European Journal of Cardiovascular Medicine

In India, hypothyroidism affects approximately 11% of the population.¹ Thyroid hormones play a critical role in regulating basal metabolism and influencing autonomic nervous system balance.² Research has shown that overt hypothyroidism is often accompanied by a sympatho-vagal imbalance(SVI), characterized by increased sympathetic activity and reduced vagal tone—factors linked to heightened cardiac morbidity.^3,4

Heart-Rate variability(HRV) provides a non-invasive measure of fluctuations in cardiac rhythm that reflect autonomic modulation, and it is increasingly used to evaluate cardiovascular involvement in endocrine disorders such as hypothyroidism.⁵ Autonomic function tests, including postural changes, sustained handgrip, the cold pressor test, the valsalva maneuver, and deep breathing, provide valuable insights into cardiac autonomic responses.⁶

Experimental animal studies have identified that Orexins are synthesized by a distinct population of hypothalamic neurons located in the lateral and adjacent nuclei. These orexin-producing neurons, or orexinergic neurons, influence a broad spectrum of physiological processes ranging from arousal, appetite and thermoregulation to

autonomic control, as stimulators of sympathetic cardiovascular activity, elevating both heart rate and blood pressure.^7,8

Experimental models suggest that orexin signaling may down-modulate hypothalamic TRH release, thereby altering pituitary TSH secretion, although human data remain limited.⁸ This suggests that hypothyroidism may be associated with reduced orexin levels, potentially resulting in elevated TSH; however, the evidence supporting this relationship remains inconclusive. Given the hypothesis that orexins and thyroid hormones may influence autonomic functions—such as appetite regulation, basal metabolic rate, and cardiac autonomic activity—through a shared pathway, this study aims to investigate the association between orexin levels and thyroid hormone levels, TSH, autonomic function, and cardiac parameters in patients diagnosed with overt hypothyroidism.

AIM:

To estimate the changes in Serum levels of orexin and its impact on the variations in autonomic function tests in patients diagnosed with overt hypothyroidism.

OBJECTIVES:

1. To estimate and compare the changes in serum levels of orexin amongst the study group
2. To estimate and compare the changes in parameters of heart rate variability among the study group
3. To find any association between the serum orexins with the parameters of heart rate variability amongst the hypothyroid patients.

Ethical Consideration: This study was started after institutional ethics clearance and written informed consent from all the participants. After ensuring adequate time, separate dates were allotted for the examination aspects of this study. Study Design and Duration: The study design is an analytical cross-sectional study. The duration of our study was 2 years from January 2021 to January 2023. Sampling method and the sample size: Sample size was calculated using the formula for mean, with an estimated mean (σ) for LF(nu) ratio as 20.1, an estimated difference (€) of 5 and 95%CI.10 The calculated sample size was n = 60, in which the hypothyroid patients were taken as Group-1(n=30) and clinically normal healthy individuals were as Group-2(n=30) using convenient sampling method. Inclusion and Exclusion criteria: All participants with 18-60 years of age, both gender, who were clinically diagnosed as hypothyroid with treatment, were included in this study. Those participants with age less than 18 years and more than 60 years, if with history of total thyroidectomy, hypertension, any chronic neurological illness, cardiovascular illness, pulmonary illness, psychiatric illness, renal insufficiency, coagulopathy, any metabolic disorders, women on hormone replacement therapy, pregnancy and lactation were excluded from this study. Selection of Participants: In Group-1, 30 clinically diagnosed hypothyroid patients(>6 months duration) undergoing treatment were recruited from the OPD/ward of Endocrinology department, Rajiv Gandhi government General Hospital, Madras Medical College, Chennai. In Group-2, 30 clinically healthy normal individuals were recruited from the Master health-checkup department of our hospital. Baseline Procedure: Participants who consented to take part in the study were instructed to report to the Research Laboratory at the Institute of Physiology and Experimental Medicine, Madras Medical College, at 8:00 AM following an overnight fast. A venous blood sample (5 mL) was collected to analyze serum orexin levels and the thyroid profile. After sample collection, participants were provided with a standardized breakfast excluding tea or coffee. Subsequently, they completed a case study proforma capturing sociodemographic details and clinical history of illness. Thorough clinical examination was conducted, followed by a series of autonomic function tests(AFT). For female participants, menstrual history was recorded, and AFT was scheduled two days after the completion of their menstrual phase to minimize hormonal influences on autonomic parameters. Autonomic Function testing: After a period of standardized rest, short-term HRV recordings were obtained following guidelines commonly employed for autonomic function assessment. The complete autonomic function testing session for each participant lasted to approximately two hours. Table-1: To minimize the influence of factors and ensure the accuracy of test results, the following precautions were observed prior to conducting the autonomic assessments: 1. The subject must not be stressed or anxious. They should be relaxed and comfortable. 2. The subject should not have any acute illness such as fever, upper respiratory infection and gastro-intestinal disturbances in the past one week. 3. The subject should wear loose fitting garments and should empty the bladder before starting the test. 4. The tests were performed two hours after a light breakfast while avoiding coffee and tea. 5. The AFT were performed in a dimly lit, quiet room. 6. The ambient temperature for conducting AFT was 25 – 28°C. 7. All electronic devices including mobile phones were switched off to nullify electrical interference. 8. The different maneuvers involved in AFT were taught to all the participants and a trial run was done before the actual recording. 9. Then the subjects were given 10 minutes rest in supine position and each maneuver was carried out with 15 minutes break-time between procedures. Table-2: Maneuvers & Recording of Heart Rate Variability. 1. Resting: All maneuvers done after adequate rest of 15 minutes with patient in sitting posture which was taken as baseline value. 2. Posture: After 15 minutes of adequate rest, short term analysis of heart rate variability using Lead II ECG was recorded for a period of 5 minutes with the participant in lying comfortably in supine position using the AD Instruments Lab Chart Pro software. Then baseline blood pressure was recorded after 15 minutes period of rest. 3. Handgrip test: Each participant was asked to use dominant hand and apply maximum force to the grip force transducer of AD instrument and instructed to apply 30% of the maximum force to the transducer for 2 minutes. During this procedure, the blood pressure was recorded in the opposite limb at 1 and 2 minutes. The average of diastolic blood pressure recorded at 1 and 2 minutes was calculated. After 15minutes rest to the participants, the baseline respiration, ECG and R-R interval were recorded each for 30 seconds. 4. Valsalva maneuver was tested using a 10ml disposable syringe connected to sphygmomanometer as mouth piece and each participant was asked to blow through mouthpiece maintaining a pressure of 40 mmHg for 15 seconds. During this procedure the ECG and baseline recordings were done continuously. The parameters assessed in the study: Thyroid hormone concentrations and TSH values were quantified using commercially available ELISA assays according to manufacturer specifications. Serum orexin levels were estimated(ELISA) in Virology Research and Diagnostic Lab. The HRV parameters such as the time domain variables-mean RR, RMSSD and pNN50 and the frequency domain variables such as TP, LF (nu), HF (nu), and LF:HF ratio were evaluated from short term analysis of heart rate variability (ADIns-Lab-Chart-Pro) after eliminating artifacts and ectopic beats. From isometric hand grip method, the difference in diastolic blood pressure above the baseline was evaluated. The Valsalva ratio was calculated from HRV tracings by comparing the peak RR interval in phase IV with the minimum RR interval observed during phase II. Statistical Analysis: All the data obtained were tabulated and analyzed. Data analyses were performed with SPSS 24.0. Normality distribution of all the variables was tested with Kolmogorov-Smirnov test. The variables were represented as mean, standard deviation and percentage. Comparison of variances between the groups were analyzed using unpaired t-test. The association between the serum orexin levels and other variables were analyzed using Pearson test. The mean difference of variables between groups and within groups is considered significant at 0.05(p) in all analysis.

30 patients clinically diagnosed as hypothyroid as Group-1 and 30 clinically normal healthy individuals as Group-2 were assessed for their clinical history of illness, serum orexin levels and autonomic function tests.

1. The Socio-demographic, Anthropometric details of the study group:

The sociodemographic details and anthropometric measurements of the study group were shown in Table-3 and the participants were of varied occupation status. The clinical history of the illness amongst the hypothyroid patients (Group-1) were discussed in Table-4. Amongst the Group-2 with clinically healthy normal individuals, we found nil family H/O of hypothyroidism.

1. Clinical examination findings and serum levels tested in the study group:

The vital parameters of the study groups were discussed in Table-5. We had excluded hypertensive patients in our study. Yet we had a slight increase in the mean systolic blood pressure, mean diastolic blood pressure and the mean arterial pressure of the hypothyroid patients (Group-1) that did not affect the study results. The mean free T₄, mean free T₃ and mean TSH levels has been discussed in Table-6. The mean serum orexin levels did not show significant difference between the two groups.

1. Autonomic function test parameters in the study group:

The time domain, frequency domain in response to the maneuvres of the autonomic function test has been discussed in Table-7. The mean of time domain variables such as SDNN, RMSSD, pNN50 showed a slight increase in group-1. The mean frequency domain variables such as LFnu, HFnu, LF/HF did not show any significant difference between the two groups. Valsalva ratio obtained by dividing longest RR interval by shortest RR interval did not show any significant difference between the two groups. During isometric handgrip test, a slight increase in diastolic blood pressure was observed in the hypothyroid group. Valsalva maneuver reflects the parasympathetic activity on heart and Isometric hand grip test reflects the sympathetic activity on heart.

1. Association of serum orexin levels with thyroid hormone levels and AFT parameters in hypothyroid patients:

                The association of Serum orexin with thyroid profile amongst group-1 was discussed in Table-8. Significant positive correlation was found between serum orexin with TSH levels, mean RR, LFnu, LF/HF and Valsalva ratio. Significant negative correlation was found between serum orexin with free T₃ levels, free T₄ levels, time domain variables (SDNN, RMSSD and pNN50), VLF, HFnu implying an impaired parasympathetic activity on heart.

Table-3: Socio-demographic, Anthropometric details of the study group

* p <0.05 is considered to be statistically significant.

Table-4: Clinical history of illness among the patients with hypothyroidism.

Table-5: Vital Parameters of the study groups.

* p <0.05 is considered to be statistically significant.

Table-6: Comparison of the thyroid profile and serum orexin levels in the study group.

* p <0.05 is considered to be statistically significant.

Table-7: Comparison of AFT parameters amongst the study group.

* p <0.05 is considered to be statistically significant. ms – millisecond, ms² – millisecond square, nu – normalized unit, SDNN - Standard deviation of all NN intervals, RMSSD - Root Mean Square of Successive Differences between normal heartbeats, pNN50 - NN50 count divided by the total number of all NN intervals, VLF – Very Low Frequency, LF – Low frequency, HF – High frequency

Table-8: Association of serum orexin levels with thyroid variables and AFT variables in group-1.

* p - value less than 0.05 is taken as significant. ms – millisecond, ms² – millisecond square, nu – normalized unit, SDNN - Standard deviation of all NN intervals, RMSSD - Root Mean Square of Successive Differences between normal heartbeats, pNN50 - NN50 count divided by the total number of all NN intervals, VLF – Very Low Frequency, LF – Low frequency, HF – High frequency

Figure.I: Correlation of serum Orexin with serum TSH in hypothyroid patients

Figure.II: Correlation of serum Orexin with free T₃ in hypothyroid patients

Figure.III: Correlation of serum Orexin with free T₄ in hypothyroid patients

Fig.IV: Correlation of serum orexin with LF/HF ratio in hypothyroid patients

Fig.V: Correlation of serum orexin with Valsalva ratio in hypothyroid patients

Fig.VI: Correlation of serum orexin with increase in diastolic blood pressure during isometric handgrip test in hypothyroid patients

This study was conducted by recruiting 30 clinically diagnosed hypothyroid patients and 30 clinically healthy normal individuals, aimed to estimate the levels of serum orexin and its impact on variations in autonomic functions in patients with hypothyroidism. A. Association between serum orexins and body mass index. The BMI was proven to have negative correlation with plasma orexin-A levels11 and no significant association to menopause.12 We found a significant positive correlation between serum orexin and BMI in hypothyroid patients. B. Association between serum orexins with basal heart rate and blood pressure. Animal studies show that activating orexin receptors within the RVLM augments sympathetic outflow, resulting in elevated arterial pressure and heart rate.13 It was proved that orexin when injected into Nucleus-Tractus solitarius increased the vagal activity to heart and inhibited sympathetic activity to the heart and vasculature.14 When nucleus ambiguous was injected with orexin, it produced bradycardia through activation of baroreceptor reflex.15 Another study done in narcoleptic patients showed a positive association was found between the heart rate with serum orexin levels.16 We did not find any association between serum orexin levels with basal heart rate and diastolic blood pressure. C. Association between serum orexins and thyroid functions. The effects of orexin on hypothalamic-pituitary thyroid axis remained conflicting till several animal experiments were carried out. Specifically, peripheral administration of orexin-A has been shown to suppress TSH levels, likely due to the inhibition of TRH secretion from the hypothalamus, as demonstrated in in vitro studies using rat models.8 It was reported that intracerebroventricular administration of orexin led to a marked reduction in plasma TSH levels, without any significant changes in plasma free T3 (FT3) or free T4 (FT4).17 These findings support the hypothesis that orexins inhibit the release of TRH from the hypothalamus, thereby reducing TSH secretion from the anterior pituitary. Additionally, observed was a negative correlation between orexin levels and FT3/FT4, along with a positive correlation between orexin and TSH.18 In the present study, higher circulating orexin concentrations paralleled elevated TSH values, whereas FT3 and FT4 showed an inverse pattern. D. Association between serum orexins and the duration of illness. Previous research has demonstrated that pre-treatment plasma orexin levels in premenopausal women with subclinical hypothyroidism significantly increased following four months of L-thyroxine therapy. However, to date, no studies have explored the association between orexin levels and the duration of hypothyroidism. Our study states that serum orexin levels has a significant positive correlation with the duration of illness. E. Association between serum orexins and cardiac autonomic functions. In an animal experiments, when an antagonist of orexin was injected into RVLM, there was decrease in LF/HF ratio which represents cardiac sympathetic activity.20 In patients with overt hypothyroidism, a significant positive correlation was observed between serum orexin levels and mean RR interval. Conversely, serum orexin levels showed a significant negative correlation with time-domain measures(SDNN, RMSSD, and pNN50). Among frequency-domain parameters, serum orexin was negatively correlated with VLF and HFnu, while no significant correlation was found with LFnu. Notably, the LF/HF ratio and the Valsalva ratio exhibited a significant positive correlation with serum orexin levels.

Serum orexin was elevated in hypothyroid patients than the normal participants. A mild sympathovagal imbalance was observed using heart rate variability analysis in hypothyroid patients. A negative association of serum orexin with time domain variables in hypothyroid patients, implied an impaired parasympathetic activity and a positive association with LF/HF ratio indicates a sympathovagal imbalance. Valsalva ratio an indicator of parasympathetic activity on heart and Isometric handgrip test that assess the sympathetic activity were influenced by serum orexin levels in hypothyroid patients. We could interpret that with serum orexin levels has its effects on autonomic nervous output in hypothyroid patients, yet, only further studies with larger cohorts may be helpful to substantiate our results.

F. LIMITATIONS OF THE STUDY:

             Our study sample size results are reported here. The autonomic function test with Valsalva maneuver and hand grip measured variables might provide more results if done on continual 24-hour recording and varied time period analysis. Most of the hypothyroid patients were females and very meagre male patients diagnosed of hypothyroidism, we could recruit in our study and clear demarcation of perimenopausal and postmenopausal women was not established.

ACKNOWLEDGEMENT:

All the authors thank the technicians, the patients and the normal healthy volunteers who contributed for this study. We thank and acknowledge the ICMR for providing financial support under MD/MS thesis financial assistance to complete this project.

CONFLICT OF INTEREST: Nil

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