Determination of Lead in Biological Samples of Childrenwith Different Physiological Consequences Using Cloud PointExtraction Method

Faheem Shah & Tasneem Gul Kazi & Naeem Ullah &

Hassan Imran Afridi

Received: 10 February 2013 /Accepted: 16 April 2013 /Published online: 28 April 2013# Springer Science+Business Media New York 2013

Abstract In present study, lead (Pb) level in biologicalsamples of children with physiological disorders (liver,bone, and gastrointestinal; age ranged 1–10 years) havebeen assessed. For comparison purpose, age-matchedhealthy children were also selected. Cloud point extraction(CPE) was employed for preconcentration of Pb in acid-digested biological samples prior to its determination byflame atomic absorption spectrometry (FAAS). Dithizone(diphenylthiocarbazone) and nonionic surfactant Triton X-114 (TX-114) were used as complexing reagent andextractant, respectively. The effects of several experimentalvariables on proposed CPE were evaluated. Under the opti-mum experimental conditions, the observed detection limit(LOD) and the enhancement factor (EF) were 0.08 μg L−1

and 53, respectively. Relative standard deviation (RSD) of10 μg L−1 Pb was 3.4 %. It was observed that children withliver-, bone-, and gastrointestinal-related disorders had

three- to fourfold higher Pb level in blood and scalp hairsamples.

Keywords Preconcentration . Lead . Cloud pointextraction . Dithizone . Triton X-114

Introduction

The sensible role of the different metals found in the body isuncertain. Lead (Pb) is one of the most ubiquitous noxiouselements found everywhere. Lead comprises a number ofhealth consequences particularly during childhood are still aworldwide problem [1–4]. Lead has the ability to affectindividuals of any age, but its effects are conflicting on chil-dren as their behavior place them at higher risk [5]. Severalcurrent reports have shown that exposure of Pb continues to bea main communal health problem globally [6–8].

Children of underdeveloped countries are equally moresusceptible to neurodevelopmental delays and less expectedto be examined for toxic exposure of Pb [9, 10]. Leadexposure in urban regions of developing nations is highestin the world [11, 12]. Man is widely exposed to Pb fromfood and environment. Being a toxicant, Pb has the potentialto disturb many biochemical actions present in cellsthroughout the body and is responsible for a number ofhealth problems [13–15]. In children, Pb toxicity may causerespiratory disorders, anemia, oxidative damage to liver,chronic nephrotoxicity, kidney impairment, and cognitivedisturbances [16, 17]. Toxic metals play an important rolein liver disease, particularly liver degeneration. Pb inhibitsbiosynthesis and affects liver membrane permeability, re-ducing some of its functions [18]. Lead in blood has a half-life of 27 days, while its half-life in bone is 30 years.However, bone biopsies were out of the question [19]. Lead

F. ShahFaculty of Science, Department of Chemistry, Erciyes University,38039 Kayseri, Turkey

F. Shah (*) : T. G. Kazi :N. Ullah :H. I. AfridiNational Center of Excellence in Analytical Chemistry,University of Sindh, 76080 Jamshoro, Pakistane-mail: shah_ceac@yahoo.com

T. G. Kazie-mail: tgkazi@yahoo.com

N. Ullahe-mail: khannaeemullah@ymail.com

H. I. Afridie-mail: hassanimranafridi@yahoo.com

N. UllahFaculty of Science and Arts, Chemistry Department,Gaziosmanpaşa University, 60250 Tokat, Turkey

Biol Trace Elem Res (2013) 153:134–140DOI 10.1007/s12011-013-9677-9

is known to associate with osteoporosis [20]. In bone, Pb, bycompeting with calcium, is linked to hydroxyapatite crystals[21]. Once it has been stored in the skeleton, it can entersystemic circulation when physiological or pathologicalprocesses induce bone remodeling [22]. For these reasons,Pb determination in trace and ultra trace levels in biologicalsamples become a matter of concern, but due to complextemplate and, generally, its low concentration, it is a difficulttask. A number of different sample preparation steps interms of preconcentration have been developed, such asliquid–liquid microextraction (LLE) [23], coprecipitation[24], ion exchange [25], cloud point extraction (CPE), andsolid phase extraction [26, 27]. CPE is an interesting andefficient alternative, reducing solvent use and exposure,disposal costs, and the extraction time [13]. It is based onthe phase separation behavior of nonionic and zwitter ionicsurfactants in aqueous solutions, where the phase separationtakes place with the increase in temperature or the addingligand [28].

This work describes the application of CPE of Pb inbiological samples (scalp hair and blood) of children aged1–10 years that have different physiological disorders. Leadwas determined after the formation of a complex withdithizone and then extracted in Triton X-114 as surfactant.

Experimental

Reagents

All of the laboratory solutions were prepared with deionizedwater further purified through reverse osmosis. Acids andother chemicals used in this study were of analytical gradeand were obtained from Merck, Darmstadt, Germany.Dithizone was obtained from Acros Organics (NJ, USA:1-800-ACROS-01) and its 0.01 % solution was preparedby dissolving 0.01 g in 100 mL of ethanol (Merck). Thenonionic surfactant Triton X-114 was obtained from Sigma-Aldrich (St. Louis, MO, USA) and was used without furtherpurification. A range of 0.2–1.4 % (v/v) nonionic surfactantsolutions were prepared by dissolving appropriate volumesof Triton X-114 (Merck) in 100 mL of deionized water. Pbstandard solutions were prepared by the dilution of certifiedstandard solution (1,000 mg L−1; Fluka Kamica, Bush,Switzerland). Stepwise dilution of the stock standard solu-tion with 0.2 M HNO3 was made for preparation of diluteworking standard solutions.

Apparatus

A pH meter (EcoScan Ion 6, Malaysia) was employed forpH adjustments. Centrifugation was carried out using aWIROWKA Laboratoryjna type WE-1, nr-6933 centrifuge

(speed range 0–6,000 rpm, timer 0–60 min, 220/50 Hz,Mechanika Phecyzyjna, Poland). A PerkinElmer Model AAnalyst 700 (Norwalk, CT, USA) flame atomic absorptionspectrophotometer was used. The hollow cathode lamp ofPb was run under the conditions suggested by the manufac-turer. A single-element hollow cathode lamp was operated at7.5 mA and spectral bandwidth of 0.7 nm. The analyticalwavelength was set at 283.3 nm. The acetylene flow rateand the burner height were adjusted in order to obtain themaximum absorbance signal.

Selection of Subjects

We selected children aged 1–10 years and have differentphysiological disorders. All the studied children were at-tending the outpatient department (OPD) of the pediatricward of Civil Hospital Karachi. Children’s parents wereinterviewed to ascertain the baseline characteristics of chil-dren. Among the children under study, 88 were sufferingwith liver-related problems, 183 with gastrointestinal disor-ders, and 173 with bone-related disorders, while 224 healthyreferents were also selected. All the subjects have the sametype of residential areas (with least municipal facilities andunhygienic living conditions) and low socioeconomic sta-tus. The children under study <4 years were not attendingschool, while children with age >4 years were going toschool situated in the vicinity of their residential area.

In the hospital, mostly sick children were accompaniedby their mother or other woman relative. These women wereinterviewed to provide information regarding their familiesand their living conditions. Informed consent to include achild in the study was obtained from the child’s mother.Biological samples (blood and scalp hair) were collectedfrom children with the help of paramedical staff of thehospital. The study was approved by the higher educationcommission of Pakistan.

Sample Treatment

Blood was taken by venepunctures after the application ofEutectic Mixture of Local Anesthetics (EMLA) local anes-thetic cream to reduce the pain produced, collected intometal-free safety vacutainer blood-collecting tubes (Becton,Dickinson and Company, Rutherford, NJ, USA) containingK2EDTA (>1.5 mg L−1).

Scalp hair (SH) samples were taken from nape of theneck. The hair were tied together from 1 cm of scalp withTeflon thread, cut with a stainless steel scissor, andpretreated as reported in our earlier work [29]. Collectedsamples were placed in zippered polythene bags. To make arepresentative hair sample, each individual SH were cut intoapproximately 0.5 cm pieces and mixed. Washing was car-ried out to provide an accurate assessment of endogenous

Lead in Children with Different Physiological Consequences 135

metal contents. Each sample was washed with dilute TritonX-100 followed by rinsing with deionized water and acetone[29, 30]. Samples were dried at 80 °C for 6 h in an oven andthen kept in precleaned and labeled polyethylene vials [31].

In the order to achieve minimum digestion time,microwave-assisted digestion procedure was followed.Samples in triplicate (0.2 mL of whole blood and 20 mgof SH) were taken in separate polytetrafluoroethylene(PTFE) flasks. Three milliliters of freshly prepared digestionmixture of conc. HNO3 and H2O2 (2:1 v/v) was added tothose flasks and left for 10 min at room temperature. Allsamples were digested in a microwave to a semidried massas reported in our previous work [16, 29, 31]. The resultingdigested semidried mass was dissolved in 10 mL of0.1 mol L−1 HNO3.

CPE Procedure

For preconcentration method, aliquots of 10 mL Pb standardsolution (10 μg L−1) and acid-digested duplicate samples ofboth biological samples of each patients and control subjectswere transferred into centrifuge tubes (25-mL capacity) withglass stopper. For CPE, 1.0 mL of 0.1 % dithizone and1.0 mL of (0.2 %–1.4 v/v) Triton X-114 were added andthe pH range (4) was adjusted, respectively; the pH range of6–11, respectively, was adjusted by the addition of appro-priate buffer solutions. The tubes were kept in thermostatedwater bath at 30–60 °C until the solution turned cloudy andwas centrifuged at 4,000 rpm for 10 min to achieve phaseseparation. The centrifuged tubes were kept for 10 min in anice bath to increase the viscosity of surfactant-rich phasemaking the removal of upper aqueous phase easier. Beforethe introduction of a sample into the flame of flame atomicabsorption spectrometry (FAAS) by conventional aspiration,0.5 mL acidic ethyl alcohol (0.1 mol L−1 HNO3) was addedto reduce its viscosity.

Analytical Figure of Merit

The calibration graph for preconcentration of Pb withdithizone was linear with a correlation coefficient of 0.992at the range of 5–20 μg L−1. Regression equation for Pb–dithizone obtained as Abs=7.12 (Pb μg L−1)−0.01. To findthe enhancement factor (EF), the analytical curve of Pb wasprepared without CPE. The calibration equation obtainedwas Abs=0.134 (Pb μg L−1)+0.021 (R2=0.998). The ex-perimental enhancement factor calculated as the ratio ofslopes of the calibration graphs with and withoutpreconcentration was 53 for Pb complexed with dithizone.The limit of detection (LOD) and limit of quantification(LOQ) were calculated as under 3 and 10 s/m, respectively,where s is the standard deviation of ten measurements of theblank and m is slope of the calibration graph. The LOD and

LOQ were calculated as 0.08 and 0.27 μg L−1, respectively.The analytical characteristic, precision of methods, expressedas the % relative standard deviation (% RSD) of a minimumsix independent analyses of certified reference materials(CRMs) and a standard of Pb (10 μg L−1), after CPE of Pbwas found to be 3.4 %. The accuracy of the proposed meth-odology was also assessed by the analysis of certified refer-ence material (CRM), ClinCheck control-lyophilized humanwhole blood and CRM BCR human hair (Recipe, Munich,Germany), and the observed values for Pb were found to be ingood agreement with the certified values. Results in Table 1confirm the validity of our proposed microextractionprocedure.

Results and Discussion

Optimization of CPE Method

The variables (pH, ligand concentration, Triton X-114 volume,sample volume, centrifugation time, and rate) affecting theproposed procedure was assessed in order to get the optimizedexperimental condition.

Effect of pH

The effect of pH on our proposed CPE procedure wasinvestigated because pH has an important role in the forma-tion of metal–chelate complex. pH range of 6–11 was stud-ied for this purpose. As observed in Fig. 1, maximumrecovery of Pb occurred at pH 9. Lower recovery wasobserved at pH 6, but as pH increases, recovery also in-creases. At very high pH, decline in recovery was observedand thus, pH 9 was selected as the optimum for consequentexperiments.

Effect of Ligand Concentration

The effect of concentration of the ligand on recovery wasexamined and the results are shown in Fig. 2. Incline inrecovery was observed as ligand concentration increased

Table 1 Effect of coexistent ions on % recovery

Coexistent ions Tolerance limit (mg L−1) % recovery

Na+, K+, Ca+2 10,000 97.2±1.2

Fe3+, Mg+2 5,000 98.5±0.7

Cu+2, Mn+2, Co+2 2,000 98.1±2.1

Cd2+ 100 99.4±1.1

Cl−, F− 10,000 98.9±0.8

PO3−4 200 99.6±2.1

NO−3, SO4−2 100 99.0±2.5

136 Shah et al.

from 0.6 to 1 w/v %, while further increase in ligandconcentration adds nothing to Pb recovery, and 1 w/v %was found to be sufficient ligand concentration for totalcomplexation and maximum recovery. A concentration of1 w/v % was marked as the optimum ligand concentration.

Effect of TX-114

Triton X-114 (TX-114) is one of the important variablesaffecting CPE efficacy. The effect of TX-114 concentrationon the extraction of Pb was examined within the TX-114concentration range from 0.2 to 1.4 % (v/v). As highlightedin Fig. 3, the recovery is maximum as the TX-114 concen-tration was 0.8 % (v/v). At concentrations below this value,lower recovery was observed because there are few mole-cules of the surfactant to extract Pb–dithizone complex. ATX-114 0.8 % (v/v) was selected for subsequent studies.

Effect of Temperature

Temperature above the cloud point was the next importantparameter considered. It was desirable to utilize the lowestpossible equilibration temperature, enough for cloud forma-tion and capable of separating the two phases. The temper-ature at which the proposed procedure was carried out wasin the range 20–60 °C. Elevated temperature should beavoided, as it can decompose dithizone. As shown inFig. 4, maximum recovery was obtained at 45 °C, so45 °C was selected as optimum for further experiments.

Effect of Centrifugation Time and Centrifugation Rate

Time and rate of centrifuge are also reported as significantvariables influencing the partitioning of surfactant and aque-ous phase. Centrifugation time was studied between 5 and45 min. When the centrifugation time was 10 min, maxi-mum recovery was observed, while at lower or highercentrifuge times, the recoveries were both lower (Fig. 5).Minimum centrifugation time cannot assure perfect phaseseparation, while maximum centrifugation time redissolvesthe extractant in aqueous phase again. Additionally, the cen-trifugation rate was studied between 500 and 5,000 rpm(Fig. 6). Maximum recovery was observed when the centrifu-gation rate of 4,000 rpm and gradual decrease were seen whenthe centrifugation rate was decreasing. For further experi-ments, 10 min and 4,000 rpmwere chosen as optimum values.

Effect of Interference

Experiments were performed to find out the extent to whichour proposed CPE procedure is affected by the presence offoreign ions possibly interfering in the determination of Pb.Different quantities of foreign ions were added to standardsolution of 10 μg L−1 of Pb and the proposed procedure wasfollowed. Pb recoveries in the presence of these foreign ionstudies were equal or higher than 98 %. As shown in Table 2,Pb was almost quantitatively recovered in the presence of allinterfering ions. These results prove the applicability of our

Fig. 1 Effect of pH on Pb analytical response

Fig. 2 Effect of ligand concentration on Pb analytical response

Fig. 3 Effect of Triton X-114 concentration on Pb analytical response

Fig. 4 Effect of temperature on Pb analytical response

Lead in Children with Different Physiological Consequences 137

proposed procedure for Pb determination in biologicalsamples.

Analysis of Real Samples

The presented CPE procedure was successfully applied forthe determination of Pb in biological samples of childrenwith different physiological disorders and healthy referentsof the same age group. Levels of Pb in all samples ofdiseased children were significantly higher than those foundin referent children (Table 3). We found that the blood Pblevel (BLL) in healthy children was lower than the WHO-permissible BLL of 10 μg dL−1 that was observed to behigher in children suffering with bone, gastrointestinal, andliver disorders. All children under study who had high BLLwere also found to have elevated scalp hair Pb level (SHL).

Lead has been demonstrated to be accumulated in boneand in some soft tissues, such as liver, kidney, and brain [32,33]. Rashed et al. found that in liver-related disorders, Pbwas found at an elevated concentration in blood serum [34].Castilla et al. determined blood Pb level in a liver-diseasedgroup and compared results with a control group. Theyobserved elevated blood Pb level in diseased group ascompared with healthy controls [35]. The resulting data ofchildren who have different bone disorders (deformation,weakness, and multiple fracture) indicated that the levels ofPb in both biological samples were two to three times higherthan the control healthy children (p<0.01). It was reportedin literature that Pb exposure to children inhibits bone

vascularization and affects mineralization of cartilage,resulting in defective hydroxyapatite lattice formation dur-ing skeletal bone growth or fracture healing [36, 37].

Lead accumulates in bones throughout the human lifespan, but at the same time, it is mobilized from bone dueto remodeling system. In adults, approximately 90 % of thetotal body burden of Pb is found in bone. However, inchildren, only 70 % of the body burden is found in thebones, but the concentration increases with age. Due tochronic exposure of Pb, it becomes deposited in the formof insoluble Pb-phosphate on rapidly growing parts of theskeleton, such as radius, tibia, and femur [38]. Hepaticdamage has been reported only in a few cases followingoccupational exposure to Pb. Gastrointestinal disturbancessuch as nausea, vomiting, anorexia, constipation, and ab-dominal cramps have also been observed in workers of Pbindustry [39]. Our previous study evaluated the Pb exposureof early adolescent boys working in battery recycling work-shops, where the level of Pb in blood samples is significantlyhigher at 95 % confidence interval (277, 285 μg L−1). Most ofthe working boys had mild to moderate symptoms of ab-dominal pain, anemia, muscle pain, irritability, and sleepingdisorders [40].

The toxicity of Pb results from its avidity for the sulfhy-dryl group of proteins and various enzymes, which leads tojeopardization of their function [41–43]. Prominent signsand symptoms of Pb toxicity in children are anemia, ab-dominal pain, kidney failure, loss of appetite, constipation,weight loss, vomiting, lethargy, learning disabilities, irrita-bility, and behavioral problems [44]. Gastrointestinal

Fig. 5 Effect of centrifugation time (min) on Pb analytical response

Fig. 6 Effect of centrifugation rate (rpm) on Pb analytical response

Table 2 Determination of Pb in certified reference materials (CRM)

Certified values Found values % recovery

BLL(μg L−1)a 105.0±24.0 103.3±19.1 98.1±1.0

UL (μg L−1)a 41.0±9.9 41.1±10.4 100±0.7

SCL (μg g−1)a 33.0±1.2 32.6±0.87 98.8±0.9

aMean±standard deviation (x ∓ s)

Table 3 The concentration of Pb in biological samples of childrenwith different physiological disorders and healthy referent subjects

Disorder No. ofsubjects

BLL(μg L−1)a

ULL(μg L−1)a

SHL(μg g−1)a

Liver disorder 88 113.1±17.1 29.3±18.1 4.6±2.1

Gastrointestinaldisorder

183 139.0±40.6 25.4±9.2 3.1±0.89

Bone-relateddisorder

173 123.2±28.7 33.8±15.9 3.4±1.8

Healthyreferents

224 42.3±13.1 15.7±8.7 1.3±0.6

aMean±standard deviation (x ∓ s)

138 Shah et al.

disorders, such as diarrhea, constipation, weight loss, poorappetite, etc., are common in case of Pb toxicity. We foundhigher Pb level in children who were suffering with differentgastrointestinal disorders. Iron deficiency has a direct rela-tion with high blood lead levels in children [45].

Conclusion

CPE using Triton X-114 and dithizone has shown to be aproficient and easy preconcentration of Pb in blood samples.Our proposed procedure resulted in low detection limits andhigh enrichment factors. Additionally, use of toxic organicextractant solvents (i.e., chloroform, toluene, carbon tetra-chloride, etc.) has been replaced with Triton X-114 as agreen alternative. Coexistent ions in different samples didnot interfere in Pb determination and were found to betolerable. The present study also revealed that among thechildren with physiological disorders (liver, bone, and gas-trointestinal) have higher Pb level in their blood and scalphair samples. In conclusion, our data provided epidemiologicindication that Pb level increases the prevalence of a numberof disorders in children. Additional research is necessary toinvestigate molecular mechanisms. Although reasons for Pbexposure among children are not identified by this study,public health agencies and organizations should focus onexposure and primary prevention. For these reasons, this typeof analysis is a common requirement in the medical andtoxicological fields.

Acknowledgments Faheem Shah is grateful to the Scientific andTechnological Research Council of Turkey (TÜBİTAK) for awardinghim “2216 Research Fellowship Programme for Foreign Citizens”. Theauthors are also grateful to HEC Pakistan for financial supports.

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