Serum or tissue zinc concentrations are often used to assess body zinc status. However, all of these methods are relatively inaccurate. Thus, we investigated three different kinetic methods for the determination of zinc clearance to establish which of these could detect small changes in the body zinc status of children.
Forty apparently healthy children were studied. Renal handling of zinc was investigated during intravenous zinc administration (0.06537 mg Zn/kg of body weight), both before and after oral zinc supplementation (5 mg Zn/day for 3 months). Three kinetic methods were used to determine zinc clearance: CZn-Formula A and CZn-Formula B were both used to calculate systemic clearance; the first is a general formula and the second is used for the specific analysis of a single-compartment model; CZn-Formula C is widely used in medical practices to analyze kinetic routine.
Basal serum zinc values, which were within the reference range for healthy children, increased significantly after oral zinc supplementation. The three formulas used gave different results for zinc clearance both before and after oral zinc supplementation. CZn-Formula B showed a positive correlation with basal serum zinc concentration after oral supplementation (R2=0.1172, P=0.0306). In addition, CZn-Formula B (P=0.0002) was more effective than CZn-Formula A (P=0.6028) and CZn-Formula C (P=0.0732) in detecting small variations in body zinc status.
All three of the formulas used are suitable for studying zinc kinetics; however, CZn-Formula B is particularly effective at detecting small changes in body zinc status in healthy children.
Zinc is a micronutrient that is essential for human health. It has structural and biochemical functions at both the cellular and subcellular level; these include acting as a catalyst for more than 300 enzymes. Zinc has a role in DNA and RNA synthesis and degradation, protein synthesis, cell-mediated immunity, cell growth and differentiation and gene expression.1 Zinc deficiency is generally caused by inadequate intake or absorption, increased excretion or increased daily needs.2
Serum zinc concentration is not a reliable index for the diagnosis of marginal zinc deficiency. The amount of zinc in serum represents only a small fraction of total body zinc, and can be temporarily altered by factors such as recent food intake or muscle catabolism. However, in spite of these limitations, serum zinc concentration remains the most widely used biochemical marker for assessing population zinc status.3, 4 It has recently been suggested that zinc kinetics5, 6, 7 and molecular markers4 may prove more effective for the diagnosis of marginal zinc deficiency. Although severe primary zinc deficiency is extremely uncommon worldwide, marginal deficiency is far more prevalent.8, 9
The term ‘zinc clearance’ indicates the volume of serum or plasma from which zinc is completely removed per unit time. Clearance is one of the major underlying factors behind the age dependence of pharmacokinetic profiles.10 Nakamura et al.5 reported a relationship between zinc clearance and plasma zinc concentration. Other studies subsequently compared the relationship between zinc clearance and serum zinc in children with short stature and in normal height controls, and reported that zinc clearance was a sensitive measure of body zinc status.6, 11, 12
Adjustment of renal zinc excretion is a secondary homeostatic mechanism that occurs with extremely low or high intakes of zinc acutely, or with prolonged marginal intake. The regulation of urinary zinc excretion is poorly understood. One possible mechanism is an adjustment in renal tubular transport.13 It has been reported that increases in urinary zinc excretion, zinc clearance and the zinc clearance/creatinine clearance ratio indicate hyperzincuria in diabetic patients, although tubular reabsorption values exhibited no significant biological variation between diabetic patients and controls.14, 15
The lack of a reliable, responsive and specific indicator of zinc status hinders diagnosis of marginal zinc deficiency. Zinc kinetics may be an efficient tool for this purpose. In this study, we assess zinc clearance in healthy children using three different zinc kinetic formulas to establish which formula is best able to detect small changes in body zinc status.
Subjects and methods
Our study population consisted of 45 prepubertal children of both sexes, aged 6 to 9 years, who were selected from three municipal schools in the city of Natal, Brazil by a non-probability sampling method. Written consent was obtained from all parents or guardians. The study protocol (no. 542/11) was approved by the Ethics Committee of the Research Committee of Onofre Lopes University Hospital at the Federal University of Rio Grande do Norte (Natal, Brazil).
Inclusion and exclusion criteria
Only apparently healthy children in Tanner stage I (for genital, breast and pubic hair growth) were included in the study. We excluded those with early pubarche, thelarche or menarche, infectious or inflammatory diseases, any history of surgery and those who used any vitamin or mineral supplement or were unwilling to participate in the study.
Renal handling of zinc was investigated during venous zinc administration both before and after three months of oral zinc supplementation (Figure 1). Weight, height and age data were collected. Serum concentrations of zinc (SZn) and creatinine (SCr), and urinary zinc (UZn) and creatinine (UCr), were also determined.
Oral zinc supplementation
The children received oral supplementation with 5 mg Zn/day for 3 months. Zinc was provided in the form of a sulfate hepahydrate (ZnSO4.7 H2O, Merck, Darmstadt, Germany) syrup, which was prepared at the Pharmacotechnical Laboratory of the Department of Pharmacy, UFRN. Each drop contained 1 mg of elemental zinc. Five drops of the syrup were added to the participants’ beverage at breakfast each day. Intake was monitored every 2 weeks by the same observer to assess adherence to the protocol and to monitor for any possible adverse effects.
Venous zinc administration
The zinc clearance test was initiated at 7 am, after a 12-h fast, and concluded at 10 am. Each child remained in the dorsal decubitus position during the procedure. An antecubital forearm vein was punctured and saline solution (zinc free) was infused at 40 ml/h throughout the test. A bolus of 0.06537 mg Zn/kg of body weight (1 μmol ZnSO4.7 H2O) was injected at time 0 min. Each 5 ml ampoule contained 40 μmol ZnSO4.7 H2O. These were prepared at the Inject Center—Handling of Injectables, Ribeirão Preto, Brazil. Blood samples were collected at 0 (before zinc administration), 30, 60, 90 and 120 min after zinc administration as shown in Figure 2. Polypropylene plastic syringes were used for all blood collections.
Renal handling of zinc
All participants urinated before the test commenced (before 7 am), and the urine was discarded. Four milliliters of ultrapure water per kg body weight (Milli-Q Plus, Millipore, Billerica, MA, USA) was ingested in the middle of the test, to facilitate subsequent urine collection (Figure 2). Urine samples were collected at the end of the test for measurement of zinc and creatinine concentrations.
The kinetic parameters of zinc clearance were calculated using the following formulas:
1. CZn-Formula A. The systemic clearance calculation (1) was based on the trapezoidal area under the curve (AUC) and applied mainly to i.v. data. We used elimination and distribution phases:16
F=fraction of dose absorbed (F=1 for i.v.),
AUC(∞)=total AUC computed by combining AUC(0-t)with an extrapolated value. AUC(∞)=AUC(0-t)+Cn/λƵ, Cn represents the Y intercept and λƵ the elimination rate constant.
2. CZn-Formula B. Total body zinc clearance (2) was calculated using the following equation:17
Kel=elimination constant of serum zinc,
Kel (3), Vd (4) and ΔCo (5) were calculated as follows:
where, ΔCo=difference between Cp and Co; Cp=theoretical serum zinc concentration immediately after zinc injection, calculated from the serum concentration versus time profile; Co=basal serum zinc concentration; T1/2=biological half-life of serum zinc calculated directly from the serum concentration versus time profile; dose i.v.=amount of zinc administered intravenously.
3. CZn-Formula C. Renal zinc clearance (6), was calculated as follows:18
UZn=urinary zinc (μg/ml),
V=urine flow rate (ml/min),
SZn=serum zinc (μg/ml).
We also determined the renal clearance of creatinine (7) and tubular reabsorption (8) of zinc, which were calculated as follows:18
Ucr=urinary creatinine (mg/ml),
V=urine flow rate (ml/min),
Scr=serum creatinine (mg%).
TRZn=tubular reabsorption of zinc,
GFR=glomerular filtration rate (ml/min),
SZn=serum zinc (μg/ml),
UZn=urinary zinc (μg/ml),
V=urine flow rate (ml/min),
m2=body surface area.
Blood samples for zinc analyses were collected in Becton Dickinson tubes (Franklin Lakes, NJ, USA), and blood samples for biochemical analyses were collected in Vacuette Z serum clot activator tubes (Greiner Bio-One, Monroe, NC, USA). Zinc samples were stored in a stainless-steel incubator (502; Fanem, São Paulo, Brazil) suitable for metal. Urine samples were collected in metal-free plastic cylinders and vats (Nalgon, Itupeva, São Paulo, Brazil). Saline solutions (metal free) were acquired from Gaspar Viana S/A (Fortaleza, Brazil) and polypropylene plastic syringes from BD (Hercules, CA, USA). Plastic tips and tubes (metal free) were obtained from Bio-Rad Laboratories (Hercules, CA, USA).
Sample collection and analyses
Blood samples were collected by puncturing a forearm vein without a tourniquet. Hemolytic samples were discarded, as erythrocytes are rich in zinc.19 Zinc samples were stored for 2 h at 37 °C in an incubator for metals to allow clotting and serum separation. Serum and urine samples were frozen and stored at −20 °C until analysis. Serum and urinary zinc were measured using an atomic absorption spectrophotometer (SpectrAA-200, Varian, Melbourne, VIC, Australia) according to the manufacturer’s instructions. The sensitivity was 0.01 μg/ml, the intra-assay coefficient of variation was 2.37% and the reference values were 70–110 mg/dl.9 Serum and urinary creatinine concentrations were determined by standard clinical laboratory methods using a semiautomatic analyzer (RA-50, Bayer Diagnostics, Dublin, Ireland). All procedures related to the handling of zinc samples were performed according to international standards.3
Statistical analyses were performed using GraphPad Prism 6.0. The normality of data was assessed using the Shapiro–Wilk test. Differences in pre- and post-supplementation serum zinc levels were assessed using the Student’s t-test for paired data. Linear regression and Pearson’s correlation coefficients were used to analyze the relationship between serum zinc concentration and zinc clearance. Repeated measures analysis of variance was used to compare repeated measurements of the three clearance values obtained from each individual. P<0.05 was considered significant.
Five children withdrew during the study, and 40 children completed the full study. Basal serum zinc concentrations (1.02±0.01 μg/ml) were in the reference range for healthy subjects (0.7–1.1 μg/ml). After oral zinc supplementation these values increased to 1.22±0.03 μg/ml, P<0.0001. Serum zinc also increased at 30, 60, 90 and 120 min after intravenous zinc administration; however, there were no significant differences between the values at each time point before and after oral zinc supplementation (Figure 3).
Median clearance values before oral zinc supplementation were 4.23, 5.20 and 0.45 ml/kg/h as calculated using CZn-Formula A, CZn-Formula B and CZn-Formula C, respectively These same clearance values were 4.24, 5.93 and 0.51 ml/kg/h after oral zinc supplementation. There were significant differences between the results of the three clearance calculations both before and after oral zinc supplementation (P<0.0001). CZn-Formula C gave the lowest clearance values at both assessment points (Figure 4).
There was a positive correlation between the zinc clearance before and after oral zinc supplementation (Figure 5).
The only clearance values that were positively correlated with serum zinc concentrations after oral zinc supplementation were those calculated using CZn-Formula B (Figure 4). Thus, CZn-Formula B was more effective in detecting small changes in body zinc status, P<0.0002 (Figure 6). However, zinc tubular reabsorption (TRZn) was almost 100% in all children, both before (99.84±0.09%) and after (99.81±0.08%) oral zinc supplementation.
Clinical parameters and laboratory tests are only useful for detecting severe zinc deficiencies. The zinc concentrations in plasma, serum, erythrocytes, platelets, leukocytes, hair, sweat, urine and metalloenzymes have all been used to assess body zinc status.3 However, none of these have been universally accepted as a measure of body zinc status, which hinders the reliable detection of marginal zinc deficiency.20 Some reports in the literature suggest that zinc kinetics may be a reliable indicator of total body zinc status.
In this study, oral zinc supplementation was initially effective at significantly increasing basal serum zinc concentrations. Similar results have been previously reported by our group and by other researchers.6, 14, 15 To assess body zinc status we used a unique kinetic approach that combines intravenous and oral zinc supplementation. All values obtained from our three different clearance calculations were <20 ml/kg/h, suggesting that there was no marginal zinc deficiency in the children participating in this study. Elevated zinc levels have been reported in children with Crohn's disease, diabetes mellitus and short stature.5, 12, 21 The clearance values that we obtained revealed interindividual differences in the results obtained before and after oral zinc supplementation. The highest clearance values were calculated using CZn-Formula B and the lowest clearance values were calculated using CZn-Formula C (Figure 4). This latter kinetic method requires a perfect renal function, because the zinc is filtered by the glomeruli, secreted by the proximal tubule and absorbed by the distal tubule. That which exceeds the renal threshold is collected in the collecting tubules. Regardless of the method of calculation, zinc clearance before and after oral zinc supplementation was positively correlated (Figure 5). To the best of our knowledge no other studies examining the kinetics of zinc clearance using multiple methods of calculation have been published to date. CZn-Formula B was positively correlated with basal serum zinc concentration after oral zinc supplementation (Figure 5).
By contrast, a negative correlation between serum zinc concentrations and zinc clearance values has been previously reported in children with short stature.12 Figure 6 shows that kinetic formula B better distinguishes the small difference in serum zinc concentration before and after oral supplementation than do the other kinetic formulas (P<0.0002) and it will be useful in children with some pathology.
Understanding zinc clearance is crucial for determining renal function and tubular reabsorption. In our study, TRZn was normal both before (99.84±0.09%) and after (99.81±0.08%) oral zinc supplementation. In other words, the children in our study exhibited altered zinc clearance without any change in glomerular or tubular function. Similar results have been reported in both prepubertal children and adults.6, 14, 15 Zinc deficiency causes a reduction in the number of nephrons and glomeruli, as well as in renal blood flow.22 Thus, zinc may have a role in preserving renal function. Zinc deficiency can result from a number of factors: (i) inadequate intake or absorption, (ii) loss of zinc, mainly through urine and (iii) increased daily needs.2 Urinary zinc loss occurs only when concentrations exceed the threshold for tubular reabsorption, but the mechanism is still not well understood.13 Urinary zinc excretion and renal zinc clearance increased after zinc injection or oral zinc load in normal individuals and type I diabetes patients.14, 15 Zinc clearance has been proved effective for detecting changes in the zinc status in patients with Berardinelli–Seip syndrome, indicating that these patients have marginal zinc deficiency.7
The main limitation of this study was its relatively small sample size. However, performing both clearance tests within 3 months in healthy children younger than 9 years of age is a difficult task. Moreover, the test is slightly invasive. Nevertheless, the rate of compliance was satisfactory, given that only five children did not complete the study. As our sample was not probabilistic, other studies with a more representative population are needed.
In conclusion, all three clearance formulas that we used were suitable for assessing zinc status in healthy children. However, CZn-Formula B was better able to detect small changes in body zinc status than the other formulas. These results suggest that CZn-Formula B may be useful for investigating marginal zinc deficiency in undernourished children and in those with diabetes mellitus or short stature.
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We would like to thank Alfredo de Araújo Silva for his invaluable technical assistance. This study was supported by the National Council for Scientific and Technological Development (CNPq), grant number 472832/2011-5.
The authors declare no conflict of interest.
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Cite this article
Vale, S., Leite, L., Alves, C. et al. Zinc pharmacokinetic parameters in the determination of body zinc status in children. Eur J Clin Nutr 68, 203–208 (2014). https://doi.org/10.1038/ejcn.2013.250
- serum zinc
- renal zinc clearance
- oral zinc supplementation
- venous zinc administration