UPLC-Q-TOF-MS Based Metabolomic Analysis On Mechanism Of Treating Hypothyroidism With Kidney Yang Warming Therapy

ObjectiveIn this study,ultra-performance liquid chromatography to quadrupole time-of-flight mass spectrometry(UPLC-Q-TOF-MS)was used for a non-targeted metabolomic analysis on volunteers with normal thyroid hormones level and hypothyroidism patients.By comparing the metabolic profiles of serum samples between healthy volunteers and hypothyroidism patients,as well as analyzing the metabolic differences in patients before and after treated with Yougui pill decoction for 6 weeks,potential biomarkers related to hypothyroidism were screened and identified.Combined with KEGG pathway enrichment analysis,involved metabolic pathways were demonstrated and material basis was established for a potential way to diagnosing hypothyroidism and the mechanism of treating hypothyroidism with kidney-yang warming therapy.MethodsSerum samples were collecting from 10 healthy volunteers and 10 hypothyroidism patients with kidney-yang deficiency pattern before and after treating with Yougui pill decoction for 6 weeks.Metabolomic analysis was deducted by using the method of UPLC-Q-TOF-MS.After preprocessing the raw data with XCMS program,pattern recognition,multidimensional statistical analysis and univariate statistical analysis were performed with SIMCA-P 14.1 software.The metabolites obtained by multidimensional statistical analysis of VIP>1 and univariate statistical analysis of P<0.05 could be considered as metabolites with significant differences,while VIP>1 and 0.05<P<0.1 were considered as differential metabolites.By comparing the different metabolites between healthy volunteers and hypothyroidism patients,as well as analyzing the metabolic differences in patients before and after treated with Yougui pill decoction,the metabolic spectrum differences were described and the target biomarkers were recognized.Moreover,with the application of KEGG pathway enrichment analysis,involved metabolic pathways of those biomarkers were demonstrated.ResultsCompared with the healthy volunteers,the concentrations of D-Proline,Acetylcarnitine,Phe-Phe,L-Carnitine,Adenosine,Choline,n-Propyl cinnamate,D-Mannose,Palmitic acid,Dimethylformamide,1-Aminocyclopropanecarboxylic acid,L-Pipecolic acid,Tyramine,Nl-Methyl-2-pyridone-5-carboxamide,Sarcosine,Betaine and L-Argininecholine of serum in hypothyroidism patients before treatment changed greatly.It was also found that metabolic pathways such as protein digestion and absorption,ABC transporters,aminoacyl-tRNA biosynthesis,central carbon metabolism in cancer and mTOR signaling pathway were significantly reprogrammed,suggesting a disordered metabolic regulation network was formed in hypothyroidism patients.The serum metabolic profiles had different characteristics in hypothyroidism patient group before and after treated with Yougui pill decoction for 6 weeks.20 metabolites with significant change including 2-Ethoxyethanol,2-Hydroxyphenylacetic acid,L-Glutamine,Glycerophosphocholine,NG,NG-dimethyl-L-arginine(ADMA),D-Proline,Acetylcarnitine,Ethyl 3-hydroxybutyrate,DL-Indole-3-lactic acid,1-O-Octadecyl-sn-glyceryl-3-phosphorylcholine,L-Tryptophan,Tyramine,L-Phenylalanine,Triethanolamine,L-Arginine,L-Pyroglutamic acid,Creatinine,n-Propyl cinnamate,β-Homoproline,Sarcosine could be detected.All these metabolites are related to metabolic pathways such as protein digestion and absorption,aminoacyl-tRNA biosynthesis,central carbon metabolism in cancer,ABC transporters and mineral absorption,implying that adjusting these metabolic pathway as treating targets could become a new therapy in hypothyroidism patients with kidney-yang pattern.ConclusionIn this research,UPLC-Q-TOF-MS was used to perform non-targeted metabolomic analysis on serum samples from hypothyroidism patients before and after treated with Yougui pill decoction and healthy volunteers.By comparing and analyzing serum samples of hypothyroidism group and normal group,17 metabolites related to abnormal metabolism of hypothyroidism were found,revealing the abnormalities of amino acid metabolism,energy metabolism,carbohydrate metabolism and lipid metabolism could lead to the onset and development of hypothyroidism.Hopefully,these metabolites could be regarded as biomarkers in preliminary screening diagnosis of hypothyroidism.20 significantly changed metabolites were detected between hypothyroidism group before and after treatment,indicating the application of kidney-yang warming therapy could adjust amino acid metabolism,energy metabolism,carbohydrate metabolism,lipid metabolism and mineral metabolism in patients with hypothyroidism.Combined with KEGG Pathway enrichment analysis,metabolic pathways like protein digestion and absorption,aminoacyl-tRNA biosynthesis,central carbon metabolism in cancer,ABC transporters and mineral absorption were involved in the process of treating hypothyroidism with kidney-yang warming therapy,revealing the possible mechanism of kidney-yang warming therapy on hypothyroidism.