, Limones M, Amusquivar E, Ramos MP. Lipid metabolism during the perinatal phase, and its implications on postnatal development. Int J Vitam Nutr Res 2006;76:216e24. 32. Tomlinson JW, Sherlock M, Hughes B, Hughes SV, Kilvington F, Bartlett W, et al. Inhibition of 11beta-hydroxysteroid dehydrogenase type 1 activity in vivo limits glucocorticoid exposure to human adipose tissue and decreases lipolysis. J Clin Endocrinol Metab 2007;92:857e64. 33. Hodyl NA, Stark MJ, Osei-Kumah A, Bowman M, Gibson P, Clifton VL. Fetal glucocorticoid-regulated pathways are not affected by inhaled corticosteroid use for asthma during pregnancy. Am J Respir Crit Care Med 2011;183:716e22. 34. Wood LG, Garg ML, Simpson JL, Mori TA, Croft KD, Wark PA, et al. Induced sputum 8-isoprostane concentrations in inflammatory airway diseases. Am J Respir Crit Care Med 2005;171:426e30. 35. Horton JD, Goldstein JL, Brown MS. SREBPs: activators of the complete program of cholesterol and fatty acid synthesis in the liver. J Clin Invest 2002;109:1125e31. 36. Li J, Thorne LN, Punjabi NM, Sun CK, Schwartz AR, Smith PL, et al. Intermittent hypoxia induces hyperlipidemia in lean mice. Circ Res 2005;97:698e706.
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37. Drager LF, Li J, Shin MK, Reinke C, Aggarwal NR, Jun JC, et al. Intermittent hypoxia inhibits clearance of triglyceride-rich lipoproteins and inactivates adipose lipoprotein lipase in a mouse model of sleep apnoea. Eur Heart J 2011. 38. Szajewska H, Horvath A, Koletzko B. Effect of n-3 long-chain polyunsaturated fatty acid supplementation of women with low-risk pregnancies on pregnancy outcomes and growth measures at birth: a meta-analysis of randomized controlled trials. Am J Clin Nutr 2006;83:1337e44. 39. Smuts CM, Huang M, Mundy D, Plasse T, Major S, Carlson SE. A randomized trial of docosahexaenoic acid supplementation during the third trimester of pregnancy. Obstet Gynecol 2003;101:469e79. 40. Oken E, Kleinman KP, Olsen SF, Rich-Edwards JW, Gillman MW. Associations of seafood and elongated n-3 fatty acid intake with fetal growth and length of gestation: results from a US pregnancy cohort. Am J Epidemiol 2004;160:774e83. 41. Olsen SF, Secher NJ. Low consumption of seafood in early pregnancy as a risk factor for preterm delivery: prospective cohort study. Br Med J 2002;324:447. 42. Saedisomeolia A, Wood LG, Garg ML, Gibson PG, Wark PA. Supplementation of long chain n-3 polyunsaturated fatty acids increases the utilization of lycopene in cultured airway epithelial cells. J Food Lipids 2008;15:421e32. 43. Meydani M, Natiello F, Goldin B, Free N, Woods M, Schaefer E, et al. Effect of long-term fish oil supplementation on vitamin E status and lipid peroxidation in women. J Nutr 1991;121:484e91. 44. Schatz M, Leibman C. Inhaled corticosteroid use and outcomes in pregnancy. Ann Allergy Asthma Immunol 2005;95:234e8. 45. Clifton VL. Review: sex and the human placenta: mediating differential strategies of fetal growth and survival. Placenta 2010;31:S33e9.
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Original article
Fatty acid profile of pregnant women with asthma Penelope McLernon a, b, Lisa Wood c, Vanessa E. Murphy c, Nicolette A. Hodyl a, Vicki L. Clifton a, b, * a
Robinson Institute, University of Adelaide, Lyell McEwin Hospital, Haydown Rd, Elizabethvale, Adelaide, SA 5112, 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 25 October 2011 Accepted 14 January 2012
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. Dietary fatty acids (FA) especially polyunsaturated fatty acids (PUFA) are particularly important during pregnancy due to their role in fetal growth and development. Dietary PUFAs also have a role in clinical outcomes for non-pregnant asthmatics. The current study was designed to characterize the fatty acid profile in pregnant women with asthma to determine whether asthma severity or reduced fetal growth were associated with an altered FA profile. Methods: Maternal dietary intake and plasma fatty acid profile were examined in women with and without asthma at 18, 30 and 36 weeks gestation. Maternal fatty acids levels were related to measures of fetal growth using Doppler ultrasound and birth outcomes. Results: The data found that pregnant women with moderate/severe asthma had increased circulating plasma fatty acid levels at 36 weeks gestation but reduced dietary intake of fats compared to those women with mild asthma and healthy pregnant controls. In addition, women with moderate/severe asthma had increased circulating n-3PUFA levels at 36 weeks gestation which was associated with reduced fetal and neonatal head circumference. Conclusion: These observations suggest moderate/severe asthma may disrupt lipid metabolism, transport or cellular uptake during pregnancy which subsequently contributes to reduced fetal growth. Ó 2012 European Society for Clinical Nutrition and Metabolism. Published by Elsevier Ltd. All rights reserved.
Keywords: Pregnancy Fetal growth Asthma Fatty acids
1. Introduction Dietary fatty acids (FA) are key contributors to daily energy requirements and play a central role in regulating physiological functions including the innate immune system, glucose metabolism and endothelial function. Fatty acids are classified according to the degree of saturation, as saturated fatty acids (SFA), monounsaturated fatty acids (MUFA) and polyunsaturated fatty acids (PUFA). PUFA can be further divided into n-3PUFA and n-6PUFA. The long chain n-3PUFA include eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), which have been shown to have antiinflammatory properties, due to their ability to modify production of eicosanoids, decrease production of pro-inflammatory cytokines
* Corresponding author. Robinson Institute, University of Adelaide, Lyell McEwin Hospital, Haydown Rd, Elizabethvale, Adelaide, SA 5112, Australia. Tel.: þ61 8 81332133; fax: þ61 8 8303 4099. E-mail address: [email protected] (V.L. Clifton).
and alter immune cell function.1e6 It has been hypothesized that increased prevalence of asthma and atopy in recent years may be linked to an increased intake of n-6PUFA relative to n-3PUFA.7 PUFAs are particularly important during pregnancy due to their role in fetal growth and development especially fetal brain development.8 It has been suggested that the anti-inflammatory properties of n-3PUFA may protect against the inflammation and oxidative damage that occurs in pregnancy.9,10 Indeed, it has been shown that insufficient n-3PUFA intake during pregnancy may contribute to suboptimal fetal neurodevelopment, and later life disease.11,12 One of the most prevalent complications of pregnancy is asthma and we are interested in the impact of this complication on fetal development.13e15 Pregnancies complicated by maternal asthma are associated with an increased incidence of intrauterine growth restriction,14,16 with significant implications for both the short and long term health of the offspring.17 The use of inhaled glucocorticoids for asthma treatment during pregnancy appears protective against the growth reducing effects of the disease.13 The maternal
2212-8263/$36.00 Ó 2012 European Society for Clinical Nutrition and Metabolism. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.clnme.2012.01.004
P. McLernon et al. / e-SPEN Journal 7 (2012) e78ee85
dietary contributions to reduced fetal growth in pregnancies complicated by asthma are not known. In particular, the maternal fatty acid profile may be associated with altered fetal development. Recently we have identified that circulating antioxidants in pregnant women with asthma are significantly increased as gestation progresses relative to a healthy population of pregnant women18 suggesting a compensatory 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. The current study was designed to assess if there are any differences in the FA profile of the same population of pregnant women with asthma compared to women without asthma18 and whether these differences are related to dietary intake, asthma treatment, asthma severity or cigarette use. 2. Methods 2.1. Experimental subjects The study was approved by the Hunter New England Research Ethics and University of Newcastle Human Research Ethics Committees. Pregnant women were recruited in the first trimester (n ¼ 131, controls n ¼ 47, and asthmatics n ¼ 84) and provided written informed consent. The protocol for this study has been described previously.13,19 Using the smallest difference observed previously in our analyses of tocopherol levels in pregnant asthmatic and healthy control women,18 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 mg/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 comorbidities associated with pregnancies complicated by asthma. Complications other than asthma such as pre-eclampsia, gestational diabetes, infection or preterm delivery were excluded from the analysis (n ¼ 4). Birth weight and fetal sex were determined at birth. The characteristics of this population were also published in a paper examining circulating antioxidants in this group of women.18 2.2. Materials and methods Clinical asthma severity was rated as mild, moderate or severe using the integrated severity score described in the Australian Asthma Management Guidelines,20 which closely approximate the National Heart, Lungs and Blood Institute Guidelines.21 Proper inhaler use and compliance was assessed by the study research nurse.22 Cumulative, inhaled corticosteroid (ICS) dose was calculated for each trimester, and summarized as the mean daily dose of beclomethasone dipropionate (BDP) or equivalent used during pregnancy, where 1 mg BDP was considered equal to 1 mg budesonide or 0.5 mg fluticasone propionate.23 For data analysis, the low, moderate and high inhaled corticosteroid (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 b2 agonist, salbutamol for symptom relief when required. Current smoking status was assessed by direct questioning at recruitment. Some of the control subjects were smokers (Table 1).
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Table 1 Maternal characteristics. Maternal characteristics
Maternal age (yrs) Range Body Mass Index Range Gravidity Range Parity Range Inhaled corticosteroid use (%) Cigarette use (n)
Control
Mild
Mod-severe
n ¼ 47
n ¼ 31
n ¼ 53
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
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.
All women participating in the study underwent ultrasound assessment at 18, 30 and 36 weeks of gestation. Fetal biparietal diameter, head circumference, abdominal circumference and femur length were measured. Birth weight, length and head circumference were recorded at delivery. Customized 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 hospital-specific growth charts. Ponderal index was calculated as birth weight (kg)/length3 (m). 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. 2.3. Blood collection Twenty mL of whole blood were collected into EDTA coated tubes from the median cubital vein at each visit (18, 30 and 36 weeks). Women were advised not to eat at least 2 h prior to blood sampling. An overnight fast from pregnant women was not appropriate for a non-medical assessment. Whole blood was centrifuged at 3000 rpm at 4 C for 10 min. Plasma was stored in 500 mL aliquots at 80 C until required for analysis. 2.4. 24 h 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 analyzing mean dietary intakes.24 The questionnaire asked for detailed information on meals and snacks consumed 24 h prior to the clinic visit. For this study, the data was analyzed 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. Dietary data was also published in the study of circulating antioxidants in pregnant women with asthma.18 3. Biochemical analyses 3.1. Maternal circulating levels of fatty acids Total fatty acids were analyzed by gas chromatography using established methodology.25,26 2 mL of methanol:toluene (4:1 v/v)
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containing C19:0 (4 mg/mL) was added to 100 mL of plasma. Fatty acids were methylated by adding 200 mL acetyl chloride dropwise whilst vortexing, then heated for 60 min at 100 C. After cooling, the reaction was stopped by adding 5 mL 6% potassium chloride (K2CO3). Samples were then centrifuged at 3000 rpm at 5 C for 10 min. Supernatant was analyzed by gas chromatograph for fatty acid methyl esters (FAME), using a HewlettePackard 6890 Gas Chromatograph equipped with a 30 m (0.25 mm ID) carbon-silica capillary column coated with 50% cyanoproylphenyl and 50% dimethylpolysiloxane (0.25 mm film thickness), a flame ionization detector, autosampler and autodetector. The initial oven temperature was set at 30 C, programmed to rise to a maximum of 240 C at 3 C/min. A split ratio of 10:1 with an injection volume of 3 mL was used. The carrier gas (hydrogen) was expelled at an average velocity of 7 cm/s. Chemstation version A.04.02 was used to quantify fatty acid methyl esters based on retention time compared to lipid standards. Plasma concentrations and 24 h recall dietary intake questionnaire data from controls (n ¼ 47) and asthmatic (n ¼ 84) women (mild; n ¼ 31, moderate/severe; n ¼ 53) were analyzed. 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. 3.2. Statistical analysis 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 analyze temporal changes of fatty acids 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 fatty acid 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) during pregnancy (P > 0.05, Fisher’s Exact Test, Table 1). A higher proportion of moderate/ severe asthmatics used inhaled corticosteroids to treat their asthma (83%) when compared to the mild asthmatics (32.3%). 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). There were no babies less than the 10th birth weight centile in this cohort. 4.2. Maternal dietary intake 4.2.1. Cross sectional analysis at 36 weeks gestation The mild asthmatic group (n ¼ 25) consumed higher quantities of energy, protein, fats, carbohydrates, starch, thiamin, riboflavin,
Table 2 Neonatal characteristics. Neonatal characteristics
Gestational age (weeks) Range Birth weight (g) Range Birth weight Centile Range Birth length (cm) Range Ponderal Index Range Head circumference (cm) Range Placental weight (g) Range male/female (n/n)
Control
Mild
Mod-severe
n ¼ 47
n ¼ 31
n ¼ 53
P value
40.4 39.4e41.2 3402.5 3115e3715 35.6 19.3e51.4 51 50e53 25.3 21.7e34.9 35 34e35.5 630.4 602.8e671.7 18/25
40.3 39.6e41 3660 3220e3940 52.3 14.6e73.5 51 50e54 24.5 19.6e31.8 34.5 33.6e36 671.8 607.4e800.8 14/17
39.8 0.06 38.6e40.6 3340 0.18 3040e3647 52 0.29 22.9e63.1 52 0.24 50e53 25.3 0.81 18.4e37.2 34.5 0.26 33.5e35.5 645.1 0.27 592.8e781.6 24/29
Values reported as the median and range. *P < 0.05.
niacin equivalents, magnesium and phosphorous than the moderate/severe asthmatics (n ¼ 34) or control population (n ¼ 32) at 36 weeks gestation (P < 0.01, Fisher’s Exact Test, Table 3). The moderate-severe asthmatic group had reduced consumption of all the fats relative to the control or mild asthmatic group (P < 0.01, Fisher’s Exact Test, Table 3) at 36 weeks. 4.2.2. Temporal analysis of dietary intake at 18, 30 and 36 weeks gestation Dietary intake data from controls (n ¼ 32) and asthmatic women (n ¼ 57) was analyzed at 18, 30 and 36 weeks gestation and asthmatic groups were compared based on severity (mild; n ¼ 25, moderate/severe; n ¼ 34). A temporal analysis revealed no significant changes from 18 to 36 weeks gestation in maternal dietary intake of total fatty acids, total saturated fatty acids, total monounsaturated fatty acids, total n-6 polyunsaturated fatty acids, total n-3 polyunsaturated fatty acids in the control, mild or the moderate/severe asthmatics. There were no significant changes over pregnancy in maternal dietary intake of the essential fatty acids (EFAs; linoleic [LA, 18:2n-6] and a-linolenic [aLNA, 18:3n-3]), nor the long chain polyunsaturated fatty acids (LCPUFA; Arachidonic [AA, 20:4n-6], eicosapentaenoic [EPA, 20:5n-3] or docosahexaenoic [DHA, 22:6n-3]) acids (P > 0.05, Friedman’s Test). The only temporal differences in the fatty acid dietary intake were observed in the moderate-severe asthmatics who had a significantly reduced percentage intake of total n-6PUFA at 36 weeks gestation which was unrelated to any individual n6 PUFA, though there was a trend toward reduced linoleic acid intake in the moderate-severe asthmatic group (18:2n-6, median controls 7.2 g/L vs moderate-severe 4.7 g/L; P ¼ 0.053). The intake of lignoceric acid (24:0) was also reduced in the moderate-severe asthmatic group relative to the control at 30 and 36 weeks gestation (data not shown). 4.3. Maternal plasma fatty acids 4.3.1. Cross sectional analysis at 36 weeks gestation Plasma total fatty acids were assessed in moderate/severe asthmatic (n ¼ 39), mild asthmatic (n ¼ 26) and control (n ¼ 38) subjects at 18, 30 and 36 weeks gestation (Table 4). Most fatty acids were not significantly different between the groups until 36 weeks gestation. Circulating SFA, MUFA and n-3PUFA were significantly increased in moderate-severe asthmatics relative to the control and mild asthmatic groups (Table 4). The ratio of n-6:n-3PUFA decreased in the moderate-severe asthmatics at 36 weeks gestation.
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4.3.2. Temporal analysis at 18, 30 and 36 weeks gestation A subgroup of women from the total cohort provided blood samples at each gestational visit (control (n ¼ 21), mild asthmatics (n ¼ 16), moderate/severe asthmatics (n ¼ 22)). Data were analyzed to identify changes in circulating levels of fatty acids throughout gestation in relation to asthma severity and compared to 6 month post partum measurements. Total plasma fatty acids (Fig. 1), SFA (data not shown), MUFA (data not shown) and n-6PUFA (Fig. 2) were significantly increased with advancing pregnancy in all Table 3 Maternal total dietary intake at 36 weeks gestation. Dietary factors
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 fiber (g) Range Thiamin (mg) Range Riboflavin (mg) Range Niacin equivalents (mg) Range Vitamin C (mg) Range Total Folate (mg) Range Viamin A Equivalents (mg) Range Retinol (mg) Range Beta carotene (mg) Range Potassium (mg) Range Magnesiums (mg) Range Calcium (mg) Range Phosphorous (mg) Range Iron (mg) Range
Control
Mild
Mod-severe
n ¼ 32
n ¼ 25
n ¼ 34
9.6 7.2e11.4 86.2 58.3e113 97.9 68.8e122.3 41.5 32.3e54.1 10.9
10.7* 9e14. 96.8* 80.2e141 108.2 87.5e148.9 43.6 35.9e66.7 14.6
8.2** 6.9e9.8 76.5** 56e96.7 80.6* 56.7e100.6 33.3* 24.8e46.4 7.8*
0.02*
6.6e16.6 6.5
9.2e19.9 7.2
5.5e14.8 6
0.3
5.0e12.1 0.9
3.8e12.5 0.8
4.4e10.2 0.8
0.5
0.7e1.5 30.9
0.6e1.8 40.9
0.5e1.23 29.3*
0.03*
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
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*
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
24.6e47 135.8 50.9e238.9 280.9 178.4e366.9 970.6
34.5e59.2 128.3 41.5e289.8 304.7 233.2e403.5 1143.1
23.8e44.6 142.3 78.5e257.7 257.7 182.8e307 955.3
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
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
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
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groups (P < 0.01, Bonferroni correction). EPA and DHA n-3 fatty acids did not significantly change during pregnancy in any group (Fig. 3, p > 0.05, Fisher’s Exact test). The ratio of n-6:n-3PUFA was found to increase significantly over the course of pregnancy in the control and mild asthmatic groups Fig. 4, P < 0.01, Bonferroni correction). There was a significant increase in maternal circulating levels of the essential fatty acids: LA and aLNA over the duration of pregnancy in each of the groups (P < 0.05, Fisher’s Exact Test, data not shown). Maternal plasma levels of AA; (20:4n-6), did not change as gestation progressed in the control or asthmatic groups (P > 0.05, Fisher’s Exact Test, data not shown).
P value
0.004* 0.02* 0.01* 0.04*
0.02* 0.02* 0.1 0.04* 0.4 0.03* 0.05* 0.04*
4.3.3. Maternal smoking and ICS use Maternal circulating levels of fatty acids at 18, 30 and 36 weeks gestation 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 (p > 0.05, Bonferroni correction). Maternal circulating levels of fatty acids at 18, 30 and 36 weeks gestation were not significantly different between smokers and non smokers (p > 0.05, Bonferroni correction). 4.4. Relationships between maternal circulating fatty acids and fetal growth parameters in pregnancies complicated by asthma There was a positive correlation between fetal head circumference (HC) at 36 weeks gestation and the n-6:n-3PUFA ratio at 30 weeks gestation (r ¼ 0.481, p ¼ 0.032, n ¼ 20) and at 36 weeks gestation (r ¼ 0.716, p ¼ 0.001, n ¼ 17) in all groups. In the moderate/severe asthmatics, there was a negative correlation between fetal HC at 36 weeks gestation and the essential omega 3 a-linolenic acid (a-LNA, 18:3n-3) at 30 weeks gestation (r ¼ 0.405, p ¼ 0.032, n ¼ 28) and at 36 weeks gestation (r ¼ 0.386, p ¼ 0.043, n ¼ 28). In the moderate/severe asthmatic group, HC at birth was negatively correlated with DHA at 30 weeks gestation (r ¼ 0.415, p ¼ 0.025, n ¼ 29), LA at 18 (r ¼ 0.360, p ¼ 0.028, n ¼ 37) and 30 weeks gestation (r ¼ 0.384, p ¼ 0.040, n ¼ 29), AA at 30 weeks (r ¼ 0.436, p ¼ 0.018, n ¼ 29), total n-6PUFA at 18 weeks (r ¼ 0.331, p ¼ 0.046, n ¼ 37) and 30 weeks gestation (r ¼ 0.373, p ¼ 0.046, n ¼ 29), total n3 PUFA at 30 weeks (r ¼ 0.394, p ¼ 0.034, n ¼ 29), total MUFA at 30 weeks (r ¼ 0.448, p ¼ 0.015, n ¼ 29), total fatty acids at 30 weeks (r ¼ 0.416, p ¼ 0.025, n ¼ 29) and the ratio of n-6:n-3PUFA at 30 weeks (r ¼ 0.397, p ¼ 0.033, n ¼ 29).
0.8 0.2 0.8
Table 4 Cross sectional analysis of maternal plasma fatty acid concentrations at 36 weeks gestation. Fatty acids
0.1 0.9 0.9 0.04* 0.3 0.01* 0.04*
Values are median and inter-quartile range. The KW-ANOVA and ManneWhitney U test with Bonferroni correction were used. *P < 0.05 was considered significant compared to control population.
Saturated Fats (mg/L) Range MUFA (mg/L) Range n6 PUFA (mg/L) range n3 PUFA (mg/L) Range n6en3 ratio Range Total fatty acids (mg/L) Range
Control
Mild
Mod-severe
n ¼ 26
n ¼ 31
n ¼ 53
793.8 621e1831 665.2 548e1563 678.5 572e2109 94.7 64e351 6.9 6e8.5 2234.5 1789e5988
832.6 619e1425 647.7 503e1307 682.3 571e1825 84.3 71e268 7.7 6.5e9.4 2207.5 1808e4923
1688.9* 706e1955 1476.9* 649e1911 1918.9 625e2151 371.2* 93e439 5.4* 4.6e7.5 5635.3 1992e6452
P value
0.05* 0.03* 0.19 0.01* 0.001* 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. No significant differences were observed between controls and mild asthmatics. MUFA: monounsaturated fats; PUFA: polyunsaturated fats.
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A
B
CONTROL 8000
total fatty acids (mg/L)
MODERATE/SEVERE
8000
*
Maternal circulating plasma
C
MILD
8000
*
* *
*
6000
6000
6000
§ 4000
4000
4000
2000
2000
2000
0
0
0
18
30
36
Gestational Week
Post
18
30
36
Gestational Week
Partum
n=21
n=16
18
Post
30
36
Post
Gestational Week
Partum
Partum
n=22
Fig. 1. Maternal plasma total fatty acids (mg/L) in control pregnancies (panel A), mild (panel B) and moderate/severe asthmatic pregnancies (panel C) at 18, 30 and 36 weeks gestation, with post partum levels depicted for reference only. Data are presented as median and inter-quartile range. *P < 0.05.
None of the correlations observed in the moderate/severe asthmatics were observed in the control group or mild asthmatic group with respect to fatty acids and fetal or neonatal head growth in early, mid or late gestation.
Intake of energy (7.1e8.9 MJ/day) and protein (58e60 g/day) were above recommendations for pregnant women. Overall the total fats contributed to 37% of the total energy consumed by the women in all groups which above the recommended levels of 35% total energy and 15% of total energy was derived from saturated fats in all groups which is also above the recommendation that <10% of saturated fats should contribute to total energy. Fat intake of the women in the current study are only slightly higher than pregnant women in Finland29 and obese pregnant women in Belgium30 suggesting that the diet of these women does not differ significantly from other populations. Moderate-severe asthmatics had significantly higher concentrations of plasma FA compared to mild asthmatics and healthy controls, even though dietary intake was lower. Our findings suggest that altered maternal physiology in the presence of severe asthma greatly influences the circulating fatty acid profile. Normal pregnancy is well recognized as a hyperlipidemic state that may be induced by the increased concentrations of estrogens during pregnancy31 however the presence of moderate-severe asthma during pregnancy appears to further exacerbate hyperlipidemia. There may be numerous mechanisms contributing to hyperlipidemia in moderate/severe asthmatics. Lipolysis can be regulated by cortisol32 and possibly influenced by the use of inhaled corticosteroids (ICS) for the treatment of asthma. Glucocorticoids have been shown to increase lipase activity in adipocytes which results in increased release of circulating free fatty acids.32 Our research
5. Discussion This study demonstrates that in the final trimester of pregnancy, pregnant women with moderate/severe asthma have increased circulating plasma fatty acid levels. This is despite the fact that they have reduced dietary intake compared to those with mild asthma and healthy controls. In addition, for women with moderate/severe asthma, increased circulating n-3PUFA levels were associated with the smallest sized neonates although none of the babies in this cohort were growth restricted. Temporal analysis indicates that in moderate/severe asthma and healthy controls, levels of circulating fatty acids increase over the duration of the pregnancy, which was also independent of changes in dietary intake. These observations suggest that lipid metabolism is altered during pregnancy, particularly in those pregnancies complicated by asthma. Dietary intake in this particular population of pregnant women is comparable to data obtained in previous studies of Australian pregnant women.27 Most women had below the recommended intake of iron, calcium, magnesium, folate, retinol and dietary fiber. Riboflavin, niacin, vitamin C and vitamin A equivalents, phosphorous and potassium intake were all above recommended intakes.28
B
CONTROL
MILD
* *
2500
fatty acids (mg/L)
Maternal circulating plasma
total omega 6 polyunsaturated
A
C
*
2500
* *
2500
2000
2000
2000
MODERATE/SEVERE
§ 1500
1500
1500
1000
1000
1000
500
500
500
0
18
30
36
Gestational Week n= 21
Post Partum
0
18
30
36
Gestational Week n=16
Post Partum
0
18
30
36
Gestational Week
Post Partum
n=22
Fig. 2. Maternal plasma total omega 6 fatty acids (mg/L) in control pregnancies (panel A), mild (panel B) and moderate/severe asthmatic pregnancies (panel C) at 18, 30 and 36 weeks gestation, with post partum levels depicted for reference only. Data are presented as median and inter-quartile range. *P < 0.05.
P. McLernon et al. / e-SPEN Journal 7 (2012) e78ee85
Maternal circulating plasma
CONTROL
eicosapentaenoic acid (EPA [20:5n3] mg/L)
A
MILD
A
80
80
#
§ 40
40
40
20
20
20
0
0
0 30
36
18
Post
36
Post
18
30
400
MODERATE/SEVERE
B
400
C
#
300
300
300
Partum
n=22
MILD
A
Post
36
Gestational Week
Partum
n=16
CONTROL 400
30
Gestational Week
Partum
n=21
Maternal circulating plasma
C
60
60
60
Gestational Week
docosahexaenoic acid (DHA [22:6n3] mg/L)
MODERATE/SEVERE
B
80
18
B
e83
§ 200
200
200
100
100
100
0
18
30
0
36
Gestational Week
18
30
36
Gestational Week
n=21
Post
0
18
30
36
Post
Gestational Week
Partum
n=16
Partum
n=22
Fig. 3. Maternal plasma eicosapentaenoic acid (EPA [20:5n-3] mg/L, Fig. 3A) and docosahexaenoic acid (DHA [22:6n-3] mg/L, Fig. 3B) in control pregnancies (panel A), mild (panel B) and moderate/severe asthmatic pregnancies (panel C) at 18, 30 and 36 weeks gestation, with post partum levels depicted for reference only. Data are presented as median and interquartile range. A Friedman Test identified no significant differences in the levels of any n3 PUFAs during gestation.
has previously identified that ICS use during pregnancy for the treatment of asthma exerts systemic effects on glucocorticoid regulated pathways in the maternal system.33 In the current study 83% of moderate-severe asthmatics used ICS for the treatment of their asthma. The increased concentrations of circulating FA observed in the moderate-severe asthmatic women may be a consequence of ICS-induced lipolysis. In addition, the presence of oxidative stress in the moderate/ severe asthmatics34 may have altered lipolysis. Oxidative stress
A
*
fatty acid ratio
total n6-n3 polyunsaturated
14
Maternal circulating plasma
B
CONTROL
induces metabolic disturbances, including altered lipid metabolism. Lipid biosynthesis in the liver is regulated by a family of transcription factors: the sterol regulatory element binding proteins (SREBPs)35 which may be altered by oxidative stress.36,37 This raises the possibility that the increased levels of circulating fatty acids in the later stages of pregnancy in the moderate/severe asthmatics may occur due to increased lipolysis, induced by increased inflammation and oxidative stress that is induced by the combination of asthma and pregnancy.34
C
MILD *
14 12
12
10
10
10
8
8
8
6
6
6
4
4
4
2
2
2
18
30
36
Gestational Week n= 21
Post Partum
0
18
30
36
Gestational Week n=16
#
14
12
0
MODERATE/SEVERE
Post Partum
0
§
18
30
36
Gestational Week
Post Partum
n=22
Fig. 4. Maternal plasma n6-n3 fatty acid ratio in control pregnancies (panel A), mild (panel B) and moderate/severe asthmatic pregnancies (panel C) at 18, 30 and 36 weeks gestation, with post partum levels depicted for reference only. Data are presented as median and inter-quartile range. *P < 0.05.
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n-3PUFAs were amongst the classes of fatty acids that were elevated at 36 weeks gestation in the moderate-severe asthma group, despite PUFA intake being lower in this group. Unexpectedly, n-3PUFA were negatively associated with reduced head growth in utero and at birth. Although none of the babies in this study were growth restricted it was interesting to note that the smallest neonates were associated with the highest maternal fatty acid concentrations. n-3PUFAs are generally considered beneficial for fetal growth, though the data remains controversial.38e41 Furthermore n-3PUFA may increase susceptibility to oxidative stress, due to the presence of multiple double bonds, which are vulnerable to attack by free radicals. We42 and others,43 have shown that increased intracellular content of n-3PUFA results in increased susceptibility to lipid peroxidation. The current data suggest placental transfer of n-3PUFAs may be significantly compromised in asthmatic pregnancies and that supplementation with n-3PUFA in abnormal pregnancies may not improve fetal outcome or growth. It is not known whether placental transport of PUFAs is altered in asthmatic pregnancies or if the fetal circulation is deficient in fatty acids. This is an area that requires further investigation. It was expected from epidemiological studies of asthma during pregnancy that at least 10% of neonates would be small for gestational age or less than the 10th birth weight centile14 in this study. The absence of any growth restricted neonates in this cohort may be a result of the study protocol22 which involved women attending a respiratory nurse -led asthma clinic regularly during their pregnancy for monitoring of their asthma management. Women with well controlled asthma are known to have normal birth outcomes44 and we have previously reported that the use of inhaled glucocorticoids during pregnancy was associated with normal sized neonates in relation to both birth weight and head circumference.13 ICS do not cross the placenta33 and therefore do not exert a direct effect on fetal growth suggesting it is the inflammatory effects of the disease that has the greatest impact on fetal growth via alterations in placental function.15,45 A limitation of this study was the use of 24 h recall dietary analysis, as a measure of recent dietary intake. While alternative methods such as 4-day food records give a more accurate representation, 24 h recalls were used in order to minimize participant burden. Furthermore, this method has been shown to provide an accurate measure of group mean intakes.24 Another limitation is the use of non-fasting samples, which was ethically appropriate for a non-medical assessment of pregnant women. The fact that significant differences in groups were observed despite the potential confounders, further strengthens the importance of the observations. In summary, our data has revealed that despite lower dietary intake in moderate/severe asthmatic women, circulating fatty acid levels increase as pregnancy progresses and associated with negative fetal head growth. These observations suggest that maternal lipid metabolism and its transport to the fetus are altered in pregnant women with asthma and this may contribute to poor fetal outcomes. Conflict of interest There are no identified conflicts of interest in the production of this work or manuscript. Statement of authorship PM conducted the study and analyzed the data. NH analyzed the data. LW, VM and VC designed the study and wrote the manuscript.
Acknowledgments 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. Martin RE. Docosahexaenoic acid decreases phospholipase A2 activity in the neurites/nerve growth cones of PC12 cells. J Neurosci Res 1998;54:805e13. 2. Simopoulos AP. Omega-3 fatty acids in inflammation and autoimmune diseases. J Am Coll Nutr 2002;21:495e505. 3. Tamura Y, Terano J, Saito A, Hirai A, Shiina T. Enhancement of PGI2 formation by EPA in rat vascular smooth muscle cells. In: Ong ASH, Niki E, Packer L, editors. Nutrition, lipids, health and disease. Illinois: Champaign; 1995. p. 169. 4. Kang JX, Man SF, Brown NE, Labrecque PA, Garg ML, Clandinin MT. Essential fatty acid metabolism in cultured human airway epithelial cells. Biochim Biophys Acta 1992;1128:267e74. 5. Harbige LS. Fatty acids, the immune response, and autoimmunity: a question of n-6 essentiality and the balance between n-6 and n-3. Lipids 2003;38:323e41. 6. Calder PC. Polyunsaturated fatty acids, inflammation, and immunity. Lipids 2001;36:1007e24. 7. Black PN, Sharpe S. Dietary fat and asthma: is there a connection? Eur Respir J 1997;10:6e12. 8. Carlson SE. Docosahexaenoic acid and arachidonic acid in infant development. Semin Neonatol 2001;6:437e49. 9. Barden AE, Mori TA, Dunstan JA, Taylor AL, Thornton CA, Croft KD, et al. Fish oil supplementation in pregnancy lowers F2-isoprostanes in neonates at high risk of atopy. Free Radic Res 2004;38:233e9. 10. Dunstan JA, Mori TA, Barden A, Beilin LJ, Taylor AL, Holt PG, et al. Fish oil supplementation in pregnancy modifies neonatal allergen-specific immune responses and clinical outcomes in infants at high risk of atopy: a randomized, controlled trial. J Allergy Clin Immunol 2003;112:1178e84. 11. Schiefermeier M, Yavin E. n-3 deficient and docosahexaenoic acid-enriched diets during critical periods of the developing prenatal rat brain. J Lipid Res 2002;43:124e31. 12. Helland IB, Smith L, Saarem K, Saugstad OD, Drevon CA. Maternal supplementation with very-long-chain n-3 fatty acids during pregnancy and lactation augments children’s IQ at 4 years of age. Pediatrics 2003;111:e39e44. 13. 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. 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. Scott NM, Hodyl NA, Murphy VE, Osei-Kumah A, Wyper H, Hodgson DM, et al. Placental cytokine expression covaries with maternal asthma severity and fetal sex. J Immunol 2009;182:1411e20. 16. Schatz M, Zeiger RS. Treatment of asthma and allergic rhinitis during pregnancy. Ann Allergy 1990;65:427e9. 17. Godfrey KM, Barker DJ. Fetal nutrition and adult disease. Am J Clin Nutr 2000;71. 1344Se52S. 18. McLernon PC, Wood LG, Murphy VE, Hodyl NA, Clifton VL. Circulating antioxidant profile of pregnant women with asthma. Clin Nutr 2011. 19. Murphy VE, Clifton VL, Gibson PG. The effect of cigarette smoking on asthma control during exacerbations in pregnant women. Thorax 2010;65:739e44. 20. Gibson PG, Wilson AJ. The use of continuous quality improvement methods to implement practice guidelines in asthma. J Qual Clin Pract 1996;16:87e102. 21. National Heart LaBI. New NHLBI guidelines for the diagnosis and management of asthma. Lippincott Health Promot Lett 1997;2:8e9. 22. Murphy VE, Gibson PG, Talbot PI, Kessell CG, Clifton VL. Asthma selfmanagement skills and the use of asthma education during pregnancy. Eur Respir J 2005;26:435e41. 23. Barnes PJ, Pedersen S. Efficacy and safety of inhaled corticosteroids in asthma. Report of a workshop held in Eze, France. Am Rev Respir Dis October 1992;1993(148):S1e26. 24. Block G. A review of validations of dietary assessment methods. Am J Epidemiol 1988;115:492e505. 25. Lepage G, Roy CC. Direct transesterification of all classes of lipids in a one-step reaction. J Lipid Res 1986;27:114e20. 26. Wood LG, Fitzgerald DA, Gibson PG, Cooper DM, Garg ML. Increased plasma fatty acid concentration following respiratory exacerbations in cystic fibrosis is associated with elevated oxidative stress. Am J Clin Nutr 2002;75:668e75.
P. McLernon et al. / e-SPEN Journal 7 (2012) e78ee85 27. Hure A, Young A, Smith R, Collins C. Diet and pregnancy status in Australian women. Public Health Nutr 2009;12:853e61. 28. Council NHaMR. Nutrient reference values for Australia and New Zealand. Canberra: Commonwealth of Australia; 2006. 29. Prasad M, Lumia M, Erkkola M, Tapanainen H, Kronberg-Kippila C, Tuokkola J, et al. Diet composition of pregnant Finnish women: changes over time and across seasons. Public Health Nutr 2010;13:939e46. 30. 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. 31. Herrera E, Lopez-Soldado I, Limones M, Amusquivar E, Ramos MP. Lipid metabolism during the perinatal phase, and its implications on postnatal development. Int J Vitam Nutr Res 2006;76:216e24. 32. Tomlinson JW, Sherlock M, Hughes B, Hughes SV, Kilvington F, Bartlett W, et al. Inhibition of 11beta-hydroxysteroid dehydrogenase type 1 activity in vivo limits glucocorticoid exposure to human adipose tissue and decreases lipolysis. J Clin Endocrinol Metab 2007;92:857e64. 33. Hodyl NA, Stark MJ, Osei-Kumah A, Bowman M, Gibson P, Clifton VL. Fetal glucocorticoid-regulated pathways are not affected by inhaled corticosteroid use for asthma during pregnancy. Am J Respir Crit Care Med 2011;183:716e22. 34. Wood LG, Garg ML, Simpson JL, Mori TA, Croft KD, Wark PA, et al. Induced sputum 8-isoprostane concentrations in inflammatory airway diseases. Am J Respir Crit Care Med 2005;171:426e30. 35. Horton JD, Goldstein JL, Brown MS. SREBPs: activators of the complete program of cholesterol and fatty acid synthesis in the liver. J Clin Invest 2002;109:1125e31. 36. Li J, Thorne LN, Punjabi NM, Sun CK, Schwartz AR, Smith PL, et al. Intermittent hypoxia induces hyperlipidemia in lean mice. Circ Res 2005;97:698e706.
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37. Drager LF, Li J, Shin MK, Reinke C, Aggarwal NR, Jun JC, et al. Intermittent hypoxia inhibits clearance of triglyceride-rich lipoproteins and inactivates adipose lipoprotein lipase in a mouse model of sleep apnoea. Eur Heart J 2011. 38. Szajewska H, Horvath A, Koletzko B. Effect of n-3 long-chain polyunsaturated fatty acid supplementation of women with low-risk pregnancies on pregnancy outcomes and growth measures at birth: a meta-analysis of randomized controlled trials. Am J Clin Nutr 2006;83:1337e44. 39. Smuts CM, Huang M, Mundy D, Plasse T, Major S, Carlson SE. A randomized trial of docosahexaenoic acid supplementation during the third trimester of pregnancy. Obstet Gynecol 2003;101:469e79. 40. Oken E, Kleinman KP, Olsen SF, Rich-Edwards JW, Gillman MW. Associations of seafood and elongated n-3 fatty acid intake with fetal growth and length of gestation: results from a US pregnancy cohort. Am J Epidemiol 2004;160:774e83. 41. Olsen SF, Secher NJ. Low consumption of seafood in early pregnancy as a risk factor for preterm delivery: prospective cohort study. Br Med J 2002;324:447. 42. Saedisomeolia A, Wood LG, Garg ML, Gibson PG, Wark PA. Supplementation of long chain n-3 polyunsaturated fatty acids increases the utilization of lycopene in cultured airway epithelial cells. J Food Lipids 2008;15:421e32. 43. Meydani M, Natiello F, Goldin B, Free N, Woods M, Schaefer E, et al. Effect of long-term fish oil supplementation on vitamin E status and lipid peroxidation in women. J Nutr 1991;121:484e91. 44. Schatz M, Leibman C. Inhaled corticosteroid use and outcomes in pregnancy. Ann Allergy Asthma Immunol 2005;95:234e8. 45. Clifton VL. Review: sex and the human placenta: mediating differential strategies of fetal growth and survival. Placenta 2010;31:S33e9.