Clinica Chimica Acta 428 (2014) 9–13
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Clinica Chimica Acta journal homepage: www.elsevier.com/locate/clinchim
Distribution of aluminum in hair of Brazilian infants and correlation to aluminum-adjuvanted vaccine exposure Denise Bohrer a, Marcella Schmidt a, Rejane C. Marques b, José G. Dórea c,⁎ a b c
Departamento de Química Analítica, Universidade Federal de Santa Maria, RS, Brazil Universidade Federal do Rio de Janeiro, Campus Macaé, CEP 27930-560, RJ, Brazil Departamento de Nutrição, Faculdade de Ciências da Saúde, Universidade de Brasília, Brasília, Brazil
a r t i c l e
i n f o
Article history: Received 9 August 2013 Received in revised form 20 October 2013 Accepted 21 October 2013 Available online 29 October 2013 Keywords: Aluminum Hair Vaccine Infants
a b s t r a c t Background: Nursing children are exposed to dietary aluminum (in breast milk and/or infant formulas) and through aluminum-adjuvanted vaccines (AAVs). We studied total hair-Al concentrations of nursing children that had been immunized with hepatitis B, DTP, and meningococcal vaccines. Methods: We studied the hair of 37 young children (aged 26 to 824 days) who were exposed to cumulative doses of Al ranging from 0.63 to 6.88 mg from AAVs. Graphite furnace atomic absorption spectrometry was used to reliably measure total Al concentrations in hair samples. Results: The analytical method proved sensitive enough to quantify Al in the hair of nursing children. At current levels of exposure it is possible to determine total Al in hair sample of 1.65 mg. Cumulative doses of AAV in children ranged from 0.63 to 6.88 mg Al. Median hair-Al was 47.7 μg g−1 (ranging from 12.2 to 221.9 μg g−1). There was no statistically significant correlation between hair-Al concentration and age of child (r = −0.049; p = 0.774), total exposure from vaccine (r = −0.078; p = 0.643), or the time elapsed after the last AAVs (r = 0.015; p = 0.931). Conclusion: Aluminum in children's hair can be reliably measured but we are still uncertain how representative it can be of the Al body burden. © 2013 Elsevier B.V. All rights reserved.
1. Introduction The sensitivity of the developing central nervous system (CNS) makes infants and fetuses highly vulnerable to toxic metals, which can cause both transient and lasting damage to the system. Young children have immature or highly diminished detoxification pathways that may impair Al metabolism as compared to adults and older children [1]. Additionally, in neonates, renal function is not fully developed, making Al an important toxic challenge for pediatric patients [2]. Depending on the severity of exposure, it may result in psychomotor and mental effects. Exposure to aluminum during infancy is linked to maternal Al in breastfed babies and to Al in infants immunized with aluminumadjuvanted vaccines (AAVs) [3]. Maternal dietary sources of aluminum can increase bio-accessibility of elemental Al to young animals [4]. We have shown that exposure to an acute dose of adjuvant-Al in newborns and in infants is greater than Al exposure through breastfeeding [3]; the high acute doses of injected adjuvant-Al (250 to 1500 μg) during
Abbreviations: AAVs, aluminum-adjuvanted vaccines; DTP, diphtheria, tetanus, pertussis; HB, hepatitis B. ⁎ Corresponding author at: C.P.04322, Universidade de Brasília, 70919-970, Brasília, DF, Brazil. Tel.: +55 61 3368 3575; fax: +55 61 3368 5853. E-mail addresses: [email protected] (M. Schmidt), [email protected] (R.C. Marques), [email protected] (J.G. Dórea). 0009-8981/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.cca.2013.10.020
recommended schedules contrasted with estimated Al available after absorption from breastfeeding [3]. In exclusively breastfed infants, estimates of Al intake vary depending on maternal dietary Al exposure; however, non-breastfed infants can be exposed to even higher levels of the metal, depending on its concentration in formulas. Zeager et al. [5] summarized estimates of dietary intake for children aged 6 to 11 months (0.7 mgAl/day) and for 2-y-olds (4.6 mgAl/day) [5,6] on formulas and infant foods respectively. At young ages, starting with hepatitis B vaccine (at birth) and subsequently through the first year, immunization with AAVs elicits acute parenteral Al exposure [3,7]. The toxicity of acute exposure to low doses of adjuvanted-Al has only recently received attention. Intra-peritoneal injection of AAVs (diphtheria-tetanus, and diphtheria-tetanus-pertussis vaccine adjuvanted with aluminum hydroxide) into four-week-old female mice caused a transient rise of aluminum in brain tissue which peaked between the second and third day post-injection [8]. The studies of toxicokinetics (TK) and toxicodynamics (TD) of aluminum during early life have to rely on characteristics that help to trace its bioavailability and mobility in organ, tissues, or body compartments. Information regarding adjuvant-Al levels in hair after exposure to AAVs is scarce. Studies have shown that medical products containing Al can be detected in adult hair as a result of Al-containing medication [2], but so far there have been no attempts to determine Al in infant hair after acute exposure to AAVs. The aim of the study is to evaluate a
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D. Bohrer et al. / Clinica Chimica Acta 428 (2014) 9–13
standard analytical method for aluminum determination in hair samples of young children who received AAVs as recommended by the Brazilian immunization scheme.
Table 1 Operating parameters for graphite furnace atomic absorption spectrometry aluminum determination. Parameter
2. Materials and methods The hair samples in this study were part of a large ongoing project with riverine communities along the Madeira river basin. A detailed description of the study has appeared in a previous publication dealing with Hg exposure [9]; the protocol was approved by the Ethics Committee for Studies in Humans of the Federal University of Rondonia (Of. 001-07/CEP/NUSAU). Parental written consent was always obtained for hair sampling. The hair samples represent material that was saved after hair analysis for mercury studies [9], which took place between December 2005 and February 2007. Hair samples were cut with stainless steel scissors from the occipital area close to the scalp, bundled together, placed in a labeled envelope and stored until analysis. The sampled hair in this study was taken to the Laboratory of Analytical Chemistry of the University of Santa Maria, for Al determination. Represented in the present work are 37 children (14 boys and 23 girls) aged one to twenty-seven months, who had been exposed to AAVs (hepatitis B and DTP vaccines). The Al dose received was calculated from the vaccine formulation. The AAVs were from the same pharmaceutical company (Biomanguinhos) and had identical Al-adjuvant concentrations (2.5 mg/ml). The adjuvant-Al exposures were adjusted to represent doses of 0.25 ml (0.63 mg Al) of Hepatitis B and 0.5 ml (1.25 mg Al) for DTP vaccines. Other vaccines adjuvanted with aluminum were not used in these children at the time of the study. 2.1. Hair analysis 2.1.1. Apparatus and reagents A Zeenit atomic absorption spectrometer equipped with a transversal heated graphite furnace, a Zeeman-Effect background corrector, an autosampler (Jena Analytik), and a Trox class 100 clean bench were used. The water used throughout was distilled, deionized, and further purified with a Milli-Q high-purity water device (Millipore). An aluminum standard solution containing 1000 mg l−1 of Al in 1% HNO3 (v/v) (SepSol. SEM 3101, National Institute of Science and Technology) was used to prepare the working standard solutions. HNO3 (65%, 1.17 g/ml) from Merck was further purified by sub-boiling distillation. Acetone, ethyl ether, hexane, EDTA, and H2O2 (30%) were from Merck. Triton X-100 was furnished by Sigma–Aldrich. 2.1.2. Contamination control To avoid contamination, only plastic materials were used. All laboratory ware (pipette tips, volumetric flasks, etc.) was immersed for at least 48 h in a 10% (v/v) HNO3/ethanol solution and, shortly before use, washed with Milli-Q purified water. To avoid contamination from the air, all steps in the preparation of samples and reagents were carried out on a class 100 clean bench [10]. 2.1.3. Aluminum measurement by graphite furnace atomic absorption spectrometry The temperature program for graphite furnace measurements was optimized for aluminum in 0.5% HNO3 (v/v) aqueous solution. Pyrolysis and atomization temperatures were optimized and the analysis was carried out following the conditions displayed in Table 1.
Wavelength (nm)
Slit (nm)
309.3
0.8
6.0
Ramp (°C/s) 5 3 2 250 0 FP 500
Hold (s) 20 20 10 10 4 5 4
Lamp current (mA)
Temperature program Step Drying Drying Drying Pyrolysis AZ Atomization Cleanout
T (°C) 90 105 110 1500 1500 2650 2650
Gas (l/min) 3.0 3.0 3.0 3.0 0.0 0.0 3.0
rinsed with purified water and left to dry under laminar flow. After being washed and dried, hair samples were weighted (ca 100 mg) and transferred to a plastic container. One milliliter of concentrated nitric acid was added and the samples were gently heated in water-bath until no nitrous fumes were observed. After that, 500 μl hydrogen peroxide (30%) was added and the mixture was left for 48 h. The volume was completed to 10 ml with water, and the aluminum was directly measured in this solution by graphite furnace atomic absorption spectrometry. A blank test was carried out with a sample containing all the reagents. Recovery tests were done by adding 25 μgAl/l to 100 mg of washed hairsample, and performing the decomposition procedure as described. As the amount of hair from several children included in the study did not make up 100 mg, the total amount of hair sample was used and the volumes of nitric acid, hydrogen peroxide and water were adjusted accordingly. Thus, samples were treated as follows: All samples were washed with 50 ml 3% Triton X-100 solution (24 h), rinsed with water and left to dry under laminar flow. For samples weighing b 10 mg, 500 μl HNO3 and 100 μl H2O2 were used and the volume was completed to 5 ml with water. For samples weighing N10 mg, 750 μl HNO3 and 150 μl H2O2 were used and the final volume was 7.5 ml. 2.1.5. Quality control Aluminum was not detected in any of the solvents or solutions after washing hair samples. As this result is probably related to the cleanness of the hair sample used in the test, a washing procedure, using a solution of Triton X-100, was adopted throughout the work. Blank samples (n = 3) presented an Al concentration of 2.6 ± 0.1 μg/l, which was discounted from the concentration of each analyzed sample. Limits of detection and quantification were calculated as described in Skoog et al., [11], and were 1.0 μg/l and 3.5 μg/l, respectively. Recoveries varied from 90.9% to 95.9% (n = 3), and the result for 6 replicates of the test hair sample revealed an average Al concentration of 4.70 ± 0.30 μg/g. Under these working parameters and a LOD of 3.5 μgAl/l it is possible to work with a hair sample of 1.65 mg. 2.1.6. Statistical analysis Data summarization and correlation analysis were done by statistical packages; Prism software (Prism, version 10IC; GraphPad Software Inc.) was used to generate graphs and determine Pearson (p) correlation between the variables of interest. We tested for the data normality using the Kolmogorov–Smirnov one-sample test in order to apply appropriate statistical tests using XLSTAT (Adinsoft, version 1.01, 2013). 3. Results
2.1.4. Sample treatment To define the method for hair analysis, experiments were carried out with a sample of hair from a 5-year-old child. To eliminate exogenous Al, different washing solutions were tested. Hair samples (500 mg) were submerged in 200 ml hexane, ether, acetone, 3% (v/v) Triton X-100, and 5% (m/v) EDTA for 24 and 48 h. After this, aliquots of the washing solutions were taken and analyzed for their Al content. Hair samples were
The analytical results for infants' hair are summarized in Table 2. All hair samples could be measured with precision and reliability. The median Al concentration was 47.7 μg/g while the median age of children was 205 days, thus suggesting that Al may reflect the heavy exposure of the immunization schedule (0.63 to 6.88 mg Al). However, the range of hair-Al concentration (12.2 to 221.9 μg/g) represents both
D. Bohrer et al. / Clinica Chimica Acta 428 (2014) 9–13
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Table 2 Total aluminum received (Vaccine Al) and concentrations of aluminum [Al] in hair samples of young children immunized with aluminum-adjuvanted vaccines (AAVs). Sample #
Sex
Body Weight, kg
Age, d
AAVs
Vaccine Al (mg)a
Time after AAV, d
Hair [Al] μg/g
397 398 354 466 472 326 305 301 290 324 376 396 335 378 361 316 408 449 346 435 431 294 295 350 353 291 399 371 473 357 327 313 405 278 281 437 369
M F F F F M F F F F F M F F M F F M F F M M M M M F F F F M M F M M F F F
5.5 4 6.6 3.8 10 7.1 8.5 10 6.9 8.1 11 9.8 5.5 9.5 7.8 10 7 8.1 7.6 9.5 8.7 7.2 6 8 7.5 6.5 9 7.7 9.9 13.3 5.2 10 8.9 7 8.1 8.2 12.3
26 58 146 37 205 65 431 523 149 220 448 436 39 414 162 471 102 204 205 477 180 235 122 82 107 214 157 130 322 824 50 380 448 241 244 439 422
HB (0); DTP (1) HB (0); DTP (2) HB (2); DTP (2) HB (0); DTP (2) HB (3); DTP (3) HB (2); DTP (0) HB (3); DTP (3) HB (3); DTP (4) HB (2); DTP (1) HB (2); DTP (2) HB (3); DTP (3) HB (3); DTP (3) HB (1); DTP (0) HB (3); DTP (2) HB (2); DTP (2) HB (2); DTP (3) HB (2); DTP (0) HB (2); DTP (3) HB (2); DTP (2) HB (3); DTP (4) HB (2); DTP (2) HB (2); DTP (1) HB (2); DTP (0) HB (2); DTP (0) HB (2); DTP (1) HB (3); DTP (3) HB (1); DTP (0) HB (2); DTP (0) HB (3); DTP (2) HB (3); DTP (4) HB (2); DTP (0) HB (3); DTP (3) HB (4); DTP (3) HB (3); DTP (3) HB (3); DTP (3) HB (3); DTP (3) HB (3); DTP (3)
0.63 1.25 3.75 1.25 5.63 1.25 5.63 6.88 2.50 3.75 5.63 5.63 0.63 4.38 3.75 5.00 1.25 5.00 3.75 6.88 3.75 2.50 1.25 1.25 2.50 5.63 0.63 1.25 4.38 6.88 1.25 5.63 6.88 5.63 5.63 5.63 5.63
26 28 2 33 16 2 176 67 52 46 121 228 40 226 33 254 32 25 25 9 53 168 89 46 46 25 84 24 121 342 8 196 73 6 14 241 197
53.9 71.19 25.2 19.07 47.74 64.64 34.58 70.49 146.59 157.74 46.59 37.49 54.51 46.03 59.4 29.15 33.96 83.5 99.28 50.29 218.56 79.36 51.27 63.72 58.39 40.49 38.37 149.11 38.39 72.92 37.4 36.55 16.63 31.8 28.09 221.97 12.2
a
Estimated from values declared by manufacturer; d, days; HB, hepatitis B(doses); DTP, diphtheria, tetanus and pertussis (doses); M, male; F, female.
enteral (Al in diet) and parenteral adjuvant-Al sources. There was a wide range of concentrations of aluminum in hair. The distribution of values showed that results could be divided into two groups (Fig. 1). Among 37 results, the lower 32 results (86.5% of results, for which Al b 100 μg/g) showed essentially a normal distribution (Al = 47.9 +/− 20.5 μg/g); the upper 5 results could be considered outliers relative to this distribution (deviation from mean N 3 SD).The wide variation in the concentrations of hair-Al as a function of AAVs and other variables is presented in Fig. 2a to c. It is worth noting that 4 of 5 outliers are within 60 days of latest vaccine exposure. However, the lack of control of exposure variables makes this impossible to interpret with respect to vaccine exposure. Fig. 2 illustrates the correlation between hair-Al and several variables related to AAVs. There was no significant correlation (r = −0.049; p = 0.774) between age of child and hair-Al concentration (Fig. 2a). Although considerable amounts of Al exposure are derived from intramuscular AAVs, correlation between total exposure to adjuvant-Al and hair-Al concentrations (Fig. 2b) was not statistically significant (r = −0.078; p = 0.643). Also, when the hair-Al concentration was plotted as a function of time elapsed after the last exposure to AAVs (Fig. 2c), the correlation was not statistically significant (r = 0.015; p = 0.931). 4. Discussion This is the first attempt to trace residual Al in hair of young children after AAVs. The work shows that it is feasible to use infants' hair to monitor Al exposure allowing insight into a) the minimum hair mass that can produce useful results, and b) the detection windows likely to capture hair-Al during peak exposure to AAVs. Our results showed a normal distribution of hair-Al concentrations, but did not show any significant
correlation with vaccine parameters (adjuvant-Al taken, and time after vaccination) and age of infants. This reliable data is useful in furthering our understanding of Al metabolism as intra-muscular administered in infants and at quantities far higher than that found in human milk and infant formulas — the only sources of enteral exposure at this early age. There are no hair-Al data in studies addressing post immunization status with AAVs with which we can compare our results. As for Al exposure after immunizations, given the variation in immunization schemes and brand of vaccines, the estimated exposure to adjuvant-Al was variable. In our tested children the maximum Al exposure at 2 years of age was almost twice that attained by children of comparable age (3700 μg) in the USA [7,12,13]. However, given the lack of significant correlation between hair-Al and doses of AAVs it is unlikely that hair is a sensitive matrix that accumulates adjuvant-Al from vaccines. We know little about the metabolism of injected Al in the immature or young organism. The intramuscular route of Al exposure is essential for the adjuvant to boost the immunologic response; however, under such conditions (100% absorption) we do not know how it is distributed in tissues and how it reaches hair. In the present case it is impossible to ascertain the origin of Al in hair (environmental or iatrogenic). We tested children ranging from a few weeks (still nursing) to 2 y. Environmental Al from water and air, as well as from food (which includes maternal sources in breastfed infants) makes it difficult to ascertain the origin of Al; only exposure originating from the child's vaccine schedule could be estimated. Neonatal hair starts growing in utero during late pregnancy (approximately 28 weeks) and remains until 3–5 months post-partum [14]. The transition from fetal hair (lanugo) to permanent hair is accompanied by structural changes that may affect its metal cumulative properties [14].
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D. Bohrer et al. / Clinica Chimica Acta 428 (2014) 9–13
100 90
300
80
Frequency, %
Hair-Al concentration, µg.g-1
a)
200
100
70 60 50 40 30 20 10 0 0
0 0
250
500
750
50
100
150
200
250
Al concentration, µg.g-1
1000
Age of children, days Fig. 2. Aluminum (Al) concentrations in hair of infants as a function of age (a), total aluminum in Al-adjuvanted vaccines - AAVs (b), and time elapsed after the last shot of an AAV (c).
Hair-Al concentration, µg.g-1
b) 300
200
100
0 0
1
2
3
4
5
6
7
8
Total adjuvanted-Al, mg
Hair-Al concentration, µg.g-1
c) 300
200
100
0 0
100
200
300
400
Time elapsed after last AAV shot, days Fig. 1. Cumulative frequency distribution of total hair-Al concentrations.
During the growing phase of hair, keratinocytes (sulfur-keratin proteins) take up metals. However, hair components such as melanin can also accumulate metals [15,16]. Kauf et al. [17] reported that inorganic elements increased from post-natal day 1 to 90 days. Nevertheless, a small sample of newborn hair showed Al concentrations (n = 6; 52 μg/g) comparable [18] to and higher (n = 15; 76.24 μg/g) [19] than our median values. Al intake in infancy varies and it is assumed that it is much higher in formulas (soy based N cow's milk based) than in breast milk [20], but Al is considered to have a low absorption in drinking water (0.28%) and food (0.1%) in general (1). In fact, Litov et al. [20] reported that infants fed soy-based formula (1,700 μg/l) showed non-significant differences in blood Al when compared with breastfed infants (b5 to 45 μg/l). Therefore, the relatively high Al concentrations (median, 47.7 μg/g) suggest that 100% systemic available Al from injected doses of AAV could end up in hair. Indeed, experimental studies with injected Al showed a dose response of Al in hair of rabbits [21]. The total Al concentrations in hair in this study are much higher than the body retentions estimated by Mitkus et al. [12] but closer to the
model developed by Keith et al. [13]. Attempts to estimate body burden from AAVs yielded calculations of an estimated body burden of approximately 0.4 mg Al at birth; they estimated that body burden from vaccines was 2-fold higher than from dietary sources [12]. However, body burdens of Al derived from Keith et al. [13] were 20-fold higher than Mitkus et al.'s [12] estimate. Keith et al. [13] estimated that the Al body burden from human milk and formulas during the first year was around 0.1 mg and that the Al attributed to vaccines would be expected to fall below 4 mg. Al is a ubiquitous element, but only trace amounts are generally present in human tissues [22]. Hair is a convenient and non-invasive tissue that is easy to collect and store. In the context of Al body burden we are still uncertain how representative it can be [22]. Although we are confident about infant hair data shown here, we can not ascertain its origin (dietary or Al-adjuvanted vaccines). Future studies can now be designed to capture hair-Al response that might be related to specific sources using a larger number of subjects with different exposure loads. In our study infants of b7 weeks showed hair-Al of 0.63 to 1.25 mg/g. We do not know the kinetics of adjuvanted-Al in young children, or what proportion is taken up by hair. However, it has recently been shown that intraperitoneal injection of Al in newborn animals results in increased Al levels compared to adults [23]. It is worth noting that route of administration is an important parameter for both toxicokinetics and toxicodynamics [24]. There are concerns related to the use of Al materials with physicochemical properties different from adjuvant-Al in experimental studies [25]; indeed, there are differences in Al-adjuvant body retention depending on its chemical form [26]. Our results should reflect the true nature of exposure derived from Al-adjuvanted vaccines [25]. Analytical techniques to quantify Al in non-invasive tissues are fundamental to understand its metabolism as well as toxicodynamics. To date, existing models have shown discrepancies in predicting kinetics of Al in vaccines [12,13]. Therefore, besides the clinical and forensic interest, there is a need for specific methods to identify Al from vaccines in both health and environmental studies. The principal constraint of the study was that we did not measure Al concentration in the children's diet. Maternal transfer (through placenta or breast milk) or from AAV cannot be distinguished from these analyses. Although it is feasible to conduct Al exposure studies in hair samples, no interpretations of Al origin associated with toxicity risks can be made without establishing its origin. In the present work we show the only data on hair-Al after Al-adjuvanted vaccines; additionally, the study presents reliable data on the Al content of babies' hair. References [1] Willhite CC, Ball GL, McLellan CJ. Total allowable concentrations of monomeric inorganic aluminum and hydrated aluminum silicates in drinking water. Crit Rev Toxicol 2012;42:358–442. [2] Winterberg B, Bertram H, Rolf N, et al. Differences in plasma and tissue aluminum concentrations due to different aluminum-containing drugs in patients with renal insufficiency and with normal renal function. J Trace Elem Electrolytes Health Dis 1987;1:69–72.
D. Bohrer et al. / Clinica Chimica Acta 428 (2014) 9–13 [3] Dórea JG, Marques RC. Infants' exposure to aluminum from vaccines and breast milk during the first 6 months. J Expo Sci Environ Epidemiol 2010;20:598–601. [4] Domingo JL. Reproductive and developmental toxicity of aluminum: a review. Neurotoxicol Teratol 1995;17:515–21. [5] Zeager M, Woolf AD, Goldman RH. Wide variation in reference values for aluminum levels in children. Pediatrics 2012;129:e142–7. [6] Pennington JA, Schoen SA. Estimates of dietary exposure to aluminium. Food Addit Contam 1995;12:119–28. [7] Tomljenovic L, Shaw CA. Do aluminum vaccine adjuvants contribute to the rising prevalence of autism? J Inorg Biochem 2011;105:1489–99. [8] Redhead K, Quinlan GJ, Das RG, Gutteridge JM. Aluminium-adjuvanted vaccines transiently increase aluminium levels in murine brain tissue. Pharmacol Toxicol 1992;70:278–80. [9] Marques RC, Dórea JG, McManus C, et al. Hydroelectric reservoir inundation (Rio Madeira Basin, Amazon) and changes in traditional lifestyle: impact on growth and neurodevelopment of pre-school children. Public Health Nutr 2011;14:661–9. [10] Bohrer D, Dessuy MB, Kaizer R, et al. Tissue digestion for aluminum determination in experimental animal studies. Anal Biochem 2008;377:120–7. [11] Skoog DA, West DM, Holler F, Crouch SR. Fundamentals of analytical chemistry. 8th ed. Cengage Learning; 2003 199–204. [12] Mitkus RJ, King DB, Hess MA, Forshee RA, Walderhaug MO. Updated aluminum pharmacokinetics following infant exposures through diet and vaccination. Vaccine 2011;29:9538–43. [13] Keith LS, Jones DE, Chou CH. Aluminum toxicokinetics regarding infant diet and vaccinations. Vaccine 2002;20(Suppl. 3):S13–7. [14] Moller M, Gareri J, Koren G. A review of substance abuse monitoring in a social services context: a primer for child protection workers. Can J Clin Pharmacol 2010;17: e177–93.
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[15] Dórea JG, Pereira SE. The influence of hair color on the concentration of zinc and copper in boys' hair. J Nutr 1983;113:2375–81. [16] Al-Saleh I, Shinwari N, El-Doush I, et al. Comparison of mercury levels in various tissues of albino and pigmented mice treated with two different brands of mercury skin-lightening creams. Biometals 2004;17:167–75. [17] Kauf E, Wiesner W, Niese S, Plenert W. Zinc, copper, manganese and gold content of the hair of infants. Acta Paediatr Hung 1984;25:299–307. [18] Savabieasfahani M, Hoseiny M, Goodarzi S. Toxic and essential trace metals in first baby haircuts and mother hair from Imam Hossein Hospital Tehran, Iran. Bull Environ Contam Toxicol 2012;88:140–4. [19] Ramírez-Altamirano Mde J, Fenton-Navarro P, Sivet-Chiñas E, et al. The relationship of aluminium and silver to neural tube defects; a case control. Iran J Pediatr 2012;22: 369–74. [20] Litov RE, Sickles VS, Chan GM, et al. Plasma aluminum measurements in term infants fed human milk or a soy-based infant formula. Pediatrics 1989;84:1105–7. [21] Yokel RA. Hair as an indicator of excessive aluminum exposure. Clin Chem 1982;28(4 Pt 1):662–5. [22] Exley C. Human exposure to aluminium. Environ Sci Process Impacts 2013;15: 1807–16. [23] Veiga M, Bohrer D, Banderó CR, et al. Accumulation, elimination, and effects of parenteral exposure to aluminum in newborn and adult rats. J Inorg Biochem 2013;128:215–20. [24] Shaw CA, Li Y, Tomljenovic L. Administration of aluminium to neonatal mice in vaccine-relevant amounts is associated with adverse long term neurological outcomes. J Inorg Biochem 2013;128:237–44. [25] Exley C. When an aluminium adjuvant is not an aluminium adjuvant used in human vaccination programmes. Vaccine 2012;30:2042. [26] Hem SL. Elimination of aluminum adjuvants. Vaccine 2002;20(Suppl. 3):S40–3.
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Clinica Chimica Acta journal homepage: www.elsevier.com/locate/clinchim
Distribution of aluminum in hair of Brazilian infants and correlation to aluminum-adjuvanted vaccine exposure Denise Bohrer a, Marcella Schmidt a, Rejane C. Marques b, José G. Dórea c,⁎ a b c
Departamento de Química Analítica, Universidade Federal de Santa Maria, RS, Brazil Universidade Federal do Rio de Janeiro, Campus Macaé, CEP 27930-560, RJ, Brazil Departamento de Nutrição, Faculdade de Ciências da Saúde, Universidade de Brasília, Brasília, Brazil
a r t i c l e
i n f o
Article history: Received 9 August 2013 Received in revised form 20 October 2013 Accepted 21 October 2013 Available online 29 October 2013 Keywords: Aluminum Hair Vaccine Infants
a b s t r a c t Background: Nursing children are exposed to dietary aluminum (in breast milk and/or infant formulas) and through aluminum-adjuvanted vaccines (AAVs). We studied total hair-Al concentrations of nursing children that had been immunized with hepatitis B, DTP, and meningococcal vaccines. Methods: We studied the hair of 37 young children (aged 26 to 824 days) who were exposed to cumulative doses of Al ranging from 0.63 to 6.88 mg from AAVs. Graphite furnace atomic absorption spectrometry was used to reliably measure total Al concentrations in hair samples. Results: The analytical method proved sensitive enough to quantify Al in the hair of nursing children. At current levels of exposure it is possible to determine total Al in hair sample of 1.65 mg. Cumulative doses of AAV in children ranged from 0.63 to 6.88 mg Al. Median hair-Al was 47.7 μg g−1 (ranging from 12.2 to 221.9 μg g−1). There was no statistically significant correlation between hair-Al concentration and age of child (r = −0.049; p = 0.774), total exposure from vaccine (r = −0.078; p = 0.643), or the time elapsed after the last AAVs (r = 0.015; p = 0.931). Conclusion: Aluminum in children's hair can be reliably measured but we are still uncertain how representative it can be of the Al body burden. © 2013 Elsevier B.V. All rights reserved.
1. Introduction The sensitivity of the developing central nervous system (CNS) makes infants and fetuses highly vulnerable to toxic metals, which can cause both transient and lasting damage to the system. Young children have immature or highly diminished detoxification pathways that may impair Al metabolism as compared to adults and older children [1]. Additionally, in neonates, renal function is not fully developed, making Al an important toxic challenge for pediatric patients [2]. Depending on the severity of exposure, it may result in psychomotor and mental effects. Exposure to aluminum during infancy is linked to maternal Al in breastfed babies and to Al in infants immunized with aluminumadjuvanted vaccines (AAVs) [3]. Maternal dietary sources of aluminum can increase bio-accessibility of elemental Al to young animals [4]. We have shown that exposure to an acute dose of adjuvant-Al in newborns and in infants is greater than Al exposure through breastfeeding [3]; the high acute doses of injected adjuvant-Al (250 to 1500 μg) during
Abbreviations: AAVs, aluminum-adjuvanted vaccines; DTP, diphtheria, tetanus, pertussis; HB, hepatitis B. ⁎ Corresponding author at: C.P.04322, Universidade de Brasília, 70919-970, Brasília, DF, Brazil. Tel.: +55 61 3368 3575; fax: +55 61 3368 5853. E-mail addresses: [email protected] (M. Schmidt), [email protected] (R.C. Marques), [email protected] (J.G. Dórea). 0009-8981/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.cca.2013.10.020
recommended schedules contrasted with estimated Al available after absorption from breastfeeding [3]. In exclusively breastfed infants, estimates of Al intake vary depending on maternal dietary Al exposure; however, non-breastfed infants can be exposed to even higher levels of the metal, depending on its concentration in formulas. Zeager et al. [5] summarized estimates of dietary intake for children aged 6 to 11 months (0.7 mgAl/day) and for 2-y-olds (4.6 mgAl/day) [5,6] on formulas and infant foods respectively. At young ages, starting with hepatitis B vaccine (at birth) and subsequently through the first year, immunization with AAVs elicits acute parenteral Al exposure [3,7]. The toxicity of acute exposure to low doses of adjuvanted-Al has only recently received attention. Intra-peritoneal injection of AAVs (diphtheria-tetanus, and diphtheria-tetanus-pertussis vaccine adjuvanted with aluminum hydroxide) into four-week-old female mice caused a transient rise of aluminum in brain tissue which peaked between the second and third day post-injection [8]. The studies of toxicokinetics (TK) and toxicodynamics (TD) of aluminum during early life have to rely on characteristics that help to trace its bioavailability and mobility in organ, tissues, or body compartments. Information regarding adjuvant-Al levels in hair after exposure to AAVs is scarce. Studies have shown that medical products containing Al can be detected in adult hair as a result of Al-containing medication [2], but so far there have been no attempts to determine Al in infant hair after acute exposure to AAVs. The aim of the study is to evaluate a
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standard analytical method for aluminum determination in hair samples of young children who received AAVs as recommended by the Brazilian immunization scheme.
Table 1 Operating parameters for graphite furnace atomic absorption spectrometry aluminum determination. Parameter
2. Materials and methods The hair samples in this study were part of a large ongoing project with riverine communities along the Madeira river basin. A detailed description of the study has appeared in a previous publication dealing with Hg exposure [9]; the protocol was approved by the Ethics Committee for Studies in Humans of the Federal University of Rondonia (Of. 001-07/CEP/NUSAU). Parental written consent was always obtained for hair sampling. The hair samples represent material that was saved after hair analysis for mercury studies [9], which took place between December 2005 and February 2007. Hair samples were cut with stainless steel scissors from the occipital area close to the scalp, bundled together, placed in a labeled envelope and stored until analysis. The sampled hair in this study was taken to the Laboratory of Analytical Chemistry of the University of Santa Maria, for Al determination. Represented in the present work are 37 children (14 boys and 23 girls) aged one to twenty-seven months, who had been exposed to AAVs (hepatitis B and DTP vaccines). The Al dose received was calculated from the vaccine formulation. The AAVs were from the same pharmaceutical company (Biomanguinhos) and had identical Al-adjuvant concentrations (2.5 mg/ml). The adjuvant-Al exposures were adjusted to represent doses of 0.25 ml (0.63 mg Al) of Hepatitis B and 0.5 ml (1.25 mg Al) for DTP vaccines. Other vaccines adjuvanted with aluminum were not used in these children at the time of the study. 2.1. Hair analysis 2.1.1. Apparatus and reagents A Zeenit atomic absorption spectrometer equipped with a transversal heated graphite furnace, a Zeeman-Effect background corrector, an autosampler (Jena Analytik), and a Trox class 100 clean bench were used. The water used throughout was distilled, deionized, and further purified with a Milli-Q high-purity water device (Millipore). An aluminum standard solution containing 1000 mg l−1 of Al in 1% HNO3 (v/v) (SepSol. SEM 3101, National Institute of Science and Technology) was used to prepare the working standard solutions. HNO3 (65%, 1.17 g/ml) from Merck was further purified by sub-boiling distillation. Acetone, ethyl ether, hexane, EDTA, and H2O2 (30%) were from Merck. Triton X-100 was furnished by Sigma–Aldrich. 2.1.2. Contamination control To avoid contamination, only plastic materials were used. All laboratory ware (pipette tips, volumetric flasks, etc.) was immersed for at least 48 h in a 10% (v/v) HNO3/ethanol solution and, shortly before use, washed with Milli-Q purified water. To avoid contamination from the air, all steps in the preparation of samples and reagents were carried out on a class 100 clean bench [10]. 2.1.3. Aluminum measurement by graphite furnace atomic absorption spectrometry The temperature program for graphite furnace measurements was optimized for aluminum in 0.5% HNO3 (v/v) aqueous solution. Pyrolysis and atomization temperatures were optimized and the analysis was carried out following the conditions displayed in Table 1.
Wavelength (nm)
Slit (nm)
309.3
0.8
6.0
Ramp (°C/s) 5 3 2 250 0 FP 500
Hold (s) 20 20 10 10 4 5 4
Lamp current (mA)
Temperature program Step Drying Drying Drying Pyrolysis AZ Atomization Cleanout
T (°C) 90 105 110 1500 1500 2650 2650
Gas (l/min) 3.0 3.0 3.0 3.0 0.0 0.0 3.0
rinsed with purified water and left to dry under laminar flow. After being washed and dried, hair samples were weighted (ca 100 mg) and transferred to a plastic container. One milliliter of concentrated nitric acid was added and the samples were gently heated in water-bath until no nitrous fumes were observed. After that, 500 μl hydrogen peroxide (30%) was added and the mixture was left for 48 h. The volume was completed to 10 ml with water, and the aluminum was directly measured in this solution by graphite furnace atomic absorption spectrometry. A blank test was carried out with a sample containing all the reagents. Recovery tests were done by adding 25 μgAl/l to 100 mg of washed hairsample, and performing the decomposition procedure as described. As the amount of hair from several children included in the study did not make up 100 mg, the total amount of hair sample was used and the volumes of nitric acid, hydrogen peroxide and water were adjusted accordingly. Thus, samples were treated as follows: All samples were washed with 50 ml 3% Triton X-100 solution (24 h), rinsed with water and left to dry under laminar flow. For samples weighing b 10 mg, 500 μl HNO3 and 100 μl H2O2 were used and the volume was completed to 5 ml with water. For samples weighing N10 mg, 750 μl HNO3 and 150 μl H2O2 were used and the final volume was 7.5 ml. 2.1.5. Quality control Aluminum was not detected in any of the solvents or solutions after washing hair samples. As this result is probably related to the cleanness of the hair sample used in the test, a washing procedure, using a solution of Triton X-100, was adopted throughout the work. Blank samples (n = 3) presented an Al concentration of 2.6 ± 0.1 μg/l, which was discounted from the concentration of each analyzed sample. Limits of detection and quantification were calculated as described in Skoog et al., [11], and were 1.0 μg/l and 3.5 μg/l, respectively. Recoveries varied from 90.9% to 95.9% (n = 3), and the result for 6 replicates of the test hair sample revealed an average Al concentration of 4.70 ± 0.30 μg/g. Under these working parameters and a LOD of 3.5 μgAl/l it is possible to work with a hair sample of 1.65 mg. 2.1.6. Statistical analysis Data summarization and correlation analysis were done by statistical packages; Prism software (Prism, version 10IC; GraphPad Software Inc.) was used to generate graphs and determine Pearson (p) correlation between the variables of interest. We tested for the data normality using the Kolmogorov–Smirnov one-sample test in order to apply appropriate statistical tests using XLSTAT (Adinsoft, version 1.01, 2013). 3. Results
2.1.4. Sample treatment To define the method for hair analysis, experiments were carried out with a sample of hair from a 5-year-old child. To eliminate exogenous Al, different washing solutions were tested. Hair samples (500 mg) were submerged in 200 ml hexane, ether, acetone, 3% (v/v) Triton X-100, and 5% (m/v) EDTA for 24 and 48 h. After this, aliquots of the washing solutions were taken and analyzed for their Al content. Hair samples were
The analytical results for infants' hair are summarized in Table 2. All hair samples could be measured with precision and reliability. The median Al concentration was 47.7 μg/g while the median age of children was 205 days, thus suggesting that Al may reflect the heavy exposure of the immunization schedule (0.63 to 6.88 mg Al). However, the range of hair-Al concentration (12.2 to 221.9 μg/g) represents both
D. Bohrer et al. / Clinica Chimica Acta 428 (2014) 9–13
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Table 2 Total aluminum received (Vaccine Al) and concentrations of aluminum [Al] in hair samples of young children immunized with aluminum-adjuvanted vaccines (AAVs). Sample #
Sex
Body Weight, kg
Age, d
AAVs
Vaccine Al (mg)a
Time after AAV, d
Hair [Al] μg/g
397 398 354 466 472 326 305 301 290 324 376 396 335 378 361 316 408 449 346 435 431 294 295 350 353 291 399 371 473 357 327 313 405 278 281 437 369
M F F F F M F F F F F M F F M F F M F F M M M M M F F F F M M F M M F F F
5.5 4 6.6 3.8 10 7.1 8.5 10 6.9 8.1 11 9.8 5.5 9.5 7.8 10 7 8.1 7.6 9.5 8.7 7.2 6 8 7.5 6.5 9 7.7 9.9 13.3 5.2 10 8.9 7 8.1 8.2 12.3
26 58 146 37 205 65 431 523 149 220 448 436 39 414 162 471 102 204 205 477 180 235 122 82 107 214 157 130 322 824 50 380 448 241 244 439 422
HB (0); DTP (1) HB (0); DTP (2) HB (2); DTP (2) HB (0); DTP (2) HB (3); DTP (3) HB (2); DTP (0) HB (3); DTP (3) HB (3); DTP (4) HB (2); DTP (1) HB (2); DTP (2) HB (3); DTP (3) HB (3); DTP (3) HB (1); DTP (0) HB (3); DTP (2) HB (2); DTP (2) HB (2); DTP (3) HB (2); DTP (0) HB (2); DTP (3) HB (2); DTP (2) HB (3); DTP (4) HB (2); DTP (2) HB (2); DTP (1) HB (2); DTP (0) HB (2); DTP (0) HB (2); DTP (1) HB (3); DTP (3) HB (1); DTP (0) HB (2); DTP (0) HB (3); DTP (2) HB (3); DTP (4) HB (2); DTP (0) HB (3); DTP (3) HB (4); DTP (3) HB (3); DTP (3) HB (3); DTP (3) HB (3); DTP (3) HB (3); DTP (3)
0.63 1.25 3.75 1.25 5.63 1.25 5.63 6.88 2.50 3.75 5.63 5.63 0.63 4.38 3.75 5.00 1.25 5.00 3.75 6.88 3.75 2.50 1.25 1.25 2.50 5.63 0.63 1.25 4.38 6.88 1.25 5.63 6.88 5.63 5.63 5.63 5.63
26 28 2 33 16 2 176 67 52 46 121 228 40 226 33 254 32 25 25 9 53 168 89 46 46 25 84 24 121 342 8 196 73 6 14 241 197
53.9 71.19 25.2 19.07 47.74 64.64 34.58 70.49 146.59 157.74 46.59 37.49 54.51 46.03 59.4 29.15 33.96 83.5 99.28 50.29 218.56 79.36 51.27 63.72 58.39 40.49 38.37 149.11 38.39 72.92 37.4 36.55 16.63 31.8 28.09 221.97 12.2
a
Estimated from values declared by manufacturer; d, days; HB, hepatitis B(doses); DTP, diphtheria, tetanus and pertussis (doses); M, male; F, female.
enteral (Al in diet) and parenteral adjuvant-Al sources. There was a wide range of concentrations of aluminum in hair. The distribution of values showed that results could be divided into two groups (Fig. 1). Among 37 results, the lower 32 results (86.5% of results, for which Al b 100 μg/g) showed essentially a normal distribution (Al = 47.9 +/− 20.5 μg/g); the upper 5 results could be considered outliers relative to this distribution (deviation from mean N 3 SD).The wide variation in the concentrations of hair-Al as a function of AAVs and other variables is presented in Fig. 2a to c. It is worth noting that 4 of 5 outliers are within 60 days of latest vaccine exposure. However, the lack of control of exposure variables makes this impossible to interpret with respect to vaccine exposure. Fig. 2 illustrates the correlation between hair-Al and several variables related to AAVs. There was no significant correlation (r = −0.049; p = 0.774) between age of child and hair-Al concentration (Fig. 2a). Although considerable amounts of Al exposure are derived from intramuscular AAVs, correlation between total exposure to adjuvant-Al and hair-Al concentrations (Fig. 2b) was not statistically significant (r = −0.078; p = 0.643). Also, when the hair-Al concentration was plotted as a function of time elapsed after the last exposure to AAVs (Fig. 2c), the correlation was not statistically significant (r = 0.015; p = 0.931). 4. Discussion This is the first attempt to trace residual Al in hair of young children after AAVs. The work shows that it is feasible to use infants' hair to monitor Al exposure allowing insight into a) the minimum hair mass that can produce useful results, and b) the detection windows likely to capture hair-Al during peak exposure to AAVs. Our results showed a normal distribution of hair-Al concentrations, but did not show any significant
correlation with vaccine parameters (adjuvant-Al taken, and time after vaccination) and age of infants. This reliable data is useful in furthering our understanding of Al metabolism as intra-muscular administered in infants and at quantities far higher than that found in human milk and infant formulas — the only sources of enteral exposure at this early age. There are no hair-Al data in studies addressing post immunization status with AAVs with which we can compare our results. As for Al exposure after immunizations, given the variation in immunization schemes and brand of vaccines, the estimated exposure to adjuvant-Al was variable. In our tested children the maximum Al exposure at 2 years of age was almost twice that attained by children of comparable age (3700 μg) in the USA [7,12,13]. However, given the lack of significant correlation between hair-Al and doses of AAVs it is unlikely that hair is a sensitive matrix that accumulates adjuvant-Al from vaccines. We know little about the metabolism of injected Al in the immature or young organism. The intramuscular route of Al exposure is essential for the adjuvant to boost the immunologic response; however, under such conditions (100% absorption) we do not know how it is distributed in tissues and how it reaches hair. In the present case it is impossible to ascertain the origin of Al in hair (environmental or iatrogenic). We tested children ranging from a few weeks (still nursing) to 2 y. Environmental Al from water and air, as well as from food (which includes maternal sources in breastfed infants) makes it difficult to ascertain the origin of Al; only exposure originating from the child's vaccine schedule could be estimated. Neonatal hair starts growing in utero during late pregnancy (approximately 28 weeks) and remains until 3–5 months post-partum [14]. The transition from fetal hair (lanugo) to permanent hair is accompanied by structural changes that may affect its metal cumulative properties [14].
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D. Bohrer et al. / Clinica Chimica Acta 428 (2014) 9–13
100 90
300
80
Frequency, %
Hair-Al concentration, µg.g-1
a)
200
100
70 60 50 40 30 20 10 0 0
0 0
250
500
750
50
100
150
200
250
Al concentration, µg.g-1
1000
Age of children, days Fig. 2. Aluminum (Al) concentrations in hair of infants as a function of age (a), total aluminum in Al-adjuvanted vaccines - AAVs (b), and time elapsed after the last shot of an AAV (c).
Hair-Al concentration, µg.g-1
b) 300
200
100
0 0
1
2
3
4
5
6
7
8
Total adjuvanted-Al, mg
Hair-Al concentration, µg.g-1
c) 300
200
100
0 0
100
200
300
400
Time elapsed after last AAV shot, days Fig. 1. Cumulative frequency distribution of total hair-Al concentrations.
During the growing phase of hair, keratinocytes (sulfur-keratin proteins) take up metals. However, hair components such as melanin can also accumulate metals [15,16]. Kauf et al. [17] reported that inorganic elements increased from post-natal day 1 to 90 days. Nevertheless, a small sample of newborn hair showed Al concentrations (n = 6; 52 μg/g) comparable [18] to and higher (n = 15; 76.24 μg/g) [19] than our median values. Al intake in infancy varies and it is assumed that it is much higher in formulas (soy based N cow's milk based) than in breast milk [20], but Al is considered to have a low absorption in drinking water (0.28%) and food (0.1%) in general (1). In fact, Litov et al. [20] reported that infants fed soy-based formula (1,700 μg/l) showed non-significant differences in blood Al when compared with breastfed infants (b5 to 45 μg/l). Therefore, the relatively high Al concentrations (median, 47.7 μg/g) suggest that 100% systemic available Al from injected doses of AAV could end up in hair. Indeed, experimental studies with injected Al showed a dose response of Al in hair of rabbits [21]. The total Al concentrations in hair in this study are much higher than the body retentions estimated by Mitkus et al. [12] but closer to the
model developed by Keith et al. [13]. Attempts to estimate body burden from AAVs yielded calculations of an estimated body burden of approximately 0.4 mg Al at birth; they estimated that body burden from vaccines was 2-fold higher than from dietary sources [12]. However, body burdens of Al derived from Keith et al. [13] were 20-fold higher than Mitkus et al.'s [12] estimate. Keith et al. [13] estimated that the Al body burden from human milk and formulas during the first year was around 0.1 mg and that the Al attributed to vaccines would be expected to fall below 4 mg. Al is a ubiquitous element, but only trace amounts are generally present in human tissues [22]. Hair is a convenient and non-invasive tissue that is easy to collect and store. In the context of Al body burden we are still uncertain how representative it can be [22]. Although we are confident about infant hair data shown here, we can not ascertain its origin (dietary or Al-adjuvanted vaccines). Future studies can now be designed to capture hair-Al response that might be related to specific sources using a larger number of subjects with different exposure loads. In our study infants of b7 weeks showed hair-Al of 0.63 to 1.25 mg/g. We do not know the kinetics of adjuvanted-Al in young children, or what proportion is taken up by hair. However, it has recently been shown that intraperitoneal injection of Al in newborn animals results in increased Al levels compared to adults [23]. It is worth noting that route of administration is an important parameter for both toxicokinetics and toxicodynamics [24]. There are concerns related to the use of Al materials with physicochemical properties different from adjuvant-Al in experimental studies [25]; indeed, there are differences in Al-adjuvant body retention depending on its chemical form [26]. Our results should reflect the true nature of exposure derived from Al-adjuvanted vaccines [25]. Analytical techniques to quantify Al in non-invasive tissues are fundamental to understand its metabolism as well as toxicodynamics. To date, existing models have shown discrepancies in predicting kinetics of Al in vaccines [12,13]. Therefore, besides the clinical and forensic interest, there is a need for specific methods to identify Al from vaccines in both health and environmental studies. The principal constraint of the study was that we did not measure Al concentration in the children's diet. Maternal transfer (through placenta or breast milk) or from AAV cannot be distinguished from these analyses. Although it is feasible to conduct Al exposure studies in hair samples, no interpretations of Al origin associated with toxicity risks can be made without establishing its origin. In the present work we show the only data on hair-Al after Al-adjuvanted vaccines; additionally, the study presents reliable data on the Al content of babies' hair. References [1] Willhite CC, Ball GL, McLellan CJ. Total allowable concentrations of monomeric inorganic aluminum and hydrated aluminum silicates in drinking water. Crit Rev Toxicol 2012;42:358–442. [2] Winterberg B, Bertram H, Rolf N, et al. Differences in plasma and tissue aluminum concentrations due to different aluminum-containing drugs in patients with renal insufficiency and with normal renal function. J Trace Elem Electrolytes Health Dis 1987;1:69–72.
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