SHORT COMMUNICATION
Dietary Nucleotide and Nucleoside Exposure in Infancy and Atopic Dermatitis, Recurrent Wheeze, and Allergic Sensitization Marijke J.C. Timmermans, Pieter C. Dagnelie, Esther H.E. Theunisz, Doreen Ewalds, Carel Thijs, Monique Mommers, and Ilja C.W. Arts ABSTRACT We hypothesized that early life exposure to nucleotides and nucleosides lowers the risk of recurrent wheeze, atopic dermatitis, and allergic sensitization among n ¼ 429 children. Concentrations in breast milk were established by high-performance liquid chromatography; concentrations in formula milks were obtained from manufacturers. Questionnaires and home visits were used to assess outcomes. Adjusted odds ratios in the highest tertile compared with those in the lowest tertile of exposure ranged from 1.11 to 1.99 in predominantly formula-fed children, and from 0.40 to 0.53 in predominantly breast-fed children, but were not significant. Thus, we found no evidence for association between nucleotide and nucleoside exposure and the development of atopic outcomes in children up to 2 years. Key Words: allergy, breast milk, formula milk, infants, nucleotides
(JPGN 2015;60: 691–693)
A
topic diseases are common in childhood. They significantly worsen the quality of life of the child affected, and have a large impact on the child’s physical, social, and emotional health (1). Dietary nucleotides are believed to play an important role in the modulation of the immune system and may therefore help to prevent atopic diseases (2–4). We therefore set out to investigate the association between nucleotides and nucleosides in breast and formula milk and the incidence of atopic diseases in infants. Research on the effects of nucleotides in infants was so far limited to intervention studies comparing supplemented formula milk with
unsupplemented formula milk. Studies including the natural variation in breast milk nucleotides are lacking. The KOALA Birth Cohort Study, initiated between 2000 and 2003, provides a unique opportunity to prospectively study nucleotide exposure from both breast milk and formula milk at a time when some but not all of the formula milks were supplemented with nucleotides. The objective of our study was to investigate whether a high intake of nucleotides and nucleosides reduces the risk of recurrent wheeze, atopic dermatitis, and allergic sensitization in children up to 2 years of age.
METHODS The study design of the KOALA Birth Cohort Study has been described in detail previously (5). Briefly, this cohort consists of n ¼ 2834 pregnant women, enrolled between October 2000 and September 2003 at 34 weeks of gestation. Mothers filled out weekly diaries providing detailed information on feeding type and frequency of feedings during the first 3 months after birth. No information about milliliters per day was registered. From October 2002 onward, breast milk was collected from 315 breast-feeding mothers at 1 month postpartum, as described previously in detail (6). Information on determinants and atopic outcomes was collected by questionnaires at 34 weeks of gestation, and at 3, 7, 12, and 24 months postpartum. Home visits were made at 1 and 2 years after birth. For the present study, we included children with complete data on feeding practice for the first 4 weeks of life and a breast milk sample if the child was breast-fed.
Main Determinant Received May 3, 2014; accepted December 20, 2014. From the Department of Epidemiology, Maastricht University, Maastricht, The Netherlands. Address correspondence and reprint requests to Ilja C.W. Arts, PhD, Department of Epidemiology, Maastricht University, PO Box 616, 6200 MD Maastricht, The Netherlands (e-mail: ilja.arts@maastrichtuni versity.nl). Collection of breast milk was supported by a grant from the Netherlands Organisation for Health Research and Development (NWO) program of Innovative Prevention Research (ZONmw Prevention program 1 (grant 210-00-090). Follow-up until age 2 years was cofinanced by the Netherlands Asthma Foundation (grant no. 3.2.03.48). Other parts of the baseline data collection of the KOALA study were financially supported by Royal Friesland Foods (Leeuwarden, The Netherlands), Triodos Foundation (Zeist, The Netherlands), Phoenix Foundation, Raphae¨l Foundation, Iona Foundation, and the Foundation for the Advancement of Heilpedagogie (all in The Netherlands). The authors report no conflicts of interest. Copyright # 2015 by European Society for Pediatric Gastroenterology, Hepatology, and Nutrition and North American Society for Pediatric Gastroenterology, Hepatology, and Nutrition DOI: 10.1097/MPG.0000000000000689
JPGN
Volume 60, Number 5, May 2015
Concentrations of the nucleotides adenosine 50 -monophosphate, cytidine 50 -monophosphate, guanosine 50 -monophosphate, inosine 50 -monophosphate, and uridine 50 -monophosphate, and the corresponding nucleosides adenosine, cytidine, guanosine, inosine, and uridine in breast milk and formula milk were considered. The type and brand of formula milk were ascertained by questionnaire at 1 month of age. For each brand of formula milk, we obtained the concentrations of nucleotides and nucleosides at the time of consumption by contacting the manufacturers. The content of nucleotides and nucleosides in breast milk was assessed by highperformance liquid chromatography (HPLC) (7). We calculated for each child the weighted average concentration of nucleotide and nucleoside exposure, based on the type of feeding the child received during the first month of life. When children used only breast milk during the first month, the measured concentration in the breast milk sample was taken as the exposure. In case children were only formula-fed, the concentration in the specific brand consumed was taken as the exposure. For children fed both breast and formula milk, or a combination of several brands
691
Copyright 2015 by ESPGHAN and NASPGHAN. Unauthorized reproduction of this article is prohibited.
Timmermans et al
JPGN
of formula milk, the weighted average concentration was calculated using information from the weekly diaries. For example, when a child used breast milk (89.3 mmol/L) for 3 weeks and formula milk brand X (98.6 mmol/L) for 1 week, exposure to nucleotides was calculated as (0.75 89.3 mmol/L) þ (0.25 98.6 mmol/L) ¼ 91.6 mmol/L. Children who were breast-fed for 50% of the first 4 weeks after birth were considered predominantly breast-fed; otherwise, they were considered predominantly formula-fed.
Outcome Measures Our outcome measures have been described in detail previously (8). Briefly, wheezing and eczema were assessed through parent report using the questions from the validated International Study of Asthma and Allergies in Childhood at 7, 12, and 24 months postpartum (9). Recurrent wheeze was defined as the presence of wheezing with at least 4 attacks between the age 0 and 7 months, between the age 7 and 12 months, or between the age 13 and 24 months. Eczema was defined as ever having had an itchy rash that was coming and going for at least 6 months. Infants with only diaper rash, rash around the eyes, or scalp scaling were excluded. Atopic dermatitis was observed by a trained research nurse during home visits at the age 2 years, using the UK working party criteria for atopic dermatitis. Infants with a UK working party atopic dermatitis probability score >0.9 were considered to be having atopic dermatitis. For the determination of allergic sensitization, specific immunoglobulin E (IgE) concentrations were measured in capillary blood collected during home visits at 1 and 2 years postpartum. Infants were classified sensitized if specific serum IgE concentrations were >0.3 IU/mL against at least 1 of the food or inhalant allergens tested (8).
Statistical Analysis Univariable and multivariable logistic regression was performed with adjustment for the potential confounders sex, recruitment group, maternal education, parental history of atopic disease,
Volume 60, Number 5, May 2015
number of siblings with atopic history, breast-feeding duration, mode and place of delivery, birth weight, maternal age at delivery, smoking during pregnancy, postnatal environmental tobacco smoke, and presence of pets. Covariates were included in the final model only if they modified the regression coefficient of nucleotide exposure by >10%. All of the analyses were adjusted for the confounder ‘‘batch’’ (10), to eliminate laboratory drift between the 2 years in which the HPLC analyses of the breast milk samples were conducted. In additional analyses, we adjusted for immunomodulating factors in breast milk, that is, cytokines (transforming growth factor-b1, interleukin 12), immunoglobulin A, and CD14. Furthermore, we investigated the confounding effect of breast milk fatty acids, that is, the sum of the main n-3 long-chain polyunsaturated fatty acids (eicosapentaenoic acid, docosapentaenoic acid, docosahexaenoic acid) and the sum of ruminant fatty acids (vaccenic and rumenic acids) (11). We also performed additional analyses for nucleotides and nucleosides separately.
Ethical Considerations The KOALA Birth Cohort Study was approved by the medical ethics committee of Maastricht University, and all of the parents gave written informed consent before participation. The present study has been performed in accordance with the ethical standards laid down in the 1964 Declaration of Helsinki and its later amendments.
RESULTS Of the 429 children included in the present study, 286 (67%) were classified as predominantly breast-fed during the first month. The total nucleotide and nucleoside concentration in the breast milk samples varied from 22.2 to 259.7 mmol/L (mean 89.3 42.4 mmol/L). Nucleotides made up >70% of the total nucleotides and nucleosides (65.3 31.7 mmol/L), with cytidine 50 -monophosphate as the main constituent (43.6 29.4 mmol/L). For predominantly breast-fed children, the risk for all of the outcomes tended to be lower in the higher tertiles of nucleotide
TABLE 1. Association between nucleotide and nucleoside exposure during the first month and atopic outcomes in children up to 2 years of age who were predominantly breast-fed during the first month Category of nucleotide and nucleoside concentration , mmol/L, in predominantly breast-fed children
Atopic outcome Recurrent wheeze n/N cORy (95% CI) aORz (95% CI) Eczema n/N cORy (95% CI) aORz (95% CI) Atopic dermatitis n/N cORy (95% CI) aORz (95% CI) Atopic sensitization n/N cORy (95% CI) aORz (95% CI)
Low (0–67.4)
Middle (67.4–100.9)
High (101.0–260.00)
Ptrend
7/91 1.00 1.00
7/88 1.23 (0.40–3.76) 1.13 (0.33–3.85)
2/88 0.53 (0.10–2.78) 0.40 (0.06–2.53)
0.499 0.370
36/94 1.00 1.00
26/95 0.62 (0.33–1.14) 0.60 (0.31–1.15)
23/92 0.56 (0.29–1.08) 0.53 (0.26–1.08)
0.100 0.091
10/63 1.00 1.00
10/72 0.82 (0.32–2.14) 0.72 (0.27–1.96)
7/60 0.60 (0.20–1.84) 0.44 (0.13–1.47)
0.375 0.183
19/63 1.00 1.00
16/70 0.67 (0.31–1.47) 0.61 (0.27–1.38)
12/59 0.53 (0.22–1.35) 0.47 (0.18–1.24)
0.199 0.131
aOR ¼ adjusted odds ratio; CI ¼ confidence interval; cOR ¼ crude odds ratio; n/N ¼ number of patients/sample size. Nucleotides: adenosine 50 -monophosphate, cytidine 50 -monophosphate, guanosine 50 -monophosphate, inosine 50 -monophosphate, and uridine 50 -monophosphate. Nucleosides: adenosine, cytidine, guanosine, inosine, and uridine. y Adjusted for the confounder batch. z Adjusted for the confounders: batch, duration of breast-feeding, parental history of atopic diseases, and number of siblings with atopic disease.
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JPGN
Volume 60, Number 5, May 2015
Nucleotide Exposure in Infancy and Atopy
and nucleoside exposure, although none of the odds ratios (ORs) were significantly different from 1 (Table 1). The remaining 143 children (33%) were predominantly formula-fed. Of the 13 different brands of formula milk used, 3 brands were supplemented with nucleotides, all at a concentration of 98.6 mmol/L. These were used by 52 (36%) of the 143 formulafed children, whereas 91 (62%) used nonsupplemented formula milk. In contrast to the results for breast-fed children, the predominantly formula-fed group tended to have a higher risk of atopic outcomes with a higher nucleotide and nucleoside exposure, although again not statistically significant (Table 2). For allergic sensitization, the adjusted OR in the third tertile was approximately 2.0 (adjusted OR 1.99, 95% confidence interval 0.74–5.38, P for trend 0.17). Additional adjustment for immunomodulating factors and fatty acids in breast milk did not materially alter the results. Finally, in the predominantly breast-fed group, the tendency toward inverse associations was found for both nucleotides and nucleosides when considered separately (data not shown).
DISCUSSION Nucleotide and nucleoside exposure during the first month of life was not clearly associated with the development of atopic outcomes in children up to 2 years of age. A previous meta-analysis included 9 randomized controlled trials studying the effects of nucleotide-supplemented formula milk on the immune response. Supplemented formula milk was significantly associated with a better antibody response to immunization and fewer episodes of
TABLE 2. Association between nucleotide and nucleoside exposure during the first month and atopic manifestations in children up to 2 years of age who were predominantly formula fed during the first month
REFERENCES
Category of nucleotide and nucleoside concentration , mmol/L, in predominantly formula-fed children Atopic outcome Recurrent wheeze n/N cORy (95% CI) aORz (95% CI) Eczema n/N cORy (95% CI) aORz (95% CI) Atopic dermatitis n/N cORy (95% CI) aORz (95% CI) Atopic sensitisation n/N cORy (95% CI) aORz (95% CI)
Low (0.00)
High (0.01–99.0)
Ptrend
15/91 1.00 1.00
5/42 0.69 (0.23–2.03) 1.11 (0.33–3.70)
0.494 0.864
27/92 1.00 1.00
17/46 1.41 (0.67–2983) 1.81 (0.81–4.07)
0.367 0.150
6/64 1.00 1.00
4/35 1.25 (0.33–4.76) 1.69 (0.38–7.43)
0.746 0.489
12/60 1.00 1.00
11/34 1.91 (0.73–4.98) 1.99 (0.74–5.38)
0.184 0.173
aOR ¼ adjusted odds ratio; CI ¼ confidence interval; cOR ¼ crude odds ratio; n/N ¼ number of patients/sample size. Nucleotides: adenosine 50 -monophosphate, cytidine 50 -monophosphate, guanosine 50 -monophosphate, inosine 50 -monophosphate, and uridine 50 -monophosphate. Nucleosides: adenosine, cytidine, guanosine, inosine, and uridine. y Unadjusted. z Adjusted for the confounders: parental history of atopic diseases and number of siblings with atopic disease.
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diarrhea compared with formula without nucleotides (4). We did not find a significant reduction in the risk of atopic outcomes. It is, however, difficult to compare our findings with the studies in this meta-analysis because different outcome measures were used and the study designs were also different. Nucleotide concentrations of supplemented formula milks in clinical trials ranged from 12 to 104 mg/L, and formulae with approximately 72 mg/L nucleotides showed greater beneficial effects on endpoints in approximately half of the studies (4). The exposure in our study (32–82 mg/L in the highest tertile of the breast-fed group) was therefore relatively low, which may explain the lack of effect. Nucleotide and nucleoside exposure in the predominantly breast-fed children showed a tendency toward a lower risk for all of the atopic outcomes, whereas in predominantly formula-fed children the opposite was true. An important difference between the breast-fed and formula-fed groups is that breast milk contains nucleosides (ranging from 0 to 142.7 mmol/L in our study), whereas formula milk does not. When we conducted separate analyses for exposure to nucleotides or nucleosides, however, the tendency toward inverse associations was found for both. This suggests that other factors associated with feeding practice may actually explain the observed tendencies, although additional adjustments for cytokines, fatty acids, and IgE did not change the results. We are the first to study a range of atopic outcomes in a population with a broad range in exposure to both breast and formula milk. Although we had no information about the exact volume of milk intake, we had detailed information on type and frequency of feeding, which kept misclassification bias to a minimum. Because the present study was initiated approximately 10 years after the actual exposure had taken place, no formula milk samples were available anymore and we could perform only HPLC analysis on the breast milk samples. For this reason, we were not able to validate the concentrations of nucleotides and nucleosides in the formula milks.
1. O’Connell EJ. The burden of atopy and asthma in children. Allergy 2004;59:7–11. 2. Niers L, Stasse-Wolthuis M, Rombouts FM, et al. Nutritional support for the infant’s immune system. Nutr Rev 2007;65:347–60. 3. Gil A. Modulation of the immune response mediated by dietary nucleotides. Eur J Clin Nutr 2002;56 (S3):S1–4. 4. Gutie´rrez-Castrello´n P, Mora-Magan˜a I, Dı´az-Garcı´a L, et al. Immune response to nucleotide-supplemented infant formulae: systematic review and meta-analysis. Br J Nutr 2007;98 (S1):S64–7. 5. Kummeling I, Thijs C, Penders J, et al. Etiology of atopy in infancy: the KOALA Birth Cohort Study. Pediatr Allergy Immunol 2005;16:679–84. 6. Snijders BE, Damoiseaux JG, Penders J, et al. Cytokines and soluble CD14 in breast milk in relation with atopic manifestations in mother and infant (KOALA Study). Clin Exp Allergy 2006;36:1609–15. 7. Coolen EK, Arts IC, Swennen EL, et al. Simultaneous determination of adenosine triphosphate and its metabolites in human whole blood by RP-HPLC and UV-detection. J Chromatogr B Analyt Technol Biomed Life Sci 2008;864:43–51. 8. Snijders BE, Thijs C, Dagnelie PC, et al. Breast-feeding duration and infants atopic manifestations, by maternal allergic status, in the first 2 years of life (KOALA study). J Pediatr 2007;151:347–51. 9. Asher MI, Keil U, Anderson HR, et al. International Study of Asthma and Allergies in Childhood (ISAAC): rationale and methods. Eur Respir J 1995;8:483–91. 10. Tworoger SS, Hankinson SE. Use of biomarkers in epidemiologic studies: minimizing the influence of measurement error in the study design and analysis. Cancer Causes Control 2006;17:889–99. 11. Thijs C, Mu¨ller A, Rist L, et al. Fatty acids in breast milk and development of atopic eczema and allergic sensitization in infancy. Allergy 2011;66:58–67.
693
Copyright 2015 by ESPGHAN and NASPGHAN. Unauthorized reproduction of this article is prohibited.
Dietary Nucleotide and Nucleoside Exposure in Infancy and Atopic Dermatitis, Recurrent Wheeze, and Allergic Sensitization Marijke J.C. Timmermans, Pieter C. Dagnelie, Esther H.E. Theunisz, Doreen Ewalds, Carel Thijs, Monique Mommers, and Ilja C.W. Arts ABSTRACT We hypothesized that early life exposure to nucleotides and nucleosides lowers the risk of recurrent wheeze, atopic dermatitis, and allergic sensitization among n ¼ 429 children. Concentrations in breast milk were established by high-performance liquid chromatography; concentrations in formula milks were obtained from manufacturers. Questionnaires and home visits were used to assess outcomes. Adjusted odds ratios in the highest tertile compared with those in the lowest tertile of exposure ranged from 1.11 to 1.99 in predominantly formula-fed children, and from 0.40 to 0.53 in predominantly breast-fed children, but were not significant. Thus, we found no evidence for association between nucleotide and nucleoside exposure and the development of atopic outcomes in children up to 2 years. Key Words: allergy, breast milk, formula milk, infants, nucleotides
(JPGN 2015;60: 691–693)
A
topic diseases are common in childhood. They significantly worsen the quality of life of the child affected, and have a large impact on the child’s physical, social, and emotional health (1). Dietary nucleotides are believed to play an important role in the modulation of the immune system and may therefore help to prevent atopic diseases (2–4). We therefore set out to investigate the association between nucleotides and nucleosides in breast and formula milk and the incidence of atopic diseases in infants. Research on the effects of nucleotides in infants was so far limited to intervention studies comparing supplemented formula milk with
unsupplemented formula milk. Studies including the natural variation in breast milk nucleotides are lacking. The KOALA Birth Cohort Study, initiated between 2000 and 2003, provides a unique opportunity to prospectively study nucleotide exposure from both breast milk and formula milk at a time when some but not all of the formula milks were supplemented with nucleotides. The objective of our study was to investigate whether a high intake of nucleotides and nucleosides reduces the risk of recurrent wheeze, atopic dermatitis, and allergic sensitization in children up to 2 years of age.
METHODS The study design of the KOALA Birth Cohort Study has been described in detail previously (5). Briefly, this cohort consists of n ¼ 2834 pregnant women, enrolled between October 2000 and September 2003 at 34 weeks of gestation. Mothers filled out weekly diaries providing detailed information on feeding type and frequency of feedings during the first 3 months after birth. No information about milliliters per day was registered. From October 2002 onward, breast milk was collected from 315 breast-feeding mothers at 1 month postpartum, as described previously in detail (6). Information on determinants and atopic outcomes was collected by questionnaires at 34 weeks of gestation, and at 3, 7, 12, and 24 months postpartum. Home visits were made at 1 and 2 years after birth. For the present study, we included children with complete data on feeding practice for the first 4 weeks of life and a breast milk sample if the child was breast-fed.
Main Determinant Received May 3, 2014; accepted December 20, 2014. From the Department of Epidemiology, Maastricht University, Maastricht, The Netherlands. Address correspondence and reprint requests to Ilja C.W. Arts, PhD, Department of Epidemiology, Maastricht University, PO Box 616, 6200 MD Maastricht, The Netherlands (e-mail: ilja.arts@maastrichtuni versity.nl). Collection of breast milk was supported by a grant from the Netherlands Organisation for Health Research and Development (NWO) program of Innovative Prevention Research (ZONmw Prevention program 1 (grant 210-00-090). Follow-up until age 2 years was cofinanced by the Netherlands Asthma Foundation (grant no. 3.2.03.48). Other parts of the baseline data collection of the KOALA study were financially supported by Royal Friesland Foods (Leeuwarden, The Netherlands), Triodos Foundation (Zeist, The Netherlands), Phoenix Foundation, Raphae¨l Foundation, Iona Foundation, and the Foundation for the Advancement of Heilpedagogie (all in The Netherlands). The authors report no conflicts of interest. Copyright # 2015 by European Society for Pediatric Gastroenterology, Hepatology, and Nutrition and North American Society for Pediatric Gastroenterology, Hepatology, and Nutrition DOI: 10.1097/MPG.0000000000000689
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Volume 60, Number 5, May 2015
Concentrations of the nucleotides adenosine 50 -monophosphate, cytidine 50 -monophosphate, guanosine 50 -monophosphate, inosine 50 -monophosphate, and uridine 50 -monophosphate, and the corresponding nucleosides adenosine, cytidine, guanosine, inosine, and uridine in breast milk and formula milk were considered. The type and brand of formula milk were ascertained by questionnaire at 1 month of age. For each brand of formula milk, we obtained the concentrations of nucleotides and nucleosides at the time of consumption by contacting the manufacturers. The content of nucleotides and nucleosides in breast milk was assessed by highperformance liquid chromatography (HPLC) (7). We calculated for each child the weighted average concentration of nucleotide and nucleoside exposure, based on the type of feeding the child received during the first month of life. When children used only breast milk during the first month, the measured concentration in the breast milk sample was taken as the exposure. In case children were only formula-fed, the concentration in the specific brand consumed was taken as the exposure. For children fed both breast and formula milk, or a combination of several brands
691
Copyright 2015 by ESPGHAN and NASPGHAN. Unauthorized reproduction of this article is prohibited.
Timmermans et al
JPGN
of formula milk, the weighted average concentration was calculated using information from the weekly diaries. For example, when a child used breast milk (89.3 mmol/L) for 3 weeks and formula milk brand X (98.6 mmol/L) for 1 week, exposure to nucleotides was calculated as (0.75 89.3 mmol/L) þ (0.25 98.6 mmol/L) ¼ 91.6 mmol/L. Children who were breast-fed for 50% of the first 4 weeks after birth were considered predominantly breast-fed; otherwise, they were considered predominantly formula-fed.
Outcome Measures Our outcome measures have been described in detail previously (8). Briefly, wheezing and eczema were assessed through parent report using the questions from the validated International Study of Asthma and Allergies in Childhood at 7, 12, and 24 months postpartum (9). Recurrent wheeze was defined as the presence of wheezing with at least 4 attacks between the age 0 and 7 months, between the age 7 and 12 months, or between the age 13 and 24 months. Eczema was defined as ever having had an itchy rash that was coming and going for at least 6 months. Infants with only diaper rash, rash around the eyes, or scalp scaling were excluded. Atopic dermatitis was observed by a trained research nurse during home visits at the age 2 years, using the UK working party criteria for atopic dermatitis. Infants with a UK working party atopic dermatitis probability score >0.9 were considered to be having atopic dermatitis. For the determination of allergic sensitization, specific immunoglobulin E (IgE) concentrations were measured in capillary blood collected during home visits at 1 and 2 years postpartum. Infants were classified sensitized if specific serum IgE concentrations were >0.3 IU/mL against at least 1 of the food or inhalant allergens tested (8).
Statistical Analysis Univariable and multivariable logistic regression was performed with adjustment for the potential confounders sex, recruitment group, maternal education, parental history of atopic disease,
Volume 60, Number 5, May 2015
number of siblings with atopic history, breast-feeding duration, mode and place of delivery, birth weight, maternal age at delivery, smoking during pregnancy, postnatal environmental tobacco smoke, and presence of pets. Covariates were included in the final model only if they modified the regression coefficient of nucleotide exposure by >10%. All of the analyses were adjusted for the confounder ‘‘batch’’ (10), to eliminate laboratory drift between the 2 years in which the HPLC analyses of the breast milk samples were conducted. In additional analyses, we adjusted for immunomodulating factors in breast milk, that is, cytokines (transforming growth factor-b1, interleukin 12), immunoglobulin A, and CD14. Furthermore, we investigated the confounding effect of breast milk fatty acids, that is, the sum of the main n-3 long-chain polyunsaturated fatty acids (eicosapentaenoic acid, docosapentaenoic acid, docosahexaenoic acid) and the sum of ruminant fatty acids (vaccenic and rumenic acids) (11). We also performed additional analyses for nucleotides and nucleosides separately.
Ethical Considerations The KOALA Birth Cohort Study was approved by the medical ethics committee of Maastricht University, and all of the parents gave written informed consent before participation. The present study has been performed in accordance with the ethical standards laid down in the 1964 Declaration of Helsinki and its later amendments.
RESULTS Of the 429 children included in the present study, 286 (67%) were classified as predominantly breast-fed during the first month. The total nucleotide and nucleoside concentration in the breast milk samples varied from 22.2 to 259.7 mmol/L (mean 89.3 42.4 mmol/L). Nucleotides made up >70% of the total nucleotides and nucleosides (65.3 31.7 mmol/L), with cytidine 50 -monophosphate as the main constituent (43.6 29.4 mmol/L). For predominantly breast-fed children, the risk for all of the outcomes tended to be lower in the higher tertiles of nucleotide
TABLE 1. Association between nucleotide and nucleoside exposure during the first month and atopic outcomes in children up to 2 years of age who were predominantly breast-fed during the first month Category of nucleotide and nucleoside concentration , mmol/L, in predominantly breast-fed children
Atopic outcome Recurrent wheeze n/N cORy (95% CI) aORz (95% CI) Eczema n/N cORy (95% CI) aORz (95% CI) Atopic dermatitis n/N cORy (95% CI) aORz (95% CI) Atopic sensitization n/N cORy (95% CI) aORz (95% CI)
Low (0–67.4)
Middle (67.4–100.9)
High (101.0–260.00)
Ptrend
7/91 1.00 1.00
7/88 1.23 (0.40–3.76) 1.13 (0.33–3.85)
2/88 0.53 (0.10–2.78) 0.40 (0.06–2.53)
0.499 0.370
36/94 1.00 1.00
26/95 0.62 (0.33–1.14) 0.60 (0.31–1.15)
23/92 0.56 (0.29–1.08) 0.53 (0.26–1.08)
0.100 0.091
10/63 1.00 1.00
10/72 0.82 (0.32–2.14) 0.72 (0.27–1.96)
7/60 0.60 (0.20–1.84) 0.44 (0.13–1.47)
0.375 0.183
19/63 1.00 1.00
16/70 0.67 (0.31–1.47) 0.61 (0.27–1.38)
12/59 0.53 (0.22–1.35) 0.47 (0.18–1.24)
0.199 0.131
aOR ¼ adjusted odds ratio; CI ¼ confidence interval; cOR ¼ crude odds ratio; n/N ¼ number of patients/sample size. Nucleotides: adenosine 50 -monophosphate, cytidine 50 -monophosphate, guanosine 50 -monophosphate, inosine 50 -monophosphate, and uridine 50 -monophosphate. Nucleosides: adenosine, cytidine, guanosine, inosine, and uridine. y Adjusted for the confounder batch. z Adjusted for the confounders: batch, duration of breast-feeding, parental history of atopic diseases, and number of siblings with atopic disease.
692
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JPGN
Volume 60, Number 5, May 2015
Nucleotide Exposure in Infancy and Atopy
and nucleoside exposure, although none of the odds ratios (ORs) were significantly different from 1 (Table 1). The remaining 143 children (33%) were predominantly formula-fed. Of the 13 different brands of formula milk used, 3 brands were supplemented with nucleotides, all at a concentration of 98.6 mmol/L. These were used by 52 (36%) of the 143 formulafed children, whereas 91 (62%) used nonsupplemented formula milk. In contrast to the results for breast-fed children, the predominantly formula-fed group tended to have a higher risk of atopic outcomes with a higher nucleotide and nucleoside exposure, although again not statistically significant (Table 2). For allergic sensitization, the adjusted OR in the third tertile was approximately 2.0 (adjusted OR 1.99, 95% confidence interval 0.74–5.38, P for trend 0.17). Additional adjustment for immunomodulating factors and fatty acids in breast milk did not materially alter the results. Finally, in the predominantly breast-fed group, the tendency toward inverse associations was found for both nucleotides and nucleosides when considered separately (data not shown).
DISCUSSION Nucleotide and nucleoside exposure during the first month of life was not clearly associated with the development of atopic outcomes in children up to 2 years of age. A previous meta-analysis included 9 randomized controlled trials studying the effects of nucleotide-supplemented formula milk on the immune response. Supplemented formula milk was significantly associated with a better antibody response to immunization and fewer episodes of
TABLE 2. Association between nucleotide and nucleoside exposure during the first month and atopic manifestations in children up to 2 years of age who were predominantly formula fed during the first month
REFERENCES
Category of nucleotide and nucleoside concentration , mmol/L, in predominantly formula-fed children Atopic outcome Recurrent wheeze n/N cORy (95% CI) aORz (95% CI) Eczema n/N cORy (95% CI) aORz (95% CI) Atopic dermatitis n/N cORy (95% CI) aORz (95% CI) Atopic sensitisation n/N cORy (95% CI) aORz (95% CI)
Low (0.00)
High (0.01–99.0)
Ptrend
15/91 1.00 1.00
5/42 0.69 (0.23–2.03) 1.11 (0.33–3.70)
0.494 0.864
27/92 1.00 1.00
17/46 1.41 (0.67–2983) 1.81 (0.81–4.07)
0.367 0.150
6/64 1.00 1.00
4/35 1.25 (0.33–4.76) 1.69 (0.38–7.43)
0.746 0.489
12/60 1.00 1.00
11/34 1.91 (0.73–4.98) 1.99 (0.74–5.38)
0.184 0.173
aOR ¼ adjusted odds ratio; CI ¼ confidence interval; cOR ¼ crude odds ratio; n/N ¼ number of patients/sample size. Nucleotides: adenosine 50 -monophosphate, cytidine 50 -monophosphate, guanosine 50 -monophosphate, inosine 50 -monophosphate, and uridine 50 -monophosphate. Nucleosides: adenosine, cytidine, guanosine, inosine, and uridine. y Unadjusted. z Adjusted for the confounders: parental history of atopic diseases and number of siblings with atopic disease.
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diarrhea compared with formula without nucleotides (4). We did not find a significant reduction in the risk of atopic outcomes. It is, however, difficult to compare our findings with the studies in this meta-analysis because different outcome measures were used and the study designs were also different. Nucleotide concentrations of supplemented formula milks in clinical trials ranged from 12 to 104 mg/L, and formulae with approximately 72 mg/L nucleotides showed greater beneficial effects on endpoints in approximately half of the studies (4). The exposure in our study (32–82 mg/L in the highest tertile of the breast-fed group) was therefore relatively low, which may explain the lack of effect. Nucleotide and nucleoside exposure in the predominantly breast-fed children showed a tendency toward a lower risk for all of the atopic outcomes, whereas in predominantly formula-fed children the opposite was true. An important difference between the breast-fed and formula-fed groups is that breast milk contains nucleosides (ranging from 0 to 142.7 mmol/L in our study), whereas formula milk does not. When we conducted separate analyses for exposure to nucleotides or nucleosides, however, the tendency toward inverse associations was found for both. This suggests that other factors associated with feeding practice may actually explain the observed tendencies, although additional adjustments for cytokines, fatty acids, and IgE did not change the results. We are the first to study a range of atopic outcomes in a population with a broad range in exposure to both breast and formula milk. Although we had no information about the exact volume of milk intake, we had detailed information on type and frequency of feeding, which kept misclassification bias to a minimum. Because the present study was initiated approximately 10 years after the actual exposure had taken place, no formula milk samples were available anymore and we could perform only HPLC analysis on the breast milk samples. For this reason, we were not able to validate the concentrations of nucleotides and nucleosides in the formula milks.
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