Amino Acid Biosignature in Plasma among Ischemic Stroke Subtypes

Abstract

Ischemic stroke is a neurovascular disorder caused by reduced or blockage of blood flow to the brain, which may permanently affect motor and cognitive abilities. The diagnostic of stroke is performed using imaging technologies, clinical evaluation, and neuropsychological protocols, but no blood test is available yet. In this work, we analyzed amino acid concentrations in blood plasma from poststroke patients in order to identify differences that could characterize the stroke etiology. Plasma concentrations of sixteen amino acids from patients with chronic ischemic stroke (n = 73) and the control group (n = 16) were determined using gas chromatography coupled to mass spectrometry (GC-MS). The concentration data was processed by Partial Least Squares-Discriminant Analysis (PLS-DA) to classify patients with stroke and control. The amino acid analysis generated a first model able to discriminate ischemic stroke patients from control group. Proline was the most important amino acid for classification of the stroke samples in PLS-DA, followed by lysine, phenylalanine, leucine, and glycine, and while higher levels of methionine and alanine were mostly related to the control samples. The second model was able to discriminate the stroke subtypes like atherothrombotic etiology from cardioembolic and lacunar etiologies, with lysine, leucine, and cysteine plasmatic concentrations being the most important metabolites. Our results suggest an amino acid biosignature for patients with chronic stroke in plasma samples, which can be helpful in diagnosis, prognosis, and therapeutics of these patients.

Figure 1

Differences in the amino acid concentrations between stroke subtypes and control individuals. Control individuals (Ctl), atherothrombotic stroke (Ath), cardioembolic stroke (Car), lacunar stroke (Lac), and undetermined stroke (Und). Kruskal-Wallis test followed by Dunn's multiple comparison test: ∗ p ≤ 0.05, ∗∗ p ≤0.01, and ∗∗∗p ≤ 0.0001.

Figure 2

PLS-DA predictions to classify ischemic stroke and control samples (a); studentized residuals for this model at the 99.7% confidence level (b); two samples (red circles) being detected as outliers. Variable importance in the projection (VIP scores) (c) and regression coefficients (d).

Figure 3

PLS-DA predictions for discriminating ischemic stroke from control samples. This PLS-DA model was built with reduced dataset, containing only the three most discriminant amino acids: Met, Ala, and Pro.

Figure 4

PLS-DA models built with 16 amino acids for classifying ischemic stroke subtypes. Models for discriminating atherothrombotic ischemic stroke from other samples (a) and for cardioembolic plus lacunar ischemic stroke (b). VIP scores (c) for atherothrombotic ischemic stroke. VIP scores (d) for cardioembolic plus lacunar ischemic stroke.

Figure 5

Metabolic pathways activated in chronic ischemic stroke. (a) The increase of proline is promoted by activation of extracellular matrix degradation. High levels of proline can inhibit ALT enzyme and cause alanine concentration reduction. The reduction in alanine concentration can also be related to donation of this amino group for glutamine synthesis, which can be converted to glutamic acid in the neurons and, then, it can be used to increase the levels of leucine. (b) Decrease of methionine concentration due to its conversion to homocysteine; homocysteine increases as a result of the cofactor pyridoxal 5′-phosphate inhibition by inflammatory mediators (C-reactive protein); decrease of pyridoxal 5′-phosphate due to its migration to inflammatory sites. Blue arrows: activated pathways. ECM: extracellular matrix. MMPs: matrix metalloproteinases.