Metabonomic fingerprints of fasting plasma and spot urine reveal human pre-diabetic metabolic traits

Abstract

Impaired glucose tolerance (IGT) which precedes overt type 2 diabetes (T2DM) for decades is associated with multiple metabolic alterations in insulin sensitive tissues. In an UPLC-qTOF-mass spectrometry-driven non-targeted metabonomics approach we investigated plasma as well as spot urine of 51 non-diabetic, overnight fasted individuals aiming to separate subjects with IGT from controls thereby identify pathways affected by the pre-diabetic metabolic state. We could clearly demonstrate that normal glucose tolerant (NGT) and IGT subjects clustered in two distinct groups independent of the investigated metabonome. These findings reflect considerable differences in individual metabolite fingerprints, both in plasma and urine. Pre-diabetes associated alterations in fatty acid-, tryptophan-, uric acid-, bile acid-, and lysophosphatidylcholine-metabolism, as well as the TCA cycle were identified. Of note, individuals with IGT also showed decreased levels of gut flora-associated metabolites namely hippuric acid, methylxanthine, methyluric acid, and 3-hydroxyhippuric acid. The findings of our non-targeted UPLC-qTOF-MS metabonomics analysis in plasma and spot urine of individuals with IGT vs NGT offers novel insights into the metabolic alterations occurring in the long, asymptomatic period preceding the manifestation of T2DM thereby giving prospects for new intervention targets. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1007/s11306-010-0203-1) contains supplementary material, which is available to authorized users.

Fig. 1

Representative UPLC-ESI-qTOF-MS base peak intensity chromatograms (BPC) (m/z 100–700; 0–25 min) from a human plasma sample analyzed in positive (a) and in negative ionization mode (b), and from a spot urine in positive (c) and in negative ionization mode (d). Methodological details are given in the “Method” section

Fig. 2

Two dimensional scores plot of a partial least squares-discriminant analysis (PLS-DA) with orthogonal signal correction (OSC) data filter of the first and second PLS component of (a) plasma and (b) spot urine. Normal glucose tolerant (NGT) individuals are labelled by open squares and impaired glucose tolerant (IGT) subjects are labelled with triangles

Fig. 3

S-plot of (a) plasma and (b) spot urine. The variables are labeled with m/z values. Potential metabolic biomarkers including the corresponding m/z values are presented in Tables 2 and 3

Fig. 4

(a) Heat map of plasma free fatty acids (FFA) and lysophsphatidylcholines (LPCs) of 39 healthy subjects with normal glucose tolerance (NGT) and 12 individuals with impaired glucose tolerance (IGT). The row above the heat map shows the m/z values of these metabolites corresponding to Table 2. Individual 2-h oGTT plasma glucose level in increasing order are given in the column to the right. Cells are colored based on the signal intensity measured in plasma. Yellow represents high plasma levels and blue showed low signal intensity (see color scale above the heat map). A red line is drawn at a 2-h plasma glucose concentration of 7.8 mmol/l representing the WHO definition for the separation of NGT and IGT. Furthermore, arrows are drawn at the 2-h plasma glucose >5 mmol/l and >6.4 mmol/l where the heat map pattern shows distinct changes. (b) and (c) differences in FFA and LPCs levels between controls and pre-diabetic subjects were examined using Student’s t-test. (b) comparison of the plasma signal intensities of free fatty acids FFA (mean peak area ± standard error; * P < 0.05, IGT vs. the subgroup of NGT with a 2-h plasma glucose level below 6.4 mmol/l) and (c) lysophosphatidylcholines LPC (mean peak area ± standard error; * P < 0.05, IGT vs. subgroup of NGT with a 2-h plasma glucose level below 6.4 mmol/l). blue filled square IGT group (n = 12), red filled square NGT group (n = 39), yellow filled square subgroup of NGT with a 2-h plasma glucose level below 6.4 mmol/l (n = 22). (Color figure online)

Fig. 5

Suggestion for a mechanistic model of metabolic alterations in the pre-diabetic state with a special focus on lipid metabolism. Insulin mediated inhibition of lipolysis is attenuated in adipose tissue in the pre-diabetic metabolic state. Consequently circulating plasma free fatty acids (FFA) are increased leading to a hepatic and muscular lipid oversupply. Hyperlipidemia, hyperglycemia and hepatic insulin resistance augments the triglyceride (TG) storage in the liver and the output of very-low-density lipoproteins (VLDL). In skeletal muscle the overload of mitochondrial lipid oxidation results in accumulation of β-oxidation intermediates and depletion of TCA intermediates which may lead to increased urinary excretion of acyl-carnitines and decreased excretion of phenylacetyl-glutamine