In this study, we identified metabolites that are associated with AIS by simultaneous comparison of the serum metabolome in the acute stage of stroke with that of controls and the chronic stage of stroke. Overall, ketone bodies, BCAAs, energy, and inflammatory compounds were elevated/decreased in the acute stage and could have been driven by stroke acuity. In contrast, most amino acids, phosphatidylcholines, phosphoglycerides, and sphingomyelins were elevated/decreased in stroke patients compared with controls, but did not change between the acute and chronic stages, likely being unrelated to stroke acuity. These findings clarify changes in the metabolome between stages of stroke and may indicate a direction for future research investigating stroke biomarkers.
Our findings validated previously described changes in serum levels of ketone bodies, BCAAs, and energy compounds in acute stroke 4,18,19. Involvement of these metabolite subclasses should come from interplay of different metabolic pathways, all triggered by cerebral vessel occlusion (Fig. 2). In our view, oxidative stress in the brain slows down the Krebs cycle; activates anaerobic glycolysis, leading to elevation of pyruvate and glycerol; and, at the same time, activates production of ketone bodies to support brain metabolic requirements. Acetone, acetoacetate, and β-hydroxybutyrate are more energetically efficient than glucose, and can provide up to 70% of the brain’s energy needs 20. Further, β-hydroxybutyrate may ameliorate the disruption of cerebral energy metabolism after ischemia, when the anaerobic glycolytic pathway is activated 21. In addition, studies suggest that cerebral uptake of ketones significantly increases during acute brain stress 20. All these findings correspond to our observed increase in acetone, acetoacetate, β-hydroxybutyrate, pyruvate, and glycerol in the acute stage of stroke, followed by a decrease in the chronic stage to levels similar to controls. Acute ketosis may also increase levels of ketogenic amino acids by preventing their degradation to Acetyl-CoA, which, in our study, corresponds to an elevated acute level of isoleucine and relative decrease of leucin in the chronic stage 22. Besides slowing of the Krebs cycle, pyruvate could have increased by synthesis from alanine through the glucose/alanine cycle, which also corresponds to decreased alanine levels. Although activation of anaerobic glucose metabolism is overall associated with increased lactate levels, previous experimental studies showed a decrease in lactate levels after stroke in the brain parenchyma 23 and blood 24, same as in our study. This decrease may be explained by utilization of lactate as an alternative energy source by still-viable brain tissue within the infarction 25. Production of lactate from pyruvate may also be inhibited by β-hydroxybutyrate through inhibition by increased acetyl-CoA 21. In the present study, however, decreased levels of lactate were observed in the acute and chronic stages of stroke, suggesting prolonged action of those mechanisms, or other, yet unknown, pathway.
Glycoprotein acetyl is an inflammatory biomarker previously associated with the risk of cardiovascular disease 26. In the present study, glycoprotein acetyl was elevated in the acute stage of stroke compared with controls, but not in the chronic stage. Therefore, it may simply reflect acute reaction to stroke, rather than stroke risk.
Although in case-control studies AIS was associated with changes in metabolism of lipids and amino acids, the question of how long these changes continue could not be answered due to lack of follow-up data 2,27. In the present study, follow-up (chronic stage) metabolome changes of lipids and most amino acids were similar to the acute stage. Although prolonged alteration of metabolism after stroke is possible, the absence of significant differences in lipids and most amino acid levels between the acute stage and the chronic stage is more logically explained by the baseline differences in metabolism, unrelated to stroke acuity. This corresponds to unconvincing pathophysiological explanations of amino acid and lipid metabolism after acute cerebral ischemia given in previous investigations 2.
Serum levels of apolipoproteins, lipoproteins, and fatty acids are closely related to patient diet; therefore, their decrease may be explained by the dietary trends of stroke patients 28. Two major factors that affect diet after stroke are dysphagia and implementation of healthy lifestyles. Persistent dysphagia after stroke may lead to deficiencies of essential metabolites, such as omega-6 fatty acids, while healthier diets with decreased consumption of meat result in decreased saturated fatty acids and apolipoprotein B, and increased monounsaturated fatty acids. The latter was not observed in our study, likely because of patient failure to increase consumption of vegetables or dysphagia. In addition, some controls in our study were recruited from the cardiology clinic and could have diets rich in saturated fat, explaining higher levels of cholesterol in controls than in stroke patients.
Strengths of our study include its unique design in which the acute stage of stroke metabolome was compared with that of controls and the chronic stage, and the comparatively reasonable sample size. Validation of data from other laboratories that used liquid chromatography mass-spectroscopy for metabolome analysis adds to the credibility of our results. Limitations include a comparatively low follow-up rate, some inequalities between the stroke and control groups, and the lack of long-term follow-up data. Using multiple covariates for regression analysis helped to address disparities in the stroke and control groups, but at the same time could significantly change the results of our or any other metabolome study. Particularly, adjusting for use of statins affected lipidomic compounds. In the future, development of standardized protocols for evaluation of the metabolome may help to improve interpretation of the results.
In summary, our pilot study showed differences in metabolism between stroke patients in the acute and chronic stages and controls, and helped to differentiate metabolites altered in the acute stage of stroke from those altered in the acute and chronic stages. Further validation of these findings in a larger, independent cohort is needed to provide a solid background for future stroke metabolome research.