Automated Speciation of Mercury in the Hair of Breastfed Infants Exposed to Ethylmercury from Thimerosal-Containing Vaccines
Automated Speciation of Mercury in the Hair of Breastfed Infants Exposed to Ethylmercury from Thimerosal-Containing Vaccines
Biol Trace Elem Res
José G. Dórea & Wade Wimer & Rejane C. Marques & Christopher Shade
Received: 7 December 2009 / Accepted: 31 March 2010 Springer Science+Business Media, LLC 2010
Abstract A simplified thiourea-based chromatography method, originally developed for methyl and inorganic mercury, was adapted to separate methylmercury (MeHg), methylmercury (EtHg), and inorganic mercury (HgII) in infants’ hair. Samples were weighed and leached with an acidic thiourea solution. Leachates were concentrated on a polymeric resin prior to analysis by Hg-thiourea liquid chromatography/cold vapor atomic fluorescence spectrometry. All but one sample showed small amounts of EtHg, and four of the six analyzed samples had proportionally higher HgII as a percent of total Hg. Breastfed infants from riverine Amazonian communities are exposed to mercury in breast milk (from high levels of maternal sources that include both fish consumption and dental amalgam) and to EtHg in vaccines (fromthimerosal). The method proved sensitive enough to detect and quantify acute EtHg exposure after shots of thimerosal-containing vaccines. Based on work with MeHg and HgII, estimated detection limits for this method are 0.050, 0.10, and 0.10 ng g−1 for MeHg, HgII, and EtHg, respectively, for a 20-mg sample. Specific limits depend on the amount of sample extracted and the amount of extract injected.
Keywords Ethylmercury . Methylmercury . Thimerosal . Hair . Vaccines . Speciation
Mercury and a gamut of neurotoxic substances are capable of causing both transient and lasting damage to the central nervous system; depending on the severity of such exposure, a great number of mental injuries can result. Environmental exposure to mercury occurs mainly through fish consumption (methylmercury (MeHg)), but young children are also exposed to ethylmercury (EtHg) as a result of immunization with thimerosal-containing vaccines (TCV). Because of physiological characteristics, young infants have highly diminished detoxification pathways and may handle Hg differently than older children and adults . Due to this increased sensitivity to environmental mercury exposure, it is crucial to identify the Hg forms which infants are exposed to during critical neurodevelopmental periods. The toxicokinetics and toxicodynamics of specific forms of mercury (e.g., inorganic mercury vs. MeHg vs. EtHg) vary according to their respective chemical characteristics such as solubility, bioavailability, and mobility. Therefore, species-specific Hg analysis is crucial. Additionally, genetic disposition for detoxification abilities may further modify response to exposures .
Current interpretation of total hair Hg in population studies assumes that most of it is derived from fish MeHg; indeed it has been shown that inorganic Hg (HgII) from occupational exposure is eliminated in urine while fish MeHg is trapped in the hair and eliminated in the feces . Though studies exist indicating that thimerosal-derived Hg can turn up in the hair as a result of occupational and chronic exposure [4, 5], none show Hg in hair from acute exposure to TCV . To accurately discern TCV-Hg exposure in hair in the presence of other forms of Hg, a sensitive speciation system is needed.
The most common mercury speciation method, ethylation/gas chromatography, can only measure monomethylmercury since monoethylmercury results in the same ethylated analog (diethylmercury) as does HgII. Other alkylating techniques such as propylation  and butylation  coupled to gas chromatography (GC) separation and isotope dilution/ICP-MS or to GC/atomic fluorescence spectrometry (AFS) allow accurate and sensitive analysis of MeHg, EtHg, and HgII, but the derivatizations are laborious and not easily scalable; additionally, due to strong matrix interference, solvent extraction is needed to obtain low detection limits; the solvent extraction further lengthens the procedure and eliminates measurement of HgII. Gas chromatography/electron capture detection  and CG/AFS  methods can measure both MeHg and EtHg as halide complexes after solvent extraction, but this approach also requires a solvent extraction step, lacks a preconcentration capability, and cannot analyze HgII. Selective reduction techniques have also been used to measure EtHg as the difference between HgII and HgT [9, 10] but this technique cannot make the important distinction between MeHg and EtHg and is not sensitive enough to use very small samples (<10 mg).
Recent developments with liquid chromatography, specifically Hg-thiourea liquid chromatography [11–13], allow scalable, automated analysis of MeHg and HgII, with quantitative pre-concentration of sample extracts possible. This separation system uses liquid chromatography to separate cationic Hg-thiourea complexes. The present work describes a modification of the method described by Shade  to allow ion-pairing reversed phase chromatographic separation of MeHg, EtHg, and HgII (forms crucial for understanding their metabolism in young infants) after acidic thiourea leaching of hair samples and on-line preconcentration of leachates. The on-line preconcentration coupled to atomic fluorescence detection allows use of extremely low sample sizes (to <1 mg if necessary), which is essential for large scale studies that look at multiple elements. Accurate analysis of these three forms of Hg is crucial to proper understanding of potential toxicological effects from vaccine-derived Hg.
Materials and Methods
Hair samples were part of a large ongoing project with riverine communities along the Rio Madeira, the main tributary of the Amazon. These communities lead a traditional lifestyle Dórea et al. with a heavy dependence on fish to complement their traditional starchy diet. The children that took part in the study were preschoolers (aged 2 to 59 months) for which we had parental consent and obtained approval of the ethics committee of the Universidade of Rondonia. Represented in the present work are five (two girls and three boys) infants (aged 2 to 6 months) that had been exposed to TCV (hepatitis B and DTP vaccines) representing exposure ranging from 25 to 100 μg of TCV-Hg. Hair samples were cut with stainless steel scissors from the occipital area close to the scalp. The sampled hair was bundled together, placed in a labeled envelope and taken to the Laboratory of Radioisótopos of the Universidade Federal do Rio de Janeiro, stored in a cool and dark place until analysis. Some of these samples were shipped to Quicksilver Scientific, LLC (Lafayette, CO 80026, USA) for mercury speciation. One additional sample was supplied by an American individual exposed to TCV (Fig. 2b) to Quicksilver Scientific.
The method of Shade  was adapted with minor adjustments to permit separation of the three forms of Hg (HgII, MeHg, and EtHg) likely to be present in breastfed infants that have been inoculated with TCV. The method, Hg-thiourea complex liquid chromatography (HgTu-LC) cold vapor atomic fluorescence, and its extensive validation are described elsewhere . Briefly, the method is based on separation of ionic mercury forms by the difference in charge of their respective thiourea S = C(NH2)2 (Tu) complexes; thiourea being a neutral ligand yields cationic Hg-Tu complexes with ionic Hg forms. The original HgTu-LC system  was based solely on charge separation (ionchromatography); the addition of ion-pairing reversed phase separation allowed the addition of alkyl group selectivity to the charge selectivity, thereby allowing separation of the monovalent complexes of different monoorganomercurials. The system features a quantitative on-line preconcentration step prior to separation; the preconcentration is based on forming neutral iodide complexes (MeHgI, HgI2, and EtHgI) and trapping them on a hydrophobic polydivinylbenzene resin. Iodide complexes are formed by addition of potassium iodide to the sample solution after leaching and filtration. After separation, Hg forms are sequentially oxidized and then reduced on-line to an elemental cold vapor that is stripped from the solution in a thin-film diffusion gas liquid separator, dried over a Nafion membrane, and then measured by atomic fluorescence spectrometry (Tekran 2500).
The main modification to the system was the use of a 4.6Å~150-mm 100% polydivinylbenzene resin column. The all-PEEK column was custom packed by Column Engineering (Ontario, CA, USA) with resin from Jordi FLP (Waltham, MA, USA). The column was kept in a heater (FIAtron CH30 column heater with TC-50 controller) and separation times were tuned with temperature. MeHg eluted first, then HgII, and then EtHg. The separation between MeHg and EtHg was mainly controlled by ion-pairing reagent and acetic acid concentrations, whereas the HgII peak retention time was strongly temperature dependent; run temperatures were typically 60–70°C and could be adjusted during the run if necessary.
A 1-μg mL-1 CH3Hg+-Hg standard was prepared from solid CH3HgCl (Sigma-Aldrich) by dissolving in methanol and then serially diluting into a solution of 0.2% v/v conc. HCl and 0.5% v/v conc. acetic acid. A 1-μg mL−1 CH3CH2Hg+-Hg standard was prepared from solid thimerosal (sodium ethylmercuric thiosalicylate, Sigma-Aldrich) by dissolving and serially diluting in methanol. The concentration of the organomercury solutions was standardized against a 1 mg mL−1 HgII SPEX Certiprep standard using EPA Method 7473 Hg Species in Infants’ Hair on a Nippon MA-2000. Certified reference materials, BCR 463 (Tunafish) and Dolt-3 (dogfish liver) were obtained from RT Corp (Laramie, WY, USA).
Thiourea is hepatotoxic and a suspected carcinogen; care should be taken in its use and disposal. However, thiourea is preferred because it is unique among the sulfur-containing ligands in that it does not protonate and is neutral but soluble. All mercury standards deserve great care in use due to their extremely toxic nature. Thiourea is hepatotoxic and a suspected carcinogen; care should be taken in its use and disposal. However, thiourea is preferred because it is unique among the sulfur-containing ligands in that it does not protonate and is neutral but soluble. All mercury standards deserve great care in use due to their extremely toxic nature.
Samples were leached in acid-washed 40-mL borosilicate glass vials with PTFE-lined polyethylene caps (I-Chem series 200) in a mixture of thiourea, HCl, and glacial acetic acid. The extraction solution removes ionic Hg forms from thiol binding sites in the protein matrix of the hair by (1) protonating the thiol sulfur group of the cysteinyl residues in the protein, and (2) forming stable hydrophilic cationic thiourea complexes. Acid concentrations and leaching time and temperature were optimized for ethylmercury stability prior to receiving the samples; ∼100% recovery was achieved for all three forms using an aqueous solution of 1% m/v thiourea (99%, Acros), 5% v/v Glacial Acetic Acid (Certified ACS Plus; Fisher Scientific), 1% v/v HCl (Trace Metal Grade
, Fisher Scientific). Samples of several strands of hair (2–16 mg) were finely chopped with stainless steel scissors leached for approximately 12 h (overnight) in 5 or 10 mL of extraction solution at 40°C and then filtered through pre-cleaned (with extraction solution) 0.45 μm PVDF syringe filters into 8-mL acidwashed borosilicate glass vials with PTFE-lined polypropylene caps. Previous work with hair had shown quantitative extraction between 3 and 24 h of leach time . After filtration and immediately prior to analysis, samples solutions were brought to 10-mM [I–] by adding 50 μL
of 1-M KI per 5 mL of sample solution; KI (ACS reagent grade, Acros) solution was preserved with 25% m/v sodium ascorbate (Pharmaceutical grade—NOW Foods) to prevent formation of I2, which can decompose organomercurials. The preserved KI solution is stable for at least one week. All mixing ratios are presented as a percentage of the final solution volume.
The preconcentration procedure is detailed in Shade . Briefly, it entails an automated sequence of rinsing the resin column with 50% MeOH in DI, loading the sample, and then rinsing with 7.5% m/v HCl prior to elution into the analytical system. Detailed account of the procedure can be found elsewhere [11–13].
The calibration curves for all three forms were highly linear over the range examined with very similar slopes (Fig. 1). The modifications to the Shade method  provided clean baseline separation for MeHg, HgII, and EtHg (Fig. 2) without excessive peak spreading in the second and third peaks. Quality control data demonstrated efficiency of extraction and good accuracy (97.7–105.1% recovery for certified reference materials (CRMs)) with precise (1.5% CV) and high recovery (94.8–96.9%) of spikes (Table 1). No CRMs exist for EtHg, but we assume complete recovery of EtHg based on the combination of (1) full recovery of MeHg and HgII from CRM’s and (2) full recovery of MeHg and EtHg spiked onto hair samples before extraction (Table 1). Due to limited sample size we could not check the sum of MeHg + HgII + EtHg for consistency with total Hg, but extensive testing in Shade  validates quantitative extraction of MeHg and HgII on similar matrices.
Samples examined showed a wide range of concentrations of all three analytes with MeHg ranging over three orders of magnitude (Table 2). EtHg was low but detectable in all but one sample, varying from 0.8 to 9.5 ng g−1. Inorganic mercury as a percentage of HgT varied from 6.6% to 72.9% of HgT.
Sample pretreatment is an important step of the analytical procedure to speciate and quantify mercury in biological samples. EtHg is known to be less stable than MeHg [7, 14] and that was born out in our development work, where we had to lower the HCl in our extraction solution in order to preserve EtHg during the extraction and analysis (data not shown). Due to the lower stability, it would be expected that EtHg, once incorporated into hair, would breakdown to HgII more rapidly than MeHg would. In our analysis work on hair and nails, HgII is usually only 5–20% of THg and is always tightly correlated with MeHg, indicating that most HgII is a breakdown product of MeHg. Thus, thimerosal exposure is expected to show both directly as residual EtHg and indirectly as elevated HgII (higher than average—typically 5–15%—percentage of THg). This pattern of enhanced breakdown of EtHg to HgII (compared with MeHg to HgII) has been clearly demonstrated before in brains of monkeys exposed to either thimerosal or MeHg  and in the kidney, liver, and brains, of mice exposed to either thimerosal or MeHg .
In these Amazonian breastfed infants (Table 2 and Fig. 3), samples showed less obvious EtHg than the United States sample (Fig. 2) and some had very high MeHg.
Even though MeHg has a slower breakdown rate than EtHg, the sheer mass of it compared to EtHg can obscure the elevation of HgII due to TCV exposure. The elevation of HgII was very ob
vious in samples 144, 152 (Fig. 3), and 179, was less obvious in 142, and washed out in 215. Sample 207A (Fig. 3) showed no evidence, either as EtHg or elevated HgII, of thimerosal exposure. With the exception of sample #144, the samples showed a good correlation between EtHg and HgII as a percentage of total Hg (Fig. 4).One limitation of the study is that the infants’ hair samples were not collected for a proper toxicokinetic study of ethylmercury, rather we took advantage of an ongoing sampling program that was monitoring the growth and development of preschool children.
Gibicar et al.  determined EtHg directly in hair samples from dialysis patients and nursing staff from a nephrology ward where thiomersal was used as an antimicrobial agent, apparently characterizing a chronic situation different from an acute exposure as a result of TCV
inoculation. Thimerosal has many uses in the healthcare industry in addition to its place in both infantile and adult vaccines as an antimicrobial preservative. In the health care industry, high rates of allergic sensitization to thimerosal occur among health care workers  and this sensitivity coincides, in the majority of cases, with polymorphisms in glutathione S-transferase genes ; clearly such genetic predispositions could cause complications in infants exposed to TCV. Thus thorough understanding of its metabolism in humans (of various genetic detoxification capacities) is needed, and accurate and reliable speciation techniques are required to develop this understanding.
Amazonian breastfed infants can be exposed to all forms of maternal-Hg present in human milk plus an additional acute EtHg load as a result of inoculation with TCV . To our knowledge, this is the first time that one analytical method is used to speciate all forms of Hg exposure during infancy.
The method presented here features sensitivity, accuracy, and precision, with a simple scalable sample preparation, and an automated analysis, facilitating the necessary largescale epidemiological studies for a complete understanding of the multifactorial mercury exposures encountered by infants receiving TCV.
Acknowledgements We thank the National Research Council of Brasil-CNPq for the research grant (CTHIDRO, project-555516/2006-7) that paid for the analytical work.
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