Quantitative metabolomic profiling of serum, plasma, and urine by (1)H NMR spectroscopy discriminates between patients with inflammatory bowel disease and healthy individuals

Abstract

Serologic biomarkers for inflammatory bowel disease (IBD) have yielded variable differentiating ability. Quantitative analysis of a large number of metabolites is a promising method to detect IBD biomarkers. Human subjects with active Crohn's disease (CD) and active ulcerative colitis (UC) were identified, and serum, plasma, and urine specimens were obtained. We characterized 44 serum, 37 plasma, and 71 urine metabolites by use of (1)H NMR spectroscopy and "targeted analysis" to differentiate between diseased and non-diseased individuals, as well as between the CD and UC cohorts. We used multiblock principal component analysis and hierarchical OPLS-DA for comparing several blocks derived from the same "objects" (e.g., subject) to examine differences in metabolites. In serum and plasma of IBD patients, methanol, mannose, formate, 3-methyl-2-oxovalerate, and amino acids such as isoleucine were the metabolites most prominently increased, whereas in urine, maximal increases were observed for mannitol, allantoin, xylose, and carnitine. Both serum and plasma of UC and CD patients showed significant decreases in urea and citrate, whereas in urine, decreases were observed, among others, for betaine and hippurate. Quantitative metabolomic profiling of serum, plasma, and urine discriminates between healthy and IBD subjects. However, our results show that the metabolic differences between the CD and UC cohorts are less pronounced.

Figure 1

Typical 600 MHz ¹H NMR spectra of serum from patients with ulcerative colitis (UC) and Crohn’s disease (CD) and from a healthy control subject. Aromatic region (5.0–9.5 ppm) magnified ×4 compared with the aliphatic region (0.7–4.3 p.p.m). Metabolites: (1) 2-hydroxy-butyrate, (2) arginine, (3) citrate, (4) glucose, (5) isoleucine, (6) lysine, (7) mannose, (8) methanol, (9) creatinine, (10) tyrosine, (11) urea.

Figure 2

Changes in OPLS-DA coefficients of serum metabolites from patients with (A) ulcerative colitis (UC) and (C) Crohn’s disease (CD) as compared to control subjects. Positive bars (± SEM) illustrate metabolites significantly increased in UC and CD, whereas negative bars (± SEM) denote metabolites significantly higher in control subjects. Panels B and D depict the respective OPLS scores plots.

Figure 3

Changes in OPLS-DA coefficients of plasma metabolites from patients with (A) ulcerative colitis (UC) and (C) Crohn’s disease (CD) as compared to control subjects. Positive bars (± SEM) illustrate metabolites significantly increased in UC and CD, whereas negative bars (± SEM) denote metabolites significantly higher in control subjects. Panels B and D depict the respective scores plots demonstrating good separation of metabolites between IBD patients and control subjects.

Figure 4

OPLS-DA coefficients (± SEM) obtained from serum and plasma samples compared between CD and UC patients (A) to demonstrate significant differences in metabolites that could help differenciating between these two diseases. Panels B and C show the respective OPLS scores plots for CD versus UC in (B) serum and (C) plasma.

Figure 5

Changes in OPLS-DA coefficients of urine metabolites from patients with (A)ulcerative colitis (UC) and (C) Crohn’s disease (CD) as compared to control subjects. Positive bars (± SEM) illustrate metabolites significantly increased in UC and CD, whereas negative bars (± SEM) denote metabolites significantly higher in control subjects. Panels B and C show the respective OPLS scores plots for (B) UC compared to control subjects and (D) CD compared to control subjects.

Figure 6

Hierarchical orthogonal projection to latent structure-discriminant analysis (hierarchical OPLS-DA) scores plots (A, B, C) obtained from scores of sub-PCA models that were separately derived from corresponding disease cohorts in the three different biofluids from patients with Crohn’s disease (CD, red), ulcerative colitis (UC, blue), and matched healthy individuals (control, green).