The effect of vegetarian diet on selected essential nutrients in children

Wp³yw diety wegetariañskiej na wybrane niezbêdne sk³adniki ¿ywieniowe dzieci

Teresa Laskowska-Klita¹, Magdalena Che³chowska ¹, Jadwiga Ambroszkiewicz¹, Joanna Gajewska¹, Witold Klemarczyk²

¹1Screening Test Department
Chief of Department: dr n. biol. M. O³tarzewski ²Department of Nutrition
Chief of Department: dr hab. n. med. H. Weker
Institute of Mother and Child in Warsaw
Director: S. Janus

Abstract
Introduction: Vegetarian diets are considered to promote health and reduce the risk of some chronic diseases. It is also known that restriction or exclusion of animal foods may result in low intake of essential nutrients.
The aim of the presented study was to assess the intake and serum status of vitamin B12, folate, vitamins A, E and D, as well as concentrations of homocysteine, total antioxidant status and iron balance in Polish vegetarian children.
Material and methods: The study included 50 children, aged 511 who had been referred to the Institute of Mother and Child for dietary consultation. From those, 32 were vegetarians (aged 6.5±4.2 years) and 18 omnivores (aged 7.9±2.7 years). Dietary constituents were analyzed using the nutritional programme Dietetyk2®. Folate and vitamin B12 were determined with a chemiluminescence immunoassay, total homocysteine with a fluorescence polarization immunoassay and TAS (total antioxidant status) by colorimetric method. Vitamin A and E in serum were determined by the highpressure liquid chromatography method (HPLC) and vitamin D by immunoenzymatic assay (ELISA). Concentrations of iron, ferritin, transferrin and total ironbinding capacity (TIBC) in serum were determined by commercially available kits.
Results: In vegetarian children daily intake of vitamin B12 (1.6 μg) was in the recommended range, that of folate (195 μg) and vitamin A (1245 μg) higher, but vitamin E slightly lower (6.6 μg) and threefold lower vitamin D (1.1 μg) than references allowance. Serum concentrations of vitamin B12 (548 pg/ml), folate (12.8 ng/ml), vitamin A (1.2 μmol/L), vitamin E (15.6 μmol/l) were within physiological range, but that of vitamin D (13.7 μg/L) was only half of the lowest limit of the reference value. In vegetarian children in comparison to omnivorous similar levels of homocysteine (6.13 μmol/L vs 5.45 μmol/L) and vitamin A (1,17 μmol/L vs 1.32 μmol/L) were observed. Lower (p<0.05) values of vitamin E (15.6 μmol/L vs 18.4 μmol/L) and TAS (1.21 mmol/L vs 1.30 mmol/L; p<0.0001) were found. Concentrations of iron markers were in physiological range.
Conclusion: Obtained results indicated that intakes of vitamin B12 and folic acid from vegetarian diets are sufficient to maintain serum concentrations of both homocysteine and iron in the range observed in omnivorous children. High consumption of vitamin A and low vitamin E only slightly affected their serum values. Significantly lower concentration of serum vitamin E in vegetarian children in comparison to nonvegetarians may be reflected with statistically significant lowering of total antioxidant status. Insufficient intake of vitamin D and its low serum concentration should be under close monitoring in vegetarian children. In order to prevent vitamin D deficiency appropriate agedependent supplementation should be considered.

Key words: vegetarian children, homocysteine, antioxidants, vitamin D, folate, vitamin B12

Streszczenie
Wprowadzenie: Dieta wegetariañska uznawana jest za zdrowy sposób od¿ywiania obni¿aj¹cy ryzyko wyst¹pienia niektórych chorób. Jednak¿e ograniczenie lub wykluczenie ¿ywnoœci pochodzenia zwierzêcego mo¿e byæ powi¹zane z niskim spo¿yciem niezbêdnych sk³adników ¿ywieniowych.
Celem przedstawianej pracy by³a ocena spo¿ycia witaminy B12, folianu, witamin A, E i D oraz ich stê¿eñ we krwi u dzieci na diecie wegetariañskiej. Badano równie¿ poziom homocysteiny, ca³kowit¹ aktywnoœæ przeciwutleniaj¹c¹ krwi oraz status ¿elaza.
Materia³ i metody: Badaniami objêto 50 dzieci w wieku 5-11 lat, konsultowanych w Poradni Gastroenterologicznej Instytutu Matki i Dziecka. Wœród badanych by³o 32 wegetarian (wiek: 6,5±4,2 lat) i 18 dzieci ¿ywionych tradycyjnie (wiek: 7,9±2,7 lat). Wartoœæ od¿ywcz¹ diety oceniano przy zastosowaniu programu ¿ywieniowego Dietetyk2®. Poziom witaminy B12 i folianu oznaczano metod¹ chemiluminescencji, homocysteiny metod¹ immunochemiczn¹ z pomiarem spolaryzowania fluorescencji, natomiast TAS (ca³kowita aktywnoœæ przeciwutleniajaca) metod¹ kolorymetryczn¹. Witaminy A i E oznaczano przy u¿yciu wysokociœnieniowej chromatografii cieczowej (HPLC) a witaminê D metod¹ immunoenzymatyczn¹ (ELISA). Stê¿enia ¿elaza, ferytyny, transferyny i ca³akowit¹ zdolnoœæ wi¹zania ¿elaza (TIBC) w surowicy oznaczano przy u¿yciu gotowych zestawów handlowych.
Wyniki: U dzieci na diecie wegetariañskiej dzienne spo¿ycie witaminy B12 (1,6 μg) by³o w zakresie wartoœci rekomendowanych. Spo¿ycie folianu (195 μg) i witaminy A (1245 μg) by³o wy¿sze, witaminy E nieznacznie mniejsze (6,6 μg), natomiast witaminy D (1,1 μg) trzykrotnie ni¿sze ni¿ wartoœci zalecane. W surowicy krwi stê¿enia witaminy B12 (548 pg/ml), folianu (12,8 ng/ml), witaminy A (1,2 μmol/L) oraz witaminy E (15,6 μmol/L) by³y w zakresie wartoœci fizjologicznych. Stê¿enie witaminy D (13,7 μg/L) wynosi³o po³owê dolnej granicy stê¿eñ referencyjnych. U dzieci na diecie wegetariañskiej w porównaniu do dzieci ¿ywionych tradycyjnie obserwowano zbli¿ony poziom homocysteiny (6,13 μmol/L vs 5,45 μmol/L) oraz witaminy A (1,17 μmol/L vs 1,32 μmol/L). Natomiast obni¿one by³y wartoœci witaminy E (15,6 μmol/L vs 18,4 μmol/L; p<0.05) i TAS (1,21 mmol/L vs 1,30 mmol/L; p<0.0001). Stê¿enia markerów statusu ¿elaza zawarte by³y w zakresie wartoœci referencyjnych.
Wnioski: Otrzymane wyniki wskazuj¹, ¿e dieta wegetariañska dostarcza witaminê B12 i kwas foliowy w iloœciach wystarczaj¹cych do zabezpieczenia stê¿eñ homocysteiny i ¿elaza na poziomie obserwowanym u dzieci ¿ywionych w sposób tradycyjny. Wysokie spo¿ycie witaminy A i niskie witaminy E wp³ywa nieznacznie na ich stê¿enie w surowicy krwi. Statystycznie istotne ni¿sze stê¿enie witaminy E u wegetarian w porównaniu z grup¹ dzieci na diecie mieszanej mo¿e istotnie obni¿aæ status przeciwutleniaczy. Niezadowalaj¹ce spo¿ycie i niskie stê¿enia w surowicy witaminy D powinny byæ œciœle kontrolowane. W celu zapobie¿enia deficytom tej witaminy powinna byæ stosowana w³aœciwa dla wieku suplementacja diety wegetariañskiej.

S³owa kluczowe: dzieci na diecie wegetariañskiej, homocysteina, przeciwutleniacze, witamina D, folian, witamina B12

INTRODUCTION
Current knowledge indicates positive but also negative consequences of vegetarianism on health status. The basic principle of vegetarian diet is avoiding animal products. Vegetarians are classified into subgroups: lacto-ovo vegetarians (diet includes dairy products and eggs), ovo vegetarians (diet excludes milk products), lacto vegetarians (diet excludes eggs) and vegans (do not eat any animal food) (1, 2).
A well-planned diet is consistent with good health and can reduce the risk of some diseases such as coronary heart disease, diabetes, some cancers and obesity. However, exclusion of animal products may result in inadequate intake of some nutrients such as vitamins, both water and fat soluble, and also some minerals (3-5).
Since vegetarianism has became increasingly popular, low intake of vitamin B12, vitamin B6 and/or folic acid as well as vitamins A, E and D may results in some deficiencies and lead to severe disturbances of important metabolic pathways. Adverse effects of vegetarian diets should be taken into account as a possible contribution factor in the etiology of some degenerative diseases especially those related to oxidative stress, hyperhomocysteinemia and bone-turnover (6-8).

Several studies indicate that diets which exclude animal products may result in low intake of nutrients which in plant food are available only in limited amounts. In vegetarians, particularly vegans in comparison to omnivores reduced level of vitamin B12 could be observed (9, 10). In animal food vitamin B12 is bound to protein. Passive absorption of cobalamin across gastrointestinal tract is poor and amounts only to about 2% of oral dose. Active absorption of vitamin B12 demands transport proteins (haptocorrin and transcobalamine) as well as some specific receptors. In mitochondria vitamin B12 in form 5-deoxyadenosylcobalamin is required for converting methylmalonyl-CoA to succinyl-CoA in reaction catalyzed by methylmalonyl-CoA mutase. In cell cytosol it participates as methylcobalamin in methylation of homocysteine to methionine. Vegetarian status of vitamin B12 depends on geographical area but everywhere it is lower than in the omnivorous. In high percentage of vegan population this status is deficient. Studies from different countries indicate that in vegetarians deficiency of cobalamin is correlated with elevation of plasma concentration of homocysteine (Hcy) (9, 11, 12).
Homocysteine derived from methionine which is present as an essential amino acid in the diet. Homocysteine metabolism is modulated by availability of vitamin B12, folate and vitamin B6. Methylcobalamine acting as coenzyme of methionine synthase is required in remethylation of homocysteine into methionine. In this pathway methylenetetrahydrofolate as a methyl donor is necessary for the activity of methylenetetrahydrofolate reductase. Homocysteine, in transulphuration reaction requiring vitamin B6 is converted to cysteine.
Deficiencies of cobalamine, folate and vitamin B6 and also polymorphism of methylenetetrahydrofolate reductase gene, may lead to increase of plasma homocysteine concentration to value above 12 μmol/L and therefore to homocysteinemia (9).
In vegetarians, vitamin B12 and folate are correlated negatively with plasma homocysteine. However, the protective effect of folate do not counteract with low cobalamine on high level of homocysteine. Vitamin B6, which in 30% of vegetarians is low, did not significantly influence plasma homocysteine and is considered as a weak determinant of its plasma values (9, 14).
Vitamins B12 and B6 are found mainly in animal products. Vegetarian diets are rich in folic acid due to high intake of fruits and vegetables. In comparison with omnivores, vegetarians consume more folic acid and values of this vitamin in their plasma are high. In plant food folate and its derivatives occur as polyglutamates which undergo hydrolysis to monoglutamates before absorption. Folate absorption occurs by the jejunum carrier mediated process. Absorbed folate is metabolized in intestinal mucosa and converted to 5-methyltetrohydrofolate. Folate as the main one-carbon donor, supports synthesis of some key- metabolites, among them precursors of nucleic acids (15, 16).
Homocysteine autooxidation leads to increase of toxic hydroxyradicals and determines its prooxidative activity (17, 18). High plasma concentration of homocysteine might be associated with oxidative stress and in consequence in damage of lipids, proteins and DNA. Ullegaddi et al. (19) observed that efficient antioxidant therapy inhibited the effect of elevated plasma homocysteine.
Total antioxidant status (TAS) represents a mixed antioxidant capacity which among others contributes mainly to fat soluble vitamins E, A and â-carotene. High consumption of vegetables and/or fish in vegetarian diet results in low levels of saturated fat, long-chain n-3 fatty acids and retinol (8, 20). Those diets are rich in n-6 fatty acids, carotenoids and vitamin E. It has been established that requirement of vitamin E is related to the amount of dietary polyunsaturated fatty acids which may impair absorption of these vitamin (21, 22).
Absorption of vitamin E is in the range of 20-70% of daily dose. Emulsification and solubilization of vitamin E in bile salt micelles are involved in its absorption in small intestine. This process is in reversely related to its intake, and is low when dietary fat is insufficient. Vitamin E is transported in blood within lipoproteins and its levels varies with total lipids, cholesterol and β-lipoproteins. Large amounts of vitamin E are found in adipose tissue. It is mostly located in mitochondria and endoplasmic reticulum. Little is found in cytosol. Vitamin E resides primarily within biomembranes.
Deficiency of vitamin E is associated with malabsorption syndromes. Depletion of dietary vitamin E has been shown to be related to increased formation of lipid peroxides. Vitamin E is considered to be a scavenger of free radicals by mechanism resulting in inactivation of two radicals per one molecule of α-tocopherol. The most important product of vitamin E oxidation in tissues is α-tocopheryl quinoine (8, 23).
Vitamin A – retinol (or vitamin A alcohol) is present in food as esters of long-chain fatty acids, mainly palmitic. Esters combine with specific transport proteins and are then absorbed in the ileum. Plasma-retinol binding protein and low density lipoproteins are responsible for transport and regulation of its blood level. Excessive load leads to toxic effect on the cell membrane because there is lack of an efficient mechanism to excrete or destroy it.
Dietary carotenoids are transported to the tissues mainly as non-polar lipids. Vitamin A is essential for erythropoiesis and is also vital for vision, cellular differentiation, immune system, for bone growth and reproduction. The most important provitamin in foods is β-carotene. It is absorbed in gastrointestinal tract and within the intestinal wall it is converted into vitamin A. Vitamin A and β-carotene may form a complex with iron which in the intestinal lumen is soluble and active against the inhibitory effect of phytates and polyphenols on absorption of non-haem iron. Vegetarians have high dietary intakes of β-carotene (8, 24, 25).
Vitamin D exists in two naturally occurring forms: cholecalciferol (vitamin D3) and ergocalciferol (vitamin D2). Cholecalciferol is absorbed from the diet. It is present in high amounts in animal products, mainly in meat, fish, oils and fat. Plant sources of this vitamin are limited to some types of mushrooms. Cholecalciferol is synthesized in the skin from 7- dehydrocholesterol after adequate sunlight exposure. After hydroxylation in the liver vitamin D is present in serum mainly as 25-hydroxyvitamin [25(OH)D]. In the kidney it is converted to 1,25-dihydroxyvitamin D which is the biologically active form and acts as a steroid hormone affected absorption of calcium, homeostasis of phosphorus and regulated bone metabolism. Vitamin D is removed from circulation by sequestration in the adipose tissue (26).
The findings from several studies have shown that vegetarians have lower serum concentration of 25-hydroxyvitamin D than omnivorous not only because of restricted dietary intake but also, particularly during the winter, insufficient sun exposure. Deficiency of this vitamin is associated in children with rickets and in adults with bone loss, osteopenia and osteoporosis (27-30).
Vegetarian diets, especially of vegans, may result in poor iron status due to its bioavailability. Dietary iron utilization depends on its form in the food (31-33).
In animal products it is the haem-iron which is well absorbed. Non-haem iron as Fe-salts derived mainly from plant and is absorbed a few times less effectively. Iron is absorbed in the duodenum and jejunum actively and is transported into the cell by several transfer proteins and specific receptors. Absorption depends on iron status and iron stores and is regulated by peptide hormone – hepcidine. Iron store deficiency leads to negative iron balance and as a result to iron deficient erythropoesis. Low iron supply,insufficient to maintain normal concentration of haemoglobin, may be a risk of anaemia.Hypoferemia is characterized by reduced iron serum level and by low value of ferritin. Hypoferemia, when iron release from proteins in excessive ammounts may generate free radicals and affects antioxidant capacity.
Several studies of Fe status have shown that in vegetarians serum ferritin is lower than in the omnivorous. Despite this, similar or only slightly lower levels of haemoglobin were observed (10, 32, 33).
According to the American Dietetic Association and Dietitians of Canada, “vegetarian diets are appropriate for all stages of the life-cycle including pregnancy, lactation, infancy, childhood and adolescence” (31). e aim of presented studies was to assess the intake and serum status of vitamin B12, folate, vitamins A, E and D, as well as concentrations of homocysteine, total antioxidant status and iron balance in Polish vegetarian children.

MATERIAL AND METHODS
We investigated 32 vegetarian children (14 girls, 18 boys) aged 6.5±4.2 years, who had been referred to the Department of Nutrition of the Institute of Mother and Child (Warsaw) for dietary consultation. The principal di erence among various vegetarian diets was the extent to which certain products were avoided. In this tested group there were:
– lacto-ovo vegetarians (n=21), who did not consume meat, poultry or !sh, but had no restrictions to eggs and dairy products consumption,
– lacto vegetarians (n=1), who also excluded eggs, – ovo vegetarians (n=5), who had no restrictions to eggs, but excluded milk products,
– vegans (n=5), who excluded all foods of animal origin.
As a control group 18 healthy children (8 girls, 10 boys, mean age 7.9±2.7 years) with normal lipids pro!le on omnivorous diets were recruited from subjects being under temporary medical supervision. Dietary constituents were analyzed using the nutritional programme Dietetyk 2(National Food and Nutrition Institute, Warsaw) and completed with supplementation data.
This research was approved by the institutional review board and informed consent was obtained from parents of the examined children.
Venous blood samples were obtained after overnight fast. Serum was prepared by centrifugation at 1000 x g at 4°C. Serum samples were frozen and collected for biochemical parameters analysis. Folate and vitamin B12 were determined with a chemiluminescence immunoassay (Elecsys, Roche, Switzerland). Total Hcy was measured with a %uorescence polarization immunoassay on IMX analyzer (Abbott, USA). TAS was measured by the colorimetric method using kits from Randox Laboratories Ltd (GB). Vitamins A and E in serum were determined by the high-pressure liquid chromatography method (HPLC) (Knauer, Germany) (34), and 25-OH Vitamin D by immunoenzymatic assay (ELISA) using kits from Biomedica (Austria). Concentrations of iron, ferritin, transferrin and total iron-binding capacity (TIBC) in serum were determined by commercially available kits (Roche, Switzerland).
Data are presented as means ± standard deviation (SD) and range, when appropriate. Student’s t-test was used for comparison of normally distributed data and non-parametric Wilcoxon test for not normally distributed variables. Shapiro- Wilk test was used for evaluation normality of data distribution prior to statistical analysis. Statistical analysis was done using the STATISTICA 6.0 (StatSoft, Poland). The di erences were considered statistically signi!cant at p<0.05.

RESULTS
As it is shown in table I average daily energy intake and the percentage of energy from protein, fat and carbohydrates in the diets of vegetarian children were similar to recommended amounts. Consumption of the carbohydrates exceeds but that of fat was below dietary allowance. Intake of PUFA remains within physiological range.
Results presented in table I indicate that in vegetarian children mean daily intake of vitamin B12 was in the reference range. However in 28% of studied group it was below the lower recommended values.
Average intake of folate was high and amounted to 160% of recommended value (tab. I). Intake of vitamin A was two-times higher than reference daily allowance, whereas that of vitamin E was at the value of lower limit. Consumption of vitamin D with vegetarian diet was low and reached only 20% of recommended daily intake.
In serum of vegetarian children concentrations of vitamin B12, folate and vitamins A and E were in the physiological range, whereas that of vitamin D was below (tab. II). Mean level of vitamin D reached only half of the lowest limit of the reference value.
Concentrations of homocysteine and vitamin A in serum of vegetarian children were similar to that found in the omnivorous group. Serum levels of vitamin E and also TAS were signi!cantly lower in vegetarian than in omnivorous children (tab. III). In vegetarian serum iron markers were within physiological range (tab. IV).

DISCUSSION
Current knowledge point to a bene!cial role of vegetarian diet in prevention of certain diseases such as coronary heart disease, diabetes and obesity. It is suggested, that these antioxidant-rich diets can be associated with lowering incidents of free radical diseases. ese diseases a ecting adults have been established to begin in childhood. However, exclusion of animal products may aect the status of some nutrients and lead to their de ciencies (1, 2, 8).
Hyperhomocysteinemia may contribute to increased oxidative stress and diminishes antioxidative capacity. In adult vegetarians high serum concentration of homocysteine is related to dietary restriction (9, 14, 35). Elevated values of this compound can be observed also in young vegetarians. Data concerning homocysteine in children are limited. In the population of omnivorous children concentration of homocysteine amounted to half of that found in adults (36). The presented results, similar to others, indicate that also in vegetarian children the level of homocysteine is 40% lower than in adult (15, 37).
Several studies have shown that elevated value of homocysteine in serum can be a consequence of low vitamin B12 and/or high intake of folate. Vegetarian diets are typically low in vitamin B12. However the body is able to store sufficient quantities of this vitamin, therefore it may take years to develop its de ciency (2, 11, 16, 38).
Low body store of vitamin B12 usually manifests in neurological and gastrointestinal disorders as well as in megaloblastic anaemia. Elevated serum folate could mask de ciency of vitamin B12 which resulted in late diagnosis of anaemia. Vegetarian diets are rich in folate because of high intake of vegetables and fruits. In our studied group of vegetarian children daily intake of vitamin B12 was suitable and that of folic acid was high. Concentrations of these vitamins in serum were in the middle of the reference range. Our results indicated that vegetarian children, contrary to adults, had no imbalance in vitamin B12 and folate. Therefore, it seems that consuming these diets are sufficient to maintain serum concentration of homocysteine at the value below 10 μmol/L which excluded suspicion of hyperhomocysteinemia. It is well known that hyperhomocysteinemia generates formation of highly toxic hydroxyl radicals, which may lead to oxidative damage of macromolecules and to oxidative stress (17, 18, 22). Some authors suggested that in adult vegetarians high serum concentration of homocysteine affects the antioxidative potential and correlates positively with TAS values (9). Haldar et al. (21) observed similar status of antioxidants in adult vegetarians as in omnivorous. Normal level of antioxidative parameters in vegetarians were also shown by Krajcovicova et al. (18, 39). No systematic studies have been carried out on TAS status in children on vegetarian diet. Our results indicated that TAS values in vegetarian children were in the reference range. However, the mean value was slightly lower than in the omnivorous (1.22 μmol/L versus 1.32 μmol/L), the difference was statistically signi cant (p<0.0001). In evaluation of antioxidant potential of vegetarian diet measurement of TAS seems to be more adequate than determination of single antioxidant nutrient. But, vitamins A and E are important parts in antioxidant defense systems and their low level may be insufficient for scavenging of free radicals in the cells. Vegetarian diets, contrary to omnivorous, maintain higher status of antioxidant vitamins A and E. It was therefore reasonable to estimate their serum concentrations (8, 17, 35).
Several studies reported that in adult vegetarians daily intake of carotenoids was higher than reference allowance (8, 20). Despite of high β-carotene dietary intake, serum vitamin A concentrations were within threshold range, probably because of poor ƒÀ-carotene intestinal absorption.
In our studied group of children the same as in adults dietary intake of vitamin A was above reference daily allowance. Serum concentration of vitamin A in children on vegetarian diet has been previously reported only by Krajcovicova-Kudlackova et al. (39). The authors observed in adolescence vegetarians higher level of vitamin A than in omnivorous. Our previous results did not con rm this observation (27, 40). In the presented studies we have shown decreased mean values of this vitamin by about 10% in vegetarians in comparison to prepubertal non-vegetarian children.
In population of adult vegetarians, the dietary intake of vitamin E exceed recommended daily amounts by 130-315% (8). Also in prepubertal vegetarian children consumption of α-tocopherol was about 20% higher than in the omnivorous. Some authors reported that concentrations of vitamin E in serum were signi cantly higher in adult vegetarians than in their omnivorous counterparts (17, 18, 35, 39). However, in studies of Proñczuk (41) serum values of α-tocopherol were lower in vegetarian than in control subjects. It can be related to high consumption of polyunsaturated fatty acids. With increased intake of these acids the requirement of vitamin E increases.
In prepubertal vegetarian children mean serum level of vitamin E was about 20% lower than in nonvegetarians but was in the reference range (35, 39). In the presented studies we con rmed these observations. Our results indicate that children consumed enough vitamin A and E in vegetarian diets, but concentrations of these nutrients in serum are below the lowest values observed in omnivorous. Therefore to meet individual needs dietary modi cations (supplementation) could be considered.
Vegetarian diets could be a risk factor for iron de ciency. Intake of iron in vegetarians and omnivorous is comparable, but bioavailability is lower because of the absence of haem-iron. Low iron concentration and low level of ferritin observed in vegetarians Alexander et al. (42), Hua et al. (32) and Hunt et al. (4).
Low iron status is common among premenopausal women and is rarely observed in men. Thane et al. (43) described low intake of iron in vegetarian girls which resulted in lowering of ferritin and Hb concentrations. Similar observations reported Bergstrom et al. (44) and Rangam et al. (45). We showed that in vegetarian children intake of iron was slightly below of recommended values (46). However concentrations of iron and transferrin saturation were lower only in 20% of subjects. Markers of iron status were in the recommended range. Our results are in agreement with Leung et al. (47). We confirmed their suggestion that vegetarian diet is su cient to maintaining normoferemia in children.
Dietary vitamin D is an important determinant of this vitamin status. !e serum 25-hydroxyvitamin concentration is the marker of vitamin D nutritional adequacy. Concentration of 25(OH)D should exceed 75 nmol/L. Significant positive association between calciferol intake and plasma concentration of 25(OH) D was found (48).
Crowe et al. (30) reported that intake of calciferol in adult vegetarians was lower than in omnivorous. Lower was also 25(OH)D plasma concentration. The difference was smaller during summer as compared with the winter months. However, studies of Chan et al. (29) suggested that serum 25(OH)D concentrations were not associated with the vegetarian status. Higher influence than diet has vitamin D supplementation, skin pigmentation and sun exposure. In the presented study we observed a two-fold lower dietary intake of vitamin D in vegetarian children than the recommended allowance. Serum concentrations of 25(OH) D in vegetarians were as low as half of the lower limit of reference values. This suggests that in vegetarian children consumption of vitamin D is not adequate and requires supplementation of diets especially during wintertime.

CONCLUSIONS
Obtained results indicated that intakes of vitamin B12 and folic acid from vegetarian diets are su cient to maintain serum concentration of both homocysteine and iron in the range observed in omnivorous children.
High consumption of vitamin A and low of vitamin E only slightly affected their serum values. Significantly lower concentration of serum vitamin E in vegetarian children in comparison to non-vegetarians may be related to statistically signiffcant lowering of total antioxidant status.
Insufficient intake of vitamin D and its low serum concentration should be under close monitoring in vegetarian children. In order to prevent vitamin D deficiency appropriate age-dependent supplementation should be considered.

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