- Email: [email protected]
Updating of normal levels of copper, zinc and selenium in serum of pregnant women
ARTICLE IN PRESS
Journal of Trace Elements in Medicine and Biology 21 (2007) S1, 49–52 www.elsevier.de/jtemb
THIRD INTERNATIONAL FESTEM SYMPOSIUM
Updating of normal levels of copper, zinc and selenium in serum of pregnant women Silvia Izquierdo A´lvarez, Sandra Garcı´ a Castan˜o´n, Maria Luisa Calvo Ruata, Elena Fuertes Aragu¨e´s, Pilar Bocos Terraz, Yolanda Gonza´lez Irazabal, Elena Garcı´ a Gonza´lez, Beatriz Garcı´ a Rodrı´ guez Servicio de Bioquı´mica Clı´nica, Hospital Universitario Miguel Servet, Paseo Isabel la Cato´lica 1-3, 50009 Zaragoza, Spain Received 29 June 2007; accepted 30 August 2007
Abstract Selenium, copper and zinc status is important in pregnant women. The aim of this study was to establish updated normal ranges for these elements in serum of pregnant women from the Spanish region of Aragon, and to study variation in levels with respect to gestational period and maternal age. The study group consisted of 159 pregnant women who did not suffer from serious pathologies. These samples were classified into four gestational-period groups. Zn and Cu determinations were obtained by flame atomic absorption spectroscopy in a Perkin-Elmer 1100B apparatus, and Se was determined by electrothermal atomic absorption spectrometry with Zeeman correction, in a Perkin-Elmer 4110 ZL apparatus. The concentrations of Cu, Zn and Se averaged 73.61743.67 mg/dL, 65.37712.87 mg/dL and 99.59721.74 mg/L, respectively. The Cu/Zn ratio increased from first trimester to the third trimester (2.07–3.49). There was no significant correlation between Zn and Se levels, but a significant correlation was found between Cu and Se levels (po0.05) and between Cu and Zn levels (po0.001). Serum Zn and Se levels decreased over gestation, while serum Cu concentrations increased; in all cases the variation occurred mostly in the first 3 or 4 months, with mean levels then remaining fairly stable until the end of pregnancy. Maternal age did not influence levels of any of the three metals. r 2007 Elsevier GmbH. All rights reserved. Keywords: Copper; Zinc; Selenium; Serum; Pregnancy
Introduction Trace elements are well known to play an important role in the maintenance of health. Alterations in maternal–fetal disposition of some essential trace elements could be a potential health risk for the mother as well as the fetus. Corresponding author. Tel.: +34 976765544/+34 650292192; fax: +34 976765543. E-mail address: [email protected] (S. Izquierdo A´lvarez).
0946-672X/$ - see front matter r 2007 Elsevier GmbH. All rights reserved. doi:10.1016/j.jtemb.2007.09.023
Copper is an essential trace element for enzyme systems, such as the catalase, superoxide dismutase and cytochrome oxidase systems, and its deficiency can lead to a variety of nutritional and vascular disorders. Zinc is an essential micronutrient that participates in carbohydrate and protein metabolism, nucleic acid synthesis and other vital functions. Zinc is required for cellular division and differentiation, and is an essential nutrient for normal embryogenesis. Micronutrient deficiency is a serious problem in many countries, so continuing study of zinc levels is required. Zinc is a trace element with a great importance for intrauterine growth
ARTICLE IN PRESS 50
S. Izquierdo A´lvarez et al. / Journal of Trace Elements in Medicine and Biology 21 (2007) S1, 49–52
and it is used during pregnancy in order to improve fetal growth. Selenium has attracted particular attention in recent years. Its biochemistry, toxicology and nutritional importance have been reviewed regularly and thoroughly over the last decade. Selenium and its determination in biological materials may be clinically relevant in situations of excessive or insufficient intake. Selenium status is particularly important in neonates and infants, and therefore in pregnant women. It is well known that serum concentrations of certain essential trace elements such as Zn and Cu are altered during pregnancy [1]. The aim of this study was to evaluate serum Cu, Zn and Se concentrations in pregnant women from the Spanish region of Aragon, taking into account gestational period (GP, weeks) and maternal age (MA, years), as a basis for establishment of normal ranges for serum levels of these metals in pregnant women.
Mean serum Cu, Zn and Se levels in the 159 pregnant women were 73.61743.67 mg/dL, 65.37712.87 mg/dL and 99.59721.74 mg/L, respectively. There was no significant correlation between Zn and Se levels [2]; however, a significant correlation was found between Cu and Se levels (po0.05) and between Cu and Zn levels (po0.001). Cu, Zn and Se levels all varied significantly with gestational period (Table 1); all three metals showed a tendency to vary during early gestation, then to level off at term of pregnancy (Cu, Fig. 1A; Zn, Fig. 1B; Se, data not shown). The Cu/Zn ratio was 42.00. It was obtained the following copper/zinc ratio by gestational weeks: 2.07 (o15 weeks), 3.22 (15–25 weeks), 3.26 (25–35 weeks) and 3.49 (435 weeks of gestation). None of the metals showed significant variation with maternal age. The Cu/Zn ratio by maternal age was 2.63 (o30 years), 2.60 (30–40 years) and 2.70 (440 years).
Material and methods
Discussion and conclusion
The initial study group consisted of 166 pregnant women who did not suffer from significant pathologies. Blood samples were collected from 159 pregnant women. These were classified into four groups of 73, 30, 18 and 38 subjects with gestational periods of 5–15, 15–25, 25–35 or 35–40 weeks, respectively. Serum Zn and Cu levels were determined by flame atomic absorption spectroscopy (AAS) in a PerkinElmer 1100B apparatus (Atomic Absorption Spectrophotometer). Analytical and instrumental parameters were: LCH lamp for copper and zinc, wavelength 324.7 and 213.8 nm, respectively, air/acetylene flame, HNO3 0.2% (diluyent), precision o8.5 (Zn) and o5% (Cu), inaccuracy o6.97 (Zn) and o1.98 (Cu). Serum Se level was determined by electrothermal atomic absorption spectrometry (ETAAS) with Zeeman correction and a graphite furnace (THGA Graphite Tubes with End Caps) in a Perkin-Elmer 4110 ZL apparatus. The conditions of lecture for selenium were: EDL lamp for selenium with wavelength of 196.0 nm, lamp current: 290 mA, slit width: 2.0 nm, matrix modifier was Paladium modificator. The detection limit of the method was less than 10 mg/L, precision (o11%) and inaccuracy (o1%). Samples were diluted in proportion 1:3 with HNO3 0.2%+TRITON 0.2%. All samples were analyzed in duplicate. The results were expressed as mg/dL (Cu and Zn) and mg/L (Se) and it was calculated the relation Cu/Zn. Statistical analyses (Pearson correlation analysis, and analysis of variance) were done with SPSS version 13.0 for Windows. The results were considered significant if po0.05.
Serum Cu, Zn and Se levels in pregnant women varied markedly with gestational period. Previous studies have likewise detected a significant relationship between gestational period and serum levels of Cu and Zn [3,4].
Results
Table 1. Data for serum copper, zinc and selenium levels classified by gestational period (A) Serum copper Metal GP (week) Mean (mg/dL) SD Median Range Sig. ANOVA
Copper 415 147.53 34.61 142.00 152.00 0.00000
15–25 197.14 24.04 200.00 108.00
25–35 195.11 34.97 190.00 140.00
o35 204.22 41.80 204.00 172.00
(B) Serum zinc Metal GP (week) Mean (mg/dL) SD Median Range Sig. ANOVA
Zinc 415 71.30 12.92 72.00 64.00 0.00000
15–25 61.14 8.63 60.00 36.00
25–35 59.78 10.03 60.00 36.00
o35 58.49 11.52 60.00 48.00
(C) Serum selenium Metal Selenium GP (week) 415 Mean (mg/L) 108.60 SD 20.07 Median 106.40 Range 137.00
15–25 99.01 24.41 96.30 152.30
25–35 87.31 16.12 85.95 54.00
o35 85.50 12.81 85.95 57.60
Sig. ANOVA
0.00000
ARTICLE IN PRESS S. Izquierdo A´lvarez et al. / Journal of Trace Elements in Medicine and Biology 21 (2007) S1, 49–52
300.00
COPPER (µg/dL)
250.00
200.00
150.00
100.00
50.00 <15
15-25
25-35
>35
GESTATIONAL PERIOD (WEEKS)
110.00 100.00
ZINC (µg/dL)
90.00 80.00 70.00
51
There is an increased requirement for selenium during pregnancy, presumably for fetal growth, which manifests as decreasing maternal blood selenium concentrations. The maximum decreased in Zn and maximum increase in Cu were observed during weeks (35–36) of pregnancy, these results are similar to the studies of another authors. Such changes could be due to the requirements of the fetus for growth [1]. The copper/zinc ratio increased from first trimester to the third trimester (2.07–3.49). These results are the same as the conclusions of some previous studies in other countries [1]. It is concluded that an early and progressive decline in serum Zn occurs during gestation and a poor maternal zinc status may limit the metabolic adaptation capacity of women especially during pregnancy. Some previous studies demonstrated that deficiency of zinc during pregnancy in early weeks can produced abortion and congenital malformations [10–12]. If this deficiency continues during all pregnancy, the growth of fetus could be disordered and could be appear complications in birth. The effect of supplementation with zinc can be result beneficial in case of deficiency pregnant women. There are not dates that confirm a relation between deficiency of selenium with disordered in growth [13]. Future studies should investigate these patterns in relation to the clinical courses of individual pregnancies, taking into account dietary supplements and medical treatments received during the pregnancy.
60.00 50.00
References
40.00 <15
15-25
25-35
>35
GESTATIONAL PERIOD (WEEKS)
Fig. 1. Boxplots (median at center) of serum copper and zinc by gestational period.
Our results show that Zn and Se levels decreased with increasing gestational period, while Cu levels increased. High levels of copper in maternal serum could be dangerous for the fetus and could cause cerebral disorder and could induce abortion in the mother. In all cases, and as reported previously [5], metal levels varied mainly during the first 15 weeks of pregnancy, and then remained fairly stable until term. The results of this study concluded that in normal pregnant women the levels of serum copper and the Cu/Zn ratio are increased with decreased serum levels of Zn. Previous studies confirmed these results [6–9].
[1] Al-Bader A, Hussain T, Al-Mosawi M, Otaibi M, Abul H, Khalifa D, et al. Serum zinc and copper concentrations in pregnant women from Kuwait. J Trace Elem Exp Med 1997;10:209–15. [2] Scheplyagina LA. Impact of the mother’s zinc deficiency on the woman’s and newborn’s health status. J Trace Elem Med Biol 2005;19(1):29–35. [3] Al-Saleh E. Maternal–fetal status of copper, iron, molybdenum, selenium and zinc in insulin-dependent diabetic pregnancies. Arch Gynecol Obstet 2005;271: 212–7. [4] Kundu N, Parke P, Petersen LP, Palmer IS, Olson O. Distribution of serum selenium, copper, and zinc in normal human pregnancy. Arch Environ Health 1985;40: 268–73. [5] Castillo-Dura´n C, Cassoria F. Trace minerals in human growth and development. J Pediatric Endocr Metab 1999;12:589–601. [6] Adeniyi FA. The implications of hypozincemia in pregnancy. Acta Obstet Gynecol Scand 1987;66:579–82. [7] Halsted JA, Smith JC. Plasma-zinc in health and disease. Lancet 1970;1:322–4.
ARTICLE IN PRESS 52
S. Izquierdo A´lvarez et al. / Journal of Trace Elements in Medicine and Biology 21 (2007) S1, 49–52
[8] Hunt IF, Murphy NJ, Martner-Hewes PM, et al. Zinc, vitamin B-6, and other nutrients in pregnant women attending prenatal clinics in Mexico. Am J Clin Nutr 1987;46:563–9. [9] Makinde OO, Amole F, Ogunniyi SO. Serum copper, zinc and magnesium in maternal and cord blood at delivery. West Afr J Med 1991;10:168–70. [10] Lorenzo MJ. Selenium levels in related biological samples: human placenta, maternal and umbilical cord blood, hair and nails. J Trace Elem Med Biol 2005;19(1):49–54.
[11] Cherry FF, Bemnett EA, Bazzano GS, Johnson LK, Fosmire GJ, Batson HK. Plasma zinc in hypertension/toxemia and other reproductive variables in adolescent pregnancy. Am J Clin Nutr 1981;34: 2367–75. [12] Swanson CA, King JC. Zinc and pregnancy outcome. Am J Clin Nutr 1987;46:763–71. [13] Pombo M, Castro L, Barreiro J. El crecimiento, el desarrollo y los elementos traza. An Esp Pediatr 2001; 54:63–71.
Journal of Trace Elements in Medicine and Biology 21 (2007) S1, 49–52 www.elsevier.de/jtemb
THIRD INTERNATIONAL FESTEM SYMPOSIUM
Updating of normal levels of copper, zinc and selenium in serum of pregnant women Silvia Izquierdo A´lvarez, Sandra Garcı´ a Castan˜o´n, Maria Luisa Calvo Ruata, Elena Fuertes Aragu¨e´s, Pilar Bocos Terraz, Yolanda Gonza´lez Irazabal, Elena Garcı´ a Gonza´lez, Beatriz Garcı´ a Rodrı´ guez Servicio de Bioquı´mica Clı´nica, Hospital Universitario Miguel Servet, Paseo Isabel la Cato´lica 1-3, 50009 Zaragoza, Spain Received 29 June 2007; accepted 30 August 2007
Abstract Selenium, copper and zinc status is important in pregnant women. The aim of this study was to establish updated normal ranges for these elements in serum of pregnant women from the Spanish region of Aragon, and to study variation in levels with respect to gestational period and maternal age. The study group consisted of 159 pregnant women who did not suffer from serious pathologies. These samples were classified into four gestational-period groups. Zn and Cu determinations were obtained by flame atomic absorption spectroscopy in a Perkin-Elmer 1100B apparatus, and Se was determined by electrothermal atomic absorption spectrometry with Zeeman correction, in a Perkin-Elmer 4110 ZL apparatus. The concentrations of Cu, Zn and Se averaged 73.61743.67 mg/dL, 65.37712.87 mg/dL and 99.59721.74 mg/L, respectively. The Cu/Zn ratio increased from first trimester to the third trimester (2.07–3.49). There was no significant correlation between Zn and Se levels, but a significant correlation was found between Cu and Se levels (po0.05) and between Cu and Zn levels (po0.001). Serum Zn and Se levels decreased over gestation, while serum Cu concentrations increased; in all cases the variation occurred mostly in the first 3 or 4 months, with mean levels then remaining fairly stable until the end of pregnancy. Maternal age did not influence levels of any of the three metals. r 2007 Elsevier GmbH. All rights reserved. Keywords: Copper; Zinc; Selenium; Serum; Pregnancy
Introduction Trace elements are well known to play an important role in the maintenance of health. Alterations in maternal–fetal disposition of some essential trace elements could be a potential health risk for the mother as well as the fetus. Corresponding author. Tel.: +34 976765544/+34 650292192; fax: +34 976765543. E-mail address: [email protected] (S. Izquierdo A´lvarez).
0946-672X/$ - see front matter r 2007 Elsevier GmbH. All rights reserved. doi:10.1016/j.jtemb.2007.09.023
Copper is an essential trace element for enzyme systems, such as the catalase, superoxide dismutase and cytochrome oxidase systems, and its deficiency can lead to a variety of nutritional and vascular disorders. Zinc is an essential micronutrient that participates in carbohydrate and protein metabolism, nucleic acid synthesis and other vital functions. Zinc is required for cellular division and differentiation, and is an essential nutrient for normal embryogenesis. Micronutrient deficiency is a serious problem in many countries, so continuing study of zinc levels is required. Zinc is a trace element with a great importance for intrauterine growth
ARTICLE IN PRESS 50
S. Izquierdo A´lvarez et al. / Journal of Trace Elements in Medicine and Biology 21 (2007) S1, 49–52
and it is used during pregnancy in order to improve fetal growth. Selenium has attracted particular attention in recent years. Its biochemistry, toxicology and nutritional importance have been reviewed regularly and thoroughly over the last decade. Selenium and its determination in biological materials may be clinically relevant in situations of excessive or insufficient intake. Selenium status is particularly important in neonates and infants, and therefore in pregnant women. It is well known that serum concentrations of certain essential trace elements such as Zn and Cu are altered during pregnancy [1]. The aim of this study was to evaluate serum Cu, Zn and Se concentrations in pregnant women from the Spanish region of Aragon, taking into account gestational period (GP, weeks) and maternal age (MA, years), as a basis for establishment of normal ranges for serum levels of these metals in pregnant women.
Mean serum Cu, Zn and Se levels in the 159 pregnant women were 73.61743.67 mg/dL, 65.37712.87 mg/dL and 99.59721.74 mg/L, respectively. There was no significant correlation between Zn and Se levels [2]; however, a significant correlation was found between Cu and Se levels (po0.05) and between Cu and Zn levels (po0.001). Cu, Zn and Se levels all varied significantly with gestational period (Table 1); all three metals showed a tendency to vary during early gestation, then to level off at term of pregnancy (Cu, Fig. 1A; Zn, Fig. 1B; Se, data not shown). The Cu/Zn ratio was 42.00. It was obtained the following copper/zinc ratio by gestational weeks: 2.07 (o15 weeks), 3.22 (15–25 weeks), 3.26 (25–35 weeks) and 3.49 (435 weeks of gestation). None of the metals showed significant variation with maternal age. The Cu/Zn ratio by maternal age was 2.63 (o30 years), 2.60 (30–40 years) and 2.70 (440 years).
Material and methods
Discussion and conclusion
The initial study group consisted of 166 pregnant women who did not suffer from significant pathologies. Blood samples were collected from 159 pregnant women. These were classified into four groups of 73, 30, 18 and 38 subjects with gestational periods of 5–15, 15–25, 25–35 or 35–40 weeks, respectively. Serum Zn and Cu levels were determined by flame atomic absorption spectroscopy (AAS) in a PerkinElmer 1100B apparatus (Atomic Absorption Spectrophotometer). Analytical and instrumental parameters were: LCH lamp for copper and zinc, wavelength 324.7 and 213.8 nm, respectively, air/acetylene flame, HNO3 0.2% (diluyent), precision o8.5 (Zn) and o5% (Cu), inaccuracy o6.97 (Zn) and o1.98 (Cu). Serum Se level was determined by electrothermal atomic absorption spectrometry (ETAAS) with Zeeman correction and a graphite furnace (THGA Graphite Tubes with End Caps) in a Perkin-Elmer 4110 ZL apparatus. The conditions of lecture for selenium were: EDL lamp for selenium with wavelength of 196.0 nm, lamp current: 290 mA, slit width: 2.0 nm, matrix modifier was Paladium modificator. The detection limit of the method was less than 10 mg/L, precision (o11%) and inaccuracy (o1%). Samples were diluted in proportion 1:3 with HNO3 0.2%+TRITON 0.2%. All samples were analyzed in duplicate. The results were expressed as mg/dL (Cu and Zn) and mg/L (Se) and it was calculated the relation Cu/Zn. Statistical analyses (Pearson correlation analysis, and analysis of variance) were done with SPSS version 13.0 for Windows. The results were considered significant if po0.05.
Serum Cu, Zn and Se levels in pregnant women varied markedly with gestational period. Previous studies have likewise detected a significant relationship between gestational period and serum levels of Cu and Zn [3,4].
Results
Table 1. Data for serum copper, zinc and selenium levels classified by gestational period (A) Serum copper Metal GP (week) Mean (mg/dL) SD Median Range Sig. ANOVA
Copper 415 147.53 34.61 142.00 152.00 0.00000
15–25 197.14 24.04 200.00 108.00
25–35 195.11 34.97 190.00 140.00
o35 204.22 41.80 204.00 172.00
(B) Serum zinc Metal GP (week) Mean (mg/dL) SD Median Range Sig. ANOVA
Zinc 415 71.30 12.92 72.00 64.00 0.00000
15–25 61.14 8.63 60.00 36.00
25–35 59.78 10.03 60.00 36.00
o35 58.49 11.52 60.00 48.00
(C) Serum selenium Metal Selenium GP (week) 415 Mean (mg/L) 108.60 SD 20.07 Median 106.40 Range 137.00
15–25 99.01 24.41 96.30 152.30
25–35 87.31 16.12 85.95 54.00
o35 85.50 12.81 85.95 57.60
Sig. ANOVA
0.00000
ARTICLE IN PRESS S. Izquierdo A´lvarez et al. / Journal of Trace Elements in Medicine and Biology 21 (2007) S1, 49–52
300.00
COPPER (µg/dL)
250.00
200.00
150.00
100.00
50.00 <15
15-25
25-35
>35
GESTATIONAL PERIOD (WEEKS)
110.00 100.00
ZINC (µg/dL)
90.00 80.00 70.00
51
There is an increased requirement for selenium during pregnancy, presumably for fetal growth, which manifests as decreasing maternal blood selenium concentrations. The maximum decreased in Zn and maximum increase in Cu were observed during weeks (35–36) of pregnancy, these results are similar to the studies of another authors. Such changes could be due to the requirements of the fetus for growth [1]. The copper/zinc ratio increased from first trimester to the third trimester (2.07–3.49). These results are the same as the conclusions of some previous studies in other countries [1]. It is concluded that an early and progressive decline in serum Zn occurs during gestation and a poor maternal zinc status may limit the metabolic adaptation capacity of women especially during pregnancy. Some previous studies demonstrated that deficiency of zinc during pregnancy in early weeks can produced abortion and congenital malformations [10–12]. If this deficiency continues during all pregnancy, the growth of fetus could be disordered and could be appear complications in birth. The effect of supplementation with zinc can be result beneficial in case of deficiency pregnant women. There are not dates that confirm a relation between deficiency of selenium with disordered in growth [13]. Future studies should investigate these patterns in relation to the clinical courses of individual pregnancies, taking into account dietary supplements and medical treatments received during the pregnancy.
60.00 50.00
References
40.00 <15
15-25
25-35
>35
GESTATIONAL PERIOD (WEEKS)
Fig. 1. Boxplots (median at center) of serum copper and zinc by gestational period.
Our results show that Zn and Se levels decreased with increasing gestational period, while Cu levels increased. High levels of copper in maternal serum could be dangerous for the fetus and could cause cerebral disorder and could induce abortion in the mother. In all cases, and as reported previously [5], metal levels varied mainly during the first 15 weeks of pregnancy, and then remained fairly stable until term. The results of this study concluded that in normal pregnant women the levels of serum copper and the Cu/Zn ratio are increased with decreased serum levels of Zn. Previous studies confirmed these results [6–9].
[1] Al-Bader A, Hussain T, Al-Mosawi M, Otaibi M, Abul H, Khalifa D, et al. Serum zinc and copper concentrations in pregnant women from Kuwait. J Trace Elem Exp Med 1997;10:209–15. [2] Scheplyagina LA. Impact of the mother’s zinc deficiency on the woman’s and newborn’s health status. J Trace Elem Med Biol 2005;19(1):29–35. [3] Al-Saleh E. Maternal–fetal status of copper, iron, molybdenum, selenium and zinc in insulin-dependent diabetic pregnancies. Arch Gynecol Obstet 2005;271: 212–7. [4] Kundu N, Parke P, Petersen LP, Palmer IS, Olson O. Distribution of serum selenium, copper, and zinc in normal human pregnancy. Arch Environ Health 1985;40: 268–73. [5] Castillo-Dura´n C, Cassoria F. Trace minerals in human growth and development. J Pediatric Endocr Metab 1999;12:589–601. [6] Adeniyi FA. The implications of hypozincemia in pregnancy. Acta Obstet Gynecol Scand 1987;66:579–82. [7] Halsted JA, Smith JC. Plasma-zinc in health and disease. Lancet 1970;1:322–4.
ARTICLE IN PRESS 52
S. Izquierdo A´lvarez et al. / Journal of Trace Elements in Medicine and Biology 21 (2007) S1, 49–52
[8] Hunt IF, Murphy NJ, Martner-Hewes PM, et al. Zinc, vitamin B-6, and other nutrients in pregnant women attending prenatal clinics in Mexico. Am J Clin Nutr 1987;46:563–9. [9] Makinde OO, Amole F, Ogunniyi SO. Serum copper, zinc and magnesium in maternal and cord blood at delivery. West Afr J Med 1991;10:168–70. [10] Lorenzo MJ. Selenium levels in related biological samples: human placenta, maternal and umbilical cord blood, hair and nails. J Trace Elem Med Biol 2005;19(1):49–54.
[11] Cherry FF, Bemnett EA, Bazzano GS, Johnson LK, Fosmire GJ, Batson HK. Plasma zinc in hypertension/toxemia and other reproductive variables in adolescent pregnancy. Am J Clin Nutr 1981;34: 2367–75. [12] Swanson CA, King JC. Zinc and pregnancy outcome. Am J Clin Nutr 1987;46:763–71. [13] Pombo M, Castro L, Barreiro J. El crecimiento, el desarrollo y los elementos traza. An Esp Pediatr 2001; 54:63–71.