Characterization of selenium status of inhabitants in the region Ústi nad orlici,czech republic by inaa of blood serum and hair and fluorimetric analysis of urine

Selenium is an essential trace element and its isufficient status may cause serious health complications for both individuals and the whole populations. To investigate the selenium status of the subpop-ulation in northeastern Bohemia represented by the region ústí nad Orlicí, 253 serum, 469 urine, and 31 hair samples from 470 randomly selected volunteers between 6 and 65 yr of age have been analyzed for selenium concentration. Serum and hair Se were detected by instrumental neutron activation analysis (means: 55 ±11 Μg Se/L sera, 0.268 ±0.040 Μg Se/g hair). Urine Se was measured by fluorimetry (12 ±5 Μg Se/L urine) with coanalyses of Lyphocheck urine, SRM Urine 2670, and Seronorm urine for quality control of the method. Results proved significant age-dependent differences, but gender differences were not significant. The frequency plot of serum Se proved maximal frequencies in adults between 55 and 70 Μg Se/L and in children in the range 45–55 Μg Se/L. The same plots of urine Se for both age groups showed maximal frequency in the limits 8–15 Μg Se/L. All indices used (Se in serum, urine, and hair) confirmed mild to severe selenium deficiency in the population of the region.     

Increased serum copper and decreased serum zinc levels in children with iron deficiency anemia

In order to evaluate serum copper and zinc status in children with iron deficiency anemia (IDA), 60 children with IDA aged 1–14 yr and 64 healthy children as controls aged 1–14 yr were included the study. Serum copper levels were higher in children with IDA (189 ± 49 (Μg/dL) than those of controls (163 ± 37 Μg/dL) (p = 0.001). Serum zinc levels were lower in the patient group (109 ± 59 Μg/dL) than those of control subjects (135 ± 56 Μg/dL) (p = 0.017). In addition, there were statistically significant negative correlations between hematological parameters and serum copper levels in the patient group, but not in controls. No correlation between hematological parameters and serum zinc levels were found in both patient and control groups, except positive correlation between mean corpuscular volume (MCV) and serum zinc level in patients. It was concluded that at the time of managing children with IDA, zinc deficiency must be borne in mind and if necessary treatment should be initiated with zinc.     

Determination of copper and zinc levels in human hair

The Cu and Zn levels of both 607 men (1–85 y old) and 649 women (1–92 y old) were determined by atomic absorption spectrometry. Sex does not influence Cu (14.89±0.89 μg/g and 15.26±0.79 μg/g hair for males and females, respectively) and Zn contents (200.97±9.68 μg/g for men and 209.81±9.49 μg/g hair for women). Age influences Cu and Zn concentrations, but only significantly in females: Cu levels decrease over 60 y of age; whereas Zn levels increase significantly from age groups 2–5 to 20–40 years. Hair color influences Cu concentrations in both males and females. In males, white hair containes less Cu than black hair; in females, white hair's Cu levels are significantly lower than those of dark blond, red, light brown, and brown hair. There are no significant differences in Zn concentrations with respect to different hair colors, in either males or females.     

Serum extracellular superoxide dismutase activity as an indicator of zinc status in humans

The present study focused on whether serum extracellular superoxide dimutase (EC-SOD) activity can be used as a functional indicator of marginal zinc deficiency in humans. Subjects in this study were 444 healthy adults over 30 yr of age living a normal rural life in Kyunggi province, Korea. The mean dietary zinc intake of subjects obtained from one 24-h recall was 6.41 ± 4.35 mg and the average serum zinc concentration of the subjects was 11.06 ± 2.44 (μmol/L. Subjects were divided into three groups by serum zinc concentrations: adequate (serum zinc >10.7 (μmol/L), low (serum zinc 9.0–10.7 μmol/L), and very low (serum zinc <9.0 μmol/L) groups. A total of 50 subjects were selected from the three groups for analysis of EC-SOD activities. The EC-SOD activity of subjects increased with increasing serum zinc concentrations, and the activities of the three groups were significantly different as indicated by the Kruskal-Wallis test (p = 0.0239). Also, serum EC-SOD activities were significantly correlated with serum zinc concentrations (r = 0.289,p = 0.04). Serum EC-SOD activities, however, were not significantly correlated to the dietary zinc intakes. In conclusion, these results show that EC-SOD activities are decreased in subjects with low serum zinc concentrations and suggest that EC-SOD activity may be a functional indicator of zinc nutritional status in humans.     

Differences in the cellular zinc content and 5′-nucleotidase activity of normal and acrodermatitis enteropathica (AE) fibroblasts

The acrodermatitis enteropathica (AE) mutation affects zinc (Zn) metabolism in human fibroblasts. We hypothesize that the mutation affects the cell Zn content, which subsequently affects the activity of various zinc-dependent enzymes, such as 5′-nucleotidase. Therefore, normal and AE fibroblasts were grown in normal medium containing physiological levels of Zn (16 Μmol/L) for ∼24 h. The medium was replaced by normal medium (16 Μmol/L Zn), Zn-depleted medium (1.5 Μmol/L Zn), or Zn-supplemented medium (200 Μmol/L Zn) for another 24 h. Regardless of the Zn concentration of the growth medium, the AE fibroblasts contained significantly less Zn than normal fibroblasts grown in comparable medium. Nevertheless, growth of the fibroblasts in 200 Μmol/L Zn medium significantly increased the cell Zn content fourfold of both normal and AE fibroblasts. The activity of 5′-nucleotidase in the AE fibroblasts grown in 16 Μmol/L Zn or 1.5 Μmol/L Zn medium was also significantly lower than in normal fibroblasts. Changing the growth medium from 16 Μmol/L Zn to 1.5 Μmol/L Zn medium did not affect the activity of the enzyme in either genotype. Cells grown in 200 Μmol/L Zn medium exhibited threefold greater 5′-nucleotidase activity in AE fibroblasts, but had no affect on enzyme activity in normal cells. In summary, altering the cell Zn content of normal fibroblasts did not result in a significant change in their 5′ -nucleotidase activity. However, AE fibroblasts grown in 200 Μmol/L Zn medium exhibited recovery of their 5′-nucleotidase activity to normal levels. These results support the hypothesis that the AE mutation affects the cellular Zn content. The lower cell Zn content subsequently affects the activity of 5′-nucleotidase.     

Significance of serum trace element status in patients with rheumatic heart disease

It is known that certain trace elements can affect various heart diseases. In this study, we aimed to evaluate the changes in concentrations of certain serum trace elements in patients with chronic rheumatic heart disease (RHD). Serum analysis of selenium (Se), zinc (Zn), and copper (Cu) trace elements was assayed by atomic absorption spectrophotometry. RHD patients had significantly lower serum concentrations of Se and Zn than control subjects (p<0.05 and p<0.001, respectively). However, the serum Cu concentration was significantly higher in RHD patients than in controls (1.93±0.59 μg/L vs 1.06±0.29 μg/L; p<0.001). Similarly, the Cu/Zn ratio in RHD patients was higher than in control subjects (4.70±0.92 vs 1.68±0.45; p<0.001). Additionally, no significant correlation was found among these trace element concentrations and the functional capacity classes (p>0.05). RHD patients had decreased serum Se and Zn element concentrations and increased serum Cu element concentration. We suggest that Se and Zn deficiency might be contributory factors in the development of rheumatic heart disease, and a high Cu concentration and a high Cu/Zn ratio might reflect an ongoing inflammatory process in this disease.     

Changes in brain metallothionein and Zinc during development in transgenic mice

The developmental alterations in metallothionein (MT) proteins and zinc (Zn) were investigated in brains of two transgenic strains of mice. MT protein was measured by a cadmium binding assay and Zn by atomic absorption spectrophotometry. MT proteins were expressed at birth (day 1) both in MT-I overexpressing transgenic mouse (MT-I*) and MT-null (expressing only brain specific isoform, MT-III) transgenic mouse. MT proteins level (mainly MT-I) in MT-I* was 16.1 Μ-g/g at birth, and thereafter increased with age to a maximal adult level of 55.3 Μg/g (day 60). Zn level in MT-I* also increased from 8.43 Μg/g (day 1) to 20.7 Μg/g (day 60) with age. MT protein (MT-III) in MT-null mouse was 9.71 Μg/g at birth and remained relatively unchanged during development. Zn level in MT-null mouse at birth was 9.46 Μg/g and also remained unchanged during development. The similar alterations in MT isoforms and Zn in brain during development suggest that MT isoforms may act as a Zn binding protein.     

Effect of Therapy on Serum Zinc and Copper in Primary Complex of Children

Tuberculosis is a global problem especially in children, more so because of the difficulty in the diagnosis of the disease. There are about 0.5 million deaths every year by this disease, worldwide. There are some reports that, during the disease, there are changes in serum concentrations of zinc and copper, which reach towards normal levels during treatment. Such data for children are scarce, especially in reference to India. Our study indicates that there is an insignificant to significant rise in serum zinc levels in various age groups of children after antitubercular therapy (mean 61.89 ± 3.21 to 65.24 ± 3.60 μg/dl) and significant fall in serum copper levels in different age groups of children (mean 129.96 ± 3.18 to 124.91 ± 3.48 μg/dl). The Cu/Zn ratio also changed significantly from 2.11 ± 0.12 to 1.92 ± 0.12. Because evaluation of available diagnostic criteria for primary complex in children has been found to have high sensitivity, and although this criterion is presently not recommended for diagnosis of primary complex of children, further research can prove its utility in diagnosis and treatment of this disease.     

Evaluation of selenium supply and status of inhabitants in three selected rural and urban regions of the Czech Republic

Blood serum selenium of 65 men and hair selenium of 77 men from three regions of the Czech Republic (CR) were analyzed by neutron activation analysis, and 202 samples of urine from the same populations were analyzed for Se by the fluorimetric method to assess selenium status of these regions. Low status (53 μg Se/L of serum and 0.29 μg Se/g lyophilized hair as means) and very low urine selenium (8.7 μg/L urine) were detected. By these data, the CR is among the countries with the lowest Se intake. A comparison of studied regions is presented. Moreover, values of serum zinc were within the reference range, but mild to moderate deficiency in the supply of iodine was detected.     

Reversal of selenium and zinc deficiencies in chronic hemodialysis patients by intravenous sodium selenite and zinc gluconate supplementation

In six chronic dialyzed uremic patients, an intravenous sodium selenite (Se 50 μg during 5 wk and then 100 μg) and zinc gluconate (Zn 5 mg) supplementation was performed during 20 wk at each dialysis session three times weekly. Before supplementation, plasma Se and Zn, plasma and erythrocytes (RBC) antioxidant metalloenzymes glutathione peroxidase (GPX), and superoxide dismutase (SOD) were significantly decreased, whereas lipid peroxidation (as thiobarbituric acid reactants TBARs) was increased. To obtain a significative change in plasma selenium, we had to use an Se dose of 100 μg/dialysis session. Then, treatment-increased plasma Se (from 0.58 ±0.09 to 0.89±0.16 μmol/L) led to a repletion of RBC-GPX (from 29.6±6 to 43±5.8 U/g Hb) and increased plasma GPX levels (from 62±13 to 151±43 U/L). Plasma Zn and RBC-SOD did not vary significantly. The change of TBARs was not observed between wk 1 and 4. They decreased significantly between wk 4 (4.80±0.21μmol/L) and wk 20 (4.16±0.26 μmol/L). We noted a low correlation between TBARs and plasma GPX. A strong correlation was observed between Se and plasma GPX. The reversal of Se deficiencies should reduce oxidative damage observed in these patients.     

Zinc and copper in Indian patients of tuberculosis

The effectiveness and success of antitubercular therapy is mainly measured by identifying the organism in sputum. In certain patients, especially in geriatric patients, available tuberculosis tests are not satisfactory and do not provide enough information on the effectiveness of antitubercular therapy, as the symptoms might be confused with the existing symptoms of ongoing diseases. Therefore, 60 diagnosed and randomly selected patients with tuberculosis were included into this study. The patients with other associated diseases likely to influence serum copper and zinc were not included in the study. The estimations of serum copper and zinc were done in healthy volunteers and in tubercular patients before the start of treatment and after 4 wk of antitubercular treatment. The average plasma concentration of serum copper and zinc in healthy volunteers were 102±20 μg/dL and 96±18 μg/dL respectively. In tuberculosis patients, serum copper and zinc levels were 123.65±9.98 μg/dL and 64.14±3.97 μg/dL, respectively, before the start of treatment, which came down to 116.23±4.27 μg/dL and 74.31±3.60 μg/dL, respectively, after 4 wk of antitubercular treatment.     

Associations of Socio-demographic and Behavioral Variables with Hair Zinc of Vancouver Preschoolers

Marginal zinc deficiency (MZD) exists in children of industrialized societies and impairs growth and development. MZD is believed to be one of the most common deficiencies, even though there is no data available on its global prevalence. This is partly because of the lack of sensitivity and specificity of serum zinc, the most commonly used biomarker of zinc status, to detect MZD. In children, MZD is always accompanied by a decrease in hair zinc. This cross-sectional study was designed to explore if there are any associations between some socio-demographic and behavioral variables with hair zinc status in an attempt to understand the social determinants of MZD and the profile of at risk children. Our citywide survey (n = 719) indicated a mean hair zinc of 116 ± 43 μg/g with 17% below the cutoff. Logistic regression analysis indicated that age, maternal education, number of adults at home, being described as “eating unhealthy”, and Child Behavior Questionnaire scores of “activity level” as the significant predictors of hair zinc status. Our study provides important information on the hair zinc status of Vancouver preschoolers and some factors in children and their environment associated with hair zinc, which may help in better understanding of hair zinc as a biomarker of MZD.     

Maternal and neonatal scalp hair concentrations of zinc, copper, cadmium, and lead

Postpartum scalp hair samples from 82 term-pregnancy mother/neonate pairs were analyzed for their concentration of zinc (Zn), copper (Cu), cadmium (Cd), and lead (Pb), using inductively coupled plasma-mass spectrometry. Maternal and neonatal Zn concentrations had geometric means (and 99% confidence intervals) of 122.5 μg/g (117.9–131.5 μg/g) and 146.9 μg (141.5–156.7 μg/g) respectively. Corresponding Cu values were 18.4 μg/g (17.6–23.8 μg/g) and 6.7 μg/g (6.3–7.6 μg/g). Those of Cd were 0.49 μg/g (0.47–0.69 μg/g) in the mothers and 0.57 μg/g (0.55–0.86 μg/g) in the neonates. For Pb, they were 7.95 μg/g (7.60–9.32 μg/g) and 4.56 μg/g (4.39–5.56 μg/g). Cigaret smoking, despite its relatively low prevalence (19.5%), was associated with lower Zn and higher Cd and Pb concentrations and in lower Zn/Cd and Zn/Pb molar concentration ratios. Smoking also altered interelemental relationships, particularly those of Zn with Cd and Pb and those between Cd and Pb. Smoking frequency appeared to show negative dose-response effects on maternal and neonatal Zn concentrations, Zn/Pb molar concentration ratios, and birth weight. Mothers with a history of oral contraceptive (OC) usage had significantly higher Cu concentrations and lower Zn/Cu molar concentration ratios than nonusers, with the highest Cu concentrations and lowest Zn/Cu values being associated with third-generation OCs. No similar effects were elicited in the respective neonatal Cu concentrations. Neither alcohol consumption nor prenatal supplementation with iron and/or folic acid had discernible effects on the maternal or neonatal elemental concentrations. The data from this study suggest that in a given population of term-pregnancy mothers and neonates, significant interindividual variations in hair trace element concentrations can occur, irrespective of commonality of general environment, and that lifestyle factors, including cigaret smoking and OC usage history, can be significant contributory factors to such variations. The data are discussed in relation to the effects of smoking-associated exposure to Cd and Pb exposure on Zn availability for placental transfer, as well as on the quantitative maternal Zn supply levels to the fetus resulting from the known tendency of smokers to have lower dietary intakes of Zn. The higher Cu concentrations in OC users are discussed in relation to altered Cu metabolism, characterized by increased synthesis of the Cu-binding protein, ceruloplasmin, as an acute-phase antioxidant response to altered lipid profile and increased lipid oxidation.     

Serum Levels of Cu,Se, and Zn in Adult Rural/Urban Residents in Ghana: Paradigm Shift?

Deficiencies in Cu, Se, and Zn impair one or more biochemical functions, and excess are associated with toxicity. Baseline studies on the Ghanaian population are scanty. The study was undertaken to determine whether significant rural/urban differences in the serum levels of Cu, Se, and Zn did exist. Forty males/60 females from rural and 50 males/50 females from urban Ghanaian communities were sampled. Serum Cu, Se, and Zn were determined using flame atomic absorption spectrometry. Cu level for rural and urban subjects was 997 ± 333 and 979 ± 290 μg/L, respectively (p = 0.68). However, Cu levels were significantly higher in the rural females (1,063 ± 367 μg/L) than the rural males (898 ± 249 μg/L; p = 0.0085). Se levels for rural/urban subjects were 97 ± 36 and 87 ± 31 μg/L, respectively (p = 0.03). Zn levels in the rural/urban subjects were 312 ± 218 and 150 ± 102 μg/L, respectively (p = 0.002). Additionally, Zn was significantly higher in rural females (428 ± 204 μg/L) than the urban females (166 ± 103 μg/L; p = 0.0002). Finally, Zn was significantly higher in rural females (428 ± 204 μg/L) than males (172 ± 116 μg/L; p = 0.0028). In conclusion, Cu, Se, and Zn were higher in the rural group compared to the urban group, and the generally low Zn levels were confirmed in another cohort follow-up study.     

Zinc Nutritional Status in Adolescents with Down Syndrome

Studies have evidenced that zinc metabolism is altered in presence of Down syndrome, and zinc seems to have a relationship with the metabolic alterations usually present in this syndrome. In this work, the Zn-related nutritional status of adolescents with Down syndrome was evaluated by means of biochemical parameters and diet. A case–control study was performed in a group of adolescents with Down syndrome (n = 30) and a control group (n = 32), of both sexes, aged 10 to 19 years. Diet evaluation was accomplished by using a 3-day dietary record, and the analysis was performed by the NutWin program, version 1.5. Antropometric measurements were performed for evaluation of body composition. The Zn-related nutritional status of the groups was evaluated by means of zinc concentration determinations in plasma and erythrocytes, and 24-h urinary zinc excretion, by using the method of atomic absorption spectroscopy. The diet of both groups presented adequate concentrations of lipids, proteins, carbohydrates, and zinc. The mean values found for zinc concentration in erythrocytes were 49.2 ± 8.5 μg Zn/g Hb for the Down syndrome group and 35.9 ± 6.1 μg Zn/g Hb for the control group (p = 0.001). The average values found for zinc concentration in plasma were 67.6 ± 25.6 μg/dL for the Down syndrome group and 68.9 ± 22.3 μg/dL for the control group. The mean values found for zinc concentration in urine were 244.3 ± 194.9 μg Zn/24 h for the Down syndrome group and 200.3 ± 236.4 μg Zn/24 h for the control group. Assessment of body composition revealed overweight (26.7%) and obesity (6.6%) in the Down syndrome group. In this study, patients with Down syndrome presented altered zinc levels for some cellular compartments, and the average zinc concentrations were low in plasma and urine and elevated in erythrocytes.     

Differences in selected zinc metabolism parameters in obese and normal-weight hypertensive patients following treatment with spironolactone

Twenty-three hypertensive outpatients aged 18–53 yr (average: 39.8±10.4 yr) were classified into two groups according to body mass index (BMI). Six patients exceeded the BMI limit, set at 30 kg/m². All were treated with 100 mg/d spironolactone and were subject to before and after measurements of their arterial pressure, efflux rate constants of zinc from lymphocytes (total ERCt-Zn and ouabain-dependent ERCos-Zn), serum zinc (Zn-s), lymphocyte zinc (Zn-l), serum aldosterone (Ald-s), plasma renin activity (PRA), serum sodium (Na-s), and potassium (K-s). After 7 d of spironolactone treatment, the ERCt-Zn change in normal-weight patients was +0.78±0.57, and −0.22±0.69 in obese patients. In the same manner, the change of ERCos-Zn was +0.59±0.94 and −0.025±0.32 in normal and obese patients, respectively. Serum Zn was increased in normal-weight patients but remained unchanged in the obese. The initial lymphocyte zinc values were significantly lower in obese patients, but increased up to normal values after spironolactone treatment.     

Change in apparent and true absorption and retention of dietary zinc with age in rats

Two groups of 16 rats each were fed the same diet with 12.9 ppm Zn. Nine days after each animal was injected with⁶⁵Zn for assessing fecal zinc of endogenous origin, zinc intake and excretion were determined for a six-day period at the age of about five (group I) and nine (II) weeks. At mean growth rates of 5.1 and 5.2 g/day, food consumption per gram of gain was 2.01 g in group I vs 2.86 g in II. Overall, zinc retention amounted to 21 vs 25 μg Zn/g of gain. Apparent absorption averaged 92 vs 74% of Zn intake (132 vs 189 μg/day), while true absorption averaged 98 vs 92%. It was concluded that endogenous fecal zinc excretion was limited to the indispensable loss (F _em) in group I (7 μg/day), while it exceeded this minimum loss in group II (33 μg/day). True retention, which reflected total zinc utilization (true absorption times metabolic efficiency), was derived from apparent absorption plusF _em (11 μg/day for group II according to the greater metabolic body size of the rats). It averaged 98% of Zn intake in group I vs 80% in group II. The mean metabolic efficiency was 100% vs 87%. The conclusion was that these marked differences between age groups in utilizing the dietary zinc reflected the efficient homeostatic adjustments in absorption and endogenous excretion of zinc to the respective zinc supply status.     

Zinc and copper concentrations in serum from Spanish women during pregnancy

A cross-sectional study of serum zinc (Zn) and copper (Cu) levels in 31 healthy pregnant women and 51 healthy, nonpregnant controls living in the Mediterranean area of Granada, Spain, was performed. The subjects were divided into two groups: Group A, consisted of pregnant women in three categories according to the trimester of pregnancy, and Group B consisted of nonpregnant women acting as controls. In pregnant women, serum Zn levels were found from 0.300-1.340 mg/L and serum Cu from 0.936-2.304 mg/L, whereas in the nonpregnant women group, the mean serum levels were 0.947 ±0.265 mg/L for Zn and 1.092 ±0.365 mg/L for Cu. Serum Zn progressively decreased with gestation. Mean Zn levels were 0.829 ±0.253, 0.846 ±0.329, and 0.620 ±0.142 mg/L, corresponding to the first, second, and third trimesters of pregnancy, respectively. Serum Zn concentrations were significantly lower in pregnant women as compared to controls: 0.712 ±0.236 mg/L vs 0.947 ±0.265 mg/L, respectively (p < 0.05). In contrast, Cu levels increased with period of gestation from 1.053 ±0.498 mg/L in the first trimester to 1.616 ±0.304 mg/L in the second and 1.689 ±0.344 mg/L in the third. Serum Cu levels in the second and third trimesters of pregnancy were significantly higher (p < 0.05) than those determined during the first trimester and for nonpregnant controls. Both Zn and Cu during pregnancy did not appear to be dependent on the subject’s age (p > 0.05).     

Determination of iodide amounts in urine and water by isotope dilution analysis

Urinary iodide and iodine in drinking water were determined in 318 healthy children aged 0 to 18 yr living in Izmir and environmental rural and urban areas in the western part of Turkey. The method is based on substochiometric isotope dilution analysis. Iodide was precipitated by substoichiometric amounts of AgNO₃. Iodide-131 was used as a tracer. Electrophoresis was performed to separate Ag¹³¹I from excess¹³¹I-. The Ag¹³¹I zone was cut off the electrophoresis paper and counted with a Nal(Tl) scintillation counter. Count rates were plotted versus added KI concentrations. The unknown iodide amount was found by using these linear plots. Iodide concentration ranges were within 1.8 –100.45 Μg/L in the analyzed drinking water samples. The mean value was 44.14 ±17.33 Μg/L and the median was 58.08 Μg/L. Urinary iodide concentration ranges were 0.22 –142.22 Μg/L. The median of the distribution was 37.71 Μg/L and the mean was 40.30 ±24.05 Μg/L. The results show that the examined area suffers moderate iodine deficiency.     

Hair and serum calcium, iron, copper, and zinc levels during normal pregnancy at three trimesters

The hair and serum levels of calcium, iron, copper, and zinc levels were measured in a group of 70 healthy pregnant women and in 66 age-matched healthy controls living in the Tianjin city of the People’s Republic of China. The study subjects were classified into three subgroups according to gestational age. The hair concentrations were measured by X-ray fluorescence spectrometry, whereas those in sera were established by atomic absorption spectrometry. The hair concentrations of Ca, Fe, Cu, and Zn in the three groups of gravida were lower or significantly lower than those in controls. In sera, the differences did not show statistical significance in most cases. A deficiency of calcium was observed in subjects in the last trimester of gestation. This reinforces the importance of supplementation with calcium during pregnancy.