4.1 Significant changes in concentration levels of the plasma metabolites in Non-CVD subjects, patients with IHD and patients with AMI
The presented data is the continuing of our previous study of plasma metabolites in IHD patients [7]. Based on the conducted metabolomic profiling, it may be revealed, that significant alterations in concentration levels of kynurenine, kynurenic acid, as well as kynurenine / tryptophan ratio, were found only in IHD group, whereas in AMI patients its levels did not differ from the plasma levels of non-CVD subjects. At the same time concentration of xanthurenic acid and 3-OH-kynurenine did not differ among the non-CVD and IHD subjects, but was significantly decreased in patients with AMI. Overall, the above mentioned metabolites are related to the kynurenine metabolic pathway (KMP) – main route of the tryptophan catabolism. KMP is presumably responsible for cellular energetic homeostasis, being the principal regulator of the immune system [8]. Therefore, KMP may reflect the inflammation processes being occurred in the body during the disease. Numerous studies suggest the application of KMP metabolites, as potential prognostic biomarkers of IHD [9]. In this regard, significant changes in concentration levels of key KMP intermediates (kynurenine and kynurenic acid) may be related to the activation of the indoleamine 2,3-dioxygenase (IDO) and tryptophan 2,3-dioxygenase (TDO) enzymes. These enzymes are mainly induced by pro-inflammatory stimuli and T-cell cytokines during the pro-inflammatory states and immune stress associated with IHD, thus suggesting, that the perturbations in IHD are mostly related to the activation of kynurenine degradation to the formation of kynurenic acid. At the same time, it may be concluded, that completely different metabolic pathways are activated in patients with myocardial infarction, presumably resulting in the decrease of the enzymes responsible for production of xanthurenic acid and 3-OH kynurenine – metabolites of an alternative to kynurenine degradation pathway. Xanthurenic acid is a well-known vasorelaxant, thus its decreased levels may indicate the need for prescription of medicines containing xanthurenic acid in patients with AMI [10]. In this case, the mechanism of action of xanthurenic acid is based on its influence on endothelial levels of nitric oxide, thus causing hypertension.
Plasma serotonin levels were found to be significantly altered in patients, suffering from IHD and AMI in comparison to non-CVD subjects. It should be mentioned, that serotonin itself possesses numerous complex effects, acting as peripheral hormone and neurotransmitter [11]. Prevalent part of serotonin is deposited in dense granules of platelets, being released in the bloodstream, as the result of its activation by thrombus formation in coronary arteries. As a result, it may lead to aggregation of platelets and constriction of coronary smooth cells [12, 13].
Several amino acids were significantly changed in IHD and AMI patients in comparison to the non-CVD subjects. However, these alterations were not linear. For example, there was found significant elevation in concentration levels of branch-chained amino acids (BCAA) metabolites in IHD patients, whereas in AMI group their level was equal to the concentration in the non-CVD group.
Overall, amino acids play a crucial role in the energy metabolism and are directly involved in various metabolic pathways, including Krebs cycle and gluconeogenesis, therefore significant changes in their concentration levels may reflect disturbances in the energy metabolism. Due to the relatively large amount of significantly altered metabolites in IHD and AMI groups of patients, application of an amino acid metabolic profiling may be used as a novel biomarker diagnostic and prognostic panel for identification of IHD and AMI patients.
Endogenous neurotransmitter norepinephrine was significantly decreased in patients with AMI, whereas its end-product – vanillylmandelic acid (VMA) – significantly increased. Plasma norepinephrine is mainly derived from sympathetic nerves, thus indicating activity of the sympathetic nervous system. Through specific binding to α1 and α2-adrenoreceptors it acts as vasoconstrictor. Significant decrease in concentration of plasma norepinephrine may be associated with increase of its cellular uptake in AMI patients. In this case, such cellular uptake leads to its deamination with the formation of methoxyhydroxyphenylglycol (MHPG), followed by its oxidation to VMA [14]. According to [15] 94% of VMA is synthesized in liver, among which in 87% it represents the results of hepatic extraction and metabolism of 3-Methoxy-4-hydroxyphenylglycol (MHPG) and DHPG. In this regard, we may hypothesize, that patients from the AMI group had more activated norepinephrine cellular uptake, that resulted in extra accumulation of VMA. However, for verification of this hypothesis it is also needed to quantify intermediate products of norepinephrine, including MHPG and dihydroxyphenylglycol.
Acetylcholine represents another neurotransmitter which is released in response to innervation of the blood vessels by the parasympathetic cholinergic or sympathetic cholinergic nerves. Binding of acetylcholine with the muscarinic M3 receptor leads to the release of NO, resulting in smooth muscle relaxation. However, it is known, that in case of NO absence, M3 receptor action is opposite, resulting in constriction of the smooth muscles [16]. In the presented study we have identified the significant alteration in the concentration levels of acetylcholine in patients with IHD and AMI, therefore suggesting the lack of NO in patients with the considered CVD disorders. At the same time, acetylcholine precursor – choline – was significantly elevated only in IHD patients, while in AMI patients its levels became equal to those from the non-CVD group. Elevated choline levels are the markers of unfavorable cardiovascular risk profile and CVD incidences [17].
Intermediates of the homocysteine-methionine cycle are directly connected with choline through its derivative – betaine, and are responsible for amino acid metabolism and cellular methylation. In the presented study we have identified a strongly impaired regulation of this cycle, including significantly increased levels of cystathionine and decreased levels of methionine in the AMI patients. Interestingly, that the methionine derivative was significantly altered in AMI patients, that may be the result of methionine oxidation. Elevated levels of cystathionine identified in the presented study are known to be linked to oxidative damage and impaired endothelial function [18].
Dimethylglycine, that was significantly altered in IHD and AMI patients, is usually produced from betaine upon remethylation of homocysteine to methionine [19]. Previously, numerous studies identified the dimethylglycine as a major risk marker of myocardial infarction in patients with suspected or established coronary heart disease [20]. At the same time, the decreased levels of glycine may confirm the accumulation of dimethylglycine in plasma.
The presented study identified, that short-chain and long-chain acylcarnitines were significantly increased in both IHD and AMI groups. Acylcarnitines play a significant role in the myocardial metabolism, being mainly responsible for mitochondrial β-oxidation of long‐chain fatty acids, and, therefore, for energy production. Numerous studies have linked disturbance in blood acylcarnitines to various cardiovascular disorders [3, 21–23]. Deprivation of oxygen and nutrients in myocardium during IHD and AMI reveals breakdown of fatty acids. In this case, elevated levels of acylcarnitines reflect disbalance in mitochondrial function and oxidative stress.
Levels of symmetric dimethylarginine (SDMA) were significantly increased in IHD and AMI patients. SDMA is known as an endogenous inhibitor of NO-synthase, therefore, its elevated levels possess extra inhibition of NO production. At the same time, increased levels of biopterin in the AMI patients may underline an alternative NO-generating pathway in patients after AMI, which is presumably caused by the compensatory mechanisms in the body, due to the acute cardiac event. In general, NO is one of the key vasodilators in the body, which is responsible for blood pressure control, therefore, its inhibition may lead to endothelial dysfunction and oxidative stress.
Citrulline, an intermediate of the urea cycle, was significantly decreased in patients with myocardial infarction in comparison to non-CVD subjects and IHD patients. Recyclization of citrulline to arginine is one of the principle steps of the endothelial NO synthesis. In this case decreased levels of citrulline may contribute to the impaired NO production in AMI patients.
In comparison to non-CVD individuals, patients with AMI had increased plasma levels of Carnitine, Isovalerylcarnitine, long-chain acylcarnitines (Palmitoylcarnitine, Palmitoleyl carnitine, Oleoylcarnitine, Hydroxystearoylcarnitine), amino acids (phenylalanine, glutamine), biopterin, symmetric dimethylarginine, tryptophan metabolism derivatives (Tryptophol, Serotonin, Anthranilic acid), Vanillylmandelic acid, Methionine sulfoxide, acetylcholine, and Dimethylglycine. At the same time, plasma concentration levels of glycine, aspartic acid and indole-3-propionic acid were significantly decreased. These data correspond to our previously obtained results [7].
In comparison to patients with IHD, patients with AMI had significant increased plasma levels of Dimethylglycine, Vanillylmandelic acid, Tryptophol, biopterin, glutamine, proline, asparagine, Isovalerylcarnitine and Hydroxystearoylcarnitine. In contrary, plasma levels of Norepinephirne, Xanthurenic acid, 3-Hydroxykynurenine, Citrulline, valine, leucine, N-methylmalonamic acid, Aspartic acid, Methionine, Glycine and Decenoylcarnitine were significantly decreased.
Figure 7 summarizes metabolic pathways, affected by the identified significantly altered metabolites.
4.3 Differences in the weighted correlation network analysis of the considered metabolites
The above presented pathway analysis and interpretation of the metabolomic profiling were based on the mapping of the identified significantly changed metabolites into preliminary defined pathways, obtained from metabolic databases, such as KEGG [24] or MetaCyc [25]. At the same time, weighted correlation network analysis represents an alternative powerful tool for identification of systemic metabolic changes, that are often undetectable, when using solely changes in metabolite levels and database pathway information [26]. In the presented study, there was utilized a DSPC algorithm, the regularized approach mainly created for handling high dimensional MS-based metabolomic profiling data [27]. Its main principle is based on the application of the de-sparsified graphical lasso model, taking into account, that the amount of true connections among the metabolites is significantly smaller than the utilized sample size.
It was found, that the first module mainly consisting of medium and long chain acylcarnitines was relatively the same in all three considered groups of patients, as well, as the principal nodes. At the same time, short chain acylcarnitines were separated from the module forming short chains presumably containing C5, C3 and C0 metabolites. Also, it should be mentioned, that this module in all groups of patients was connected with VMA, showing unchangeable relations of medium and long chain acylcarnitines with the end-product of the norepinephrine degradation pathway. However, we may conclude, that the number of intra edges of this module in AMI was significantly lower compared to IHD and non-CVD subjects, that may be explained by the alternative activity of these metabolites after AMI.
The second module is mainly consisted of kynurenines. The key nodes were Indole-3-acrylic acid, kynurenine and Indole-3-carboxaldehyde in non-CVD, IHD and AMI groups, respectively. In non-CVD group the kynurenine module showed strong relation with choline, SDMA and ADMA, whereas in patients with IHD and AMI these connections were absent. In AMI patients the tryptophan pathway metabolites were “diveded” into two parts: one connected with amino acids, and the second – with neopterine.
The third module is primarily comprised of amino acids. Among these metabolites it should be mentioned, that strong correlation between BCAA was relatively the same in all three considered groups of patients. Moreover, in non-CVD and IHD groups the amino acid module is also containing the relations with NO-cycle intermediates.
4.4 Advantages and limitations of the study
The main advantage of the study is that the presented approach provides new insights into the development of AMI from the metabolic point of view. There were found new significant ratios of the metabolites, which may further be utilized as new potential biomarkers of AMI.
This study is not without limitations. The main of it is the necessity of confirmation the results obtained, which need a large number of patients and future studies.