Relationships of Lead, Copper, Zinc, and Cadmium Levels versus Hematopoiesis and Iron Parameters in Healthy Adolescents

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1 Available online at Relationships of Lead, Copper, Zinc, and Cadmium Levels versus Hematopoiesis and Iron Parameters in Healthy Adolescents Jong Weon Choi 1 and Soon Ki Kim 2 1 Department of Laboratory Medicine and 2 Department of Pediatrics, College of Medicine, Inha University, Inchon, South Korea Abstract. To investigate the relationships of trace element concentrations vs hematopoiesis and iron parameters, we measured lead, copper, zinc, cadmium, and ferritin levels in 251 healthy adolescents. Concentrations of trace metals were determined by atomic absorption spectrophotometry. There were no significant gender-related differences in serum copper or serum cadmium concentrations. However, blood lead and serum zinc levels were significantly higher in males than females (3.82 ± 1.24 and ± 43.7 µg/dl vs 2.86 ± 1.06 and 83.5 ± 35.2 µg/dl, p <0.05, respectively). Subjects with elevated lead and copper concentrations exhibited significantly higher leukocyte counts and significantly lower serum iron levels than those with decreased lead and copper concentrations, but no significant differences were observed in blood erythrocyte counts or hemoglobin levels between the 2 groups. Blood lead concentrations were 2- fold higher in male adolescents with leukocytes >9.1 x 10 3 /µl than in those with leukocytes <4.3 x 10 3 /µl (5.04 ± 1.67 µg/dl vs 2.51 ± 0.75 µg/dl, p <0.05). Leukocyte counts had significant correlations with blood lead (r = 0.39, p <0.05) and serum copper (r = 0.26, p <0.05) in males and zinc (r = 0.28, p <0.05) in females. Serum iron levels were inversely correlated with blood lead and serum copper concentrations but were not correlated with serum zinc or cadmium levels. In short, blood lead and serum copper concentrations have important relationships to leukocyte counts and iron parameters in adolescents. Keywords: lead, copper, zinc, cadmium, iron, ferritin, hematopoiesis, leukocytes Introduction comprise metals in biological fluids at concentrations <1 µg/g of wet weight [1]. Among the trace elements, iron, copper, and zinc are involved in the function of several enzymes and are essential for maintaining health throughout life; lead and cadmium are non-essential toxic metals [2,3]. can cause diseases through deficiency, imbalance, or toxicity. Trace mineral deficiencies usually occur when dietary intake is Address correspondence to Jong Weon Choi, M.D., Ph.D., Department of Laboratory Medicine, Inha University Hospital, 7-206, 3-ga Shinheung-dong, Jung-gu, Inchon, , South Korea; tel ; fax ; jwchoi@inha.ac,kr. inadequate or result from metabolic imbalances produced by antagonistic or synergistic interactions among metals [4,5]. For instance, zinc absorption in small intestine is decreased by calcium, phosphate, and copper. On the other hand, excess zinc ingestion is a cause of copper deficiency [6]. There is an increasing recognition of the coexistence of multiple micronutrient deficiencies. Several investigators reported that blood lead concentrations are high in iron-deficient children [7], and that high dietary iron intake is associated with lower blood lead concentrations [8]. However, other researchers showed that iron depletion does not affect blood lead concentrations [9]. Interactions among trace elements and the relationships between blood lead concentration and iron parameters have /05/ $ by the Association of Clinical Scientists, Inc. 09 Choi indd /11/05 8:22:46 AM

2 Trace metals and hematopoiesis in adolescents 429 been extensively studied [7-9], but few studies have closely examined associations of copper, zinc, and cadmium concentrations vs hematopoietic activities in healthy adolescents, especially in relation to body iron status. In the present study, we determined which hematologic index is most closely related to lead, copper, zinc, and cadmium levels in subjects who had no evidence of iron deficiency or irondeficiency anemia. We also investigated whether the concentrations of trace metals show genderrelated differences in adolescents. Materials and Methods This study was approved by the Committee of Ethics of the Inha University Hospital, and informed consent was obtained from all subjects. A total of 251 adolescents (122 males, 129 females) with a median age of 14 yr (range, yr) were investigated by measurements of hemograms, serum iron parameters, and trace elements, including lead, copper, zinc, and cadmium. The investigation was conducted between 1 September 2004 and 15 October Subjects were all Korean students residing in municipalities without contaminated environments or industrial areas. To minimize the influence of age, smoking habits, and alcohol consumption on the concentrations of trace elements, only middle school students who had no histories of smoking or drug usage were enrolled. Non-anemic adolescents with a normal serum iron (>50 µg/dl) and serum ferritin levels (>12 µg/dl) were investigated because some trace elements can be affected by anemia or iron depletion [7]. Adolescents with a history of vitamin supplementation (n = 3) were excluded, as were those with recent infections (n = 2), since serum zinc levels can decrease in infections such as hepatitis [10]. After the subjects had fasted for >12 hr, blood samples were obtained by venipuncture using Vacutainer tubes (Becton-Dickinson, Franklin Lakes, NJ, USA). Blood lead levels were determined in 100 µl of EDTA-anticoagulated whole blood samples and the concentrations of copper, zinc, and cadmium were measured in serum specimens with an atomic absorption spectrophotometry (model 2380, Perkin- Elmer, Oak Brook, IL, USA). These assays are based on graphite furnace atomic absorption and have a high degree of accuracy [11]. Prior to assays of trace metals, the samples were refrigerated for no longer than 24 hr at 4 C. Complete blood cell counts were measured with EDTA-anticoagulated blood using an electronic counter (SE 9000, Sysmex, Kobe, Japan). Serum iron was assayed with a chemical analyzer (Hitachi 747; Hitachi, Tokyo, Japan) and ferritin was measured by a chemiluminescent method (ACS 180; Bayer Diagnostics, Tarrytown, NY, USA). To investigate differences in blood cell counts and iron parameters in relation to trace element concentrations, subjects were assigned to the following groups: (i) males with lead <3.82 µg/dl (n = 64) and 3.82 µg/dl (n = 58), copper <98.2 µg/dl (n = 62) and 98.2 µg/dl (n = 60), and zinc <118.4 µg/dl (n = 59) and µg/dl (n = 63); (ii) females with lead <2.86 µg/dl (n = 68) and 2.86 µg/dl (n = 61), copper <95.8 µg/dl (n = 63) and 95.8 µg/dl (n = 66), and zinc <83.5 µg/dl (n = 60) and 83.5 µg/dl (n = 69). The provisional cut-off levels applied in this categorization were based on mean concentrations of lead, copper, and zinc in male and female adolescents, respectively. To evaluate more strictly, we further stratified the adolescents into 2 groups based on those with leukocyte counts <10th percentile or >90th percentile leukocyte counts of all subjects: males with leukocytes <4.3 x 10 3 /µl (10th percentile) and leukocytes >9.1 x 10 3 /µl (90th percentile); females with leukocytes <4.2 x 10 3 /µl (10th percentile) and leukocytes >9.2 x 10 3 /µl (90th percentile). Data analysis was performed using SAS statistical software (version 6.12, SAS Institute, Cary, NC, USA). A nonparametric test (Wilcoxon rank-sum test) was used to test the statistical significance of inter-group differences. Correlation coefficients were calculated by the Spearman method. All p values <0.05 were considered statistically significant. Results Mean values of trace element concentrations, iron parameters, and hemograms in subject populations are summarized in Table 1. Serum copper and serum cadmium concentrations showed a propensity toward high values in males compared to females, but the tendency was not statistically significant. However, blood lead and serum zinc concentrations averaged 3.82 ± 1.24 and ± 43.7 µg/dl in males, which were significantly above the corresponding values (2.86 ± 1.06 and 83.5 ± 35.2 µg/dl, p < 0.05) in females. Males with elevated blood lead ( 3.82 µg/dl) and serum copper ( 98.2 µg/dl) concentrations exhibited significantly higher leukocyte counts [7.6 ± 1.7 and 7.8 ± 1.6 (x 10 3 /µl) vs 5.9 ± 1.5 and 6.0 ± 1.4 (x 10 3 /µl), p <0.05] and showed significantly lower serum iron levels (91.0 ± 28.1 and 89.9 ± 29.7 µg/dl vs ± 33.1 and ± 32.9 µg/dl, p< 0.05) than those with decreased values of the corresponding parameters (lead <3.82 and copper <98.2 µg/dl). However, there were no significant differences in erythrocyte counts and hemoglobin levels between the two groups (Table 2). Serum iron concentrations were 85.1 ± 31.8 and 89.2 ± 29.6 µg/dl in females with elevated blood lead ( 2.86 µg/dl) or serum copper levels ( 95.8 µg/dl), respectively, which were significantly lower than in those (117.9 ± 36.5 and ± 33.2 µg/dl, p <0.05) with decreased blood lead (<2.86 µg/dl) or serum copper levels (< 95.8 µg/dl) (Table 09 Choi indd /11/05 8:22:47 AM

3 430 Table 1. Trace element concentrations, iron parameters, and hematologic markers in healthy adolescents. The data are reported as means ± SD, with medians in brackets. Parameters All subjects Male subjects Female subjects (n = 251) (n = 122) (n = 129) Age (yr) 14.3 ± 0.4 [14] 14.2 ± 0.4 [14] 14.3 ± 0.5 [14] Blood lead (µg/dl) 3.34 ± 1.22 [3.31] 3.82 ± 1.24 [3.87] a 2.86 ± 1.06 [2.92] Serum copper (µg/dl) ± [98.15] ± [99.23] ± [96.34] Serum zinc (µg/dl) ± 50.7 [105.9] ± 43.7 [120.1] a 83.5 ± 35.2 [85.2] Serum cadmium (µg/dl) ± [0.062] ± [0.067] ± [0.063] Iron parameters Serum iron (µg/dl) ± 34.9 [98.6] ± 31.5 [101.7] ± 32.8 [96.9] Serum ferritin (ng/dl) 28.9 ± 14.1 [30.1] 32.6 ± 14.9 [34.5] 25.4 ± 13.5 [26.7] Blood leukocytes (10 9 /L) 6.5 ± 1.8 [6.4] 6.7 ± 1.6 [6.8] 6.4 ± 1.8 [6.5] Blood erythrocytes(10 12 /L) 4.71 ± 0.35 [4.69] 4.87 ± 0.34 [4.81] 4.56 ± 0.32 [4.43] Blood hemoglobin (g/dl) 13.7 ± 1.0 [13.7] 14.0 ± 1.1 [14.1] 13.4 ± 0.9 [13.5] a Statistically significant (p <0.05), compared to females, computed by Wilcoxon rank sum test. Table 2. Iron parameters and hematologic markers in relation to lead, copper, and zinc concentrations in male adolescents. The data are reported as means ± SD, with medians in brackets. Parameters Blood lead (µg/dl) Serum copper (µg/dl) Serum zinc (µg/dl) < < < Subjects (n) Age (yr) 14.3± ± ± ± ± ±0.4 [14] [14] [14] [14] [14] [14] Blood lead (µg/dl) 2.58± ± ± ± ± ±1.16 [2.35] [4.85] a [3.45] [3.79] [3.38] [4.05] Serum copper (µg/dl) 96.6± ± ± ± ± ±20.7 [95.2] [97.4] [81.3] [116.5] b [93.8] [98.1] Serum zinc (µg/dl) 99.1± ± ± ± ± ±54.7 [92.5] [129.8] a [106.1] [115.2] [85.4} [160.3] c Serum cadmium (µg/dl) 0.067± ± ± ± ± ±0.038 [0.065] [0.064] [0.068] [0.061] [0.068] [0.062] Iron parameters Serum iron (µg/dl) 122.4± ± ± ± ± ±31.9 [124.5] [90.7] a [122.1] [86.5] b [107.3] [95.2] Serum ferritin (ng/dl) 35.1± ± ± ± ± ±13.8 [36.9] [25.6] [39.8] [29.3] [35.8] [30.1] Leukocytes (10 9 /L) 5.9± ± ± ± ± ±1.5 [5.7] [7.8] a [5.9] [7.9] b [6.5] [7.1] Erythrocytes (10 12 /L) 4.88± ± ± ± ± ±0.34 [4.85] [4.84] [4.88] [4.86] [4.85] [4.84] Hemoglobin (g/dl) 14.0± ± ± ± ± ±1.1 [14.1] [14.0] [14.0] [14.0] [14.1] [13.9] a Statistically significant (p <0.05), vs males with blood lead <3.82 µg/dl, computed by Wilcoxon rank sum test. b Statistically significant (p <0.05) vs males with serum copper <98.2 µg/dl, computed by Wilcoxon rank sum test. c Statistically significant (p <0.05) vs males with serum zinc <118.4 µg/dl, computed by Wilcoxon rank sum test. 09 Choi indd /11/05 8:22:48 AM

4 Trace metals and hematopoiesis in adolescents 431 Table 3. Iron parameters and hematologic markers in relation to lead, copper, and zinc concentrations in female adolescents. The data are reported as means ± SD, with medians in brackets. Parameters Blood lead (µg/dl) Serum copper (µg/dl) Serum zinc (µg/dl) < < < Subjects (n) Age (yr) 14.4± ± ± ± ± ±0.4 [14] [14] [14] [14] [14] [14] Blood lead (µg/dl) 1.64± ± ± ± ± ±1.34 [1.79] [4.35] a [2.69] [3.41] [2.93] [3.62] Serum copper (µg/dl) 91.7± ± ± ± ± ±18.1 [90.2] [95.4] [80.5] [117.6] b [95.1] [102.7] Serum zinc (µg/dl) 71.1± ± ± ± ± ±51.8 [75.4] [105.2] a [80.1] [92.5] [64.8} [125.9] c Serum cadmium (µg/dl) 0.064± ± ± ± ± ±0.021 [0.063] [0.061] [0.064] [0.063] [0.062] [0.061] Iron parameters Serum iron (µg/dl) 117.9± ± ± ± ± ±29.9 [124.1] [82.7] a [120.5] [87.3] b [105.9] [97.3] Serum ferritin (ng/dl) 27.8± ± ± ± ± ±11.8 [26.4] [23.9] [28.1] [26.8] [27.1] [26.2] Leukocytes (10 9 /L) 5.5± ± ± ± ± ±1.8 [5.4] [7.6] a [5.5] [7.4] b [5.9] [7.0] Erythrocytes (10 12 /L) 4.58± ± ± ± ± ±0.30 [4.57] [4.53] [4.56] [4.53] [4.53] [4.56] Hemoglobin (g/dl) 13.4± ± ± ± ± ±1.1 [13.5] [13.3] [13.5] [13.4] [13.3] [13.5] a Statistically significant (p <0.05), vs females with blood lead <2.86 µg/dl, computed by Wilcoxon rank sum test. b Statistically significant (p <0.05) vs females with serum copper <95.8 µg/dl, computed by Wilcoxon rank sum test. c Statistically significant (p <0.05) vs females with serum zinc <83.5 µg/dl, computed by Wilcoxon rank sum test. Table 4. Concentrations of trace elements in relation to leukocyte counts in male and female adolescents. The data are reported as means ± SD, with medians in brackets. Blood leukocyte counts Males <4.3 x 10 3 /µl (10th percentile) >9.1 x 10 3 /µl (90th percentile) Blood lead (µg/dl) 2.51 ± 0.75 [2.36] 5.04 ± 1.67 [4.95] a Serum copper (µg/dl) 89.1 ± 19.6 [90.5] ± 21.5 [112.3] a Serum zinc (µg/dl) ± 50.3 [102.4] ± 56.2 [135.6] Serum cadmium (µg/dl) ± [0.064] ± [0.065] Females <4.2 x 10 3 /µl (10th percentile) >9.2 x 10 3 /µl (90th percentile) Blood lead (µg/dl) 2.71 ± 1.03 [2.75] 3.02 ± 1.41 [3.24] Serum copper (µg/dl) 90.5 ± 19.8 [86.4] ± 16.5 [102.5] Serum zinc (µg/dl) 67.1 ± 40.9 [70.5] ± 45.4 [104.1] a Serum cadmium (µg/dl) ± [0.063] ± [0.062] a Statistically significant (p <0.05), compared to corresponding data for subjects with blood leukocytes <10th percentile, computed by Wilcoxon rank sum test. 09 Choi indd /11/05 8:22:48 AM

5 432 Table 5. Correlation coefficients of hematologic markers and iron parameters vs trace element concentrations. Parameter Blood lead Serum copper Serum zinc Serum cadmium Males Females Males Females Males Females Males Females Leukocytes (10 9 /l) 0.39 a 0.21 a 0.26 a 0.19 a a Erythrocytes (10 12 /l) Hemoglobin (g/dl) Iron markers Serum iron (µg/dl) a a a Serum ferritin (ng/dl) Blood lead (µg/dl) NA NA a 0.27 a 0.21 a Serum copper (µg/dl) a NA NA Serum zinc (µg/dl) 0.27 a 0.21 a NA NA Serum cadmium (µg/dl) NA NA NA = not applicable. a Statistically significant (p <0.05), computed by Spearman method. 3). As shown in Table 4, males with increased leukocyte counts >9.1 x 10 3 /µl (90th percentile) showed markedly high blood lead (5.04 ± 1.67 µg/ dl) and serum copper (108.7 ± 21.5 µg/dl) concentrations, vs those with decreased leukocyte counts <4.3 x 10 3 /µl (10th percentile) (2.51 ± 0.75 and 89.1 ± 19.6 µg/dl, p <0.05, respectively). Correlation coefficients of hematologic markers and iron parameters vs trace element concentrations are summarized in Table 5. Leukocyte counts had significant correlations with blood lead (r = 0.39, p <0.05) and serum copper (r = 0.26, p <0.05) in males and serum zinc levels (r = 0.28, p <0.05) in females. Blood lead concentrations had negative correlation with serum iron levels (r = -0.32, p <0.05) and positive correlation with serum zinc concentrations (r = 0.27, p <0.05). Correlation coefficients between trace elements and serum iron levels were higher in males than in females for copper (r = vs -0.15) and lead (r = vs -0.19). No significant relations were noted between trace metal concentrations and hemoglobin levels. Discussion This study shows that blood lead and serum copper concentrations have important relationships to blood leukocyte counts and serum iron parameters in adolescents. Our data are in partial agreement with a previous study, which demonstrated that white blood cell counts are significantly high in dogs and cats with increased blood lead levels [12]. Counter et al [13] reported that blood lead concentrations were higher in males than in females among children with chronic lead intoxication. Similarly, in our study blood lead and serum zinc concentrations were significantly higher in males than females. Clark et al [14] found that blood hemoglobin levels were significantly low in subjects with elevated blood lead concentrations [14]. Cohen et al [15] reported that elevated blood lead levels caused anemia by inhibition of hemoglobin production and shortened red cell survival. In the present study, no significant differences were observed in blood hemoglobin levels between subjects with and without increase in blood lead levels. These apparent discrepancies may reflect the differences in blood lead concentrations of subject populations in the various studies. In general, blood lead levels in the range of 10 to 20 µg/dl are linked with adverse health effects, especially deleterious influences on hematopoietic, renal, endocrine, and reproductive systems [16]. On the other hand, in our study, blood lead concentrations ranged from µg/dl in adolescents and there were no subjects with lead levels >10.0 µg/dl. Interestingly, blood lead and serum copper levels correlated significantly with white blood cell counts but not with blood erythrocyte and hemoglobin levels. In particular, lead concentrations 09 Choi indd /11/05 8:22:49 AM

6 Trace metals and hematopoiesis in adolescents 433 were 2-fold higher in male adolescents with moderately increased leukocyte counts than in those with decreased leukocyte counts. These results suggest that elevated blood lead and serum copper concentrations may be more closely associated with alteration in peripheral leukocyte numbers than with erythropoietic activities in healthy adolescents. To explain these findings, it is possible that subjects in this study included only non-anemic adolescents without iron depletion. Nutritional deficiencies have been shown to increase the absorption and toxicity of orally ingested lead and cadmium [17]. Gastrointestinal lead absorption is enhanced by deficiency of iron, calcium, and zinc [18]. Oral ingestion of cadmium or lead perturbs the metabolism of zinc, copper, and iron and these changes may be the earliest manifestations of the toxicity of lead and cadmium [17]. In this study, subjects with elevated lead and copper concentrations revealed significantly lower serum iron levels than those with decreased lead and copper levels. Serum lead concentrations had negative correlations with serum iron levels and positive relationships with serum zinc concentrations. These data corroborate the results of our previous study, which demonstrated that iron deficiency constitutes a predisposing factor for lead poisoning and that elevated blood lead concentrations significantly diminished after iron supplementation in patients with iron-deficiency anemia [19]. The present study shows that blood lead concentration has a close association with serum iron levels in adolescents with no evidence of iron depletion or iron-deficiency anemia. These results suggest that minimal change in serum iron levels, even while subjects are in adequate iron balance, has an impact on blood lead concentrations. Taking these findings into consideration, it is plausible that gender-related difference in lead and zinc concentrations may be attributable to slight differences in serum iron and ferritin levels between males and females, although such differences were not statistically significant. Toxic metals, such as lead and cadmium, interact metabolically with essential trace elements [20]. Chronic exposure to lead or cadmium contributes to a decrease of serum zinc levels in adult men [21]. Serum zinc levels have a negative effect on anemia by blocking the utilization of iron in the iron reserves of anemic subjects [22]. Cengiz et al [23] reported inverse correlations between serum zinc and copper levels in pregnant women during the second trimester. On the contrary, in the present study, no significant correlations were observed between serum zinc and copper levels, nor between serum cadmium and zinc levels. However, blood lead concentrations had significant correlations with serum zinc levels. Relationships among the trace metals may be influenced by characteristics of subject populations, including age, gender, nutritional status, degree of exposure to environmental contamination, or behavioral habits. In fact, exposure to cadmium occurs mostly through smoking, while admitting that cadmium intoxication is related to industrial inhalation [21]. In our study, we investigated only non-smoking adolescents who had no history of chronic exposure to trace metals. In conclusion, blood lead and serum copper concentrations were correlated significantly with blood leukocyte counts and serum iron levels but were not correlated with blood hemoglobin, erythrocyte counts, or serum cadmium levels, suggesting that the trace metals have important relationships to the lineage of hematopoietic cells and to iron metabolism. Correlation coefficients between trace elements and serum iron levels were higher in males than in females, indicating genderrelated differences of trace metal metabolism in adolescents. References 1. Reinhold JG. : a selective survey. Clin Chem 1975;21: Delves HT. Assessment of trace element status. Clin Endocrinol Metab 1985;14: Papageorgiou T, Zacharoulis D, Xenos D, Androulakis G. Determination of trace elements (Cu, Zn, Mn, Pb) and magnesium by atomical absorption in patients receiving total parenteral nutrition. Nutrition 2002; 18: Harraki B, Guiraud P, Rochat MH, Alary J, Favier A. Interactions related to trace elements in parenteral nutrition. Pharm Acta Helv 1995;70: Goldhaber SB. Trace element risk assessment: essentiality vs toxicity. Regul Toxicol Pharmacol 2003;38: Choi indd /11/05 8:22:49 AM

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