Biol Trace Elem Res DOI 10.1007/s12011-015-0262-2
Concentration of Selected Metals in Whole Blood, Plasma, and Urine in Short Stature and Healthy Children Maria Klatka & Anna Błażewicz & Małgorzata Partyka & Witold Kołłątaj & Ewa Zienkiewicz & Ryszard Kocjan
Received: 2 December 2014 / Accepted: 29 January 2015 # Springer Science+Business Media New York 2015
Abstract The short stature in children is defined as height below the third percentile from the mean for age and gender. This problem affects about 3 % of young people. More than 20,000 children in Poland have problems with short stature. There is not much information available in the literature on the study of metals in blood, plasma, and urine in children with short stature. The study was conducted on a group of 56 short stature Polish children and 35 healthy children. The content of metals was determined using high-performance ion chromatography and inductively coupled plasma mass spectrometry methods. The study revealed significant differences between the content of selected metals in body fluids between a short stature group and healthy children. There were significant differences in the Fe, Cu, and Ni concentrations between the groups with respect to the hormonal therapy. There were no significant differences between the groups with respect to the area where the children lived. The results showed no statistically significant differences between metal concentration and
M. Klatka (*) : W. Kołłątaj Department of Pediatric Endocrinology and Diabetology, Medical University of Lublin, Gębali 6, 20-093 Lublin, Poland e-mail: [email protected] A. Błażewicz : R. Kocjan Department of Analytical Chemistry, Medical University of Lublin, Chodźki 4A, 20-093 Lublin, Poland M. Partyka Department of Jaw Orthopedics, Medical University of Lublin, Karmelicka 7, 20-081 Lublin, Poland E. Zienkiewicz Department of Pediatric Neurology, Medical University of Lublin, Gębali 6, 20- 093 Lublin, Poland
age, body weight, and height. The study demonstrated statistically significant differences between the content of metals in body fluids in short stature children compared with the healthy children. It seems that the difference in the concentration of certain elements may also be the result of growth hormone therapy and the interaction between various metals. Both the alterations in the content of metals and their mutual interactions may play an important role in the pathogenesis of short stature children. Keywords Short stature in children . Metals . Body fluids . Ion chromatography
Introduction The short stature in children is defined as the height below the third percentile from the mean for age and gender. This problem affects about 3 % of the population. In the USA, short stature affects more than one million children aged 4–15 years, and in Europe, more than half a million children of similar age. More than 20,000 children in Poland have problems with short stature. In Poland, about 3,000 children are treated with a growth hormone [1]. Children with this condition reveal no identifiable systemic or endocrine disease but have short stature and a delayed bone age [2]. Most short stature children are not affected by hormonal disorders or genetically conditioned syndromes [3, 4]. Short stature due to endocrine factors affects no more than 5 % of short stature children, while short stature due to disturbances in somatotrophin axis affects about 3 % of such children (1:4000 to 1:10,000). According to Ruz, growth is a complex process in which several nutritional and nonnutritional factors play roles and interact, but the extent and
Klatka et al.
consequences of such interactions are not still completely understood [5]. There is a strong evidence to support the concept that the outcome of human pregnancy is significantly affected by the nutritional status of the mother. The consumption of “poor diets” has been associated with an increased risk for pregnancy complications, including gross structural birth defects, prematurity, low birth weight, and an increased risk for neurobehavioral and immunological abnormalities after birth [6]. Our knowledge of the amount of trace elements in human tissues and fluids has increased significantly due to the recent advances in analytics. Analytical methods can be employed in the study of the relation between the basic composition of body fluids and tissues, and the pathological conditions and general nutritional status in humans. The content of essential elements and toxic metals in human tissues and organs has been studied by many authors [7, 8]. Heavy metals are known to cause a variety of health problems/diseases/injury, and mechanisms of their toxicity have been persistently investigated [9, 10]. The elements studied in the presented study have been divided into four groups [11]: (a) metals of greatest toxicological significance (arsenic, cadmium, lead, mercury, and uranium) that are widespread in the human environment, (b) essential trace metals (chromium, cobalt, manganese, selenium, and zinc), (c) a group of elements of interest to biological sciences (nickel and vanadium), and (d) a group of elements that may be applied in pharmacology (aluminum, gallium, and lithium).
Material and Methods Sample Preparation Diagnostic tests and treatment were performed at the Department of Pediatric Endocrinology and Diabetology at the Medical University in Lublin, southeast Poland. Informed consent was obtained from the parents. The study was approved by the Bioethical Committee at the Medical University in Lublin. The study material consisted of 56 samples of blood, plasma, and urine obtained from 29 males and 27 females, in age from 5 to 14 years. The control group consisted of 35 healthy children (15 males and 20 females), in age from 5 to 14 years. Children did not take any mineral supplements or drugs before the samples for analyses were collected. All samples were transported and stored in polypropylene containers. All kinds of samples taken from healthy and short stature patients were treated in the same way, i.e., the same reagents were used, the same ratio of chemical reagents was used to mineralize the samples, the same time and number of stages of the procedures, and the same analytical method for their determination was applied. First, 1 mL of each type of
sample was divided into two parts (each 0.5 mL) in order to have two independent solutions prior to the mineralization procedure. Blood, plasma, and urine samples were mineralized using a microwave-assisted high-pressure digestion system (UniClever BM-1, Plazmotronika, Poznań, Poland). Each time, the acidic digestion with 99 % HNO3 (Sigma-Aldrich, Germany) water solution was applied (1 mL of HNO3 : 9 mL H2O). A program of microwave digestion consisted of the following four stages: (1) 40 % of power of a microwave generator, heating for 3 min, (2) 60 % of power of a microwave generator; heating for 5 min, (3) 100 % of power of a microwave generator, heating for 8 min, and (4) cooling for 10 min. The conditions of mineralization procedure had been previously optimized [8]. After digestion, the Teflon vessels (with screw caps) were cleaned thoroughly twice with diluted digestion mixture in order to avoid memory effects (adsorption by the walls of containers). The obtained solutions were poured into volumetric flasks (PTFE), and when it was necessary, they were tenfold diluted with deionized water (18 MΩ cm) before final analysis. Ion Chromatography (IC) After the mineralization, each sample was analyzed at least in triplicate using IC and inductively coupled plasma mass spectrometry (ICP MS) techniques. Chromatographic analyses were performed on a Dionex DX-500 ion chromatograph (Dionex, Sunnyvale, CA, USA). Metal cations were analyzed using an isocratic elution with 7.0 mmol of PDCA (pyridine2,6 dicarboxylic acid), 66 mmol of potassium hydroxide, 5.6 mmol of potassium sulfate, and 74 mmol of formic acid as mobile phase; IonPac CS5A (250×4 mm I.D., Dionex, S u nn y va l e , U S A ) a s t h e se pa r at i o n co l u m n a n d spectophotometric detection (at 530 nm) after the postcolumn derivatization with the use of 0.5 mmol of 4-(2pyridylazo)resorcinol (PAR); 1.0 mol of 2-dimethylaminoethanol, 0.3 mol of sodium bicarbonate, and 0.50 mol of ammonium hydroxide dissolved in deionized water. Appropriate concentrations of standards were prepared from 1 g/L stock standard solutions (Merck, Darmstadt, Germany). The detailed procedure of the standard solution preparation, operating IC conditions, and validation of the applied method were already described in the previous papers [8]. The limit of detection (LOD) values for all the elements used in this study were expressed in μg/mL: 0.022(Cd), 0.026 (Co), 0.048 (Cu), 0.009 (Fe), 0.006 (Mn), 0.056 (Zn), and 0.006 (Ni) [12]. Inductively Coupled Plasma Mass Spectrometry (ICP MS) ICP MS studies were conducted at the Warsaw University of Technology, Chemistry Department, Warsaw, Poland. ICP MS was carried out with Agilent 7500a ICP mass
Concentration of Selected Metals in Whole Blood
spectrometer (Agilent Technologies, Tokyo, Japan) using 10 ng/mL of yttrium (89Y) and bismuth (209Bi) as internal standards. A sample introduction system consisted of a concentric nebulizer 0.4 mL/min (SeaSpray nebulizer, Glass Expansion, Pocasset, MA, USA) attached to a commercial Scott’s spray chamber (Agilent Technologies), quartz torch with platinum shield and quartz bonnet, Pt sampling, and skimmer cones. Samples were transported via a peristaltic pump with PVC tubes. Limit of detection was calculated as standard deviations (SD) of 10-times-measured blank solution. For the studied elements, LOD values (expressed in μg/mL) were found to be 0.0001(Cd), 0.0002 (Co), 0.0005 (Cu), 0.0008 (Fe), 0.0005 (Mn), 0.0005 (Zn), and 0.0004 (Ni). Statistical Analysis Statistical analysis of the countable and measurable data was performed with the use of the SPSS 11.0 for Windows software package. The normality of the distribution was tested with the use of the Shapiro–Wilk test. A p value less than 0.05 was considered statistically significant. Differences in the concentration of metal in the blood, plasma, and urine were determined, and the influence of short stature and gender was analyzed using two-way ANOVA test. All pairwise multiple comparison procedures were done by Bonferroni test. Data are expressed as means and standard deviation. The influence of two variables, the area where patients live and hormonal therapy, was investigated using the independent sample t test. The correlations between the metal concentration in the blood, plasma, and urine and body weight, body height, and age were analyzed with the Pearson correlation coefficient test.
Results The results of determination of selected metals (Zn, Cd, Co, Mn, Cu, Fe, Ni) in body fluids (blood, plasma, and urine) in children with short stature in comparison with the control group are presented in Tables 1, 2, 3. There were no significant differences between the groups concerning the area where patients live (small city, village). The effects of therapy with growth hormone on the content of Fe, Ni, and Cu are presented in Table 4. Correlation coefficients between metal concentration in the blood, plasma, and urine in short stature children (pair of parameters) are presented in Table 5. The obtained results revealed no statistically significant differences between metal concentration and age, body weight, and height.
Table 1 Content of studied metals in the blood, plasma, and urine in short stature patients without dividing into sex groups Element Control group (n=35) Mean [mg/L]
Std. dev. [mg/L]
Concentration in the blood Cd 0.003 0.001 Co 0.034 0.003 Cu 1.686 0.053 Fe 513.7 11.66 Mn 0.016 0.002 Zn 9.258 0.159 Ni 0.007 0.001 Concentration in plasma Cd 0.006 0.0003 Co 0.0032 0.0003 Cu 1.286 0.0482 Fe 1.457 0.0441 Mn 0.0085 0.0007 Zn 1.348 0.0950 Ni 0.0023 0.0004 Concentration in urine Cd 0.0102 0.0001 Co 0.0048 0.0008 Cu 0.0164 0.0010 Fe 0.1575 0.0147 Mn 0.0024 0.0001 Zn 0.6702 0.1035 Ni 0.0177 0.0018
Short stature (n=56) Mean [mg/L]
Std. dev. [mg/L]
p value
0.005 0.026 1.258 387.3 0.012
0.002 0.019 0.161 46.70 0.002
**0.00 *0.01 **0.00 **0.00 **0.00
6.020 0.007
0.554 0.002
**0.00 0.63
0.0057 0.0038 1.303 1.382 0.0064 1.168 0.0019
0.0017 0.0016 0.1666 0.2765 0.0032 0.1817 0.0017
0.564 *0.026 0.513 0.089 **0.000 **0.000 0.057
0.0117 0.0040 0.0263 0.1350 0.0023 0.4960 0.0171
0.0024 0.0013 0.0119 0.0519 0.0009 0.1524 0.0044
**0.001 **0.002 **0.000 *0.016 0.607 **0.000 0.503
*p
Concentration of Selected Metals in Whole Blood, Plasma, and Urine in Short Stature and Healthy Children Maria Klatka & Anna Błażewicz & Małgorzata Partyka & Witold Kołłątaj & Ewa Zienkiewicz & Ryszard Kocjan
Received: 2 December 2014 / Accepted: 29 January 2015 # Springer Science+Business Media New York 2015
Abstract The short stature in children is defined as height below the third percentile from the mean for age and gender. This problem affects about 3 % of young people. More than 20,000 children in Poland have problems with short stature. There is not much information available in the literature on the study of metals in blood, plasma, and urine in children with short stature. The study was conducted on a group of 56 short stature Polish children and 35 healthy children. The content of metals was determined using high-performance ion chromatography and inductively coupled plasma mass spectrometry methods. The study revealed significant differences between the content of selected metals in body fluids between a short stature group and healthy children. There were significant differences in the Fe, Cu, and Ni concentrations between the groups with respect to the hormonal therapy. There were no significant differences between the groups with respect to the area where the children lived. The results showed no statistically significant differences between metal concentration and
M. Klatka (*) : W. Kołłątaj Department of Pediatric Endocrinology and Diabetology, Medical University of Lublin, Gębali 6, 20-093 Lublin, Poland e-mail: [email protected] A. Błażewicz : R. Kocjan Department of Analytical Chemistry, Medical University of Lublin, Chodźki 4A, 20-093 Lublin, Poland M. Partyka Department of Jaw Orthopedics, Medical University of Lublin, Karmelicka 7, 20-081 Lublin, Poland E. Zienkiewicz Department of Pediatric Neurology, Medical University of Lublin, Gębali 6, 20- 093 Lublin, Poland
age, body weight, and height. The study demonstrated statistically significant differences between the content of metals in body fluids in short stature children compared with the healthy children. It seems that the difference in the concentration of certain elements may also be the result of growth hormone therapy and the interaction between various metals. Both the alterations in the content of metals and their mutual interactions may play an important role in the pathogenesis of short stature children. Keywords Short stature in children . Metals . Body fluids . Ion chromatography
Introduction The short stature in children is defined as the height below the third percentile from the mean for age and gender. This problem affects about 3 % of the population. In the USA, short stature affects more than one million children aged 4–15 years, and in Europe, more than half a million children of similar age. More than 20,000 children in Poland have problems with short stature. In Poland, about 3,000 children are treated with a growth hormone [1]. Children with this condition reveal no identifiable systemic or endocrine disease but have short stature and a delayed bone age [2]. Most short stature children are not affected by hormonal disorders or genetically conditioned syndromes [3, 4]. Short stature due to endocrine factors affects no more than 5 % of short stature children, while short stature due to disturbances in somatotrophin axis affects about 3 % of such children (1:4000 to 1:10,000). According to Ruz, growth is a complex process in which several nutritional and nonnutritional factors play roles and interact, but the extent and
Klatka et al.
consequences of such interactions are not still completely understood [5]. There is a strong evidence to support the concept that the outcome of human pregnancy is significantly affected by the nutritional status of the mother. The consumption of “poor diets” has been associated with an increased risk for pregnancy complications, including gross structural birth defects, prematurity, low birth weight, and an increased risk for neurobehavioral and immunological abnormalities after birth [6]. Our knowledge of the amount of trace elements in human tissues and fluids has increased significantly due to the recent advances in analytics. Analytical methods can be employed in the study of the relation between the basic composition of body fluids and tissues, and the pathological conditions and general nutritional status in humans. The content of essential elements and toxic metals in human tissues and organs has been studied by many authors [7, 8]. Heavy metals are known to cause a variety of health problems/diseases/injury, and mechanisms of their toxicity have been persistently investigated [9, 10]. The elements studied in the presented study have been divided into four groups [11]: (a) metals of greatest toxicological significance (arsenic, cadmium, lead, mercury, and uranium) that are widespread in the human environment, (b) essential trace metals (chromium, cobalt, manganese, selenium, and zinc), (c) a group of elements of interest to biological sciences (nickel and vanadium), and (d) a group of elements that may be applied in pharmacology (aluminum, gallium, and lithium).
Material and Methods Sample Preparation Diagnostic tests and treatment were performed at the Department of Pediatric Endocrinology and Diabetology at the Medical University in Lublin, southeast Poland. Informed consent was obtained from the parents. The study was approved by the Bioethical Committee at the Medical University in Lublin. The study material consisted of 56 samples of blood, plasma, and urine obtained from 29 males and 27 females, in age from 5 to 14 years. The control group consisted of 35 healthy children (15 males and 20 females), in age from 5 to 14 years. Children did not take any mineral supplements or drugs before the samples for analyses were collected. All samples were transported and stored in polypropylene containers. All kinds of samples taken from healthy and short stature patients were treated in the same way, i.e., the same reagents were used, the same ratio of chemical reagents was used to mineralize the samples, the same time and number of stages of the procedures, and the same analytical method for their determination was applied. First, 1 mL of each type of
sample was divided into two parts (each 0.5 mL) in order to have two independent solutions prior to the mineralization procedure. Blood, plasma, and urine samples were mineralized using a microwave-assisted high-pressure digestion system (UniClever BM-1, Plazmotronika, Poznań, Poland). Each time, the acidic digestion with 99 % HNO3 (Sigma-Aldrich, Germany) water solution was applied (1 mL of HNO3 : 9 mL H2O). A program of microwave digestion consisted of the following four stages: (1) 40 % of power of a microwave generator, heating for 3 min, (2) 60 % of power of a microwave generator; heating for 5 min, (3) 100 % of power of a microwave generator, heating for 8 min, and (4) cooling for 10 min. The conditions of mineralization procedure had been previously optimized [8]. After digestion, the Teflon vessels (with screw caps) were cleaned thoroughly twice with diluted digestion mixture in order to avoid memory effects (adsorption by the walls of containers). The obtained solutions were poured into volumetric flasks (PTFE), and when it was necessary, they were tenfold diluted with deionized water (18 MΩ cm) before final analysis. Ion Chromatography (IC) After the mineralization, each sample was analyzed at least in triplicate using IC and inductively coupled plasma mass spectrometry (ICP MS) techniques. Chromatographic analyses were performed on a Dionex DX-500 ion chromatograph (Dionex, Sunnyvale, CA, USA). Metal cations were analyzed using an isocratic elution with 7.0 mmol of PDCA (pyridine2,6 dicarboxylic acid), 66 mmol of potassium hydroxide, 5.6 mmol of potassium sulfate, and 74 mmol of formic acid as mobile phase; IonPac CS5A (250×4 mm I.D., Dionex, S u nn y va l e , U S A ) a s t h e se pa r at i o n co l u m n a n d spectophotometric detection (at 530 nm) after the postcolumn derivatization with the use of 0.5 mmol of 4-(2pyridylazo)resorcinol (PAR); 1.0 mol of 2-dimethylaminoethanol, 0.3 mol of sodium bicarbonate, and 0.50 mol of ammonium hydroxide dissolved in deionized water. Appropriate concentrations of standards were prepared from 1 g/L stock standard solutions (Merck, Darmstadt, Germany). The detailed procedure of the standard solution preparation, operating IC conditions, and validation of the applied method were already described in the previous papers [8]. The limit of detection (LOD) values for all the elements used in this study were expressed in μg/mL: 0.022(Cd), 0.026 (Co), 0.048 (Cu), 0.009 (Fe), 0.006 (Mn), 0.056 (Zn), and 0.006 (Ni) [12]. Inductively Coupled Plasma Mass Spectrometry (ICP MS) ICP MS studies were conducted at the Warsaw University of Technology, Chemistry Department, Warsaw, Poland. ICP MS was carried out with Agilent 7500a ICP mass
Concentration of Selected Metals in Whole Blood
spectrometer (Agilent Technologies, Tokyo, Japan) using 10 ng/mL of yttrium (89Y) and bismuth (209Bi) as internal standards. A sample introduction system consisted of a concentric nebulizer 0.4 mL/min (SeaSpray nebulizer, Glass Expansion, Pocasset, MA, USA) attached to a commercial Scott’s spray chamber (Agilent Technologies), quartz torch with platinum shield and quartz bonnet, Pt sampling, and skimmer cones. Samples were transported via a peristaltic pump with PVC tubes. Limit of detection was calculated as standard deviations (SD) of 10-times-measured blank solution. For the studied elements, LOD values (expressed in μg/mL) were found to be 0.0001(Cd), 0.0002 (Co), 0.0005 (Cu), 0.0008 (Fe), 0.0005 (Mn), 0.0005 (Zn), and 0.0004 (Ni). Statistical Analysis Statistical analysis of the countable and measurable data was performed with the use of the SPSS 11.0 for Windows software package. The normality of the distribution was tested with the use of the Shapiro–Wilk test. A p value less than 0.05 was considered statistically significant. Differences in the concentration of metal in the blood, plasma, and urine were determined, and the influence of short stature and gender was analyzed using two-way ANOVA test. All pairwise multiple comparison procedures were done by Bonferroni test. Data are expressed as means and standard deviation. The influence of two variables, the area where patients live and hormonal therapy, was investigated using the independent sample t test. The correlations between the metal concentration in the blood, plasma, and urine and body weight, body height, and age were analyzed with the Pearson correlation coefficient test.
Results The results of determination of selected metals (Zn, Cd, Co, Mn, Cu, Fe, Ni) in body fluids (blood, plasma, and urine) in children with short stature in comparison with the control group are presented in Tables 1, 2, 3. There were no significant differences between the groups concerning the area where patients live (small city, village). The effects of therapy with growth hormone on the content of Fe, Ni, and Cu are presented in Table 4. Correlation coefficients between metal concentration in the blood, plasma, and urine in short stature children (pair of parameters) are presented in Table 5. The obtained results revealed no statistically significant differences between metal concentration and age, body weight, and height.
Table 1 Content of studied metals in the blood, plasma, and urine in short stature patients without dividing into sex groups Element Control group (n=35) Mean [mg/L]
Std. dev. [mg/L]
Concentration in the blood Cd 0.003 0.001 Co 0.034 0.003 Cu 1.686 0.053 Fe 513.7 11.66 Mn 0.016 0.002 Zn 9.258 0.159 Ni 0.007 0.001 Concentration in plasma Cd 0.006 0.0003 Co 0.0032 0.0003 Cu 1.286 0.0482 Fe 1.457 0.0441 Mn 0.0085 0.0007 Zn 1.348 0.0950 Ni 0.0023 0.0004 Concentration in urine Cd 0.0102 0.0001 Co 0.0048 0.0008 Cu 0.0164 0.0010 Fe 0.1575 0.0147 Mn 0.0024 0.0001 Zn 0.6702 0.1035 Ni 0.0177 0.0018
Short stature (n=56) Mean [mg/L]
Std. dev. [mg/L]
p value
0.005 0.026 1.258 387.3 0.012
0.002 0.019 0.161 46.70 0.002
**0.00 *0.01 **0.00 **0.00 **0.00
6.020 0.007
0.554 0.002
**0.00 0.63
0.0057 0.0038 1.303 1.382 0.0064 1.168 0.0019
0.0017 0.0016 0.1666 0.2765 0.0032 0.1817 0.0017
0.564 *0.026 0.513 0.089 **0.000 **0.000 0.057
0.0117 0.0040 0.0263 0.1350 0.0023 0.4960 0.0171
0.0024 0.0013 0.0119 0.0519 0.0009 0.1524 0.0044
**0.001 **0.002 **0.000 *0.016 0.607 **0.000 0.503
*p
