Sudan Journal of Medical Sciences Volume 17, Issue no. 2, DOI 10.18502/sjms.v17i2.11456 Production and Hosting by Knowledge E Original Article Association of TSH Levels in the Therapeutically Neglected Range of 6.5–8 mIU/L with Significant Changes in Liver and Kidney Function: A Retrospective Study of the Kashmiri Population Tousief Irshad Ahmed*1 and Ruqaya Aziz2 1Tutor Demonstrator, Department of Biochemistry, SKIMS Medical College and Hospital, Srinagar, J&K, India 2Professor and Head, Department of Biochemistry, SKIMS Medical College and Hospital, Srinagar, J&K, India ORCID: Tousief Irshad Ahmed: https://orcid.org/0000-0003-3037-6204 Abstract Background: The thyroid gland secretes hormones crucial for growth, differentiation, regulation of metabolic processes, and homeostasis. In response to underactivity of this gland, the pituitary secretes thyrotropin, also known as the thyroid-stimulating hormone (TSH). Medication for thyroid hypofunction is usually started when TSH levels exceed 10 mIU/L. However, we hypothesize that TSH levels much below this therapeutic threshold level may herald significant renal and hepatic dysfunction. The present study was thus conducted to assess liver and kidney function parameters in cases having TSH in the subclinical range with particular focus on the therapeutically neglected (6.5–8 mIU/L) range. Methods: Hospital laboratory archives of 297 adults with laboratory evidence of hypothyroidism, that is, TSH > 6.5 mIU/L, were retrieved and compared with data obtained from 430 euthyroid hospital controls, that is, TSH < 2.5 mIU/L, also from the same period. The thyroid profile and clinical chemistry analyses were performed on Beckman Coulter’s UniCel DxI 800 and AU 5800, respectively. SPSS version 20 was used to analyze the results. Results: Significant differences in triiodothyronine (T3), thyroxine (T4), TSH, urea, creatinine, total bilirubin, total protein (TP), and liver enzymes were observed between cases with TSH > 6.5 mIU/L and controls (P < 0.05). There was also a significant difference in T4, TSH, urea, creatinine, total bilirubin, albumin and aspartate aminotransferase (AST) among cases with TSH in the range of 6.5–8 mIU/L when compared with controls (P < 0.05). A correlation of T3 with TSH, urea, and creatinine was seen (P < 0.05). No correlations between TSH and other clinical chemistry parameters could be observed. However, in the 6.5–8 mIU/L subgroup, correlation of TSH was seen with TP and albumin only. How to cite this article: Tousief Irshad Ahmed* and Ruqaya Aziz (2022) “Association of TSH Levels in the Therapeutically Neglected Range of 6.5–8 mIU/L with Significant Changes in Liver and Kidney Function: A Retrospective Study of the Kashmiri Population,” Sudan Journal of Medical Sciences, vol. 17, no. 2, pp. 218–235. DOI 10.18502/sjms.v17i2.11456 Page 218 Corresponding Author: Tousief Irshad Ahmed; email: khagankhan@gmail.com Received 16 October 2021 Accepted 7 May 2022 Published 30 June 2022 Production and Hosting by Knowledge E Tousief Irshad Ahmed and Ruqaya Aziz. This article is distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use and redistribution provided that the original author and source are credited. Editor-in-Chief: Prof. Mohammad A. M. Ibnouf http://www.knowledgee.com mailto:khagankhan@gmail.com https://creativecommons.org/licenses/by/4.0/ https://creativecommons.org/licenses/by/4.0/ Sudan Journal of Medical Sciences Tousief Irshad Ahmed and Ruqaya Aziz Conclusion: Authors found that, as a rule, subtle renal and hepatic dysfunction were established in cases with TSH levels <8 mIU/L, which was below the typical “therapeutic cut-off” of 10 mIU/L. Accordingly, we advocate against incautiousness and suggest regular monitoring, especially in the 6.5–8 mIU/L range. Keywords: subclinical hypothyroidism, liver function test, kidney function test, thyroid- stimulating hormone 1. Introduction Thyroid hormones (THs), namely Thyroxine (T4) and 3, 5,3I L-tri-iodothyronine (T3), secreted by the thyroid gland following synthesis from the amino acid tyrosine in the thyroid follicles act as the “master regulators,” exerting a profound influence on almost every cell of the body by “canonical” and “non-canonical” mechanisms [1]. THs are crucial for regulating protein, carbohydrate, and fat metabolism. They are essential for the general processes of metabolism, development and growth, which they accomplish through various genomic as well as non-genomic routes. Of particular importance is the action of THs on the liver, where they actively modulate glucose, cholesterol, and fatty acid metabolism and stimulate de-novo lipogenesis. These hepatic actions have a bearing on basal energy expenditure, thermogenesis, and metabolic homeostasis [2]. THs act on kidneys where they regulate the renal hemodynamics by direct mechanisms and by modulating ion transport in the glomerular and tubular cells. THs also affect the organs above by influencing the cardiac output. Hypothyroidism adversely affects cardiac contractility, myocardial oxygen consump- tion, vascular resistance, blood pressure, and electrophysiological conduction. As THs have genomic effects, any reduction in their concentrations result in decline of transla- tional products involved in myocardial contractility, endothelial vasodilation, and renin synthesis. Response to β-adrenergic stimulus is also downgraded. This has a profound impact on the renal milieu [3]. Certain studies have hinted at histological changes in hypothyroidism. Several observational studies have shown that elevated thyroid-stimulating hormone (TSH) levels are significantly associated with the development of non-alcoholic fatty liver disease (NAFLD). This can be partly explained by the steatogenic changes induced by an underactive thyroid. Modulation of signal transduction pathways, impairment in lipid metabolism, increased de-novo lipogenesis, and upregulation of reactive oxygen DOI 10.18502/sjms.v17i2.11456 Page 219 Sudan Journal of Medical Sciences Tousief Irshad Ahmed and Ruqaya Aziz species (ROS) and inflammatory cytokines can all be triggered by disturbances in thyroid function [4]. Hypothyroidism and hyperthyroidism indicate underactivity and overactivity of the thyroid gland, respectively [5]. Subclinical hypothyroidism (SCH) is a laboratory diagnosis wherein TSH levels are higher than normal, while levels of T3 and T4 remain in the normal range. Hypothyroidism is the most prevalent among thyroid disorders in South Asia and especially so in the northern Himalayan states of India, where iodine deficiency has been a historical concern. Coastal Indian cities have a lower prevalence of hypothyroidism (both SCH and overt) than northern inland territories. In fact, a north Indian study suggested that the prevalence of SCH could be as high as 19.3% [6]. Of late, questions on thyroid gland underactivity have been arising, primarily whether cases of TH values falling marginally outside of normal limits should be pharmacolog- ically addressed, or is overprescription of T4 for the treatment of these “laboratory derangements” a genuine concern in that, are we treating the lab reports or the patient themselves?[7]. SCH is usually characterized by laboratory evidence of increased TSH (5–10 mIU/L or more) along with average T4 values. Older studies did not provide sufficient evidence for the benefits of treatment at TSH values of 4.5–10 mIU/L [8]. In fact, treatment was only recommended for TSH values higher than the threshold value of 10 mIU/L [9]. A comprehensive perusal of recent studies shows that levothyroxine is generally prescribed only in manifest hypothyroidism. Therapy for SCH is usually discouraged unless TSH values exceed 10 mIU/L [10]. However, there is a possibility that undue focus on the 10 mIU/L threshold may potentially leave out many who are otherwise worthy of the medication. Specific ailments, such as psychiatric disorders [11], unexplained infertility [12], and metabolic syndrome may have a thyroid basis. Patho- logical alterations in crucial analytes as seen in hyperinsulinemia, insulin resistance, dyslipidemia, hypercoagulability, cardiovascular status (as gauged by elevated hsCRP), and hyperuricemia are often closely related to thyroid function. These alterations may manifest themselves even at TSH levels <10 mIU/L [13]. In many such cases, despite mid- to high-normal TSH values, subtle thyroid dysfunction is evident beyond doubt. Evidently, there is a possibility of the setting in of indiscreet biochemical changes which may benefit from early therapy. All these questions lend more weight to the concept of individualized assessment of thyroid function status [5]. Some persons may not have any (obvious) symptoms, and clues obtained from measuring biochemical parameters despite borderline TH measurements may herald a less than assuring prognosis. In DOI 10.18502/sjms.v17i2.11456 Page 220 Sudan Journal of Medical Sciences Tousief Irshad Ahmed and Ruqaya Aziz such persons, therapy initiation at high-normal to moderately raised TSH levels may provide both tangible and intangible benefits. 1.1. Renal effects of thyroid hypofunction The thyroid influences both the kidney and liver (Figure 2). Bulur et al. observed that T4 therapy in previously hypothyroid patients caused renal function to improve significantly as the raised creatinine and TSH levels normalized. They also attributed the raised creatinine levels in hypothyroidism to a reduction in GFR and renal plasma flow. This was in turn due to a “hypodynamic state” of the circulatory system in addition to the lack of TH-induced inotropic and chronotropic stimulus. Creatinine was thus not cleared from the circulation in such individuals with the same vigor as seen in euthyroid individuals [14]. Either that or actual effects on glomerular physiology or both may be the reason for creatinine elevation. Animal studies on hypothyroid rats showed histological evidence of a reduction in glomerular capillary density, which indicated a pro-angiogenic role for THs [15]. These hormones may also be instrumental in increasing vascularity. In addition, they exert an activatory effect on the renin–angiotensin system (RAS), both with and without the involvement of the sympathetic nervous system. In a study by Ichihara et al., T3 was found to increase renin secretion and renin mRNA in juxtaglomerular cell cultures by calcium-dependent and independent mechanisms [16]. Certain indirect (endocrine/paracrine) effects mediated via signal proteins like vascular endothelial growth factor (VEGF) and insulin-like growth factor type 1 (IGF-1) are also contributory to the renal effects seen in SCH [17]. 1.2. Hepatic effects of thyroid hypofunction Historically, the liver has always been an indispensable organ for medical research. It is probably the most influenced by the thyroid. Preliminary studies on rat liver and kidneys during the early second world war period revealed that the administration of T4 and TSH had definitive effects on tissue respiration and organ weight, with the former hormone consistently elevating the oxygen consumption rates (QO2) as compared to the latter [18]. The effect of TH perturbations on liver function was demonstrated in another study during the cold war period, utilizing electrophoresis. The said study showed alterations in serum protein patterns in thyroid disorders, with hypothyroid patients exhibiting lower albumin levels and elevated β globulin fractions. Treatment with T4, however, tended to reduce levels of both these fractions [19]. A seminal work DOI 10.18502/sjms.v17i2.11456 Page 221 Sudan Journal of Medical Sciences Tousief Irshad Ahmed and Ruqaya Aziz on biochemical changes underlying cellular differentiation during T3-induced metamor- phosis in tadpoles observed a significant increase in specific activities of liver nucleic acids and proteins when administered the TH. The Liver RNA: DNA ratio also increased indicating active transcription triggered by T3 [20]. The same year, while studying the effect of T3 on the growth of the liver in thyroidectomized rats, an Indian researcher, Jamshed Tata, observed accelerated incorporation of amino acids into nuclear protein. He also reported an increased turnover of basic nuclear proteins [21]. An early animal experiment to assess the role of THs on hepatic metabolism revealed an increased efficiency in lactate utilization in hypothyroid rats administered T3. The sensitivity to glucagon improved and gluconeogenesis was also found to be markedly enhanced. The gluconeogenic enzyme pyruvate carboxylase appeared to be highly responsive to T3. The latter hormonewas also effective in reducing urea formation. Redox equilibrium of the perfused hypothyroid liver, which showed a more reduced state, possibly due to underutilization of NADH in gluconeogenesis, was normalized by the administration of T3[22]. In the early 1970s, the pioneer of TH action, J.H. Oppenheimer, identified high-affinity receptors for T3 in nuclei of rat liver and kidney [23]. In one of the earliest reviews on TH action, he mentioned that the interaction of T3 (and to a much lesser extent T4) with the receptors resulted in significant modulation of gene activity. Enzymes vital to carbohydrate and lipid metabolism, such as α-glycerophosphate dehydrogenase and malic enzyme, were particularly affected by T3(by that time, they were already being used as resourceful indices for studying the hormone’s effects in rat liver). The rat liver tissue had a high binding capacity and a larger number of binding sites per nucleus compared to several other organs [24]. The liver affects circulating TH concentrations as well. Carrier proteins that bind T4 and transport it to different targets in the body are synthesized and degraded by the liver. Only 0.04% of T4 circulates freely. Most of it is complexed with TH carrier proteins like thyroxine-binding globulin (TBG), thyroxine- binding pre-albumin (TBPA), albumin, and other plasma proteins. The hepatocyte thus represents a central control point. Peripheral deiodination is also accomplished by deiodinases which synthesized in the liver. The liver–thyroid relationship is thus a two- way interaction [25]. The purpose of this study was to assess derangements in routine liver and kidney biochemical parameters in subclinical and overt hypothyroidism with respect to controls. Special attention was paid to the analysis of such derangements in the therapeutically neglected TSH range of 6.5–8 mIU/L. We also attempted to determine the correlation between T3, T4, TSH levels and these parameters. DOI 10.18502/sjms.v17i2.11456 Page 222 Sudan Journal of Medical Sciences Tousief Irshad Ahmed and Ruqaya Aziz 2. Materials and Methods 2.1. Study population Hospital laboratory archives of adult OPD patients visiting SKIMS (Sher-i-Kashmir Insti- tute of medical sciences) Medical College and Hospital, Srinagar, Jammu & Kashmir, India between January 2017 and March 2021 were accessed and scanned for all records with laboratory evidence of hypothyroidism, that is, TSH > 6.5 mIU/L (including mild/significant SCH and OH). After several rounds of exclusion, records of 297 adults (78 males [26.26%] and 219 females [73.74%]) were retrieved and compared with data obtained from 430 euthyroid hospital controls, that is, TSH < 2.5 mIU/L (57 males [13.26%] and 373 females [86.74%]), also from the same period. 2.2. Inclusion and exclusion criteria Adults of either sex were included. Those with a history of thyroid surgery or use of T4, amiodarone, or lithium were excluded. We also excluded all records with TSH levels between 2.51 and 6.49 mIU/L or <0.5 mIU/L for both cases and controls. The scheme of enrollment of data is given in Figure 1. 2.3. Data collection methodology The authors’ laboratory currently uses a reference range for TSH of 0.45 to 6.0 mIU/L. Thus, 4997 lab archives with adequate quality control (QC) results were retrospectively scanned, and data of all individuals with TSH levels > 6.5 mIU/L were considered. After exclusion, it was narrowed down to 297 records. These included routine LFT (n = 281) and KFT (n = 297) records. Cases were divided into two groups for statistical analysis. The first group had TSH in the “therapeutically neglected” range of 6.5–8 mIU/L (n = 44 for LFT and n = 48 for KFT), while the second group had TSH in the “therapeutically important” range of >8 mIU/L (n = 237 for LFT and n = 249 for KFT). Hospital controls were used, also from the lab archive database, wherein data having TSH in the range of 0.5–2.5 mIU/L (n = 430) were selected. 2.4. Protocol and procedure All retrospective records we obtained were of patients who, as per routine protocol, were advised to report for sampling after an overnight fast. A single blood draw was used to DOI 10.18502/sjms.v17i2.11456 Page 223 Sudan Journal of Medical Sciences Tousief Irshad Ahmed and Ruqaya Aziz obtain a 4–6 mL venous blood sample and divided into two aliquots immediately post phlebotomy. One aliquot was sent to the biochemistry laboratory, and another aliquot was sent to the immunology section, which performs the thyroid panel. Immediately after receipt of the aliquots, centrifugation for 5 min at 3000 g was performed. Serum was subsequently separated. While the biochemistry aliquots were immediately processed and analyzed after a brief (15–30 min) delay to allow for data entry, the thyroid panel aliquot was subject to a moderate (30–60 min) delay for sufficient batch size to form and then processed. Generally, the average turnaround time (TAT; from sample registration to report authentication) of biochemistry samples is in the 60–120 min range, while thyroid panel TAT averages around 120–180 min. Different technical staff performed the analyses of each aliquot and results were automatically uploaded on the hospital LAN (local area network). 2.5. Measurement of T3, T4, and TSH T3, T4, and TSH (3𝑟𝑑 gen) levels were estimated on Beckman Coulter’s UniCel DxI 800 Access Immunoassay System. This analyzer utilizes chemiluminescent detection and magnetic particle-separation technology. Reference intervals provided by the manufac- turer were TSH 0.45–5.33 mIU/L for TSH, 0.87–1.78 ng/ml for T3, and 6.09–12.23 μg/dl for T4. The sensitivities of TSH, T3, and T4 were 0.01 mIU/L, 0.1 ng/mL, and 0.50 μg/dL, respectively. 2.6. Measurement of biochemical parameters The biochemical parameters, namely urea, creatinine, total bilirubin, total protein, albu- min, aspartate aminotransferase (AST), alanine aminotransferase (ALT), and alkaline phosphatase (ALP) were examined on Beckman Coulter’s AU5800 clinical chemistry system. This analyzer is based on the principles of spectrophotometry and potentiom- etry. 2.7. Quality control Apart from stringent daily maintenance and calibration protocols, internal controls for clinical chemistry and immunoassay parameters provided by Bio-rad® laboratories were run at least twice daily. Any nonconformity or errors reported were investigated and root cause analysis performed. Outliers or results exceeding linearity were subject to repeat DOI 10.18502/sjms.v17i2.11456 Page 224 Sudan Journal of Medical Sciences Tousief Irshad Ahmed and Ruqaya Aziz testing with suitable dilution. The intraassay coefficients of variation (CV) for T3, T4, and TSH were <10%. The clinical chemistry parameters also had acceptable CVs (Table 1). 2.8. Statistical analysis The recorded data were compiled and entered in a spreadsheet (Microsoft Excel) and then exported to data editor of SPSS Version 20.0 (SPSS Inc., Chicago, Illinois, USA). Continuous variables were expressed as Mean ± SD, and categorical variables were summarized as frequencies and percentages. The Student’s independent t-test was employed for comparing continuous variables. Karl Pearson’s correlation coefficient was applied to determine the correlation of TSH, T3 and T4 with various parameters among study cases. A P-value of < 0.05 was considered statistically significant. All P-values were two-tailed. 3. Results In the present study, on comparison of liver and kidney function parameters, it was found that the total bilirubin, total protein, globulin, liver enzymes aspartate aminotrans- ferase (AST), alanine aminotransferase (ALT) and alkaline phosphatase (ALP), urea and creatinine were significantly elevated in the cases which had TSH > 6.5 mIU/L when compared to the controls (Table 2). The same effect was seen when comparing the group with TSH > 8 mIU/L to controls (Table 3). Albumin levels were not statistically different in the cases vis-à-vis controls. Globulin levels were higher nonetheless, and this resulted in lower A/G ratios in the cases. However, on comparing cases with TSH levels in the 6.5–8 mIU/L range to controls, we found that the total protein, ALT and ALP ceased to show a statistically significant difference. At the same time, albumin and the other LFT and KFT parameters remained significantly elevated (Table 4). Thus, the rise in urea, creatinine, total bilirubin, albumin, globulin, and AST appears to have established itself in this “therapeutically neglected” range, even though T3 and T4 levels, despite being significantly lower, were still mainly in the normal to low normal range. No significant correlations were found between TSH/T4 and any biochemical parameter. Nonetheless, we did find a significant correlation between serum T3 and urea, creatinine (P =< 0.001), and a moderately significant correlation between serum T3 and ALP (P = 0.059). Interestingly, when we limited the analysis to TSH in the range of 6.5–10, we found a significant correlation between TSH on the one hand and total protein (P = 0.004), albumin (P = 0.013) on the other. Urea levels also showed a DOI 10.18502/sjms.v17i2.11456 Page 225 Sudan Journal of Medical Sciences Tousief Irshad Ahmed and Ruqaya Aziz moderate correlation in this TSH range (P = 0.086) (Table 5). Thus, the fluctuations in TSH levels observed in individuals within this range may be accompanied by corresponding changes in the above parameters. Figure 1: Scheme of data enrollment. Table 1: Assay specifications of hepatic and renal parameters. Analyte Method Reference range Sensitivity Intra-assay CV Urea Adaptation of the enzymatic method uti- lizing glutamate-dehydrogenase (GLDH) 17–43 mg/dL 5 mg/dL ≤5% Creatinine Kinetic modification of the Jaffe procedure 0.6–1.3 mg/dL 0.2 mg/dL ≤3% Total bilirubin 3,5-dichlorophenyldiazonium tetrafluoroborate (DPD) modification of Diazo method 0.3–1.0 mg/dL 0.01 mg/dL ≤3% Total protein Weichselbaum modification of biuret 6.4–8.9 g/dL 3 g/dL ≤3% Albumin Modification of Doumas and Rodkey Bromocresol green method 3.5–5.7 g/dL 1.5 g/dL ≤3% AST Modification of the International Federa- tion of Clinical Chemistry (IFCC) method 13–39 U/L 3 U/L ≤10% ALT Wroblewski and LaDue modification of the International Federation of Clinical Chemistry (IFCC) method 7–52 U/L 3 U/L ≤10% ALP Bowers and McComb method 30–120 U/L 5 U/L ≤10% 4. Discussion We found substantial derangements in almost all of the recorded kidney and liver function parameters in those with TSH > 6.5 mIU/L. Most of these derangements DOI 10.18502/sjms.v17i2.11456 Page 226 Sudan Journal of Medical Sciences Tousief Irshad Ahmed and Ruqaya Aziz Figure 2: Underlying mechanisms for the effects of thyroid hypofunction on the liver and kidney. were evident even at TSH levels <8 mIU/L. We could also establish a significant negative correlation between Tri-iodothyronine levels and the renal function markers. Liver function indicators such as total bilirubin, albumin, globulin, some of the liver enzymes, as well as kidney function parameters were affected even in the narrow TSH range of 6.5–8 mIU/L. In the 6.5–10 mIU/L range, the presence of significant correlations between TSH levels and total protein, albumin, and urea were evident. Thus, TSH levels far below the conventional “therapeutic boundary” of 10 mIU/L were often associated with laboratory evidence of incipient organ impairment. Our findings compare with the Indian study by Arora et al., who reported significantly higher levels of creatinine (albeit not exceeding the reference range) in hypothyroid subjects compared to euthyroid controls (P < 0.001) and also with another Indian study by Yadav et al. which observed significantly raised serum ALT, ALP, and total protein levels in SCH subjects (TSH 6–9.9 mIU/L) [26, 27]. A third Indian study by Saini et al. demonstrated higher urea and creatinine levels in SCH and OH patients than controls DOI 10.18502/sjms.v17i2.11456 Page 227 Sudan Journal of Medical Sciences Tousief Irshad Ahmed and Ruqaya Aziz Table 2: Comparison of LFT parameters in cases and controls. Parameter Cases (TSH > 6.5) (n = 281) Controls (TSH < 2.5) (n = 425) P-value Mean SD Mean SD T3 1.20 0.53 1.41 0.35 <0.001* T4 8.41 2.31 10.09 1.95 <0.001* TSH 16.73 39.58 1.70 0.51 <0.001* Total bilirubin 0.74 0.43 0.59 0.28 <0.001* TP 7.65 0.72 7.44 0.52 <0.001* Albumin 4.09 0.59 4.08 0.50 0.822 Globulin 3.57 0.56 3.371 0.39 <0.001* A/G ratio 1.17 0.26 1.23 0.21 0.002* AST 37.22 25.90 30.95 15.78 <0.001* ALT 37.03 36.53 31.57 24.17 0.017* ALP 124.62 55.52 115.25 65.73 0.035* Comparison of KFT parameters in cases and controls Parameter Cases (TSH > 6.5) (n = 297) Controls (TSH < 2.5) (n = 430) P-value Mean SD Mean SD Urea 26.45 11.04 19.71 8.55 <0.001* Creatinine 0.74 0.39 0.51 0.19 <0.001* Table 3: Comparison of LFT parameters in cases with TSH > 8 and controls. Parameter Cases (n = 237) Controls (TSH < 2.5) (n = 425) P-value Mean SD Mean SD T3 1.18 0.51 1.41 0.35 <0.001* T4 8.37 2.41 10.09 1.95 <0.001* TSH 18.39 42.69 1.70 0.51 <0.001* Total bilirubin 0.72 0.42 0.59 0.28 <0.001* TP 7.68 0.72 7.44 0.52 <0.001* Albumin 4.13 0.60 4.08 0.50 0.311 Globulin 3.57 0.583 3.371 0.39 < .001* A/G ratio 1.11 0.2 1.23 0.21 0.02* AST 37.21 23.49 30.95 15.78 <0.001* ALT 38.16 38.34 31.57 24.17 0.007* ALP 126.89 58.24 115.25 65.73 0.023* Comparison of KFT parameters in cases with >8 mIU/L and controls Parameter Cases (TSH > 8) (n = 249) Controls (TSH < 2.5) (n = 430) P-value Mean SD Mean SD Urea 27.16 11.35 19.71 8.55 <0.001* Creatinine 0.75 0.41 0.51 0.19 <0.001* [17]. Arora et al., however, reported no significant differences in urea levels of cases and controls. They reported a positive correlation between serum TSH on the one DOI 10.18502/sjms.v17i2.11456 Page 228 Sudan Journal of Medical Sciences Tousief Irshad Ahmed and Ruqaya Aziz Table 4: Comparison of LFT parameters in cases with TSH 6.5–8 mIU/L and controls. Parameter Cases (n = 44) Controls (TSH < 2.5 mIU/L) (n = 425) P-value Mean SD Mean SD T3 1.32 0.60 1.41 0.35 0.211 T4 8.63 1.64 10.09 1.95 <0.001* TSH 7.25 0.48 1.70 0.51 <0.001* Total bilirubin 0.85 0.49 0.59 0.28 <0.001* TP 7.46 0.71 7.44 0.52 0.803 Albumin 3.90 0.49 4.08 0.50 0.022* Globulin 3.55 0.46 3.371 0.39 .00588* A/G ratio 1.11 0.2 1.23 0.21 0.00416* AST 37.28 36.84 30.95 15.78 0.035* ALT 30.83 23.67 31.57 24.17 0.847 ALP 111.76 34.26 115.25 65.73 0.735 Comparison of KFT parameters in cases with TSH 6.5–8 mIU/L and controls Parameter Cases (n = 49) Controls (TSH < 2.5 mIU/L) (n = 430) P-value Mean SD Mean SD Urea 22.72 8.38 19.71 8.55 0.022* Creatinine 0.70 0.27 0.51 0.19 <0.001* Table 5: Correlation of TSH with various parameters among study cases for TSH values 6.5–10 mIU/L. Parameter Pearson correlation P-value T3 0.352 <0.001* T4 –0.382 <0.001* Urea 0.142 0.084 Creatinine 0.041 0.619 Total bilirubin –0.095 0.295 Total protein 0.242 0.004* Albumin 0.212 0.013* Globulin 0.017 0.842 AST –0.003 0.972 ALT 0.107 0.213 ALP 0.128 0.137 hand and serum ALT, AST, total protein, and albumin on the other, and a negative correlation between serum T4 and the latter four parameters. Yadav et al. found a positive correlation between TSH and the liver enzymes, AST and ALP. Saini et al. also reported a negative correlation between TSH with urea. However, as seen in the results, our study could not replicate the correlation results of the three Indian studies in cases with TSH > 6.5 mIU/L. One scholar confirmed our findings of higher bilirubin levels DOI 10.18502/sjms.v17i2.11456 Page 229 Sudan Journal of Medical Sciences Tousief Irshad Ahmed and Ruqaya Aziz in SCH subjects. He additionally reported lower albumin levels, a finding which we observed in subjects with TSH in the 6.5–8 mIU/L range [28]. Our observation is that kidney function is affected in SCH and OH, as typified in direct measurements such as creatinine and indirect measurements such as esti- mated glomerular filtration rate (eGFR). We found support for our findings in a study by Schairer et al., who studied a cohort of chronic kidney disease (CKD) patients post- transplantation, concluding that positive changes in TSH (ΔTSH) were associated with decrease in (eGFR) (to the tune of 1.34 mL/min for every 1 µIU/mL increase in TSH) [29]. Another study by Shin et al. focusing on CKD patients with SCH found that TSH reduction secondary to TH replacement therapy was helpful in preventing the deterioration of renal function [30]. Tsuda et al. reported drastic glomerular hemodynamic effects of hypothyroidism, even in the high-normal TSH range. TSH was found to have a significant positive cor- relation with afferent arteriole vascular resistance and a significant negative correlation with renal plasma flow (RPF), renal blood flow (RBF), and GFR. This may have arisen due to the direct action of TSH on its specific receptors in the kidneys. Thus, the effects of compromised thyroid function would lead to suppressed renal function [31]. Recent studies such as that of Kim et al. found that lower thyroid function was associated with higher prevalence and risk of nonalcoholic steatohepatitis (NASH) and fibrosis. They found histological evidence of extensive hepatic steatosis showing significant hepatocyte “balloon degeneration” and fibrosis. High- and high-normal TSH levels were closely related to NASH and NASH-related advanced fibrosis. This could be explained by the increasing propensity for development of insulin resistance and other metabolic disturbances brought about by dyslipidemia and obesity in hypothyroid individuals. Insulin resistance has been shown to improve with TH therapy. Other mechanisms of thyroid hypofunction-induced hepatic damage are oxidative stress, mitochondrial dysfunction, and altered TH signaling in hepatocyte fibrogenesis [32]. TSH on binding to receptors on hepatocytes has been found to upregulate sterol regulatory element-binding protein-1c (SREBP-1c) activity. This may induce steatogenic changes [4]. Our findings of higher bilirubin and liver enzymes in subjects with TSH > 8 mIU/L would be secondary to the above changes. The findings of elevated bilirubin, low albumin, high globulin, and high AST in the TSH range of 6.5–8 mIU/L suggest that thyroid dysfunction in this range of TSH profoundly induces impairment in lipid metabolism. This in turn results in steatogenic changes by the various mechanisms discussed previously, thus precipitating and/or potentiating hepatic injury [28]. In some cases, cholestatic jaundice coincident with hypothyroidism has been ascribed to the impairment in bilirubin and DOI 10.18502/sjms.v17i2.11456 Page 230 Sudan Journal of Medical Sciences Tousief Irshad Ahmed and Ruqaya Aziz bile excretion, which is secondary to the hepatic injury. The enzyme activity of UDP- glucuronyl transferase may also be reduced, thus hampering bilirubin excretion. Diminu- tion in bile flow also results from an increased membrane cholesterol–phospholipid ratio and the consequent reduction in fluidity. Membrane transporters may thus be affected [25]. Consequently bilirubin levels rise in hypothyroidism. AST may rise due to a combination of myopathies and hepatic injury. Hypothyroidism is an inflammatory state, possibly elevating liver enzyme levels and increasing total proteins, mostly the inflammatory globulins, which could partially explain our findings of raised globulin lev- els in all the cases with TSH > 6.5 mIU/L [26]. A significant positive correlation between TSH and total proteins, albumin in the 6.5–8 mIU/L range suggests that hepatic damage may have already started in this range. Putative mechanisms of this damage are hepatic congestion secondary to hypothyroidism-induced cardiac compromise and augmented state of vascular endothelial permeability, in addition to the changes mentioned above [33]. 5. Conclusion We suggest physicians exercise caution in cases having TSH in the range of 6.5–8 mIU/L without apparent signs and symptoms. The results of our study strongly emphasize that alterations possibly involving steatogenic changes in the liver, and insidious decre- ments in kidney function, among myriad other processes discussed above, establish themselves in this range, and a treatment initiation threshold of 8 or 10 mIU/L TSH may be incautious. A new diagnostic scoring system, which takes into account TSH levels for evaluating steatogenic liver changes must be envisaged [4]. Thyroid hypofunction may precipitate/worsen kidney disease, especially in hospitalized patients [17]. In cases of established liver injury, hypothyroidism should not be ruled out as a significant causative factor [33]. It is thus imperative to perform regular liver and kidney function tests for all patients, even for TSH levels <8 mIU/L. Also, the possibility of TH analogues in reversing hypothyroidism-induced fatty change (as well as other indiscreet biochemical changes) at this range of 6.5–8 mIU/L may be considered and further researched [34]. The direct positive correlation of TSH with total protein and albumin in this range suggests that efforts to reduce TSH levels even in this therapeutically neglected range may have tangible benefits. DOI 10.18502/sjms.v17i2.11456 Page 231 Sudan Journal of Medical Sciences Tousief Irshad Ahmed and Ruqaya Aziz Acknowledgements The authors would like to thank the technical staff at the central biochemistry laboratory of SKIMS Medical College for their support. Ethical Consideration Ethical clearance for the study was obtained from the institutional ethical committee of SKIMS medical college, Bemina, Srinagar, J&K, India. Competing Interests None declared. Availability of Data and Material All data and materials associated with this study are available through the corresponding author upon reasonable request. Funding None. References [1] Brix, K., Szumska, J., Weber, J., et al. (2020). Auto-regulation of the thyroid gland beyond classical pathways. Experimental and Clinical Endocrinology & Diabetes, vol. 128, no. 6–7, pp, 437–445. [2] Damiano, F., Rochira, A., Gnoni, A., et al. (2017). Action of thyroid hormones, T3 and T2, on hepatic fatty acids: Differences in metabolic effects and molecular mechanisms. International Journal of Molecular Sciences, vol. 18, no. 744, pp. 1– 19. [3] Iglesias, P., Bajo, M. A., Selgas, R., et al. (2017). Thyroid dysfunction and kidney disease: An update. Reviews in Endocrine and Metabolic Disorders, vol. 18, p. 131– 144. DOI 10.18502/sjms.v17i2.11456 Page 232 Sudan Journal of Medical Sciences Tousief Irshad Ahmed and Ruqaya Aziz [4] Liu, L., Li, P., Mi, Y., et al. (2019). Thyroid-stimulating hormone is associated with nonalcoholic steatohepatitis in patients with chronic hepatitis B. Medicine, vol. 98, no. 46, pp. 1–5. [5] Fuhrer, D., Brix, K., and Bibermann, H. (2015). Understanding the healthy thyroid state in 2015. European Thyroid Journal, vol. 4, no. 1, pp. 1–8. [6] Unnikrishnan, A. G. (2020). Thyroid disorders: A South Asian perspective. In S. Melmed, R. J. Auchus, A. B. Goldfine, R. J. Koenig, C. J. Rosen (Eds.), Williams textbook of endocrinology (pp. 1731–1733). New Delhi, India: Elsevier Inc. [7] Melville, N. A. (2021, June 24). Levothyroxine overprescribing is common, consistent over time. Medscape. Retrieved from: https://www.medscape.com/viewarticle/953652 [8] Surks, M. I., Ortiz, E., Daniels, G. H., et al. (2004). Subclinical thyroid disease: Scientific review and guidelines for diagnosis and management. JAMA, vol. 291, no. 2, pp. 228–238. [9] Almandoz, J. P. and Gharib, H. (2012). Hypothyroidism: etiology, diagnosis, and management. Medical Clinics of North America, vol. 96, pp. 203–221. [10] Calissendorff, J. and Falhammar, H. (2020). To treat or not to treat subclinical hypothyroidism. What is the evidence? Medicina, vol. 56, no. 40, pp. 1–11. [11] Cohen, B. M., Sommer, B. R., and Vuckovic, A. (2018). Antidepressant-resistant depression in patients with comorbid subclinical hypothyroidism or high-normal TSH levels. The American Journal of Psychiatry, vol. 175, no. 7, pp. 598–604. [12] Jokar, T. O., Fourman, L. T., Lee, H., et al. (2018). Higher TSH Levels within the normal range are associated with unexplained infertility. The Journal of Clinical Endocrinology and Metabolism, vol. 103, no. 2, pp. 632–639. [13] Chang, Y., Hua, S., Chang, C., et al. (2019). High TSH level within normal range is associated with obesity, dyslipidemia, hypertension, inflammation, hypercoagulability, and the metabolic syndrome: A novel cardiometabolic marker. Journal of Clinical Medicine, vol. 8, no. 817, pp. 1–15. [14] Bulur, O., Dal, K., Ertugrul, D. T., et al. (2017). Renal function improves with the treatment of hypothyroidism. Endocrine Research, vol. 42, no. 3, pp. 246–251. [15] Gomez, I. R., Banegas, I., Wangensteen, R., et al. (2013). Influence of thyroid state on cardiac and renal capillary density and glomerular morphology in rats. Journal of Endocrinology, vol. 216, no. 1, pp. 43–51. [16] Ichihara, A., Kobori, H., Miyashita, Y., et al. (1998). Differential effects of thyroid hormone on renin secretion, content, and mRNA in juxtaglomerular cells. American Journal of Physiology, vol. 274, no. 2, pp. E224–E231. DOI 10.18502/sjms.v17i2.11456 Page 233 Sudan Journal of Medical Sciences Tousief Irshad Ahmed and Ruqaya Aziz [17] Saini, V., Yadav, A., Arora, M. K., et al. (2012). Correlation of creatinine with TSH levels in overt hypothyroidism — A requirement for monitoring of renal function in hypothyroid patients ? Clinical Biochemistry, vol. 45, no. 3, pp. 212–214. [18] Belasco, I. J. (1941). The effect of thyroxin and thyrotropic hormone on liver and kidney tissue respiration of rats of various ages. Endocrinology, vol. 28, no. 2, pp. 153–160. [19] Lamberg, B. and Grasbeck, R. (1955). The serum protein pattern in disorders of thyroid function. European Journal of Endocrinology, vol. 19, no. 1, pp. 91–100. [20] Finamore, F. J. and Frieden, E. (1960). Nucleic acids and induced amphibian metamorphosis. Journal of Biological Chemistry, vol. 235, no. 6, pp. 1751–1755. [21] Tata, J. R. (1966). In vivo synthesis of nuclear protein during growth of the liver induced by hormones. Nature, vol. 212, no. 5068, pp. 1312–1314. [22] Menahan, L. A. and Wieland, O. (1969). The role of thyroid function in the metabolism of perfused rat liver with particular reference to gluconeogenesis. European Journal of Biochemistry, vol. 10, no. 1, pp. 188–194. [23] Oppenheimer, J. H., Koerner, D., Schwartz, H. L., et al. (1972). Specific-nuclear Triiodothyronine binding sites in rat liver and kidney. The Journal of Clinical Endocrinology and Metabolism, vol. 35, no. 2, pp. 330–333. [24] Oppenheimer, J. H. (1979). Thyroid hormone action at the cellular level. Science, vol. 203, no. 4384, pp. 971–979. [25] Malik, R. and Hodgson, H. (2002). The relationship between the thyroid gland and the liver. QJM, vol. 95, no. 9, pp. 559–569. [26] Arora, S., Chawla, R., Tayal, D., et al. (2009). Biochemical markers of liver and kidney function are influenced by thyroid function-a case-controlled follow up study in Indian hypothyroid subjects. Indian Journal of Clinical Biochemistry, vol. 24, no. 4, pp. 370– 374. [27] Yadav, A., Arora, S., Saini, V., et al. (2013). Influence of thyroid hormones on biochemical parameters of liver function : A case-control study in North Indian population. Internet Journal of Medical Update, vol. 8, no. 1, pp. 4–8. [28] Kim, H. J. (2020). Importance of thyroid-stimulating hormone levels in liver disease. Journal of Pediatric Endocrinology and Metabolism, vol. 33, no. 9, pp. 1133–1137. [29] Schairer, B., Jungrethmayr, V., Schuster, M., et al. (2020). Effect of thyroid hormones on kidney function in patients after kidney transplantation. Scientific Reports, vol. 10, no. 2156, pp. 1–7. [30] Shin, D. H., Lee, M. J., Lee, H. S., et al. (2013). Thyroid hormone replacement therapy attenuates the decline of renal function in chronic kidney disease patients with subclinical hypothyroidism. Thyroid, vol. 23, no. 6, pp. 654–661. DOI 10.18502/sjms.v17i2.11456 Page 234 Sudan Journal of Medical Sciences Tousief Irshad Ahmed and Ruqaya Aziz [31] Tsuda, A., Inaba, M., Ichii, M., et al. (2013). Relationship between serum TSH levels and intrarenal hemodynamic parameters in euthyroid subjects. European Journal of Endocrinology, vol. 169, no. 1, pp. 45–50. [32] Kim, D., Kim, W., Joo, S. K., et al. (2018). Subclinical hypothyroidism and low-normal thyroid function are associated with nonalcoholic steatohepatitis and fibrosis. Clinical Gastroenterology and Hepatology, vol. 16, no. 1, pp. 123–131. [33] Duong, N., Lee, A., and Lewis, J. (2018). Case of acute mixed liver injury due to hypothyroidism. BMJ Case Reports, vol. 2018, bcr2017222373. [34] Walsh, J. P. (2011). Setpoints and susceptibility: Do small differences in thyroid function really matter? Clinical Endocrinology, vol. 75, no. 2, pp. 158–159. DOI 10.18502/sjms.v17i2.11456 Page 235 Introduction Renal effects of thyroid hypofunction Hepatic effects of thyroid hypofunction Materials and Methods Study population Inclusion and exclusion criteria Data collection methodology Protocol and procedure Measurement of T3, T4, and TSH Measurement of biochemical parameters Quality control Statistical analysis Results Discussion Conclusion Acknowledgements Ethical Consideration Competing Interests Availability of Data and Material Funding References