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Analysis of Thyroid Hormones in Serum of Baikal Seals and Humans by Liquid Chromatography-Tandem Mass Spectrometry (LC-MS/MS) and Immunoassay Methods: Application of the LC-MS/MS Method to Wildlife Tissues Tatsuya Kunisue,†,* Akifumi Eguchi,‡ Hisato Iwata,‡ Shinsuke Tanabe,‡ and Kurunthachalam Kannan†,§,* †
Wadsworth Center, New York State Department of Health, and Department of Environmental Health Sciences, School of Public Health, State University of New York at Albany, Empire State Plaza, P.O. Box 509, Albany, New York 12201-0509, United States ‡ Center for Marine Environmental Studies (CMES), Ehime University, Bunkyo-cho 2-5, Matsuyama 790-8577, Japan § State Key Laboratory of Urban Water Resources & Environment, IJRC-PTS, Harbin Institute of Technology, Harbin 150090, China
bS Supporting Information ABSTRACT: Thyroid hormones (THs) are essential for the regulation of growth and development in both humans and wildlife. Until recently, TH concentrations in the tissues of animals have been examined by immunoassay (IA) methods. IA methods are sensitive, but for TH analysis, they are compromised by a lack of adequate specificity. In this study, we determined the concentrations of six THs, L-thyroxine (T4), 3,30 ,5-triiodo-L-thyronine (T3), 3,30 ,50 -triiodo-L-thyronine (rT3), 3,5-diiodo-L-thyronine (3,5-T2), 3,30 -diiodo-L-thyronine (3,30 T2), and 3-iodo-L-thyronine (3-T1), in the serum of humans (n = 79) and wild Baikal seals (n = 37), by isotope ([13C6]-T4)dilution liquid chromatography (LC)-tandem mass spectrometry (MS/MS), and compared the TH levels with those measured by an electrochemiluminescent immunoassay (ECLIA) method. T3 and T4 were detected in all serum samples of both humans and Baikal seals, whereas T1, 3,30 -T2, and 3,5-T2 were below the limit of detection (LOD). rT3 was detected in Baikal seal sera at concentrations higher than T3 in 28 seal samples, indicating an anomaly in deiodinase activity in Baikal seals. In humans, regression analyses of TH concentrations, measured by ECLIA and LC-MS/MS methods, showed significant correlations for T4 (r = 0.852) and T3 (r = 0.676; after removal of a serum sample with abnormal T3 levels). In Baikal seals, a low correlation coefficient (r = 0.466) for T4 levels and no correlation for T3 levels (p = 0.093) were found between ECLIA and LC-MS/MS methods. These results suggest that interference by a nonspecific reaction against anti-T3 and anti-T4 antibodies used in the ECLIA can contribute to inaccuracies in TH measurement in Baikal seals. When the relationship between concentrations of THs in sera and dioxin-like toxic equivalents in blubber samples of Baikal seals (n = 19) was examined, a significantly negative correlation was found for serum T4 levels measured by the LC-MS/MS method, but not for those measured by ECLIA. Thus, our results indicate that the LC-MS/MS method is more reliable and accurate for the elucidation of alteration in circulating TH levels in wildlife, as caused by environmental and physiological factors.
’ INTRODUCTION It is well-known that thyroid hormones (THs) play a critical role in the regulation of biological processes such as growth, metabolism, neurodevelopment, and protein synthesis. L-thyroxine (T4), which acts as a prohormone, is synthesized in the thyroid gland (TG) and is carried by the bloodstream to peripheral tissues. After entering peripheral tissues, T4 is biotransformed by iodothyronine deiodinases type I (D1), which can function as either an outer phenolic ring (50 -deiodination) or inner tyrosyl ring (5-deiodination) deiodinase, or type II (D2), which is exclusively outer 50 -deiodinase, into 3,30 ,5-triiodo1,2 L-thyronine (T 3). The active hormone, T3, regulates the r 2011 American Chemical Society
expression of genes associated with the biological processes described above (e.g., growth), by binding to thyroid hormone receptors (TRs). 1 It is estimated that approximately 60% (in rats) to 80% (in humans) of T 3 produced daily is formed from the metabolism of T 4 by D1 or D2, and the remainder is produced in the TG and is secreted directly into the bloodstream. 1,2 Received: August 26, 2011 Accepted: October 28, 2011 Revised: October 27, 2011 Published: October 28, 2011 10140
dx.doi.org/10.1021/es203002a | Environ. Sci. Technol. 2011, 45, 10140–10147
Environmental Science & Technology T3 and T4 measurements in blood can supply clinically important information on TH homeostasis. Immunoassay (IA) methods are widely used and are sensitive to TH determination in human sera but are compromised by a lack of adequate specificity. The interlaboratory proficiency testing conducted by the College of American Pathologists indicated a 50% variation in TH values reported among the participating laboratories, depending not only on the IA methods used but also on the serum samples tested.3 This result suggests differences in specificity of the antibodies used in IA methods. It has been recognized that circulating endogenous antibodies directed against different antigens may either falsely depress or enhance TH values through nonspecific reactions with anti-T3 or anti-T4 animal (e.g., sheep, mouse) antibodies used in IAs. Thus far, iodothyronine-binding autoantibodies, heterophilic antibodies, human antianimal antibodies, and rheumatoid factors are considered potential interferences in IA methods.4,5 For example, a study that used anti-T3 or anti-T4 sheep antibodies showed falsely elevated serum T3 and T4 concentrations in euthyroid humans, but reexamination with anti-T3 or anti-T4 mouse antibodies showed normal TH values.6 Such diagnostically false TH values have been frequently observed in humans with certain conditions such as pregnancy, estrogen therapy, and hepatic and thyroidal diseases, and in livestock handling.79 Thus, to enable proper diagnosis, it is important that clinicians be made aware of potential TH modulating conditions, and apply alternative assays for TH measurements.9 In wildlife, TH homeostasis has been assessed by measuring serum concentrations of T3 and T4 by IA methods, particularly in the examination of an association with environmental chemicals (such as dioxins, polychlorinated biphenyls [PCBs], and polybrominated diphenyl ethers [PBDEs]).10,11 More investigations that report alterations in circulating TH concentrations from exposure to organohalogen contaminants (OCs) are available for seals, in comparison to other wildlife species.10 Although it is likely that high concentrations of OCs in animal tissues are related to a reduction in circulating TH levels,10 the results of recent studies have been inconsistent.12,13 This could be due to the influence of nutritional, physiological, or environmental factors such as fasting, molting, disease, age, and season that can affect circulating TH levels.1417 Furthermore, the presence of circulating endogenous antibodies directed against the wildlife-specific antigens and their nonspecific reactivity with antibodies used in IA methods can contribute to inaccuracies in TH measurement in wildlife serum or plasma. Nevertheless, potential interferences present in the blood of wildlife species for the analysis of THs have not well been examined. Recently, our group has developed reliable methods for the measurement of six THs, namely, T 4 , T 3 , 3,3 0 ,5 0 -triiodo0 0 L-thyronine (rT3), 3,3 -diiodo-L-thyronine (3,3 -T2), 3,5-T2, and 3-monoiodo-L-thyronine (3-T1), in human and rat sera, in addition to the brain and TG, using liquid chromatography (LC)-tandem mass spectrometry (MS/MS).1820 LC-MS/MS methods, as an alternative assay, are just beginning to be used for the clinical diagnosis of TH modulation in humans because these methods are more selective as compared to IA methods.21 In this study, we determined the serum T3 and T4 concentrations in Baikal seals (Pusa sibirica) and humans, using the LC-MS/MS method20 and an ECLIA method. The ECLIA method is a modern assay that is routinely used for the clinical diagnosis of thyroid disorders in humans. The aim of this study was to compare TH concentrations in sera of seals and humans
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determined by LC-MS/MS and ECLIA methods. Along with D1 and D2, iodothyronine deiodinases type III (D3), which can function exclusively as an inner tyrosyl ring (5-deiodination) deiodinase, is also expressed in various peripheral tissues.1 Hence, in addition to 5-deiodination activity by D1, D3 catalyzes the conversion of T4 to rT3 and the conversion of T3 to 3,30 -T2; rT3 and 3,30 -T2 are biologically inactive.1,2 For a comprehensive evaluation of TH modulation, determination of not only T3 and T4 but also of rT3, T2, and T1 in serum samples is needed.
’ MATERIALS AND METHODS Samples. In this study, we utilized serum samples of Baikal seals (n = 37) and humans (n = 79) stored frozen in the Environmental Specimen Bank (es-Bank) of Ehime University, Matsuyama, Japan.22 In 2005, 13 male and 24 female Baikal seals from Lake Baikal, Russia, were captured, under a permit (annual seal culling quota) issued by the Lake Baikal Basin Committee for Protection, Reproduction of Fishery Resources and Fishing Control. In addition to blood, various tissues and organs were collected for biomonitoring studies on environmental contaminants23,24 and for toxicological studies on gene expression and biomarkers.2527 After centrifugation, the serum samples were immediately frozen in liquid nitrogen, transported to the es-Bank, and stored at 80 °C. Details of sample collection have been reported previously.2327 The ages of Baikal seals, determined by counting the growth layer groups in dentine and cementum, were in the range of 0.2541.5 years.24 Human serum samples were collected from 26 males and 53 females, aged 1354 years old, in two suburban areas (Bui Dau and Duong Quang) of Hanoi, Vietnam, during January 2010, for the purpose of the examination of biomarkers of exposure to trace elements and OCs, including THs. The results for environmental contaminants in sera will be reported elsewhere. Informed consent was obtained from all donors, and this study was approved by the Ethical Committee of Ehime University, Japan. Information on age, occupation, pregnancy, smoking habits, dietary habits, and health condition were obtained through an interview with each donor. Blood samples were collected by a certified physician, and serum was prepared immediately by centrifugation and stored at 25 °C in the es-Bank until analysis. TH Analysis by LC-MS/MS. Six THs were analyzed in serum by following the LC-MS/MS procedure reported in recent studies.19,20 The details are provided in the Supporting Information (SI). TH Analysis by ECLIA. In this study, Elecsys kits (Roche Diagnostics Ltd., Rotkreuz, Switzerland) were utilized as a backtitration ECLIA method. Serum concentrations of total T3 and T4 in Baikal seals and humans were measured by SRL Inc. (Tokyo, Japan), where these THs were routinely measured for the clinical diagnosis of thyroid disorders in humans. Elecsys T3 (anti-T3 sheep polyclonal antibodies) and Elecsys T4 (anti-T4 sheep polyclonal antibodies) were used for the measurement of serum T3 and T4, respectively. An aliquot of serum was incubated with 8-anillino-1-naphthalene sulfonic acid (ANS) and ruthenium tris (bipyridyl)32+ (Ru[bpy]32+)-labeled anti-T3 or anti-T4 sheep antibodies at 37 °C for 9 min. Then, to occupy the unbound antibodies, biotinylated-T3 or -T4 and streptavidincoated paramagnetic microparticles were added, and the mixture was incubated at 37 °C for 9 min. After the mixture was aspirated into the measurement cell, the complexes of Ru[bpy]32+-labeled anti-T3 or anti-T4 antibody + biotinylated-T3 or -T4 with a 10141
dx.doi.org/10.1021/es203002a |Environ. Sci. Technol. 2011, 45, 10140–10147
Environmental Science & Technology
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Table 1. Serum Concentrations (ng/mL) of Thyroid Hormones in Humans Measured by Electrochemiluminescent Immunoassay (ECLIA) and LC-MS/MS Methods mean ( SD
method T4
ECLIA
LC-MS/MS
T3
ECLIA
LC-MS/MS
min
25%a
median
75%a
max
total (n = 79)
83.8 ( 16
51.0
74.0
82.9
92.7
141b,f
male (n = 26) female (n = 53)
77.3 ( 13 87.0 ( 16
51.0 59.5
70.1 77.0
81.2 85.9
85.7 95.5
103 141d,e
total (n = 79)
74.9 ( 17
43.2
61.0
73.7
84.3
131
male (n = 26)
70.3 ( 15
43.2
60.1
70.7
77.9
104
female (n = 53)
77.2 ( 18
48.2
63.2
74.3
87.7
131
total (n = 79)
1.35 ( 0.47
0.840
1.17
1.25
1.42
4.80c,e
male (n = 26)
1.22 ( 0.30
0.840
1.06
1.18
1.40
2.33
female (n = 53)
1.41 ( 0.53
0.890
1.22
1.29
1.44
4.80
total (n = 79) male (n = 26)
1.44 ( 0.61 1.26 ( 0.32
0.500 0.500
1.10 1.09
1.36 1.31
1.57 1.39
5.51 2.16
female (n = 53)
1.53 ( 0.70
0.830
1.20
1.40
1.70
5.51
a
25%or75%percentile. b Significantly higher than the LC-MS/MS method. c Significantly lower than the LC-MS/MS method. d Significantly higher than males. e p < 0.01. f p < 0.001.
streptavidin-coated paramagnetic microparticle were immobilized at the electrode surface. The amount of luminescence released from Ru[bpy]32+ by reaction with tripropylamine was measured using Modular Analytics (Hitachi Ltd., Tokyo, Japan). Concentrations of T3 or T4 in serum samples were determined by the Elecsys T3 or T4 calibration curve, with the measurable ranges of 0.1956.51 ng/mL for T3 and of 4.20249 ng/mL for T4. The precision of this Elecsys ECLIA was determined using pooled human sera; within-run (n = 6) or between-run (10 days) variability was below 5% or 10% of coefficient of variation (CV). The expected reference intervals in euthyroid humans are within 0.802.0 ng/mL for T3 and 45117 ng/mL for T4. Statistical Analyses. Statistical analyses were conducted with Statistica V. 06J (StatSoft Inc., Tulsa, OK). The strength of the correlation between serum TH concentrations, measured by LCMS/MS and ECLIA methods, was evaluated using simple regression analysis. The significance of difference in serum TH concentrations between LC-MS/MS and IA methods or between males and females was evaluated by a paired or unpaired t-test, after an F-test was conducted for the unpaired t-test. Spearman’s rank correlation was used to analyze the relationship between serum TH concentrations and reported dioxin-like toxic equivalents (TEQs) in the blubber of Baikal seals.24 A p value of
