Children with Cow's Milk Protein Allergy (CMPA), treated with a milk-free diet, may be at risk of deficiency of nutrients important for appropriate development. The aim of the study was to evaluate the diets and the nutritional status of children with CMPA.
The study included 48 children, aged 4-6 years, with CMPA treated with a milk-free diet. The principles of the milk-free diet were defined and the compliance with the diet by children was monitored by a physician and a dietician. The control group consisted of 30 healthy controls, aged 4-6 years, on a traditional diet containing milk and milk products. The energy and nutritional value of the diets was determined on the basis of 3-day dietary records. Children's nutritional status was evaluated based on the normalised Body Mass Index (BMI z-score) and selected biochemical indices. The energy value of the diets of children with CMPA was higher compared to recommendations and significantly higher than the energy value of the diets of healthy controls. The intake of protein in both groups exceeded approximately three times the daily requirement. Children with CMPA consumed significantly more long-chain poly-unsaturated fatty acids, carbohydrates, iron, iodine, copper and vitamins D, E, B1, B6 and PP. Vitamin D deficiency was found in the diets of children from both groups. Calcium deficiency was recorded in the diets of 93.8% of children with CMPA and 90% of the controls. The evaluated biochemical indices in both groups did not different significantly and, except for the concentration of 25(OH) vitamin D in blood serum of children with allergy, were within the reference range. Milk-free diets in children with CMPA were adequate for most nutrients, but the children are at risk of vitamin D insufficiency despite the higher vitamin D content of their diets compared to the group of healthy children. The better balancing of the diets of children with allergy was probably due to continuous medical and nutritional care provided by paediatricians and dieticians.
Cow's milk protein allergy, Milk-free diet, Dietary intake, Nutritional status, Children
Milk and milk products play an important role not only in the diet of non-breastfed infants, but also in the diet of children in subsequent years of life. Those products are a source of complete protein, calcium, phosphorus, vitamins from B group (mainly B2) and contain also fat-soluble vitamins A and D. Milk sugar (lactose) is conducive to colonisation of intestines with beneficial bacterial flora and facilitates the absorption of calcium from the small intestine. The fat in milk consists primarily of short- and medium-chain fatty acids, which makes it easily digestible. However, milk and its products are among the most common food allergens in the population of children. Cow's milk contains more than 25 different proteins and some of them are known to be allergenic. The proteins that are most often responsible for causing allergies include casein, as well as α-lactalbumin and β-lactoglobulin of the whey fraction .
The prevalence of Cow's Milk Protein Allergy (CMPA) among children in the first year of age is approximately 2-3%, and below 1% in children aged above 6 years . The main treatment for CMPA is an elimination diet consisting in temporary or permanent exclusion of milk and its products from the mother's diet, if the child is breastfed, or from the child's diet, if the child is formula-fed. A properly balanced milk-free diet ensures optimal psychophysical and intellectual development of the child, even if it is followed for several months or several years . Nevertheless, it is sometimes difficult to balance such diet, and thus children treated with a milk-free diet may be at risk of nutritional deficiencies [4,5].
According to the nutritional programming theory, a non-balanced diet may, due to both deficiency and surplus of nutrients, permanently change metabolism and course of physiological processes and thus increase predispositions to numerous diseases, such as cardiovascular diseases, osteoporosis, obesity, cancers in later life. Therefore, appropriately early detection of abnormalities in the diet of the child and prompt introduction of proper nutritional intervention are important not only for the current health status of the child, but also for its health in adulthood .
The aim of the study was to evaluate the energy and nutritional value of the diets of children aged 4-6 years treated for allergy to cow's milk protein, and to link their diets intake with selected indices of the nutritional status.
The study covered 48 children aged 4-6 years with CMPA who were treated with a milk-free diet for at least a year and for this reason were under care of both a physician and a dietician from the Gastroenterological Outpatient Clinic of the Institute of Mother and Child in Warsaw. After the diagnosis of CMPA in a child, its parents/caregivers received nutritional counseling from a dietician on the principles of the milk-free diet with the use of the milk substitutes ordered by a physician. Then, the compliance with the milk-free diet was monitored by a physician and a dietician. The diagnosis of cow's milk protein allergy in children was made based on clinical symptoms, evidence of sensitization (food-specific IgE antibodies, skin prick tests) and was confirmed with the open food challenge. The control group consisted of 30 age-matched healthy children on a traditional diet with the share of milk and milk products.
The dietary intake was assessed using children's food diaries recorded according to instructions by parents/carers for three days .
The dietary records were used for estimating the intake of individual products using the Photo Album of Products and Meals  and the energy and nutritional value of the children's diets was calculated using the Dieta 5 software . The energy and nutrients intake were compared to the nutritional standards for Polish children population aged 4-6 years . Furthermore, the dietary records were used for estimating the amount of milk substitutes and milk-free fortified products consumed by children, and the amount of milk and milk products consumed by the control group. The amount of calcium and vitamin D consumed with milk substitutes and milk-free fortified products, as well as with milk and milk products, was calculated using the Dieta 5.0 dietary software.
The nutritional status of the children was assessed using anthropometric data (current body weight and height), the BMI (body weight [kg], divided by height squared [m2]), standardised with respect to WHO growth charts, thus receiving the BMI z-score independent from gender and age [11,12]. The nutritional status was also assessed using selected biochemical indices. Fasting peripheral blood samples (3 ml) were taken in the summer season (from June to September 2015) in the morning hours from all children. Blood in EDTA-containing tubes was analysed immediately for the determination of erythrocytes, Haemoglobin (Hb) and Mean Corpuscular Volume (MCV) using a haematology analyser (Pentra, HORRIBA ABX, and France). In order to obtain serum, the blood was centrifuged at 2500 × g, at 4 ℃ for 10 minutes and stored in small portions at -25 ℃ for subsequent biochemical analysis. Serum iron, ferritin, transferrin as well as calcium, phosphate, magnesium were determined using commercially available kits on a biochemical analyser (COBAS INTEGRA, ROCHE, Switzerland). The 25(OH) vitamin D concentrations in blood serum were determined by chemiluminescence method using DiaSorin (USA) kits.
The study was performed in accordance with the Helsinki Declaration for Human Research and the study protocol was approved by the Ethics Committee of the Institute of Mother and Child in Warsaw, Poland.
All parents of participating children were informed of the study objectives and written consent was obtained for analysis of blood samples before their participation in the study.
The t-student test for normally distributed variables and U Mann-Whitney test for variables with non-normal distribution were used to analyse differences in anthropometric and haematological and biochemical parameters as well as energy and nutritional value of diets between children with CMPA and healthy controls. Data are presented as means and standard deviations for normally distributed variables and medians and interquartile ranges for non-normally distributed variables. An assessment of the normality of data was performed with Shapiro-Wilk test. The Chi2 test was used for analysing differences in percentages of children with nutrients intake below EAR (Estimated Average Requirement) or AI (Adequate Intake). Dependencies between biochemical parameters and dietary intake of selected minerals and vitamins were assessed using the Pearson's r correlation coefficient.
For all types of tests, α = 0.05 was adopted as the statistical significance level. Statistical analyses were performed using the Statistica 12 PL package.
In 41.6% (N = 20/48) of the children with CMPA symptoms of allergy from the gastrointestinal tract were present (loose stools, constipation, stomach pain, abdominal bloating), in 16.6% (N = 8/48) it was atopic dermatitis, in 12.5% (N = 6/48) respiratory tract symptoms, while 29.1% (N = 16/48) of children had symptoms from more than one system.
The characteristics of the both groups of children are presented in (Table 1).
Table 1: Clinical and anthropometric data in children with CMPA and healthy controls. View Table 1
There were no significant differences in terms of age, body weight, height, BMI and BMI z-score between CMPA and healthy children. The value of BMI z-score pointed to appropriate nutritional status of children from both groups (BMI z-score < -1; +1 >).
The energy and nutritional value of the diets of children from both groups in reference to Polish nutritional standards (2012) are presented in (Table 2).
The energy value of the diets of children with CMPA was higher in comparison to recommendations and significantly higher compared to the energy value of the diets of healthy controls. The protein and fat intake did not differ significantly between the two groups. The protein intake was almost three times higher than EAR, while fat intake was close to recommended level. A similar percentage of children from both groups (56.3% vs. 53.7%) did not comply with the standard for fat, while the diets of children with CMPA included significantly more long-chain poly-unsaturated fatty acids (p < 0.05). The diets of children with allergy contained more carbohydrates (p < 0.05) than the traditional diets of healthy ones, and the majority of children from both groups exceeded recommended intake of digestible carbohydrates (97.9% in CMPA group and 96.7% in controls).
Calcium intake in the diets of children from both groups did not differ and was lower than recommended. The percent of children with this nutrient intake below EAR was similar of children on a milk-free and a traditional diet (93.8% vs. 90.0%). Iron content in the diets of children with allergy was significantly higher compared to its content in the diets of healthy children (p < 0.05). In all children with CMPA, the standard for this nutrient was met. Iodine intake was significantly higher in the group of children with allergy (p < 0.05). Also in this group, a lower percentage of children did not achieve the EAR recommended level for this nutrient (27.1% vs. 50.0% in healthy controls; p < 0.05). The diets of children with allergy were better balanced in terms of vitamins content, apart from vitamin D, the intake of which in the diets of all children was deficient. Vitamin D deficiency was significantly higher in the group of children on a traditional diet (p < 0.05).
The diets of 75% (N = 36/48) of children with allergy included milk substitutes (e.g. extensively hydrolysed formulas and soy, oat, almond, rice or other grain beverages and dairy substitute) fortified among others with minerals and vitamins.
From among the children with allergy, 10.4% (N = 5/48) received extensively hydrolysed formulas of whey or casein, 12.5% (N = 6/48)-extensively hydrolysed formulas and fortified milk substitutes and 52.0% (N = 25/48) -only fortified milk substitutes. Due to the lack of organoleptic acceptance, 25.0% (N = 12/48) of children with allergy did not consume such foods at all. The diets of all children from the control group included milk and milk products.
The amounts of extensively hydrolysed formulas and/or fortified milk substitutes consumed by children with CMPA, as well as the amount of milk and milk products consumed by controls are presented in (Table 3).
Table 3: Comparison of amounts of calcium and vitamin D derived from selected dietary sources in diets of children with CMPA and healthy controls. View Table 3
The median and interquartile ranges of the amounts (ml) of extensively hydrolysed formulas and/or fortified milk substitutes consumed by children with CMPA equalled 201.0 ml (88.7-413.2), whereas the amount of milk and milk products consumed by children from the control group totalled 368.1 ml (176.4-600.2).
The median daily intake of calcium derived from extensively hydrolysed formulas and/or fortified milk substitutes in children with CMPA was 154.0 mg (interquartile range 63.3-328.8 mg) versus 400.3 mg (interquartile range 168.2-634.0 mg) derived from milk and dairy products in the diets of healthy controls. The content of vitamin D derived from the median amount of extensively hydrolysed formulas and/or fortified milk substitutes consumed daily by children with CMPA was 1.7 µg (interquartile range 0.9-3.7 µg), whereas the median amount of 368.1 ml of milk and its products consumed daily by healthy children provided only 0.1 µg (interquartile range 0.1-0.3 µg) of vitamin D.
In 50% (N = 24/48) children with CMPA and 10% (N = 3/30) on a traditional diet, supplementation of vitamin D was used. Calcium supplementation was used in only 15% (N = 7/48) of children with allergy.
Biochemical and haematological parameters in both groups of children are presented in (Table 4).
Table 4: Haematological and biochemical parameters in children with CMPA and healthy controls. View Table 4
Haematological and biochemical parameters did not differ significantly in both groups of children and were within the reference range, except for 25(OH) vitamin D concentrations in blood serum in the group of children with CMPA, indicating suboptimal status of this vitamin . The correlations between the content of selected diet components and their concentration in blood serum of children with CMPA are presented in (Table 5).
Table 5: Correlations between dietary intake of selected components and their concentration in blood serum of children with CMPA. View Table 5
In the group of children with CMPA, we found a significant positive correlation between dietary intake of vitamin D and concentration of 25(OH) vitamin D in blood serum (r = 0.53, p < 0.05).
The child's diet, regardless of its type, should provide an adequate amount of energy and building components necessary for appropriate development of the growing body. The study of Mowszet, et al. demonstrated that food allergy was the most common reason for malnutrition in children aged up to 5 years . Similarly, Gębala, et al. in the study on 571 children aged 1-18 years (mean age of 4.5 years), found that 21.7% (N = 124) of these subjects were underweight of whom in 22.6% (N = 28) the identified reduced body weight resulted from food allergy . Meyer, et al. also found, in the study conducted in the UK, that children with allergy were more often underweight than the entire population of children in England, also noting that it was dependent not only on adequacy of dietary intake, but also on the course of allergy . Interesting results were obtained by Tuokkola, et al. in a study on children with CMPA treated with a milk-free diet, where nutritional intake at age 1, 2 and 3 was assessed and anthropometric measurements were made once a year until the age of 5. The authors found that, despite the balanced diets, the growth of these children was slower compared to healthy children on a traditional diet . Similarly, Flammarion, et al. proved that children with allergy (mean age 4.7 ± 2.5 years) were smaller for their age than controls even when they received similar nutrient intakes . Our work seems to confirm these observations, since the energy value of the diets of children with CMPA was higher not only compared to recommendations, but also compared to the diet of children from the control group. Nevertheless, the nutritional status of children from both groups, assessed on the basis of anthropometric parameters, was not only appropriate, but also not significantly different. Paganus, et al. believe that children with CMPA require increased energy intake due to inflammation, i.e. skin or gastrointestinal . In our study, 41.6% of children with CMPA had gastrointestinal symptoms, 16.6% atopic dermatitis, and 12.5% respiratory symptoms, while 29.1% had symptoms from more than one system.
Children with CMPA or multiple allergies are at risk of consuming less than recommended amount of calcium and vitamin D. Vitamin D deficiencies in the diets of children with food allergies (mean age 4.7 years), but also in a control group, were found by Flammarion, et al. . In the work of Christie, et al. covering 98 children with food allergy, with mean age of 3.7 ± 2.3 years, and 99 controls at a similar age, dietary records obtained in 66% and 64%, respectively, showed that more than 25% of children in both groups consumed less than 67% of RDA/RDI (Recommended Dietary Allowances/Recommended Dietary Intake) for calcium and vitamin D. The authors also point out that the nutritional counselling and introduction a safe infant/toddler formula or fortified soy beverages into the diet decrease the risk of insufficient intake of calcium and vitamin D in children with food allergy . Our study proved that consumption of vitamin D in both groups of children did not meet the EAR, but the children with allergy consumed significantly more of this vitamin, which could be due to consumption of fortified foods by 75% of these children. Such foods provided 1.7 µg of vitamin D to those children, while milk and milk products supplied only 0.1 µg of this vitamin in the control group. Therefore, our study, similarly to the study by Sidnell, et al., shows that fortified foods contributed to the better balancing of the children's diets in terms of vitamin D . Canani, et al. also observed a positive impact of supervision of a physician and a dietician on the balancing of the diets of children with food allergies. The authors argue that such supervision also results in a significant improvement of anthropometric and laboratory biomarker of nutritional status of those children . This is further confirmed by the results of the study by Medeiros, et al. . The concentration of 25(OH) vitamin D in blood serum of children with CMPA was 26.6 ng/l and was within the range of suboptimal values, although the study was performed in summer months during which skin synthesis of vitamin D is the most effective. In addition, it should be emphasized that approximately 50% of the children received vitamin D supplements. According to "Practical guidelines for the supplementation of vitamin D and the treatment of its deficits in Central Europe", blood serum concentration of 25(OH) vitamin D within the range of 0-50 nmol/l (0-20 ng/ml) is considered to indicate deficiency of the vitamin, the concentration > 50-75 nmol/l (> 20-30 ng/ml) is considered suboptimal, > 75-125 nmol/l (> 30-50 ng/ml)-optimal, while > 125-250 nmol/l (> 50-100 ng/ml)-high . The concentration of 25(OH) vitamin D in blood serum of children from the control group amounted to 30.5 ng/ml on average and was within optimal range, and vitamin D supplementation was received by only around 10% of the children. The lower concentration of 25(OH) vitamin D in blood serum of children with allergy, combined with a higher content of this vitamin in the diet, could result from its impaired bioavailability due to gastrointestinal inflammation caused by allergy. It is possible that the observed differences in the status of vitamin D in children from both groups could also result from different exposure to sun rays and application or non-application of creams with SPF filters during the exposure, which was not analysed in this work. In the group of children with CMPA, there is also a positive correlation between the vitamin D intake in the diet and the concentration of 25(OH) vitamin D in blood serum. The observed correlation may indicate that the compliant with recommendations vitamin D dietary intake in children with CMPA is important for optimal vitamin D status, although its main source is skin synthesis. The importance of consumption of foods fortified with vitamin D for achieving an appropriate concentration of 25(OH) vitamin D in blood serum of children is also confirmed by the study of Houghton, et al. .
The calcium intake in the diets of children from both groups did not differ significantly and was lower than recommended. In a similar percentage of children on a milk-free and traditional diet (93.8% vs. 90.0%) the intake of calcium was below EAR. The diet of children with CMPA, although insufficient in calcium, is more diversified in terms of dietary sources of this nutrient than the diet of healthy children where dairy products are almost the only source of calcium. It is possible that the lack of differences in calcium concentration, despite the suboptimal vitamin D status in children with CMPA could result from calcium supplementation used in 15% of these children, but also mobilization of calcium stores out from bones. Insufficient supply of calcium and vitamin D, in particular in childhood, may result in disturbances of calcium metabolism and in consequence in development of such diseases as rickets, osteopenia, or osteoporosis which lead to disability [25,26]. Ambroszkiewicz, et al. note that children with CMPA, despite the correct values of basic parameters of calcium and phosphorus balance, have a lower level of sclerostin and increased cytokine ratio RANK/OPG, which may point to disturbances of the balance between bone formation and resorption . The results of the study by Mailhot, et al. of pre-puberty children with persistent CMPA allergy showed that in the group of these children lumbar spine BMD (Bone Mineral Density) z-scores were significantly lower compared to the control group. Low bone mass was detected in 6% of children from this group compared with none in the controls. At the same time, the vitamin D status assessed based on concentration of 25(OH) vitamin D in blood serum of children with allergy was not reduced compared with control subjects, but this group had a lower calcium intake. Furthermore, in approximately 50% of children with allergy, the use of calcium and vitamin D supplements was declared . Numerous authors point to adverse effects of long-term milk-free diet in children with allergy. The studies by Infante, et al. show that approximately 50% of children (2-14 years, mean age 7 years) treated with a milk-free diet had osteopenia or osteoporosis . Hidvegi, et al. found low bone density in approximately 30% of children with CMPA . Black, et al. also note that in growing children (3-10 years) long term avoidance of cow's milk is associated with short stature and poor bone health assessed based on bone mineral density with dual-energy X-ray absorptiometry .
Bone health requires adequate intake of i.a. protein, magnesium, phosphorus, zinc and vitamin C. The intake of those nutrients was not deficient in the diets of children from both groups and did not differ significantly. Blood serum concentration of phosphate and magnesium did not reveal any differences and was within the reference range.
The diets of children with CMPA allergy may be deficient in iron. In the study by Salman, et al. in children with allergy, aged 1-9, iron consumption was below 67% of RDA (Recommended Dietary Allowances) . In our observation, iron intake in the diets of children with CMPA was higher than recommended (EAR - Estimated Average Requirement) and also significantly higher than the intake in the diets of children from the control group. Furthermore, in all children with CMPA the recommended level of intake for this nutrient was achieved. This could be due to consumption of fortified foods; the children could also consume different types of meat, including veal and beef which are a good source of easily absorbed hem iron. Adequate iron intake is necessary for erythropoiesis. Iron is a component of haemoglobin, myoglobin, it is present in tissue enzymes, participates in immune processes, is co-responsible for integrity of intestinal barrier. Children from both groups received adequate amounts of this dietary component, since none of them was diagnosed with iron deficiency or iron deficiency anemia.
Oh, et al. suggest that the increased risk of development of allergies is due to insufficient intake of foods being the source of antioxidants in the diet. They argue that in children with allergy (mean age 5.3 ± 0.9 y) risk of Atopic Dermatitis (AD) was associated negatively with intakes of antioxidant related nutrients and AD was predicted better by the intake measure than the corresponding blood biomarkers regarding vitamin E and beta-carotene . Patel, et al. observing an unselected cohort of 861 children from birth until the age of 8, found that increased beta-carotene intake was associated with a reduced risk of allergic sensitization and lower IgE levels in 5 and 8-year-old children .
Our study found that the dietary intake of vitamin E in allergic children was significantly higher, compared to the control group. We also did not observe the risk of vitamin E, A and C deficiency in group of children with CMPA.
It should also be noted that the diets of children with CMPA contained significantly more LCPUFA, and the percentage of children who did not achieve the nutritional standard for this diet component determined at AI (Adequate Intake) was lower than in control group, though insignificantly. It is important, since LCPUFA hamper excessive immune reaction and have anti-inflammatory and anti-allergic properties . Adequate intake of long-chain polyunsaturated fatty acids LCPUFA is one of dietary factors affecting the skin condition. In the group of children with allergy, in 16.6% of children atopic dermatitis was the only clinical manifestation of allergy or accompanied its other symptoms . The study by Kim, et al. on children with atopic dermatitis shows that 4-month individualised elimination diet, by improving the nutrient intake, is useful for alleviating the severity of atopic dermatitis and improving the growth status .
Milk-free diets in children with CMPA were adequate for most nutrients, but the children are at risk of vitamin D insufficiency, despite the higher vitamin D content of their diets compared to the group of healthy children. The better balancing of the diets of children with allergy was probably due to continuous medical and nutritional care provided by paediatricians and dieticians.
The authors declare no conflict of interest.
According to the order of the authorship.
The authors wish to thank Dr. Hanna Dyląg for valuable revising and improving the manuscript.