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Gaceta Sanitaria

versión impresa ISSN 0213-9111

Gac Sanit vol.36 no.5 Barcelona sep./oct. 2022  Epub 13-Mar-2023 

Original Articles

Serum metal levels in a population of Spanish pregnant women

Niveles de metales en suero en una población de mujeres embarazadas españolas

Miren Begoña-Zuberoa  b  , Study design, Methodology, Data analysis and interpretation, Writing the article, Final version approval; Sabrina Llopc  d  *  , Study design, Literature review, Writing the article, Final version approval; Amaia Irizarb  , Literature review, Data collection and clean-up, Analysis and critical review, Final version approval; Mario Murciac  d  , Study design, Data analysis and interpretation, Analysis and critical review, Final version approval; Amaia Molinuevob  , Study design, Data analysis and interpretation, Analysis and critical review, Final version approval; Ferrán Ballesterc  d  e  , Study design, Literature review, Writing the article, Final version approval; Michael Levif  , Study design, Literature review, Writing the article, Final version approval; Manuel Lozanoc  g  , Study design, Literature review, Writing the article, Final version approval; Mikel Ayerdib  h  , Literature review, Data collection and clean-up, Analysis and critical review, Final version approval; Loreto Santa-Marinab  d  h  , Study design, Methodology, Writing the article, Final version approval

aPreventive Medicine and Public Health Department, University of the Basque Country, Leioa, Bizkaia, Spain

bBiodonostia, Health Research Institute, Donostia, Gipuzkoa, Spain

cEpidemiology and Environmental Health Joint Research Unit, FISABIO-Universitat Jaume I, Valencia, Spain

dCIBER de Epidemiología y Salud Pública (CIBERESP), Spain

eDepartment of Nursing and Chiropody, Universitat de València, València, Spain

fInstitute of Environmental Medicine, Karolinska Institutet, Stockholm, Sweden

gPreventive Medicine and Public Health, Food Sciences, Toxicology and Forensic Medicine Department, Universitat de València, València, Spain

hDepartment of Health of the Basque Government, Public Health Division of Gipuzkoa, Donostia, Gipuzkoa, Spain



To describe serum levels of calcium, copper, selenium, magnesium, iron and zinc and evaluate their relationship with maternal socio-demographic characteristics and dietary variables in women in the first trimester of pregnancy.


Cross-sectional study with 1279 participants from the INMA cohorts.


The concentrations of the elements analyzed were within the normal range. Associations with higher levels of these metals were found for calcium with white meat intake (p = 0.026), for copper with excess body weight (p < 0.01), low social class (p = 0.03) and being multipara (p < 0.01), for magnesium with being over 35 years old (p = 0.001), high social class (p = 0.044), primiparous status (p = 0.002) and low daily intake of bread (p = 0.009) and legumes (p = 0.020); for zinc with university education (p = 0.039) and residence in Gipuzkoa (p < 0.01), and for selenium with residence in Valencia (p< 0.01), university education (p = 0.001), vitamin B6 supplementation (p = 0.006), fish intake (> 71 g/day) (p = 0.014) and having been born in Spain (p = 0.001). Further, lower iron levels were associated with being overweight (p = 0.021) or obese (p < 0.001) and vitamin B12 supplementation (p = 0.006).


Our results suggest that trace elements in the analyzed cohorts are adequate for this stage of pregnancy. The variability in these elements is mainly linked to socio-demographic and anthropometric variables.

Keywords: Serum; Trace elements; Pregnancy



Describir las concentraciones de calcio, cobre, selenio, magnesio, hierro y zinc en muestras de suero de gestantes en el primer trimestre y evaluar la relación con las características sociodemográficas maternas y las variables de dieta.


Estudio transversal con 1279 participantes de las cohortes INMA.


Las concentraciones de los elementos analizados estuvieron dentro de los límites de referencia. El calcio se asoció con el consumo de carne blanca (p = 0,026). Los valores elevados de cobre se asociaron con tener exceso de peso (p < 0,01), clase social baja (p = 0.03) y ser multípara (p < 0,01). Los valores más elevados de magnesio se asociaron con tener más de 35 años (p = 0,001), clase social alta (p = 0,044), ser primípara (p = 0,002) y bajo consumo diario de pan (p = 0,009) y legumbres (p = 0,020). El zinc se asoció con tener estudios universitarios (p = 0,039) y con la cohorte de Gipuzkoa (p < 0,01). Los valores más altos de selenio se asociaron con la cohorte de Valencia (p < 0,01), tener estudios universitarios (p = 0,001), tomar suplementos de vitamina B6 (p = 0,006), consumo de pescado >71 g/día (p = 0,014) y ser española (p = 0,001). Los valores más bajos de hierro se asociaron con tener exceso de peso (p = 0,021) u obesidad (p < 0,001) y con tomar suplementos de vitamina B12 (p = 0,006).


Nuestros resultados sugieren que los oligoelementos en las cohortes analizadas son adecuados para esta etapa del embarazo. La variabilidad de estos elementos está asociada principalmente a las variables sociodemográficas y antropométricas.

Palabras clave: Suero; Oligoelementos; Embarazo


Trace elements are bioelements that play a significant role in keeping the body healthy.1 The human body cannot produce them, and therefore, we need to obtain them at appropriate doses from our diet. Both deficiency and excess may have a negative impact on body functions and the body's needs vary depending on the stage of the growth. Nutrition is vital during pregnancy for both the expectant mother and the developing fetus. Notably, the nutritional requirements of pregnant women differ from those of non-pregnant women of the same age. They also vary according to the week of pregnancy, with few differences compared to non-pregnant women during the first trimester; demands increasing during the second trimester; and a sharp rise during the third trimester, due to the rapid development of the fetus. Daily dietary reference intakes (DRIs) for pregnant women in Spain, published by the Spanish Federation of Nutrition, Food and Dietetics Societies2 in 2010 were: 1000-1300 mg/d for calcium (Ca), 350-400 mg/d for magnesium (Mg), 1000 µg/d for copper (Cu), 27 mg/d for iron (Fe), 60 µg/d for selenium (Se) and 11-13 mg/d for zinc (Zn). Serum levels in the following ranges are considered normal: Ca 8.5-10.2 mg/dL, Fe 60-180 µg/dL, Mg 0.75-1.25 mmol/L, Cu 70-140 µg/dL, Se 70-150 ng/mL and Zn 0.66-1.10 µg/mL.3

Fe is necessary for proper development of the placenta, as well as bone and organ formation in the fetus. Iron-deficiency anemia is relatively common among pregnant women and may increase the risk of some complications, including preterm delivery and low infant birth weight. To prevent maternal anemia, it is recommended that pregnant women take a daily oral supplement of 30 to 60 mg of elemental Fe.4

Zn is important for protein synthesis, cell division, and nucleic acid metabolism. Deficiency in this element is manifested in different ways, depending on its severity, but in particular, may have a negative impact on the nervous and reproductive systems. Cu is essential for oxidative metabolism, cell growth, development of connective tissue and hemoglobin synthesis.5 Deficiency in this element can cause bone deformations, as well as cardiovascular problems. Se is part of the key enzyme glutathione peroxidase, seems to be involved in platelet function and helps to neutralize heavy metals such as mercury, lead, arsenic and cadmium. Further, deficiency in this element affects all components of the immune system. During pregnancy, there is active transport to the fetus through the placenta and Se deficiency is reported to be associated with a variety of adverse outcomes including miscarriage, preterm delivery, gestational diabetes and preeclampsia.6

Mg is a key element for fetal growth and is one of the most abundant nutrients in the human body. It is involved in cellular respiration, protein synthesis, maintenance of cardiovascular health, regulation of cellular function and the action of collagen, among many other functions. Deficiency in Mg during pregnancy has been associated with preeclampsia and preterm delivery, as well as low infant birth weight.7

Lastly, Ca is another element necessary for normal fetal growth.8 Among its functions, in addition to providing structure and rigidity to the bones, it plays a key role in muscle contractility, the transmission of signals from the brain nerves to the rest of the body, the circulation of the blood and the production of hormones and enzymes for various body functions. Lower maternal serum Ca levels have been associated with pregnancy-induced hypertension and preeclampsia.

The objective of this work was to describe serum levels of Ca, Fe, Mg, Se, Cu and Zn, and evaluate their relationship with maternal socio-demographic, anthropometric and dietary variables in women from cohorts from Valencia and Gipuzkoa (Spain) in their first trimester of pregnancy.


Study population

Study participants were enrolled in the INMA Project (from the Spanish: Infancia y Medio Ambiente, meaning childhood and the environment: A cross-sectional study was designed. We gathered information from women in the INMA Valencia and Gipuzkoa cohorts during their routine check-up in the first trimester of pregnancy (Valencia n = 855; Gipuzkoa n = 638). The final study population was composed of 1279 women (Valencia n = 656, Gipuzkoa n = 623). Samples were collected between February 2004 and June 2005 in Valencia and between May 2006 and February 2008 in Gipuzkoa, and Mg was only analyzed in the Valencia cohort (n = 656). Informed consent was obtained from all the participants before inclusion. The hospital ethics committees of each area approved the research protocols.

Socio-demographic and dietary characteristics

Data on socio-demographic characteristics were collected through questionnaires. Occupational status data were classified into two categories according to whether the women were in work. Social class was based on occupation and five categories were considered in accordance with the Spanish adaptation of the British Registrar General's Social class classification (I being the highest socioeconomic position and V the lowest). In order to increase the statistical power for the analysis, social class was further grouped into two categories: manual worker or lower class (IV and V) and non-manual worker or higher class (I-III). Educational level was classified according to the highest level attained into three categories: up to primary school, secondary school and university. Body mass index (BMI) before pregnancy was classified as underweight: < 18.5 km/m2; normal weight: 18.5-24.9 kg/m2; overweight: 25-29.9 kg/m2; or obese: ≥ 30 kg/m2. Smoking status information was classified as: smoker during pregnancy, quit at the beginning of pregnancy, ex-smoker (quit earlier than at the beginning of pregnancy) or never-smoker. The daily consumption of alcohol was recorded as grams of alcohol consumed daily and was subsequently categorized into two categories: ≤ 5 g/day and > 5 g/day. Food and mineral or vitamin supplement intake was also evaluated using a food-frequency questionnaire (FFQ) during pregnancy. The FFQ used was a version of Willett's questionnaire adapted for use in a Spanish population9. Regarding vitamin and mineral supplementation, information was collected on the intake of vitamin B6,vitamin B12, vitamin D,Ca,Fe,Mg and Zn supplements. The food frequency variables were categorized at the median for the analysis.

Chemical analysis

  1. Sample preparation

    Serum samples were prepared for ICP-MS analysis by a direct alkali dilution method. Briefly, samples were diluted 1:15-50 with an alkali solution consisting of 2% butanol (Honeywell Research Chemicals, Seelze, Germany), 0.05% EDTA (Sigma-Aldrich, St. Louis MO, USA), 0.05% Triton X-100 (SigmaAldrich),1% NH4OH (Romil,Cambridge,UK) and 20 µg/g of the internal standards Sc,Ge and Rh. Finally, the samples were vortexed and analyzed.

  2. Instrumentation and analysis

    An inductively coupled plasma mass spectrometry system (Agilent 7700x ICP-MS, Agilent Technologies, Tokyo, Japan) equipped with an octopole reaction system, employing a collision/reaction cell, was used for measuring concentrations of six elements in human serum: Ca, Mg, Fe, Cu, Zn, and Se. Two modes were used for the analysis of Se: collision mode using helium and reaction mode using hydrogen gas. The hydrogen mode has certain benefits over using helium for Se analysis. Hydrogen removes doubly charged species, and due to its smaller size, it can increase the sensitivity of Se measurements.

  3. Quality control

    The level of detection for each element was estimated as three times the standard deviation (SD) of blanks (alkali solution) and a signal-to-noise ratio of 3. The accuracy and precision of each measurement was verified by analyzing commercially available reference materials: Seronorm human serum lot MI0181 (SERO, Billingstad, Norway) and NIST animal serum SRM 1598a (National Institute of Standards and Technology, Gaithersburg MD, USA).

    Blanks and reference materials were treated along with the serum samples collected and analyzed at the begin ning, in the middle and at the end of each run. Metal concentrations were corrected according to the variations in the three daily measurements of the Seronorm reference material. The correction was performed by adding to each measurement the difference between the daily mean of the reference measures and the overall mean of the reference measures. These measurements were performed at the Institute of Environmental Medicine at the Karolinska Institutet (Sweden).

Statistical analysis

Descriptive statistics were calculated for each metal. A bivariate analysis was carried out between each metal and the defined variables overall and by cohort. Student's t-test or analysis of variance was performed depending on the number of categories of the independent variable (see Appendix online). Multivariable linear regression models were built to estimate the association between serum levels of metals and the independent variables. The variables with a p ≤ 0.20 in the bivariate analysis were initially included in the models and those that were statistically significant (p < 0.05) were kept in the model, following a backward selection procedure. Statistical analyses were carried out using IBM SPSS for Windows, version 24.

We also performed permutational multivariate analysis of variance to test the null hypotheses of no association between multivariate variance in serum levels of Ca, Cu, Fe, Se and Zn, and socio-demographic and dietary variables. To this end, two multivariate linear models were built: the first only considered dietary explanatory variables; and the second, only socio-demographic explanatory variables. The method for variable selection that we applied was stepwise backward regression. Finally, we also built a joint (socio-demographic-dietary) model with the variables selected in the former step and then applied the method of Borcard et al.10 to partition multivariate variance in serum levels of the metals into components explained by socio-demographic factors, by dietary factors, and by a shared fraction.


We analyzed data from serum samples of 1279 women (51.3% from the Valencia cohort). Sample size, percentages and missing values of all the variables are shown in Table 1. Overall, 92% had been born in Spain, 61.3% were 30 years old or older, with a mean age of 30.7 years (31.3 and 30.1 years for the Gipuzkoa and Valencia cohorts respectively), 50.1% lived in urban areas, 77.2% had secondary or university studies, 78.5% were active workers, 51% belonged to the lowest social class and 55% were primipara. In relation to other variables, 24.7% had excess body weight, 17.4% smoked during pregnancy, 14.9% had smoked but quit before pregnancy and 1.1% consumed more than 5 g of alcohol per day.

Table 1. Maternal anthropometric, socio-demographic, and dietary characteristics. 

Variable Categories Meana N % SDa Missing data
1279 100
Socio-demographic variables
Cohort Gipuzkoa 623 48.7 0
Valencia 656 51.3
Season Spring 332 26.0 0
Summer 350 27.4
Autumn 303 23.7
Winter 294 23.0
Country of birth Spain 1175 91.9 0
Other 104 8.1
Age (years) 30,7a 4.1a
Age-cat (years) < 25 84 6.6 0
25-29 412 32.2
30-34 570 44.6
≥35 213 16.7
Residence Urban 636 50.1 10
No urban 633 49.9
Physical activity before pregnancy < 1 hour/week 768 60.0 16
1-3 hours/week 316 24.7
>4 hours/week 179 14.0
Social class No manual 629 49.2 0
Manual 650 50.8
Educational level Up to Primary 291 22.8 2
Secondary 511 40.0
University 475 37.2
Mother's smoking habit Never-smoker 526 42.3 37
Ex-smoker 315 25.4
Quit at the beginning of pregnancy 185 14.9
Smoker in pregnancy 216 17.4
Mother alcohol consumption ≤5 g/day 1249 97.7
>5 g/day 14 1.1
Mother alcohol consumption (g/day) 0.28a 1.1a
Parity Primipara 699 54.7 0
Multipara 580 45.3
Gestation week 13.10* 1.27*
BMI (kg/m2) Underweight 50 3.9 1
Normal weight 912 71.4
Overweight 225 17.6
Obese 91 7.12
Marital situation Live with father 1263 98.7 0
No live with father 16 1.3
Occupational status Employed 1002 78.5 2
Unemployed 275 21.5
Diet variables
Calories (KJ/day) ≤2000 516 40.3 0
> 2000 747 58.4
Proteins (g/day) ≤99.50 461 36.5 16
>99.50 802 63.5
Fat (g/day) ≤89.5 726 57.5 16
>89.5 537 42.5
Carbohydrates (g/day) ≤250.50 687 54.4 16
>250.50 576 45.6
Dairy products (g/day) ≤455 676 53.5 16
>455 587 46.5
Red meat (g/day) ≤53.50 399 31.6 16
>53.50 864 68.4
White meat (g/day) ≤30.45 551 43.6 16
>30.45 712 56.4
Fish (g/day) ≤71 698 55.3 16
>71 565 44.7
Cereals and pasta (g/day) ≤90.5 723 57.2 16
>90.5 540 42.7
Legumes (g/day) ≤50 561 44.4 16
>50 702 55.6
Potatoes (g/day) ≤61.5 288 47.0 667
>61.5 324 52.9
Bread (g/day) ≤42 571 45.2 16
>42 692 54.8
Supplement variables
Supplement Vit B12 No 354 27.7 0
Yes 925 72.3
Supplement Vit B6 No 888 69.4 0
Yes 391 30.6
Supplement Vit D No 962 75.2 0
Yes 317 24.8
Supplement Ca No 915 71.5 0
Yes 354 28.5
Supplement Fe No 836 65.4 0
Yes 442 34.6
Supplement Mg No 903 70.6 0
Yes 376 29.4
Supplement Zn No 903 70.6 0
Yes 376 29.4

BMI: Body mass index; SD: standard deviation.

aMean and standard deviation.

Sample size, missing data, mean, SD, minimum, maximum and percentiles (25th, 50th and 75th) for each metal are presented in Table 2 by cohort. Mean and standard deviation of the levels were 94030.7 ± 4278.0 µg/L for Ca, 1614.8 ± 279.5 µg/L for Cu, 1113.9 ± 325.7 µg/L for Fe, 17032.5 ± 1006.4 µg/L for Mg, 79.56 ± 9.64 µg/L for Se and 642.1 ± 110.8 µg/L for Zn.

Table 2. Concentrations of Ca, Cu, Fe, Mg, Se and Zn (µg/L) in serum samples during the first trimester of pregnancy by cohort and the whole sample. t-test independent samples analysis. 

Cohort N Missing data Mean SD Min P25 P50 P75 Max p
Ca (µg/L) Gipuzkoa 620 3 93748.46 5176.31 77547.12 90629.34 93249.86 96037.01 129211.7
Valencia 656 0 94297.40 3186.93 86610.39 92255.24 94092.87 96138.34 113686.1
Total 1276 3 94030.67 4278.00 77547.12 91648.91 93668.1 96103.3 129211.7 0.022a
Cu (µg/L) Gipuzkoa 622 1 1620.35 291.36 520.29 1438.82 1604.02 1791.99 3004.92
Valencia 656 0 1609.62 267.84 642.42 1438.86 1612.59 1783.43 2563.56
Total 1278 1 1614.84 279.48 520.29 1438.74 1608.887 1787.08 3004.92 0.493
Fe (µg/L) Gipuzkoa 622 1 1113.00 319.78 160.64 889.54 1099.11 1289.85 2369.89
Valencia 654 2 1114.88 331.40 234.16 880.62 1101.78 1325.09 2514.47
Total 1276 3 1113.96 325.66 160.64 888.30 1099.46 1304.38 2514.47 0.918
Mg (µg/L) Valencia 656 0 17032.46 1006.40 13665.48 16363.53 17064.49 17700.25 20297.71 -
Se (µg/L) Gipuzkoa 623 0 77.07 10.62 37.99 70.17 76.01 82.64 145.34
Valencia 656 0 81.93 7.92 48.45 76.35 81.27 86.40 115.12
Total 1279 0 79.56 9.64 37.99 73.61 79.05 85.15 145.34 < 0.001a
Zn (µg/L) Gipuzkoa 561 59 673.79 102.80 307.75 601.87 666.28 730.34 1098.17
Valencia 629 27 613.84 110.17 349.81 544.63 600.48 667.37 1390.13
Total 1190 86 642.10 110.83 307.75 569.20 633.45 705.78 1390.13 < 0.001a

P: percentile; SD: standard deviation.

aSignificant p-values.

Table 3 summarizes the results of multiple linear regression models for each metal. Ca levels were related to daily white meat consumption (p = 0.026). Higher levels of Cu were related to excess body weight (p < 0.001), low social class (p = 0.003) and being multipara (p < 0.001). On the other hand,lower levels of Cu were related to bread intake of less than 42 g /day (p = 0.013) and residence in Gipuzkoa, after adjustment for the other variables. With reference to Se, higher levels were related to residence in Valencia (p < 0.001), having a university degree (p = 0.001),taking B6 vitamin supplements (p = 0.006) and a fish intake of more than 71 g/day (p = 0.008), after adjustment for the other variables. Women born outside Spain showed lower Se levels (p < 0.001). In regard to Fe, lower levels were related to having excess body weight (p = 0.021) or obesity (p < 0.01) and taking vitamin B12 supplements (p = 0.006). Higher levels of Mg were associated with being over 35 years old (p = 0.001), high social class (p = 0.044), primiparous status (p = 0.002) and low daily bread (p = 0.009) and legume (p = 0.020) intake, after adjustment for the other variables.

Table 3. Linear regression model for association between levels of Ca, Cu, Se, Mg, Fe, Zn and sociodemographic and dietary variables. 

Dependent variable Independent variables β (95%CI) p
Ca Cohort (Gipuzkoa vs. Valencia) 216.22 (−24.02; 456.61) 0.078
White meat consumption (≤30.45 g/day vs. >30.45 g/day) 549.44 (65.40; 1033.48) 0.026
Cu Cohort (Gipuzkoa vs. Valencia) − 29.96 (−49.36; −10.59 0.002
BMI (kg/m2)
18.5-25 -
< 18.5 −69.98 (−143.18; 11.21) 0.094
25< 30 102.62 (63.26; 141.99) < 0.001
≥35 170.40 (111.71; 229.08) < 0.001
Social class (no manual vs. manual) 45.32 (15.14; 75.50) 0.003
Parity (primipara vs. multipara) 92.31 (62.43; 122.18) < 0.001
Bread consumption (≤42 g/day vs. >42 g/day) −49.35 (−88.19; −10.52) 0.013
Se Cohort (Gipuzkoa vs. Valencia) 4.69 (3.40; 5.98) < 0.001
Country of birth (Spain vs. Other) −3.27 (−5.17; −1.40) 0.001
Educational level
Primary -
Secondary 0.99 (−0.36; 2.34) 0.149
University 2.29 (0.89; 3.69) 0.001
Fish consumption (≤71 g/day vs. >71 g/day) 1.29 (0.26; 2.33) 0.014
B6 vitamin supplementation (no vs. yes) 1.88 (0.54; 3.22) 0.006
Fe Cohort (Gipuzkoa vs. Valencia) 31.96 (−7.08; 71.00) 0.109
BMI (kg/m2)
18.5-25 -
< 18.5 56.37 (−36.17; 148.92) 0.232
25< 30 −55.48 (−102.72; −8.24) 0.021
≥35 −143.82 (−213.61; −73.93) < 0.001
B12 vitamin supplementation (no vs. yes) −60.93 (−104.61; −17.24) 0.006
Mg Age (years)
25-29 -
< 25 47.95 (−320.39; 224.50) 0.730
30-34 19.35 (−164.78; 203.07) 0.836
>35 423.22 (167.52; 678.93) 0.001
Social class (no manual vs. manual) −163.73 (−323.15; −4.31) 0.044
Parity (primipara vs. multipara) −256.18 (−421.60; −90.76) 0.002
Bread consumption (≤42 g/day vs. >42 g/day) −241.70 (−423.59; −59.82) 0.009
Legumes consumption (≤50 g/day vs. >50 g/day) −182.36 (−335.97; −28.76) 0.020
Zn Cohort (Gipuzkoa vs. Valencia) −28.21 (−34.60; −21.82) < 0.001
Age (years)
25-29 -
< 25 −4.89 (−31.07; 21.28) 0.714
30-34 −1.46 (−15.59; 12.66) 0.839
>35 −20.57 (−38.80; −2.34) 0.027
Educational level
Primary -
Secondary 4.35 (−11.72; 20.41) 0.596
University 17.85 (0.89; 34.80) 0.039

BMI: body mass index; 95%CI: 95% confidence interval.

Finally, higher levels of Zn were related to having a university degree (p = 0.039) and being from the Gipuzkoa cohort (p < 0.001), and lower levels of Zn were related to being over 35 years old (p = 0.027), after adjustment for the other variables.

Table 4 shows that the multivariate variance in serum levels of Ca, Cu, Fe, Se and Zn was associated with body mass, parity, educational level and country of origin (socio-demographic model) and also with white bread and vitamin B6 intake (dietary model). BMI and country of origin emerge as the most important among the explanatory variables considered. Although the influence of both socio-demographic conditions and diet on serum levels appears to be rather modest, the partitioning of variance in the joint model (Fig. 1) suggests that, overall, socio-demographic conditions are more influential than diet itself. Table 5 summarizes the results of previous studies.

Table 4. PERMANOVA. The response or outcome, which is constituted by multivariate variation in serum levels of Ca, Cu, Fe, Se and Zn (for both cohorts, Gipuzkoa and Valencia), is explained in terms of either a purely diet model or in terms of a purely socio-demographic model. Adjusted sums of squares and permutation-based p-values are reported. 

Variable Df SS R2 F p
Diet model
White bread intake 1 40.3 0.0075 8.20 0.001
Vitamin B6 intake 1 33.6 0.0063 6.84 0.001
Residual 1069 5246.0 0.9796 - -
Total 1071 5355.0 1.0000 - -
Socio-demographic model
Body mass index 1 61.1 0.0114 12.57 0.001
Parity 1 36.2 0.0068 7.45 0.001
Education level 2 28.4 0.0053 2.93 0.003
Country of origin 3 55.5 0.0104 3.81 0.001
Residual 1064 5166.1 0.9647 - -
Total 1071 5355.0 1.0000 - -

Figure 1. Venn diagram showing how multivariate variation in serum levels of Ca, Cu, Fe, Se and Zn was partitioned among a diet component and a socio-demographic component. Numbers are R2 values (%). The diet component includes white bread intake and vitamin B6 intake. The socio-demographic component includes body mass index, parity, education level and country of origin. 

Table 5. Trace elements concentrations in serum samples from women in different studies. 

Author Year Country Sample size Mean (SD)
Ca (µg/L) Sukonpan12 2005 Thailand 40 97000 (7000)
Ainy13 2006 Iran 48 92000 (6000)
Punthumapol15 2008 Thailand 36 89900 (3100)
Tong16 2010 China 90 21600
Kanagal14 2014 India 60 89700 (6900)
Arun7 2017 Nepal 35 95900 (6200)
Mg (µg/L) Sukonpan12 2005 Thailand 40 20700
Li19 2010 China 100 9800
Kanagal14 2014 India 60 15700 (7200)
Arun7 2017 Nepal 35 20300 (1600)
Punthumapol15 2008 Thailand 36 20400 (1900)
Cu (µg/L) Awadallah21 2004 Jordan 52 1750 (420)
Zhang23 2013 China 2380 1026
Jariwala20 2014 India 42 1614 (295)
Choi22 2016 Korea 245 1650
Polanska24 2017 Poland 539 1980 (570)
Zn (µg/L) Awadallah21 2004 Jordan 52 770 (160)
Izquierdo26 2007 Spain 159 654 (12.9)
Zhang23 2013 China 2380 920
Jariwala20 2014 India 42 514 (149)
Shen28 2015 China 1447 900
Choi22 2016 Korea 245 570
Khoushabi27 2016 Iran 60 749
Polanska24 2017 Poland 539 910 (270)
Liu34 2017 China 1400 740
Fe (µg/L) Awadallah21 2004 Jordan 52 690 (260)
Zhang23 2013 China 2380 900
Jariwala20 2014 India 42 1132 (519)
Shen28 2015 China 1447 800
Khoushabi27 2016 Iran 60 744
Liu34 2017 China 1400 1315
Se (µg/L) Kantola31 2004 Finland 216 81 (27)
Izquierdo26 2007 Spain 159 99.59 (21.7)
Ejezie32 2012 Nigeria 120 112.3
Jariwala20 2014 India 42 70 (15)
Choi22 2016 Korea 245 94
Liu34 2017 China 1400 77.6

SD: standard deviation.


We analyzed trace element levels in pregnant women in their first trimester of pregnancy. Our results reveal that the main variables related to these levels are BMI, social class and cohort. According to Abbassi-Ghanavati et al.11, serum Ca between 88,000 µg/L and 106,000 µg/L can be considered normal and our results fall within this range (94,030.7 µg/L). Further, the Ca levels in our sample of pregnant women are in line with those observed in several previous studies7,12,13 (with values of 95,900 µg/L, 97,000 µg/L and 92,000 µg/L respectively). On the other hand, they are a little higher than those obtained by Kanagal et al.14 (89,700 µg/L), and Punthumapol and Kittichotpanich15 (89,900 µg/L), and much higher than those found in a study carried out in China by Tong16 (21,600 µg/L). Some studies have shown Ca levels to be related to body weight, levels being significantly lower in those with excess body weight,17,18 but we did not find any such relationship in our study. Regarding age, unlike other authors,18 we found an association close to statistical significance, levels decreasing with increasing age (p = 0.077).

Regarding Mg, our results (17,000 µg/L) are within the normal range for Mg of between 16,000 µg/L and 22,000 µg/L11. The levels we observed were similar to those reported by Kanagal et al.14 (15,700 µg/L) and slightly lower than those observed by Punthumapol and Kittichotpanich15 (20,400 µg/L), Arun et al.7 (20,300 µg/L) and Sukonpan and Phupong12 (20,700 µg/L). In contrast, a study carried out in China by Li19, showed levels of serum Mg well below those obtained in this study (9800 µg/L).

The mean Cu level in our study population (1615 µg/L) was within the normal range of between 1120 µg/L and 1990 µg/L11. Similar levels have been observed in other studies20-22 (1614 µg/L, and 1650 µg/L and 1750 µg/L, respectively). On the other hand, Cu levels considerably different from ours have been observed in China23 and in Poland24 (1026 µg/L and 1980 µg/L, respectively). Wilson et al.25 reported significantly higher levels in obese women than underweight, normal weight or overweight women, as in our study. We found a trend related to BMI (with the highest levels in obese women), while others have observed an opposite trend.17

Again, in the case of Zn, the levels we observed (642 µg/L) lie within the normal range of between 570 µg/L and 880 µg/L.11 These results were in agreement with those found in previous studies in Spain26 and Iran27 (654 µg/L and 749 µg/L, respectively). Studies carried out in China23,28 and Poland24 showed higher levels (920 µg/L, 900 µg/L and 910 µg/L, respectively), while studies carried out in Korea22 and India20 showed lower levels (570 µg/L and 514 µg/L respectively). We found an association between Zn and educational level, which could be an indicator of social class. Nevertheless, other authors did not find this pattern29.

As for Se, our results were consistent with studies carried out in Spain30 and Finland31 with reported values of 81 µg/L, while a study from India20 found lower values of 70 µg/L. On the other hand, higher values were found in Korea,22 Spain26 and Africa32 (94 µg/L, 99.59 µg/L and 112.3 µg/L, respectively). In our study, Se levels appear to be related to fish consumption, which is consistent with other research findings.33

Fe levels between 720 µg/L and 1430 µg/L can be considered normal,11 and our results are in that range (1114 µg/L). Similar results have been reported in China23 and India20 (900 µg/L and 1132 µg/L respectively), while some studies have shown slightly lower values21,27,28 (690 µg/L, 800 µg/L and 744 µg/L respectively), and another study in China,34 found values slightly higher than ours (1500 µg/L). We observed that being overweight or obese was associated with significantly lower serum Fe levels, a pattern that has been observed previously15,25.

Social inequalities in dietary habits have been widely described and indicate that the lowest social classes tend to have less balanced and less healthy diets. Pregnant women with lower social status have fewer healthy habits, including a less healthy diet, more harmful behaviors, and poorer monitoring during pregnancy. Indeed, the role of education as a social determinant of diet has been confirmed by several authors as the most robust independent predictor of healthy dietary habits.35

The strengths of the study are the sample size (n = 1279) and the fact that we gathered data on and adjusted models for a large number of factors in the first trimester, especially socio-demographic characteristics. Regarding its limitations, the descriptive and crosssectional character of the design means that we cannot ascertain causality in the associations observed, measurements were taken at only one point during pregnancy, and the spectroscopy measurements of the metals were element-specific not species-specific. Another limitation is that the information regarding some of the behavioral variables (smoking and alcohol consumption) was selfreported, an approach which may underestimate the prevalence of socially disapproved behaviors.


Our results indicate that the Ca, Cu, Se, Fe, Mg and Zn levels in both cohorts were adequate and within the ranges considered normal for pregnant women. More than half of the sample did not take supplements, except in the case of vitamin B12 (72%). Among the variables considered, those with the best explanatory value of the levels of these trace elements were anthropometric and socio-demographic variables and within these, BMI, social class, educational level and cohort. This study will serve as a basis for future research, in particular, to identify trends in these elements through pregnancy and their possible relationship with the later development of the child.

What is known about the topic?

Poor nutritional status is known to carry a higher risk of disease. This risk is greater during pregnancy since not only maternal health is at stake, but also the proper development of the fetus. Therefore the establishment and knowledge of reference values is of vital importance.

What does this study add to the literature?

This study provides relevant data on serum metal levels in pregnant women and their relationship with socio-demographic and anthropometric characteristics, supplementation during pregnancy and diet. The follow-up of clinical variables during pregnancy and the knowledge of socio-demographic conditions, supplementation, vitamins, minerals and diet of pregnant women are vital to ensure adequate care during pregnancy. This article will serve as a basis for future work assessing the evolution of these trace elements throughout pregnancy in order to improve perinatal outcomes and subsequent child development.

What are the implications of the results?

It is necessary to implement measures to intervene in healthy lifestyles (healthy diet, not smoking, avoiding a sedentary lifestyle, etc). as well as care and attention during pregnancy. A balanced diet must be sufficient to cover the needs in a way that it contains all nutrients (proteins, carbohydrates, fats, vitamins and minerals) in adequate amounts. The requirements of minerals (Ca, Cu, Fe, Mg, Zn, Se, etc). and vitamin (B12) are adequately covered by the consumption of raw fruit and vegetables, whole grains, raw olive oil and dairy products. On the other hand, it may be advisable to pay special attention to pregnant women with excess weight in relation to Fe control, as well as vitamin B12 supplementation.

Fish consumption, avoiding the most mercurycontaminated species (swordfish, shark, bluefin tuna), has a beneficial effect on health due to its high content in proteins of great nutritional value, omega-3 fatty acids, vitamin D and antioxidant substances (including Se). Considering that the variability of all these elements is mainly associated with socio-demographic and anthropometric variables, it is necessary to promote healthy habits and lifestyles, as well as self-care during pregnancy.

Transparency declaration

The corresponding author on behalf of the other authors guarantee the accuracy, transparency and honesty of the data and information contained in the study, that no relevant information has been omitted and that all discrepancies between authors have been adequately resolved and described.


We want to acknowledge all the INMA families for their altruistic participation in the study, without which it would have not been possible to carry out this work. We also wanted to thank Nerea Urbieta for her contribution to the work.


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Supplementary data

Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.gaceta.2021.07.006.

FundingThis study is part of the INMA (Infancia y Medio Ambiente) project. It was supported by grants from Instituto de Salud Carlos III (FIS-FEDER: 06/0867, 09/00090, 13/1944, 16/1288, 19/1338; Miguel Servet-FEDER: CP15/0025; Miguel Servet-FSE: MS15/0025, by the Council of Gipuzkoa (DFG15/009) and by the Health Department of the Basque Government.

Received: November 30, 2020; Accepted: July 29, 2021; pub: October 06, 2021

* Corresponding author. E-mail address: llop (S. Llop).

Editor in charge

Juan Alguacil

Conflicts of interest

We wish to confirm that there are no known conflicts of interest associated with this publication that could have influenced its outcome.

Authorship contributions

Study design: M. Begoña Zubero, S. Llop, M. Murcia, A. Molinuevo, F. Ballester, M. Levi, M. Lozano, L. Santa-Marina. Methodology: M. Begoña Zubero, L. Santa-Marina. Literature review: S. Llop, A. Irizar, F. Ballester, M. Levi, M. Lozano, M. Ayerdi. Data collection and clean-up: A. Irizar, M. Ayerdi. Data analysis and interpretation: M. Begoña Zubero, M. Murcia, A. Molinuevo. Writing the article: M. Begoña Zubero, S. Llop, F. Ballester, M. Levi, M. Lozano, L. Santa-Marina. Analysis and critical review: A. Irizar, M. Murcia, A. Molinuevo, M. Ayerdi. Final version approval: M.B. Zubero, S. Llop, A. Irizar, M. Murcia, A. Molinuevo, F. Ballester, M. Levi, M. Lozano, M. Ayerdi, L. Santa-Marina.

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