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Rapid method for the measurement of circulating thyroid hormones in low volumes of teleost fish plasma by LC-ESI/MS/MS

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Abstract

Thyroid hormones are critical regulators of normal development and physiological functioning in all vertebrates. Radioimmunoassay (RIA) approaches have been the method of choice for measuring circulating levels of thyroid hormones in vertebrates. While sensitive, RIA-based approaches only allow for a single analyte measurement per assay, can lack concordance across platforms and laboratories, and can be prone to analytical interferences especially when used with fish plasma. Ongoing advances in liquid chromatography tandem mass spectrometry (LC/MS/MS) have led to substantial decreases in detection limits for thyroid hormones and other biomolecules in complex matrices, including human plasma. Despite these advances, current analytical approaches do not allow for the measurement of native thyroid hormone in teleost fish plasma by mass spectrometry and continue to rely on immunoassay. In this study, we developed a new method that allows for the rapid extraction and simultaneous measurement of total T4 (TT4) and total T3 (TT3) in low volumes (50 μL) of fish plasma by LC/MS/MS. Methods were optimized initially in plasma from rainbow trout (Oncorhynchus mykiss) and applied to plasma from other teleost fishes, including fathead minnows (Pimephales promelas), mummichogs (Fundulus heteroclitus), sockeye salmon (Oncorhynchus nerka), and coho salmon (Oncorhynchus kisutch). Validation of method performance with T4- and T3-spiked rainbow trout plasma at 2 and 4 ng/mL produced mean recoveries ranging from 82 to 95 % and 97 to 105 %, respectively. Recovery of 13C12-T4 internal standard in plasma extractions was: 99 ± 1.8 % in rainbow trout, 85 ± 11 % in fathead minnow, 73 ± 5.0 % in mummichog, 73 ± 1.7 % in sockeye salmon, and 80 ± 8.4 % in coho salmon. While absolute levels of thyroid hormones measured in identical plasma samples by LC/MS/MS and RIA varied depending on the assay used, T4/T3 ratios were generally consistent across both techniques. Less variability was measured among samples subjected to LC/MS/MS suggesting a more precise estimate of thyroid hormone homeostasis in the species targeted. Overall, a sensitive and reproducible method was established that takes advantage of LC/MS/MS techniques to rapidly measure TT4 and TT3 with negligible interferences in low volumes of plasma across a variety of teleost fishes.

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References

  1. Davies L, Welch HG (2006) Increasing incidence of thyroid cancer in the United States, 1973–2002. JAMA J Am Med Assoc 295(18):2164–2167

    Article  CAS  Google Scholar 

  2. Pellegriti G, Frasca F, Regalbuto C, Squatrito S, Vigneri R (2013) Worldwide increasing incidence of thyroid cancer: update on epidemiology and risk factors. J Cancer Epidemiol 2013:965212–965212

    Article  Google Scholar 

  3. Cheek AO, Kow K, Chen J, McLachlan JA (1999) Potential mechanisms of thyroid disruption in humans: interaction of organochlorine compounds with thyroid receptor, transthyretin, and thyroid-binding globulin. Environ Health Perspect 107(4):273–278

    Article  CAS  Google Scholar 

  4. Gilbert ME, Rovet J, Chen Z, Koibuchi N (2012) Developmental thyroid hormone disruption: prevalence, environmental contaminants and neurodevelopmental consequences. Neurotoxicology 33(4):842–852

    Article  Google Scholar 

  5. McNabb FMA, Fox GA (2003) Avian thyroid development in chemically contaminated environments: is there evidence of alterations in thyroid function and development? Evol Dev 5(1):76–82

    Article  CAS  Google Scholar 

  6. Zhang JS, Lazar MA (2000) The mechanism of action of thyroid hormones. Annu Rev Physiol 62:439–466

    Article  CAS  Google Scholar 

  7. Shiao JC, Hwang PP (2006) Thyroid hormones are necessary for the metamorphosis of tarpon Megalops cyprinoides leptocephali. J Exp Mar Biol Ecol 331(2):121–132

    Article  CAS  Google Scholar 

  8. Schreiber AM, Specker JL (1998) Metamorphosis in the summer flounder (Paralichthys dentatus): stage-specific developmental response to altered thyroid status. Gen Comp Endocrinol 111(2):156–166

    Article  CAS  Google Scholar 

  9. Klaren PHM, Guzman JM, Mancera JM, Geven EJW, Flik G (2005) The involvement of thyroid hormone metabolism in ilthead sea bream. (Sparus auratus) osmoregulation. In: Vaudry H, Roubos E, Schoofs L, Fiik G, Larhammar D (eds) Trends in comparative endocrinology and neurobiology, vol 1040. Annals of the New York Academy of Sciences. pp 360–362. doi:10.1196/annals.1327.062

  10. Lema SC, Nevitt GA (2004) Evidence that thyroid hormone induces olfactory cellular proliferation in salmon during a sensitive period for imprinting. J Exp Biol 207(19):3317–3327

    Article  CAS  Google Scholar 

  11. Peter MCS (2011) The role of thyroid hormones in stress response of fish. Gen Comp Endocrinol 172(2):198–210

    Article  CAS  Google Scholar 

  12. Coffin AB, Raine JC, Hawryshyn CW (2012) Exposure to thyroid hormone in ovo affects otolith crystallization in rainbow trout Oncorhynchus mykiss. Exp Biol Fish 95(3):347–354

    Article  Google Scholar 

  13. Blanton ML, Specker JL (2007) The hypothalamic-pituitary-thyroid (HPT) axis in fish and its role in fish development and reproduction. Crit Rev Toxicol 37(1–2):97–115

    Article  CAS  Google Scholar 

  14. Nelson ER, Allan ERO, Pang FY, Habibi HR (2010) Thyroid hormone and reproduction: regulation of estrogen receptors in goldfish gonads. Mol Reprod Dev 77(9):784–794

    Article  CAS  Google Scholar 

  15. Dickhoff WW, Folmar LC, Mighell JL, Mahnken CVW (1982) Plasma thyroid hormones during smoltification of yearling and underyearling Coho salmon and yearling Chinook salmon and Steelhead trout. Aquaculture 28(1–2):39–48

    Article  CAS  Google Scholar 

  16. Larsen DA, Swanson P, Dickhoff WW (2011) The pituitary-thyroid axis during the parr-smolt transformation of Coho salmon, Oncorhynchus kisutch: quantification of TSH beta mRNA, TSH, and thyroid hormones. Gen Comp Endocrinol 171(3):367–372

    Article  CAS  Google Scholar 

  17. Maclatchy DL, Eales JG (1992) Intracellular and extracellular sources of T3 binding to putative thyroid-hormone receptors in liver, kidney, and gill nuclei of immature rainbow trout, Oncorhynchus mykiss. J Exp Zool 262(1):22–29

    Article  CAS  Google Scholar 

  18. Brown S, Eales JG (1977) Measurement of l-thyroxine and 3,5,3′-triiodo-l-thyronine levels in fish plasma by radioimmunoassay. Can J Zool 55(2):293–299

    Article  CAS  Google Scholar 

  19. Crane HM, Pickford DB, Hutchinson TH, Brown JA (2004) Developmental changes of thyroid hormones in the fathead minnow, Pimephales promelas. Gen Comp Endocrinol 139(1):55–60

    Article  CAS  Google Scholar 

  20. Ghosh S, Howlett M, Boag D, Malik I, Collier A (2008) Interference in free thyroxine immunoassay. Eur J Intern Med 19(3):221–222

    Article  CAS  Google Scholar 

  21. Hoofnagle AN, Wener MH (2009) The fundamental flaws of immunoassays and potential solutions using tandem mass spectrometry. J Immunol Methods 347(1–2):3–11

    Article  CAS  Google Scholar 

  22. Tate J, Ward G (2004) Interferences in immunoassay. Clin Biochem Rev 25(2):105–120

    Google Scholar 

  23. Soldin SJ, Soukhova N, Janicic N, Jonklaas J, Soldin OP (2005) The measurement of free thyroxine by isotope dilution tandem mass spectrometry. Clin Chim Acta 358(1–2):113–118

    Article  CAS  Google Scholar 

  24. Tai SSC, Bunk DM, White EV, Welch MJ (2004) Development and evaluation of a reference measurement procedure for the determination of total 3,3′,5-trilodothyronine in human serum using isotope-dilution liquid chromatography-tandem mass spectrometry. Anal Chem 76(17):5092–5096

    Article  CAS  Google Scholar 

  25. Tai SSC, Sniegoski LT, Welch MJ (2002) Candidate reference method for total thyroxine in human serum: use of isotope-dilution liquid chromatography-mass spectrometry with electrospray ionization. Clin Chem 48(4):637–642

    CAS  Google Scholar 

  26. Butt CM, Wang DL, Stapleton HM (2011) Halogenated phenolic contaminants inhibit the in vitro activity of the thyroid-regulating deiodinases in human liver. Toxicol Sci 124(2):339–347

    Article  CAS  Google Scholar 

  27. Kunisue T, Eguchi A, Iwata H, Tanabe S, Kannan K (2011) 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. Environ Sci Technol 45(23):10140–10147

    Article  CAS  Google Scholar 

  28. Kunisue T, Fisher JW, Kannan K (2011) Determination of Six thyroid hormones in the brain and thyroid gland using isotope-dilution liquid chromatography/tandem mass spectrometry. Anal Chem 83(1):417–424

    Article  CAS  Google Scholar 

  29. Wang DL, Stapleton HM (2010) Analysis of thyroid hormones in serum by liquid chromatography-tandem mass spectrometry. Anal Bioanal Chem 397(5):1831–1839

    Article  CAS  Google Scholar 

  30. Noyes PD, Hinton DE, Stapleton HM (2011) Accumulation and debromination of decabromodiphenyl ether (BDE-209) in juvenile fathead minnows (Pimephales promelas) induces thyroid disruption and liver alterations. Toxicol Sci 122(2):265–274

    Article  CAS  Google Scholar 

  31. Little AG, Kunisue T, Kannan K, Seebacher F (2013) Thyroid hormone actions are temperature-specific and regulate thermal acclimation in zebrafish (Danio rerio). BMC Biol 11

  32. Lema SC, Dickey JT, Schultz IR, Swanson P (2009) Thyroid hormone regulation of mRNAs encoding thyrotropin beta-subunit, glycoprotein alpha-subunit, and thyroid hormone receptors alpha and beta in brain, pituitary gland, liver, and gonads of an adult teleost, Pimephales promelas. J Endocrinol 202(1):43–54

    Article  CAS  Google Scholar 

  33. Johnson KM, Lema SC (2011) Tissue-specific thyroid hormone regulation of gene transcripts encoding iodothyronine deiodinases and thyroid hormone receptors in striped parrotfish (Scarus iseri). Gen Comp Endocrinol 172(3):505–517

    Article  CAS  Google Scholar 

  34. Brown SB, Adams BA, Cyr DG, Eales JG (2004) Contaminant effects on the teleost fish thyroid. Environ Toxicol Chem 23(7):1680–1701

    Article  CAS  Google Scholar 

  35. Eales JG, Hughes M, Uin L (1981) Effect of food intake on diel variation in plasma thyroid hormone levels in rainbow trout. Gen Comp Endocrinol 45(2):167–174

    Article  CAS  Google Scholar 

  36. Todd KJ, Eales JG (2002) The effect of handling and blood removal on plasma levels and hepatic deiodination of thyroid hormones in adult male and female rainbow trout, Oncorhynchus mykiss. Can J Zool 80(2):372–375

    Article  CAS  Google Scholar 

  37. Gomez JM, Boujard T, Boeuf G, Solari A, LeBail PY (1997) Individual diurnal plasma profiles of thyroid hormones in rainbow trout (Oncorhynchus mykiss) in relation to cortisol, growth hormone, and growth rate. Gen Comp Endocrinol 107(1):74–83

    Article  CAS  Google Scholar 

  38. Plate EM, Adams BA, Allison WT, Martens G, Hawryshyn CW, Eales JG (2002) The effects of thyroxine or a GnRH analogue on thyroid hormone deiodination in the olfactory epithelium and retina of rainbow trout, Oncorhynchus mykiss, and sockeye salmon, Oncorhynchus nerka. Gen Comp Endocrinol 127(1):59–65

    Article  CAS  Google Scholar 

  39. Reddy PK, Leatherland JF (1994) Does the time of feeding affect the diurnal rhythms of plasma hormone and glucose concentration adn hepatic glycogen content of rainbow trout. Fish Physiol Biochem 13(2):133–140

    Article  CAS  Google Scholar 

  40. Feng CL, Xu YP, Zhao GF, Zha JM, Wu FC, Wang ZJ (2012) Relationship between BDE 209 metabolites and thyroid hormone levels in rainbow trout (Oncorhynchus mykiss). Aquat Toxicol 122:28–35

    Article  Google Scholar 

  41. Holloway AC, Sheridan MA, Van der Kraak G, Leatherland JF (1999) Correlations of plasma growth hormone with somatostatin, gonadal steroid hormones and thyroid hormones in rainbow trout during sexual recrudescence. Comp Biochem Physiol B 123(3):251–260

    Article  CAS  Google Scholar 

  42. Noyes PD, Lema SC, Macaulay LJ, Douglas NK, Stapleton HM (2013) Low level exposure to the flame retardant BDE-209 reduces thyroid hormone levels and disrupts thyroid signaling in fathead minnows. Environ Sci Technol 47:10012–10021

    Google Scholar 

  43. Muzzio AM, Noyes PD, Stapleton HM, Lema SC (2013) Tissue distribution and thyroid hormone effects on mRNA abundance for membrane transporters Mct8, Mct10, and organic anion-transporting polypeptides (OATPs) in a teleost fish. Comp Biochem Phys A 167:77–89

    Google Scholar 

  44. Lema SC, Dickey JT, Schultz IR, Swanson P (2008) Dietary exposure to 2,2′,4,4′-tetrabromodiphenyl ether (PBDE-47) alters thyroid status and thyroid hormone-regulated gene transcription in the pituitary and brain. Environ Health Perspect 116(12):1694–1699

    Article  CAS  Google Scholar 

  45. Crane HM, Pickford DB, Hutchinson TH, Brown JA (2006) The effects of methimazole on development of the fathead minnow, Pimephales promelas, from embryo to adult. Toxicol Sci 93(2):278–285

    Article  CAS  Google Scholar 

  46. Brown CL, Stetson MH (1983) Prolactin thyroid interaction in Fundulus heteroclitus. Gen Comp Endocrinol 50(2):167–171

    Article  CAS  Google Scholar 

  47. Zhou T, John-Alder HB, Weis JS, Weis P (2000) Endocrine disruption: thyroid dysfunction in mummichogs (Fundulus heteroclitus) from a polluted habitat. Mar Environ Res 50(1–5):393–397

    Article  CAS  Google Scholar 

  48. Munakata A, Amano M, Ikuta K, Kitamura S, Aida K (2012) Involvement of sex steroids, luteinizing hormone and thyroid hormones in upstream and downstream swimming behavior of land-locked sockeye salmon Oncorhynchus nerka. Fish Sci 78(1):81–90

    Article  CAS  Google Scholar 

  49. Eales JG, Devlin R, Higgs DA, McLeese JM, Oakes JD, Plohman J (2004) Thyroid function in growth-hormone-transgenic coho salmon (Oncorhynchus kisutch). Can J Zool 82(8):1225–1229

    Article  CAS  Google Scholar 

  50. Larsen DA, Beckman BR, Dickhoff WW (2001) The effect of low temperature and fasting during the winter on metabolic stores and endocrine physiology (Insulin, insulin-like growth factor-I and thyroxine) of coho salmon, Oncorhynchus kisutch. Gen Comp Endocrinol 123(3):308–323

    Article  CAS  Google Scholar 

  51. Young G, Bjornsson BT, Prunet P, Lin RJ, Bern HA (1989) Smoltification and seawater adaptation in coho salmon (Oncorhynchus kisutch) - Plasma prolactin, growth hormone, thyroin hormones, and cortisol. Gen Comp Endocrinol 74(3):335–345

    Article  CAS  Google Scholar 

  52. Babin PJ, Vernier JM (1989) Plasma-lipoproteins in fish. J Lipid Res 30(4):467–489

    CAS  Google Scholar 

  53. Santulli A, Cusenza L, Modica A, Curatolo A, Damelio V (1991) Fish plasma-lipoproteins—comparative observations in Serranides and Sparides. Comp Biochem Physiol B 99(2):251–255

    CAS  Google Scholar 

  54. Zhou T, John-Alder HB, Weis P, Weis JS (1999) Thyroidal status of mummichogs (Fundulus heteroclitus) from a polluted versus a reference habitat. Environ Toxicol Chem 18(12):2817–2823

    CAS  Google Scholar 

  55. Despres N, Grant AM (1998) Antibody interference in thyroid assays: a potential for clinical misinformation. Clin Chem 44(3):440–454

    CAS  Google Scholar 

  56. Manzon RG, Neuls TM, Manzon LA (2007) Molecular cloning, tissue distribution, and developmental expression of lamprey transthyretins. Gen Comp Endocrinol 151(1):55–65

    Article  CAS  Google Scholar 

  57. Bianco AC, Larsen PR (2005) Intracellular pathways of iodothyronine metabolism. In: Braverman LE, Utiger RD (eds) The thyroid: a fundamental and clinical text, 9th edn. Lippincott Williams & Wilkens, Philadelphia, p 120

    Google Scholar 

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Acknowledgments

This study was supported by a National Institute of Environmental Health Sciences research grant (R01-ES016099) and US EPA STAR graduate fellowship (FP-917145010). Findings and conclusions in this article are those of the authors and do not necessarily represent the views of the NIEHS or EPA. The authors thank Dr. Even Gallagher and Chase Williams, University of Washington for providing coho salmon plasma, and Dr. Andrew Dittman, NOAA Fisheries, Northwest Fisheries Science Center for providing sockeye salmon plasma, and the Armstrong Hatchery for their donation of rainbow trout plasma. We also thank Dr. Penny Swanson and Jon Dickey, NOAA Fisheries, Northwest Fisheries Science Center, for assistance with the RIA methods.

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  1. Nicholas School of the Environment, Duke University, Box 90328, Durham, NC, 2770, USA

    Pamela D. Noyes, Simon C. Roberts, Ellen M. Cooper & Heather M. Stapleton

  2. Department of Biological Sciences, California Polytechnic State University, San Luis Obispo, CA, 93407, USA

    Sean C. Lema

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  1. Pamela D. Noyes
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  2. Sean C. Lema
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Correspondence to Heather M. Stapleton.

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Noyes, P.D., Lema, S.C., Roberts, S.C. et al. Rapid method for the measurement of circulating thyroid hormones in low volumes of teleost fish plasma by LC-ESI/MS/MS. Anal Bioanal Chem 406, 715–726 (2014). https://doi.org/10.1007/s00216-013-7528-3

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