Thyroid cancer (TC) is responsible for over 1% of neoplasms diagnosed every year in the general population. In Europe, there are approximately 3,500 new cases each year (1). Females are involved 3–5 times more often than men. The incidence of TC has increased rapidly in the last several years. In terms of short-term prognosis, it may become the second most common malignant neoplasm in women. Papillary thyroid cancer (PTC) accounts for more than 90% of all thyroid neoplasms (2) with a global incidence of 586,000 cases (3). Although PTC is related to an indolent disease course and has a favorable prognosis, it is a major challenge to stratify patients by risk of mortality or recurrence. Currently, clinicopathologic features associated with an unfavorable prognosis include older age, large tumor size, extrathyroidal extension (ETE), lymph node metastasis (LNM) and distant metastasis. Individuals with those features require more aggressive treatment (4, 5). On the other hand, low-intensity treatment or even active surveillance may be sufficient for patients who do not have these risk factors. Although some recent studies have indicated that Hashimoto’s thyroiditis (HT) (6) may be a tumor-promoting factor, HT-related issues have barely been mentioned in current TC treatment guidelines. The link between chronic inflammation and cancer is well described (7); however, it is generally associated with indolent potential in the GI tract, liver, and skin (8). There are several theories to explain the potential relationship; for example, misbehaved follicular epithelial regeneration following chronic inflammatory damage (9) or enhanced TSH stimulus together with additional inflammatory cytokines act as potential activators of aberrant cell proliferation (10). However, the exact molecular pathomechanism remains unclear. Autoimmune thyroid diseases, including Hashimoto’s thyroiditis (HT), are T-cell‐mediated organ‐specific autoimmune diseases, and the annual incidence of Hashimoto thyroiditis worldwide is estimated to be 0.3-1.5 cases per 1000 persons (11). HT, affects women 7-10 times more often than men (12). Prevalence increases with age, especially in patients diagnosed with other autoimmune conditions. There are various signs and symptoms of HT mainly due to hypothyroidism, including cool and dry skin, coarse hair, loss of body hair, and hyperlipidaemia (13). Chronic HT-induced inflammation may be associated with an increased risk of thyroid cancer. A recent meta-analysis reported that the rate of HT in PTC patients ranged between 4.75 and 38.4%, whereas the rate of PTC in HT patients ranged between 0.12 and 64.3% (14). The immune responses against PTC and HT are different. In PTC, the immune system is more silent and allows tumor progression, while in HT, the reaction is aggressive, destroying the proper functioning of the gland. According to some authors, HT is associated with a better prognosis due to an enhanced immune response and better control of tumor progression (15). However, the role of HT in PTC seems ambiguous and should be elucidated. It has been proven that HT plays a role in protein metabolism (16). As the basic building blocks of peptides and proteins, amino acids have a variety of physiological functions. Serum levels of polyamine metabolites were found to differ between patients with autoimmune thyroid disease and healthy controls (17). Hypothyroid status is related to lower alanine, aspartate, and glutamate concentrations. It is mainly caused by decreased whole-body proteolysis and maldigestion (18). Amino acids are the primary units of proteins and are involved in multiple physiological and pathophysiological processes (19). According to many publications, amino acids (AAs) may play a critical role in cancer cell metabolism. In contrast to hypothyroidism status, it is known that in cancer, especially in the early stages, the amino acid turnover rate is increased because of hypermetabolism. Glutamine, as a nitrogen and carbon source, is involved in the metabolic reprogramming in cancer and plays a pivotal role in the growth and proliferation of cancer cells (18, 20–22). Most thyroid cancer studies have presented increased concentrations of glutamine and glutamate in tumor samples (23–25), as well as in serum (25–27). High glutamine uptake is related with upregulated glutaminase in several tumor models (28–30). Glutaminase initiates glutaminolysis by converting glutamine to glutamate. This pathway is involved in the maintenance of the TCA cycle and synthesis of non-essential amino acids, nucleotides and fatty acids (11) as well as in cell signaling (29, 30). Also, alteration of arginine metabolism is characteristic for cancer metabolism. It is necessary for growth of cancer cells, but paradoxically, arginine is important for immune surveillance (31). Alanine also is desired amino acid in most of the perturbed pathways (25). Glycine and serine provide crucial substrates for the synthesis of nucleic acids, proteins and lipids, which are essential for cancer cell growth (32). High levels of glycine have been observed in cancer thyroid tissues (24, 33) and in malignant nodules (34) compared to samples from healthy subjects. Also, other amino acid are necessary for maintenance of cellular redox homoeostasis (35). Some derivatives produced from AAs may support cancer growth, but tryptophan induces immunosuppression by weakening the ability of dendritic cells and T cells to target and eliminate cancer cells (36). Our aim was the examination of AA profile disorders in PTC1 and PTC0 and comparison of changes in AA concentrations in these 2 pathologies.

Common biochemical parameters obtained from whole blood are presented in Table 1 . PTC1 patents had elevated C-reactive protein compared with HC and PTC0 patients (although the values were within the reference range). PTC0 patients had significantly elevated concentrations of total cholesterol compared with HCs. Among the other parameters, significant differences were not observed. PTC0 patients differed statistically from PTC1 patients in thyroid peroxidase and thyroglobulin antibody levels.

The standard diagnostic tools for PTC are ultrasound and fine needle aspiration biopsies (6). In turn, HT identification is based on clinical symptoms of hypothyroidism, the presence of TPOAbs, and ultrasound features, although seronegative HT can be observed in more than 10% of cases. In such cases, diagnosis is made based on final histopathology. Additional diagnostic tools that may help to identify PTC and distinguish Hashimoto concomitant with PTC may have crucial clinical implications. Indeed, recent guidelines allow less aggressive treatment for PTC in some circumstances, which might significantly reduce postoperative complications (40). However, there is no gold standard that would allow us to distinguish between Hashimoto’s and cancer, and this distinction is of great importance in further management/treatment. In the development of cancer, AA metabolism is reprogrammed. Additionally, HT affects patient catabolism, and preliminary research suggests that increased serum TSH concentration and autoimmune thyroid inflammation are involved in thyroid tumor growth (18). Therefore, is it possible to find the difference between these two diseases based on the amino acid profiles?

To the best of our knowledge, this is the first study to determine AA profiles in serum samples from PTC0 patients and PTC1 patients compared to those of healthy controls.

Lysine was one of the AAs that was elevated in PTC1 compared to PTC0. Lysine affects the production of proteins in muscles and bones, and lysine deficiency causes chronic fatigue, irritability, hair loss, anemia, susceptibility to infection, recurrent herpes and metabolic disorders. Jiang et al. (41) studied the serum of HT patients and showed that lysine degradation pathways had an impact on different clinical stages of HT (41). Additionally, lysine was increased in the serum of HT and Graves’ disease patients (17). In our study, lysine was increased only in the PTC1 group. According to the referenced authors, alterations in lysine degradation affect the occurrence of HT (42).

Other AAs increased in the serum of PTC1 patients compared to HCs were leucine and arginine ( Table S1 ). Interestingly, in PTC0 patients, we also observed a strong increase in arginine concentration ( Table S1 ). Serum concentrations of arginine were altered in both PTC0 and PTC1 patients, which was also indicated by the MetPA analysis pathway “arginine biosynthesis” ( Figures 4 , 5 ). A positive correlation of arginine with fT4 was observed in PTC0 serum ( Table 2 ). Interestingly, Ittermann et al. (43) found that in patients with hyperthyroidism, serum concentrations of arginine and its metabolites, including asymmetric and symmetric dimethylarginines and homoarginine, were associated with serum TSH, fT3, and fT4 concentrations. Treatment of hyperthyroidism by antithyroid drugs increased arginine levels (44). In turn, Gluvic et al. (44) described many cases of increased NO bioavailability by levothyroxine therapy. Thyroid hormones stimulate L-arginine uptake by endothelial cells by upregulating L-Arg transporters (45), and arginine is a major regulator of mitochondrial activities in cancer metabolism (31). Supplementation with arginine rewires T-cell metabolism from glycolysis to oxidative phosphorylation and promotes its survival and antitumor ability (31). Lu et al. (46), in H¹ NMR analysis of plasma from papillary thyroid microcarcinoma patients, reported reduced levels of valine, lysine and leucine compared with healthy groups. In a study by Jiang et al. (41), valine, leucine, and isoleucine degradation and valine, leucine, and isoleucine biosynthesis differentiated euthyroid HT patients from HT patients with subclinical hypothyroidism. However, it should be stressed that Hashimoto disease may influence the metabolism of many other tissues, and this may affect serum AA concentrations. Indeed, we observed elevated concentrations of leucine in serum from HT patients. According to Krishnamurthy et al. (47), arginine, valine, and leucine are important in immunological responses, including the synthesis of various antibodies and the activation of T cells and macrophages. It appears that the deficiency of any essential AAs, including valine, impairs T4 production and leads to primary hypothyroidism (48). Thyroid hormones have a catabolic effect on protein metabolism. In most catabolic states, uptake of branched-chain amino acids from body proteins is reduced; therefore, the increase in their concentrations does not depend on the increase in their content in the diet but results from both their reduced peripheral metabolism and increased release from fat-free tissues (49). Additionally, in our study, the concentration of valine was elevated in both PTC groups; however, much higher differences in valine levels were observed in PTC1 patients. Plasma branched-chain amino acids are decreased in short-term profound hypothyroidism and increase in response to thyroid hormone supplementation (16). Therefore, thyroid hormone supplementation can be a reason for the higher serum concentrations of BCAAs in PTC1 patients ( Table S1 ). The next amino acid, glycine is a highly desirable compound for cancer cells (24, 33)., therefore, reduced levels of glycine in the serum of PTC patients ( Table S1 ) could be the reason for glycine participation in cancer pathogenesis (21). We observed an inverse correlation of glycine with fT4 in PTC0 serum ( Table 2 ). Glycine supplementation improves the conversion of fT4 to fT3, which contributes to the proper functioning of the thyroid gland. Mannisto et al. reported that intraperitoneal administration of glycine inhibited TSH secretion in rats (50).

In turn, glutamate and aspartate, which are excitatory amino acids, act by increasing the concentrations of TSH, fT3 and fT4 in rat serum (51). Indeed, in our study, glutamate and aspartate were positively correlated with TSH in the serum of PTC1 patients. Moreover, significantly higher concentrations of glutamate and aspartic acid were detected in the serum of PTC0 and PTC1 patients in comparison to healthy controls ( Table S1 ). Aizawa et al. (52) studied the effects of glutamic acid and glutamine on TSHβ expression in pars tuberalis (PT) slice cultures from rat brains. After 2- and 4-h treatments, glutamic acid and glutamine significantly stimulated TSHβ expression in PT slices, and the impact of glutamic acid was stronger than that of glutamine (52). TSH was also positively correlated with aspartic acid, although the correlation was weak (0.356, p<0.05) ( Table 3 ). The enzymes involved in glutaminolysis were overexpressed in thyroid cancer tissue (20, 21, 53) and promoted the transformation of glutamine to glutamate to sustain the TCA cycle and anabolic processes (27). Therefore, excess products of glutaminolysis, such as aspartic and glutamic acid, can be removed into the serum of PTC patients. Furthermore, aspartic and glutamic acid are substrates for nucleotide biosynthesis, and increased amounts could replenish the levels of the metabolites of the TCA cycle that may be decreased as a result of aerobic glycolysis (Warburg effect) (25). According to Cheng et al. (54), the increased glutamate concentrations in PTC patients are a result of increased glutamine metabolism in tumour cells. Moreover, the existing association of thyroid autoimmunity with PTC may be involved in increased serum glutamine concentrations (18). One of the amino acids with a reduced concentration in serum samples in both the PTC0 and PTC1 groups compared to HCs was alanine. Additionally, Qing Huang et al. (25) found reduced concentrations of alanine in the serum of PTC patients. A similar reason is indicated by Wojtowicz et al. (26). Decreased alanine might be evidence of its fast utilization from circulating blood as an answer for energy demands (26). In our study, alanine showed a decreasing trend in the serum of PTC1 patients compared to PTC0 patients. Additionally, we observed a strong negative correlation of alanine with TPO-Abs in the serum of PTC1 patients. Thyroid hormones control a multitude of homeostatic functions, including protein proteolysis (16). In Hashimoto, the antibody titre is significantly elevated, so this may be another factor lowering the concentration of alanine in patients with HT. When comparing different studies, the differences in the abundance of these amino acids between healthy subjects and patients with benign or malignant thyroid lesions do not always match. Certainly, observed results need further investigations. One of this reason might be the use of different sampling methods, techniques and study groups. However, there is agreement that plasma/serum levels of tyrosine, a precursor of thyroid hormones, are lower in PTC patients than in controls (26, 46, 55–57). The T3 hormone, triiodothyronine, constitutes only 10% of the total thyroid hormones, although it is considered responsible for most of the thyroid’s activities and is 3-4 times stronger than the T4 hormone. Tyrosine is necessary for synthesis of thyroxine, which is produced by the thyroid gland (47). Reduced values of fT4 and elevated values of fT3 accompany Hashimoto (18). Deficiency in tyrosine, as well as phenylalanine, results in altered levels of thyroid hormones (47). Tyrosine is considered a nonessential amino acid because it can be synthesized from phenylalanine; nonetheless, it has an important role in the production of proteins that are a part of signal transduction processes, acting as a receiver of phosphate groups transferred through tyrosine kinases. In turn, these enzymes have been associated with the regulation of cellular proliferation, survival, differentiation, function and motility, linking them to a cancer phenotype Tahara et al. (48) demonstrated the effects of amino acid deficiency on serum levels of T4, T3, fT4, and reverse T3; they reported that reduction of phenylalanine and tyrosine drastically affected the serum levels of thyroid hormones. We observed a positive correlation between tyrosine and fT3 and fT4 in the serum of PTC1 patients. Interestingly, in HT and other autoimmune thyroid diseases, other studies (17, 18) did not observe the phenomenon of decreased levels of tyrosine in serum patients. Jiang et al. (41) suggested that lysine degradation and tyrosine metabolism played an important role in the HTS group compared to the control group. However, this was not supported by measured tyrosine concentrations, only enrichment analysis (41). This may be due to the effects of the HT drugs.

The data presented in the study are deposited in the MetaboLights repository, accession number MTBLS8012; https://www.ebi.ac.uk/metabolights/editor/study/MTBLS8012.

The studies involving human participants were reviewed and approved by Independent Bioethics Committee for Scientific Research at the Medical University of Gdansk under number NKBBN/62/2021. Written informed consent to participate in this study was provided by the participants’ legal guardian/next of kin.

AH and AM, designed the study, analyzed and interpreted results, and wrote the manuscript. AH, and AT, contributed to sample and clinical data collection. JT, conducted the amino acids analysis. JK conducted the analysis of mRNA levels by real-time PCR. AZ and AM, performed the statistical analysis. All authors reviewed and edited the manuscript.