Graves’ disease (GD) is the most common cause of hyperthyroidism (HT) with an autoimmune origin in children and adults (1–3). The incidence of GD in children is about 0.9-14.1/100,000, peaking in adolescent females (4–6). A trend of surging incidence of juvenile thyrotoxicosis was observed worldwide, with two to three times higher incidence in the Chinese pediatric population compared to the Caucasians (4–6).

It is well known that thyroid hormone regulates metabolic processes essential for normal growth and development in children (7). With excessive thyroid hormone, HT facilitates the metabolism process via elevated resting energy expenditure, weight loss, upregulated lipolysis, and gluconeogenesis, as well as decreased cholesterol levels (8).

Metabolomics is the study of specific small molecule metabolites or their profiles. Untargeted metabolomics provides a global fingerprint of information through the simultaneous identification of as many metabolites as possible within a tissue, biological fluid, or even cell sample (9, 10). Different instrumental platforms, including nuclear magnetic resonance (NMR) spectroscopy, gas chromatography (GC), and liquid chromatography (LC) coupled with mass spectrometry (MS), are used to cover different features of the human metabolome (9, 10). Metabolomics has been used to explore new biomarkers of disease risk in large-scale studies. In smaller studies, metabolomics has been designed to investigate the underlying mechanisms and progression of certain diseases, or to reveal the potential roles of dietary and environmental exposures, as well as gut flora activity in chronic diseases (11, 12). Multiple studies on metabolomic changes in GD adult patients have been reported, which showed that acylcarnitine and polyamine profiles were different between GD patients and healthy controls (13, 14). Metabolic pathways, such as arginine and proline metabolism and aminoacyl-transfer ribonucleic acid (tRNA) biosynthesis have also been altered in GD patients (15). However, the metabolomic alterations of GD in pediatric population have not been fully explored.

In the present study, we used an ultra-high-performance liquid chromatography coupled with the quadruple time-of-fight mass spectrometry (UHPLC-QTOF/MS)-based untargeted metabolomics approach to explore the perturbation of metabolic process in GD children compared to age- and gender- matched healthy normal controls. We aim to extend the current knowledge beyond previously reported targeted metabolite changes by examining the global serum metabolomics profiles of GD, and propose new dietary suggestions which may improve the treatment of GD.

The present study showed that serum metabolic patterns were significantly different between GD children and healthy controls. A total of 730 metabolites were identified in all participants, among which 48 differential metabolites between GD and control groups were filtered out. To our knowledge, this is the first study analyzing the metabolic changes in GD children via an untargeted metabolomic method.

Amino acids are organic compounds that contain amine (-NH2) and carboxyl (-COOH) functional groups, along with a side chain (R group) specific to each amino acid (19). Amino acids are the monomers that make up human proteins and the second largest component of human muscle and other tissues. Besides, amino acids also actively participate in numerous biological processes, including biosynthesis and neurotransmitter transport (19). In the present study, we found that GD children had more active protein digestion/absorption and nitrogen metabolism. Quite a few amino acids, mostly essential amino acids, had much higher levels in GD children compared with controls. Interestingly, the amino acid pattern changes in GD children are quite different from those of adult patients. For example, significant changes of arginine and proline metabolism pathways were observed in GD adult patients (20), but not the children. This may be explained by the different amino acid metabolism among various age groups (20). Additionally, significantly elevated levels of phenylalanine and tyrosine were observed in GD children, but not in GD adults (20). Most phenylalanine is converted to tyrosine, a key component of thyroxine, through phenylalanine hydroxylase (21). Thus, reduction of phenylalanine intake in GD children, but not adults, may be a potential way to decrease their thyroxine production.

Tryptophan (Trp) is an essential amino acid that serves several important purposes and a precursor of the neurotransmitter serotonin. The tri-iodothyronine (T3) and thyroxine (T4) entry into different cell types depends on the aromatic amino acid transport system T, and there is a counter transportation between T3 and Trp. Thus, Trp supplementation may reduce the T3 uptake of cells (22, 23). Normal Trp metabolism has two main branches: 3-10% of Trp keeps the indole ring intact while producing chemical messengers such as serotonin, while the majority (90%) breaks the indole ring generating the kynurenine path, kynurenine, nicotinic acid, and the nicotinamide adenine dinucleotide (NAD⁺) (24). Previous studies have reported that thyroxine elevated the conversion ratio of Trp to nicotinamide, not through the kynurenine pathway but via aminocarboxymuconate semialdehyde decarboxylase (25). Our results were consistent with previous studies since no elevation of substrates in the kynurenine pathway was observed. Moreover, elevated 5-hydroy-indoleacetate were detected in the serum of GD children, indicating an intensified conversion of Trp to serotonin. Similar findings have been reported in the brain of adult GD patients, and the elevation of serotonin may contribute to the mood change of GD patients (26). Besides, we found that Trp levels in GD children were negatively correlated with TGAb levels, which may suggest a regulation role of Trp in the autoimmune process of GD. Indeed, previous studies have points to Trp degradation as a potent immunosuppressive mechanism involved in the maintenance of immunological tolerance. Therefore, Trp metabolism is quite important in the pathogenesis of GD, further studies are needed to explore the impact of supplementation and/or deprivation of Trp on GD pathogenesis.

Sphingosine 1-phosphate (S1P), a sphingolipid mediator, regulates various cellular functions via high-affinity G protein-coupled receptors, S1P1-5, and plays an important regulatory role in congenital and adaptive immune responses (27). Sphk1/S1P/S1PR signaling pathway can be used as a target for the treatment of autoimmune diseases (28). For example, the immunosuppressant Fingolimod (FTY720), a sphingosine analogue, is used as an S1PR antagonist in the treatment of multiple sclerosis (29). In addition, S1P lyase inhibitors can alleviate joint inflammation and destruction in rheumatoid arthritis mice (30). In recent years, Cheng Han et al. found that Sphk1/S1P/S1PR1 signal transduction is involved in the development of mice autoimmune thyroiditis (AIT). In AIT mice, the proportions of inflammation-related cell subtypes (such as Th1, Th17 and Tfh) are elevated, while FTY720 administration can decrease the their levels, suggesting that suppression of Sphk1/S1P/S1PR1 signaling pathway may be a potential therapeutic target of AIT (31). In this study, we found that sphinganine decreased, while S1P levels increased in the GD group. Besides, S1P levels positively correlate with TT4 levels. Thus, we suppose that FTY720 is also promising in the treatment of GD.

We detected much higher levels of pregnenolone sulfate in GD patients, which is consistent with previous studies (32). Pregnenolone sulfate is the source of steroid synthesis pathway. More importantly, pregnenolone sulfate regulates the release of multiple neurotransmitters and is crucial to multiple brain functions (33). The elevated pregnenolone sulfate levels may exert important effects on the neurodevelopment of GD children, but further studies are still needed to elucidate the consequences.

Protoporphyrin IX levels were significantly lower in GD children compared to those of controls. This can be easily explained by the fact that protoporphyrin IX is a crucial constituent of thyroid peroxidase (TPO), and more protoporphyrin IX is transported into the thyroid gland in GD patients. Interestingly, exacerbation of erythropoietic protoporphyria and acute intermittent porphyria has been reported in a few patients with HT patients (34, 35). Since these situations are quite rare and a decrease of protoporphyrin IX is observed in otherwise normal GD children, the correlation of HT and porphyria needs further investigation.

Our study has several limitations. Firstly, the sample size was relatively small, and further studies with larger sample size are needed to confirm our findings. Secondly, the main findings need to be validated by a second method. Thirdly, because this study included only local Chinese children, our results may not apply to other populations.

In conclusion, GD children have highly different serum metabolomic patterns compared to healthy controls, which may be induced either by HT or by autoimmunity. Our study is the first study addressing metabolic changes in GD children via untargeted metabolomic analysis. More importantly, our study provides comprehensive insights into the changes of metabolic processes, which is beneficial in improving the understanding and treatment of GD children.

The original contributions presented in the study are included in the article/ Supplementary Material . Further inquiries can be directed to the corresponding author.

The studies involving human participants were reviewed and approved by Ethics Committee of Children’s Hospital affiliated to Suzhou University. Written informed consent to participate in this study was provided by the participants’ legal guardian/next of kin.

Conceived and designed the experiments: TC; Performed the experiments: QX, WQ, LC, XC, RX, DZ, HW, HS, FW, JL; Analyzed the data: QX, WQ and TC; Wrote the paper: TC, QX, and WQ. All authors contributed to the article and approved the submitted version.

This study was supported by Suzhou Personnel Planning Project (project code GSWS2019051 and GSWS2020046), and Suzhou Science and Technology Development Project (SS22064) awarded to TC; a National Natural Science Foundation of China (project code 31701251) awarded to JL. This study was also supported by the Department of Pediatrics Clinical Center of Suzhou (Szzx201504).

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.