Quantitative metabolomics by H-NMR and LC-MS/MS confirms altered metabolic pathways in diabetes

Abstract

Insulin is as a major postprandial hormone with profound effects on carbohydrate, fat, and protein metabolism. In the absence of exogenous insulin, patients with type 1 diabetes exhibit a variety of metabolic abnormalities including hyperglycemia, glycosurea, accelerated ketogenesis, and muscle wasting due to increased proteolysis. We analyzed plasma from type 1 diabetic (T1D) humans during insulin treatment (I+) and acute insulin deprivation (I-) and non-diabetic participants (ND) by (1)H nuclear magnetic resonance spectroscopy and liquid chromatography-tandem mass spectrometry. The aim was to determine if this combination of analytical methods could provide information on metabolic pathways known to be altered by insulin deficiency. Multivariate statistics differentiated proton spectra from I- and I+ based on several derived plasma metabolites that were elevated during insulin deprivation (lactate, acetate, allantoin, ketones). Mass spectrometry revealed significant perturbations in levels of plasma amino acids and amino acid metabolites during insulin deprivation. Further analysis of metabolite levels measured by the two analytical techniques indicates several known metabolic pathways that are perturbed in T1D (I-) (protein synthesis and breakdown, gluconeogenesis, ketogenesis, amino acid oxidation, mitochondrial bioenergetics, and oxidative stress). This work demonstrates the promise of combining multiple analytical methods with advanced statistical methods in quantitative metabolomics research, which we have applied to the clinical situation of acute insulin deprivation in T1D to reflect the numerous metabolic pathways known to be affected by insulin deficiency.

Figure 1. Scores plot (a) and PC1 loadings plot (b1 and b2) from the PCA of ¹H NMR spectra of plasma from insulin deprived patients, treated patients and healthy people (glucose and residual water peaks were removed prior to PCA).

Intensities of the NMR variables were normalized using the total sum of intensities. The data were analyzed after being mean-center scaled. Identified metabolites: 1, allantoin; 2, acetate; 3, acetone; 4, 3-hydroxybutyrate ; 5, acetoacetate; 6, valine; 7, lactate; 8, alanine; 9, tyrosine; 10, citrate; 11, histidine; 12, formate; 13, creatinine; 14, glycoprotein N-acetyl groups; 15, lipid:CH₃; 16, lipid:CH₂; 17, lipid:CH₂CH₂-CO;18, lipid:CH₂-C = C;19, Lipid:fatty acyl groups = CH.

Figure 2. Pearson's correlations of the quantities of the 48 plasma metabolites measured by LC-MS/MS and ¹H-NMR in type 1 diabetics who were insulin treated (a) or insulin deprived (b).

Figure 3. The difference in correlation of the quantities of the 48 metabolites measured by LC-MS/MS and ¹H-NMR between type 1 diabetics during insulin deprivation and insulin treatment.