Serum metabolomic analysis reveals key metabolites in drug treatment of central precocious puberty in female children

Abstract

Precocious puberty (PP) is a common condition among children. According to the pathogenesis and clinical manifestations, PP can be divided into central precocious puberty (CPP, gonadotropin dependent), peripheral precocious puberty (PPP, gonadotropin independent), and incomplete precocious puberty (IPP). Identification of the variations in key metabolites involved in CPP and their underlying biological mechanisms has increased the understanding of the pathological processes of this condition. However, little is known about the role of metabolite variations in the drug treatment of CPP. Moreover, it remains unclear whether the understanding of the crucial metabolites and pathways can help predict disease progression after pharmacological therapy of CPP. In this study, systematic metabolomic analysis was used to examine three groups, namely, healthy control (group N, 30 healthy female children), CPP (group S, 31 female children with CPP), and treatment (group R, 29 female children) groups. A total of 14 pathways (the top two pathways were aminoacyl-tRNA biosynthesis and phenylalanine, tyrosine, and tryptophan biosynthesis) were significantly enriched in children with CPP. In addition, two short peptides (His-Arg-Lys-Glu and Lys-Met-His) were found to play a significant role in CPP. Various metabolites associated with different pathways including amino acids, PE [19:1(9Z)0:0], tumonoic acid I, palmitic amide, and linoleic acid-biotin were investigated in the serum of children in all groups. A total of 45 metabolites were found to interact with a chemical drug [a gonadotropin-releasing hormone (GnRH) analog] and a traditional Chinese medicinal formula (DBYW). This study helps to understand metabolic variations in CPP after drug therapy, and further investigation may help develop individualized treatment approaches for CPP in clinical practice.

Keywords: central precocious puberty; drug treatment; key metabolites; metabolomics; serum of female children.

FIGURE 1

(A) Correlation analysis of the identified metabolites. Positive correlations are indicated in red, whereas negative correlations are indicated in blue. (B) Network analysis of the identified metabolites. Positive correlations are indicated in red, whereas negative correlations are indicated in blue (Spearman’s correlation coefficient threshold = 0.4).

FIGURE 2

Metabolic profiling analysis of the serum of female children with central precocious puberty. (A) Score plot generated using the 3D-PCA model. (B) Score plot generated using the 3D-PLS-DA model (Supplementary Figure 1A). OPLS-DA between the N and S groups (Supplementary Figure 1B). OPLS-DA between the R and S groups (Supplementary Figure 1C). OPLS-DA between the R and N groups (Supplementary Figure 1D). Qualitative analysis of the OPLS-DA model (N vs. S) via response permutation testing (RPT) (Supplementary Figure 1E). Qualitative analysis of the OPLS-DA model (R vs. S) via RPT (Supplementary Figure 1F). Qualitative analysis of the OPLS-DA model (R vs. N) via RPT. N, healthy female children (n = 30); S, female children with central precocious puberty (n = 31); R, female children treated with drugs used in this study (n = 29).

FIGURE 3

Volcano plot demonstrating differentially expressed metabolites. A represents metabolites that are downregulated, not significant, or upregulated in different groups of female children. The abscissa is log₂(FC), the left ordinate is −log₁₀(p−value),and the right ordinate is VIP (variable importance in projection). Volcano plot demonstrating differentially expressed metabolites between the S and N groups (Supplementary Figure 2A). Volcano plot demonstrating differentially expressed metabolites between the R and S groups (Supplementary Figure 2B). Volcano plot demonstrating differentially expressed metabolites between the R and N groups. N, healthy female children (n = 30); S, female children with central precocious puberty (n = 31); R, female children treated with drugs used in this study (n = 29).

FIGURE 4

Pathway enrichment analysis. (A) Bar charts of enriched pathways among the R, S, and N groups. The abscissa is the pathway, and the ordinate is −log₁₀(p−value). The blue line indicates a p-value threshold of 0.05, and the red line indicates a p-value threshold of 0.01. (B) Bubble chart of enriched pathways among the R, S, and N groups. The abscissa is the rich factor (the detailed description is provided in section “3.4. KEGG pathway enrichment analysis” of results), and the left ordinate is the pathway.

FIGURE 5

Short peptides in box and-whisker plots. N, healthy female children (n = 30); S, female children with central precocious puberty (n = 31); R, female children treated with drugs used in this study (n = 29). Plot are expressed as the 5th–95th percentiles (*p < 0.05; **p < 0.01; ***p < 0.001; two-tailed Mann–Whitney U-test).

FIGURE 6

Amino acid pathways and lipids in box-and-whisker plots. N, healthy female children (n = 30); S, female children with central precocious puberty (n = 31); R, female children treated with drugs used in this study (n = 29). Plot are expressed as the 5th–95th percentiles (*p < 0.05; **p < 0.01; ***p < 0.001; two-tailed Mann–Whitney U-test).

FIGURE 7

Acid metabolites in box-and-whisker plots. N, healthy female children (n = 30); S, female children with central precocious puberty (n = 31); R, female children treated with drugs used in this study (n = 29). Plot are expressed as the 5th–95th percentiles (*p < 0.05; **p < 0.01; ***p < 0.001; two-tailed Mann–Whitney U-test).

FIGURE 8

Arachidonic acid and other metabolites in box-and-whisker plots. N, healthy female children (n = 30); S, female children with central precocious puberty (n = 31); R, female children treated with drugs used in this study (n = 29). Plots are expressed as the 5th–95th percentiles (*p < 0.05; **p < 0.01; ***p < 0.001; two-tailed Mann–Whitney U-test).

FIGURE 9

Serum levels and ROC analysis of altered metabolites. Healthy female children, n = 30; female children with central precocious puberty (CPP), n = 31.