| Purpose:This study uses targeted metabolomics technology to detect the terminal metabolites of steroid substances in the urine of national para-alpine skiing athletes,aiming to explore the effects of different training loads on cholesterol-related metabolism in the body and their impact on athletes’ blood lipids and serum vitamin D levels.This study provides new ideas for understanding sports training load and the status of athletes’ blood lipids and serum vitamin D levels from the perspective of steroid-related metabolism.Methods:This study focused on 16 national para-snowboard athletes.During their summer physical training period,blood and urine samples were collected three times: during the adaptation training phase,maximum strength phase and rapid strength phase.Biochemical indicators such as blood lipids,serum vitamin D,creatine kinase and urea nitrogen were detected in the blood samples,while steroid-related metabolites were detected in the urine samples using ultra-high performance liquid chromatography-mass spectrometry metabolomics technology.The enzyme-linked immunosorbent assay was used to measure the presence of 3-methylhistidine.Pairwise T-test analysis was used to analyze the inter-group differences in biochemical indicators,with P<0.05 indicating significant differences.Multivariate principal component analysis and univariate fold change analysis were used to screen for different metabolites in the urine,with FC ≥ 2 or FC ≤ 0.5 indicating significant differences.Spearman correlation analysis was used to explore the correlation between different metabolites in urine and biochemical indicators,with 0.3 ≤ |r| < 0.8and P<0.05 indicating a correlation,and |r| ≥ 0.8 and P<0.05 indicating a significant correlation.The screened different metabolites in the urine were imported into the KEGG database for metabolic pathway enrichment analysis and DA score calculation to determine the overall impact of the changes in different metabolites on the metabolic pathway they belong to.Results:(1)Differential results of biochemical indicators: significant decrease in triglycerides was observed at the maximum strength stage(P<0.05).Low-density lipoprotein cholesterol showed a significant decrease after all physical training(P<0.05).Urea nitrogen showed a significant increase after the rapid strength stage(P<0.05).3-methylhistidine showed a significant decrease after all physical training(P<0.05),while serum vitamin D showed a significant increase at the maximum strength stage(P<0.01)and after all physical training(P<0.05).(2)Urine differential metabolite screening results:a)During the maximal strength phase,13 types of urine differential metabolites were identified in male athletes and 12 types in female athletes.Differential metabolites that were significantly upregulated(FC ≥ 2)included 6metabolites in male athletes,such as 5α-dihydrotestosterone,dihydroprogesterone,11-deoxycorticosterone,estrone and 20α-hydroxycholesterol and 8 metabolites in female athletes,including dihydroprogesterone,17α-hydroxyprogesterone,testosterone,11-ketotestosterone and hydrocortisone.Differential metabolites that were significantly downregulated(FC ≤ 0.5)included 7 metabolites in male athletes,such as dehydroepiandrosterone,androgens/testosterone,corticosteroids(cortisol)and 17β-estradiol,and 4 metabolites in female athletes,including pregnenolone,androgens/testosterone,aldosterone,and17β-estradiol.b)During the rapid strength phase,9 urinary metabolites were identified as significantly different between male athletes and 8 urinary metabolites were identified as significantly different between female athletes.Metabolites upregulated with a fold change(FC)of ≥2 in male athletes included 5β-dihydrotestosterone,deoxycorticosterone,17β-estradiol,7α,27-dihydroxycholesterol,among others,while those upregulated in female athletes included pregnenolone,aldosterone,and deoxycorticosterone.Metabolites downregulated with a FC of ≤0.5 in male athletes included dehydroepiandrosterone,aldosterone,estrone,among others,while those downregulated in female athletes included 11-ketobetacholic acid,corticosterone,and dexamethasone.c)After screening all the physical training,13 different metabolites were found to be significantly upregulated(FC ≥ 2)in male athletes,including progesterone,5α-dihydrotestosterone,5β-dihydrotestosterone,deoxycorticosterone,11-dehydrocorticosterone,7a,27-dihydroxycholesterol,etc.In female athletes,14 different metabolites were found,including progesterone,testosterone,hydrocortisone/prednisolone,deoxycorticosterone,etc.Additionally,6 different metabolites were found to be significantly downregulated(FC ≤ 0.5)in male athletes,including dehydroepiandrosterone,androsterone,aldosterone,11-dehydrocorticosterone,7a,27-dihydroxycholesterol,etc.In female athletes,8different metabolites were found,including pregnenolone,11-ketotestosterone,testosterone,dihydrotestosterone,deoxycorticosterone,17β-estradiol,etc.(3)Relationship between differential metabolites in urine and differential biochemical indicators:a)In the maximum strength phase,a correlation was found between dihydroxyprogesterone and urea nitrogen,25-D3,and 5α-dihydrotestosterone in male athletes;estradiol and urea nitrogen,creatine kinase,and 25-D3;and testosterone/androstenedione and urea nitrogen,5α-dihydrotestosterone,and dehydroepiandrosterone in male athletes.A correlation was found between 25-D3 and dihydroxyprogesterone,pregnenolone,and testosterone/androstenedione in female athletes.b)In the rapid strength phase,a correlation was found between 5β-androstanedione and aldosterone,deoxycorticosterone,and 7α,27-dihydroxycholesterol in male athletes;and between 7α,27-dihydroxycholesterol and aldosterone,deoxycorticosterone in male athletes.A correlation was found between urea nitrogen and corticosteroids(cortisol),and deoxycortisone in female athletes;and between corticosteroids and 11-ketotestosterone,deoxycortisone in female athletes.c).After all physical training,a correlation was found between urea nitrogen and 25-D3,3MH,and testosterone/androstenedione in male athletes;between 25-D3 and 3MH,LDL-C,testosterone/androstenedione,5β-androstanedione,deoxycorticosterone,corticosteroids(cortisol)and 7α,27-dihydroxycholesterol in male athletes;and between 7α,27-dihydroxycholesterol and HDL,TC,testosterone/androstenedione,3MH,5β-androstanedione,deoxycorticosterone,and 11-dehydrocorticosterone in male athletes.A correlation was found between testosterone/androstenedione and deoxycorticosterone,corticosteroids(cortisol),2-methoxyestrone,and LDL-C;and between 5β-androstanedione and deoxycorticosterone,11-dehydrocorticosterone,and LDL-C in male athletes.A correlation was found between 3MH and 25-D3,and testosterone in female athletes;between dihydroxyprogesterone and 5β-pregnane-3α,20α-diol,and testosterone in female athletes;and between 11-ketotestosterone and estradiol,pregnenolone and testosterone/androstenedione in female athletes.(4)Enrichment analysis of KEGG metabolic pathways based on differential metabolites and DA scores:The significant metabolic pathways affected by differential metabolites in the athletes’ bodies during the maximum strength phase were the steroid hormone biosynthesis pathway(male athletes: DA Score=0.00,P=0.37;female athletes:DA Score=0.05,P=0.17).In addition,the significant metabolic pathway affected by differential metabolites in female athletes was the hypothalamic-pituitarygonadal axis pathway(DA Score=-0.22,P=0.47).During the rapid strength phase,the significant metabolic pathways affected by differential metabolites in male athletes were the primary bile acid synthesis pathway(DA Score=0.00,P=0.48)and the prolactin signaling pathway(DA Score=0.00,P=0.34).The significant metabolic pathway affected by differential metabolites in female athletes was the aldosterone synthesis and secretion pathway(DA Score=0.33,P=0.04).Throughout the entire period of physical training,the significant metabolic pathways affected by differential metabolites in male and female athletes were the steroid hormone biosynthesis pathway(male athletes: DA Score=0.05,P=0.21;female athletes: DA Score=-0.11,P=0.12).In addition,the significant metabolic pathway affected by differential metabolites in female athletes was the hypothalamic-pituitary-gonadal axis pathway(DA Score=0.00,P=0.20).Conclusions:(1)Summer physical training has a significant impact on athletes’ blood lipids,serum vitamin D,and urine metabolites,with the most significant effect on serum vitamin D during the maximal strength phase.(2)Summer physical training has a significant impact on the body’s synthesis and metabolism pathway of steroids in male and female athletes,and the secretion pathway of hypothalamic-pituitary-gonadal hormones in female athletes,with significant effects on the primary bile acid synthesis pathway and prolactin signaling pathway in male athletes during the fast strength phase,and the aldosterone synthesis and secretion pathway in female athletes.(3)After summer physical training,there is a correlation between different metabolites in male athletes’ urine and low-density lipoprotein,serum vitamin D levels,and 3-methylhistidine,and a correlation between different metabolites in female athletes’ urine and 3-methylhistidine. |
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