Alterations in gut microbiota and urine metabolomics in infants with yin-deficiency constitution aged 0-2 years

Abstract

Background: Based on the constitution theroy, infants are classified into balanced constitution (BC) and unbalanced constitution. Yin-deficiency constitution (YINDC) is a common type of unbalanced constitutions in Chinese infants. An infant's gut microbiota directly affects the child's health and has long-term effects on the maturation of the immune and endocrine systems throughout life. However, the gut microbiota of infants with YINDC remains unknown. Herein, we aimed to evaluate the intestinal flora profiles and urinary metabolites in infant with YINDC, find biomarkers to identify YINDC, and promote our understanding of infant constitution classification.

Methods: Constitutional Medicine Questionnaires were used to assess the infants' constitution types. 47 infants with 21 cases of YINDC and 26 cases of BC were included, and a cross-sectional sampling of stool and urine was conducted. Fecal microbiota was characterized using 16S rRNA sequencing, and urinary metabolomics was profiled using UPLC-Q-TOF/MS method. YINDC markers with high accuracy were identified using receiver operating characteristic (ROC) analysis.

Results: The diversity and composition of intestinal flora and urinary metabolites differed significantly between the YINDC and BC groups. A total of 13 obviously different genera and 55 altered metabolites were identified. Stool microbiome shifts were associated with urine metabolite changes. A combined marker comprising two genera may have a high potential to identify YINDC with an AUC of 0.845.

Conclusions: Infants with YINDC had a unique gut microbiota and metabolomic profile resulting in a constitutional microclassification. The altered gut microbiome in YINDC may account for the higher risk of cardiovascular diseases. Metabolomic analysis of urine showed that metabolic pathways, including histidine metabolism, proximal tubule bicarbonate reclamation, arginine biosynthesis, and steroid hormone biosynthesis, were altered in infants with YINDC. Additionally, the combined bacterial biomarker had the ability to identify YINDC. Identifying YINDC in infancy and intervening at an early stage is crucial for preventing cardiovascular diseases.

Keywords: Biomarker; Infant; Metabolomics; Microbiota; Traditional Chinese medicine; Yin deficiency constitution.

Fig. 1

Intestinal microbial diversity analysis. (A) Venn diagram demonstrating the existence of different OTUs in the YINDC and BC groups. (B) Species alpha diversity differences between the two groups based on the ACE, Chao 1, and Shannon indices. (C) The overall microbial structure of the two groups as represented by PCoA of unweighted (left) and weighted (right) unifrac distances.

Fig. 2

Alterations in gut microbiota associated with YINDC. (A) Relative abundance of the most prevalent bacterial phyla, families, genera and Faecalibacterium in the YINDC and BC groups. (B) Cladogram showing the distribution of intestinal microbial phylogeny. (C) Histograms of different taxa. (D) KEGG pathways of YINDC-associated intestinal flora.

Fig. 2

Alterations in gut microbiota associated with YINDC. (A) Relative abundance of the most prevalent bacterial phyla, families, genera and Faecalibacterium in the YINDC and BC groups. (B) Cladogram showing the distribution of intestinal microbial phylogeny. (C) Histograms of different taxa. (D) KEGG pathways of YINDC-associated intestinal flora.

Fig. 3

Alterations in urine metabolites associated with YINDC. (A) OPLS-DA score plot in the positive- (above) and negative-ion (below) modes with VIP >1.0 and p < 0.05. (B) Volcano map of the significantly different metabolites in the positive (left) and negative (right) ion modes. Upregulated metabolites were detected by FC > 1.5 and p < 0.05, whereas downregulated metabolites were detected by FC < 0.67 and p < 0.05. (C) KEGG pathways of YINDC-associated metabolites.

Fig. 4

Correlation analysis conducted by Spearman. (A) Heatmap of different microbiota and metabolites with |r| < 1. (B) Network diagram of significantly related microbiota and metabolites with 0.5 < |r| < 1. The red line and blue line indicate positive and negative correlation respectively. A darker color indicated a stronger correlation. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Fig. 5

ROC curves for differential genera and metabolites of YINDC. (A) Genus Bradyrhizobium. (B) Genus Delftia. (C) Metabolite 3-dehydroepiandrosterone sulfate. (D) Metabolite 2′-fucosyllactose. (E) Metabolite 4-pyridoxic acid. (F) Combined marker composed of the two genus markers.