Differences in the Plasma Proteome of Patients with Hypothyroidism before and after Thyroid Hormone Replacement: A Proteomic Analysis

Abstract

Thyroid hormone is a potent stimulator of metabolism, playing a critical role in regulating energy expenditure and in key physiological mechanisms, such as growth and development. Although administration of thyroid hormone in the form of levo thyroxine (l-thyroxine) has been used to treat hypothyroidism for many years, the precise molecular basis of its physiological actions remains uncertain. Our objective was to define the changes in circulating protein levels that characterize alterations in thyroid hormone status. To do this, an integrated untargeted proteomic approach with network analysis was used. This study included 10 age-matched subjects with newly diagnosed overt hypothyroidism. Blood was collected from subjects at baseline and at intervals post-treatment with l-thyroxine until they reached to euthyroid levels. Plasma protein levels were compared by two-dimensional difference in gel electrophoresis (2D-DIGE) pre- and post-treatment. Twenty differentially expressed protein spots were detected. Thirteen were identified, and were found to be unique protein sequences by MALDI-TOF mass spectrometry. Ten proteins were more abundant in the hypothyroid vs. euthyroid state: complement C2, serotransferrin, complement C3, Ig κ chain C region, α-1-antichymotrypsin, complement C4-A, haptoglobin, fibrinogen α chain, apolipoprotein A-I, and Ig α-1 chain C region. Three proteins were decreased in abundance in the hypothyroid vs. euthyroid state: complement factor H, paraneoplastic antigen-like protein 6A, and α-2-macroglobulin. The differentially abundant proteins were investigated by Ingenuity Pathway Analysis (IPA) to reveal their associations with known biological functions. Their connectivity map included interleukin-6 (IL-6) and tumour necrosis factor α (TNF-α) as central nodes and the pathway identified with the highest score was involved in neurological disease, psychological disorders, and cellular movement. The comparison of the plasma proteome between the hypothyroid vs euthyroid states revealed differences in the abundance of proteins involved in regulating the acute phase response.

Empowering thyroid hormone research in human subjects using OMICs technologies

“…the determination of TSH to monitor adequate secretion of TH. Despite the benefits of plasma proteomics, its application in biomarker research in general and thyroid research in particular is still limited (Geyer et al 2017, Alfadda et al 2018. The major challenge in plasma proteomics is the great dynamic range covering about ten orders of magnitude from highly abundant albumin (~0.6 mol/L) to low-abundant TSH (~5 pmol/L) (Hortin & Sviridov 2010).…”

“…However, a comparable study (Hooper et al 2012) observed no alterations in serum C3 levels (and coagulation factors) after 2 weeks of L-T 4 application. Moreover, endogenous hypothyroidism has been characterized by increased abundance of complement proteins using a plasma proteomic approach (Alfadda et al 2018). Hence, this seems to be a mid-to long-term effect with a U-shaped association with respect to the thyroid state.…”

Identification of Protein Changes in The Urine of Hypothyroid Patients Treated with Thyroxine Using Proteomics Approach

“…None of the patients recruited in the study had a history of hypertension, diabetes mellitus, inflammatory or other autoimmune. Blood samples were collected after a standard 10 h fast by venipuncture into EDTA-coated tubes and plasma was obtained by centrifugation (15 min, 3000 × g), then was aliquoted and stored in multiple aliquots at -80°C until analysis [20].…”

“…DIGE analysis was performed to determine differentially expressed proteins between the hypothyroid Vs Euthyroid groups as described [20]. Briefly, Proteins extracted (50ug) from each sample were 6 labeled with either Cy3 or Cy5, respectively.…”

“…All spots were pre-filtered and manually checked before applying the statistical criteria (ANOVA test, p ≤ 0.05 and fold ≥ 1.5). Furthermore the spots showing statistical significance between the groups were manually excised and in-gel digestion with trypsin as described [5,20] MALDI-MS(/MS) data were obtained using an UltraflexTerm time-of-flight (TOF) mass spectrometer equipped with a LIFT-MS/MS device (Bruker-Daltonics) instrument as described [5,20,22,23] briefly The reflector voltage was set to 21 kV and the detector voltage to 17 kV. Peptide mass fingerprints (PMF) were calibrated against a standard (peptide calibration standard II, Bruker Daltonics).…”