Introduction
Dysfunction of thyroid gland broadly classified as hypothyroidism and hyperthyroidism. Thyroid hormone plays indispensable role in regulating various physiological functions. Depleted Thyroid hormone production and secretion causes hypothyroidism. In contrast, unconfined and excess free thyroid hormones leads to hyperthyroidism. Thyroid peroxidase (TPO) Pendrin, sodium/iodide symporter (NIS) and monocarboxylate transporter 8 (MCT8) are some of the major bio molecules which are prerequisite for synthesis and secretion of thyroid hormone in thyrocytes.1
After carbon monoxide (CO) and nitric oxide (NO), Hydrogen Sulphide is most commonly produced endogenously through enzymatic processes such as cystathionine β-synthase (CBS), 2 cystathionine γ-lyase (CSE)3 and 3-mercaptosulphurtransferase (3-MPST).4
H2S takes part in antiatherogenesis by S-sulphydration of sirtuin-1 (SIRT1), thereby diminishes apoptosis and cellular senescence in alveolar epithelial cells by modulating SIRT1. Auxiliary information suggest elevated glucose level has an antagonistic impact on thyroid cell line with consequential thyroid hormone deficiency through SIRT1 inactivation. Based on these derivations, contribution of H2S in synchronising SIRT1 for thyroid function can be presumed. 1
This probe is stationed to analyse the change in H2S levels in hypothyroid patients. Also the regulatory effect of H2S on thyroid function and the detailed mechanism were further explored.
Background
Hypothyroidism is an endocrinopathy eventuate due to destruction of thyroid cells incapacitating synthesis of thyroid hormones. 5 Xue Zhao et al postulated that H2S, a gas signalling molecule perhaps foster and endorse the expression of thyroid hormone synthesis regulatory proteins and stimulating activity of TPO by way of SIRT1. In mammalian tissue, H2S is a by-product created by action of two enzymes, cystathionine gamma-lyase (CSE) and cystathionine beta-synthase (CBS) pivoted by pyridoxal-5’-phosphate on amino acid termed L-cysteine.6 Contemporary studies concluded a third enzyme coined 3-mercaptopyruvate sulfur transferase (3MST), along with cysteine aminotransferase (CAT), which synthesise H2 S in the brain and vascular endothelium. 7, 8 CSE manifests principally in the thoracic aorta, ileum, heart, portal vein, vascular smooth muscle, kidney and liver whereas CBS is pronounced in the peripheral and central nervous systems. 9, 10, 11, 12, 13 Bulk of H2S is metabolized to sulphate and thiosulphate in mitochondria via oxidative metabolism. Low level H2S transform to less toxic compounds through a non enzymatic course exclaimed as cytosolic detoxification pathway. Thiosulphate further remoulds to sulphate and/or sulphite catalyzed by thiosulphate cyanide sulphur-transferase (TST). 14 Thereafter, above-mentioned metabolised substances are excreted across the intestinal tract, kidney and lungs in less than 24 hours span to retain the normal magnitude of plasma H2S as it is non-toxic and dissipate.
Materials and Methods
We conducted a case control study in the department of Biochemistry and Medicine, KPC Medical College, Jadavpur, Kolkata, India & department of medicine, Techno India DAMA Healthcare & Medical Centre. Overall, 45 confirmed Hypothyroid patients were chosen of which 21 were male and 24 were female aged above 18 years as case with age matched healthy volunteers as controls consisting of 22 male and 23 females. Institutional Ethics Committee has pre-approved this piece for evaluation. After acquiring consent from all voluntary participants, blood samples to be collected aseptically in clot activator containing vials. Serum to be separated by centrifugation will be deep-frozen at -20⁰C for further analysis.
Inclusion criteria
Case
45 patients of age18 and above, both males and females, suffering from hypothyroidism (clinically evaluated and serum TSH>5.0 mIU/ml, fT4 value < 0.89 ng/dl) those who are willing to participate in the study procedure.
Control
45 healthy volunteers (euthyroid) of the same age group both males and females proposed to be included as controls in the study. Healthy controls are selected from staff, faculty and relatives of patients who are clinically healthy and euthyroid after laboratory evaluation.
Since it is a pilot study, few number of study subjects and controls have been recruited initially, later planned to workup evaluate in a larger sample size for both study subjects and controls.
Measurement of H2S concentration in plasma
Serum H2S levels were assessed following modified versions of the methods reported earlier.15, 16, 17 These procedures were calibrated in our setup. 18
Principle
On addition of Zn²+ to plasma, deposits of H2S, H²– and S²–, along with serum protein were obtained and these serum protein were again solvated using NaOH. ZnS deposits dissolved with addition of N, N -dimethyl-p-phenylenediamine, and trichloroacetic acid sediments residual protein in exist. Supernatant obtained following centrifugation was mixed with ferric chloride that produced Methylene blue. Reading was evaluated in spectrophotometer at 670nm.
Assay procedure
425 microlitres of PBS were mixed with 75 microlitres of serum and further diluted with 250 microlitres 10% trichloroacetic acid and heterogeneously stored in a well capped test tube. Subsequently, tube was centrifuged at 3000 rpm for 30 minutes. Thereafter, supernatant obtained after centrifugation was transferred to a fresh test tube. 250 microlitres of 1% zinc acetate was added to the tube containing supernatant and corked tightly. Additionally, 133 microliters of 20 mmol N, N-dimethyl- p- phenylenediamine sulphate and 133 microliters of 30 milimol of FeCl3 were put in the tube and recapped. The resulting solution was prepared by further addition of 60 microlitres of 10% NaOH and the tube was incubated for 10 minutes at room temperature. Each and every samples were triplicates and concentration of H2S in solution was measured against sodium sulphide (NaHS diluted in deionised water) calibration curve under micromol/L denomination.
Standardization of serum H2S level assay
When NaHS is dissolved in water, HS- is released to forms H2 S, with H+ ions in water. This is one of the mainstay for using Sodium Sulphide as standard for devising calibration curve plotted on a graph by dilution of stock solution under same conditions and technique for estimation of H2S (Figure 1). The linearity limit is 25-250 micromol/l for NaHS. Hereunder, calibration curve is extrapolated with plasma concentration of H2S in NaHS standards of manifold dilution. 6
Results
The clinico-biochemical parameters of the study subjects are presented in Table 1. The serum H2 S level in case in our study was 33.95 ± 4.14 micromol/l with the range from 27.82 to 43.94 micromol/l. This was significantly (P< 0.001) lower than age/sex matched healthy controls which is 64.67±4.25 micromol/l, with a range from 49.81 to 80.02 micromol/l (Table 1 and Figure 3, Figure 4). The level of TSH and FT4 in case was 61.02±21.78 and 0.78±0.067 respectively. Where as in age sex matched healthy control group, the TSH and fT4 levels are 3.85±2.23 and 1.31± 0.23 consecutively.
Discussion
Numerous proof endorse and substantiate the role and involvement of H2S in multiple physiological conditions such as angiogenesis, 19, 20 energy production, 21 vascular relaxation, 22 neuronal activity, 23 and glucose regulation. 24 Notwithstanding that, atherosclerosis, 25 diabetes, 26 asthma, 27 hypertension, and neurodegenerative diseases 28 are some of the pathological conditions linked to unusual & atypical H2S metabolism. Hypothyroid patients were reportedly manifested with depleted concentration of plasma H2S in an article done by Xeu Zhao et al. 1 The above statement was clarified by demonstrating ascending modulation of SIRT1which was enriched via H2S surge enhancing synthesis & secretion of thyroid hormones. In another investigation, Dongdong Wu et al illustrated the controlling potential of exogenous H2S on proliferation, viability, migration and invasion of thyroid carcinoma cells. Role of external H2S is crucial and of paramount importance in progression of human thyroid carcinoma cells via ROS/PI3K/AKT/mTOR signalling pathway. Moreover, H2S level regulates RAS/RAF/MEK/ERK signaling pathway and correspondingly has escalating effect on thyrocytes.29 On the basis of volatility, propensity to undergo oxidation, binding capability to organic molecules and surface adsorption to glass and rubber, it is inconvenient to accurately estimate the concentration of Sulphide in biotic (Richardson et al, 2000). 27 Sulphide concentrations are distinctively measured In biological tissues and fluids by numerous analytical procedures. Unionized sulphide can be further calculated from concentration of dissolved sulphide. 30 Analytes like blood or plasma are estimated performing specifically gas chromatography either coupled with flame photometric detection (GC/FPD) or flame ionizing detection (GC/FID), iodometric titration, potentiometry and spectrophotometry, with ion selective electrodes (ISE), and high performance liquid chromatography (HPLC). Our technique of choice for this article is modified methylene blue method originally developed by Siegel L M in 1965, and later modified by Stipanuk M H et al in 1982. 18 This colorimetric method is cost effective and easy to set-up at any simple laboratory. This method has disadvantages and limitations over higher end equipment based procedures due to interferences in the likes of viscosity, turbidity and low detection limit.
Conclusion
The current study elucidated decreased levels of serum H2S in hypothyroid patients. Thereby, this study suggest serum H2S levels are significantly correlated with level of diagnostic parameters of hypothyroidism. Further study is needed in this direction to focus on the role of H2S modulators towards the management of this disorder.
