In the study, we quantified the levels of 25 amino acids in the serum of EOCAD patients and identified eight that were related to the disease state. 4-Hydroxyproline was validated because it was the only amino acid with low serum levels in the disease group. Our results showed that 4-Hydroxyproline levels were significantly decreased in patients with early-onset MI, and that elevated serum levels were a protective factor for EOCAD.
CAD is a complex disease caused by genetic factors and environmental interactions. The relationship between CAD and endogenous/exogenous lipid metabolism, nutrition, inflammation, and immune response has been widely studied. Although metabolomics has developed rapidly in the past two decades, very few studies focus on the relationship between the serum levels of amino acids and CAD. In one of these studies, researchers from Duke University quantified plasma levels of 15 amino acids in CAD patients, and indicated that in patients referred for cardiac catheterization, levels of branched-chain amino acids and catabolites are predictors of cardiovascular events . However, the quantitative results of these 15 amino acids have not been published in the study. A subsequent study from this group quantified the plasma levels of Proline, Leucine/Isoleucine, Valine, Methionine, Glutamic acid, and Citrulline, and showed that Leucine/Isoleucine, Glutamic acid, and Methionine levels are significantly elevated in CAD patients . In 2005, Obeid also attempted to find differential amino acid levels in the plasma of CAD patients by HPLC. Limited by test methodology and sample size, he found that of the 23 amino acids tested, only cysteine was significantly elevated in the plasma of CAD patients tested . Circulating branched amino acids (BCAAs; isoleucine, leucine, and valine) have been previously identified as significant predictors of CAD. A large prospective study of American women demonstrated that circulating BCAAs levels were associated with the long-term risk of CAD, comparable in magnitude to LDL cholesterol . Similar results have also been observed in the Chinese population [15, 16]. In previous studies, we examined the effects of homocysteine (Hcy), asymmetric dimethylarginine (ADMA), L-citrulline, and L-arginine on arterial endothelial function and their associations with the risk of CAD [17–21].
To the best of our knowledge, a complete description of serum amino acid levels of CAD, especially in EOCAD, is lacking. Compared to late-onset CAD, EOCAD may have different pathogenic mechanisms and a wide range of biomarkers have already been identified for EOCAD, including genes, proteins, and other bio-macromolecules. In our previous studies the MTHFR C677T polymorphism was shown to be associated with a risk of EOCAD , serum ADMA levels were associated with the presence and severity of EOCAD , and that there is a close relationship between lipid metabolism and early-onset MI . Because of the different dietary structures, serum amino acid levels may differ in CAD patients of different ages [24, 25]. At the screening stage of this study, one sample pool was composed of eight serum samples. We quantified 25 amino acids in 24 serum sample pools of three groups (CAD without MI group, MI group, and control group) and differences in a total of eight different amino acids were identified. Significant differences in L-Phenylalanine, L-Methionine, and L-Glutamic acid levels were seen between the CAD without MI and control groups. Significant differences in L-Phenylalanine, L-Methionine, L-Tyrosine, 4-Hydroxyproline, L-Glutamic acid, L-Aspartic acid, L-Arginine, and L-Serine levels were seen between the CAD with MI and control groups. Compared to the CAD without MI group, in the CAD with MI group, L-Phenylalanine and L-Glutamic acid levels were significantly increased, and 4-Hydroxyproline levels were decreased. Notably, 4-Hydroxyproline was the only amino acid whose levels decreased in the serum of the disease group serum, suggesting that it may play a specific role in CAD pathogenesis.
Because there is a paucity of systematic studies on the correlation between non-BCAAs and CVD, it is difficult to discuss causes and possible mechanisms of changes in serum amino acid levels in CAD patients. L-Phenylalanine, a precursor of L-Tyrosine, is an essential amino acid and both are also precursors for catecholamines . Murr et al. showed that elevated serum L-phenylalanine and L-Tyrosine levels in CAD patients may be associated with immune activation and inflammatory response .
We recently focused on the relationship between one-carbon metabolism and CVD. We found that various metabolites in the metabolic pathway are closely related to the risk of CAD . Hcy is formed in the one-carbon metabolism pathway during the metabolism of methionine to cysteine, and can also be used to regenerate methionine by vitamin B12 . Other studies have shown that serum Hcy levels are significant in CAD patients and that it is an independent risk factor for the disease [30–32].
Although glutamic acid is a non-essential amino acid, almost all living beings use it in protein biosynthesis. Animal studies have shown that exogenous glutamic acid protects hypoxic cardiomyocytes and enhances anaerobic ATP synthesis in mitochondria to reduce myocardial contracture [33, 34]. Elevated serum levels of L-Glutamic acid may be a compensatory response to myocardial ischemia and can promote the formation of anaerobic ATP, enhancing the body’s resistance to severe hypoxia. Recent studies have shown that specific genetic variations can disrupt glutamic acid metabolism and lead to CVD [3, 35].
L-Glutamic acid is a precursor of L-Arginine  which is the substrate of nitric oxide synthase and the biological precursor of nitric oxide. It is vital for maintenance of vascular endothelial function, which is closely related to coronary heart disease . The results of this study indicate that L-glutamate and L-Arginine serum levels are significantly elevated in CAD patients, especially those with MI.
Only a few studies have focused on the correlation between aspartic acid, serine, and CVD. Elevated serum L-Aspartic acid levels may be related to its involvement in antiplatelet aggregation during acute MI , and L-Serine may play an antioxidant role .
As early as 2009, Allejo and co-workers observed that 4-Hydroxyproline plasma levels were significantly decreased in patients with acute coronary syndrome using plasma fingerprinting with GC-MS technology . This indicated that 4-Hydroxyproline is an attractive compound for further studies. In this study, 4-Hydroxyproline was once again identified as being associated with MI. 4-Hydroxyproline levels were significantly decreased in CAD with MI patients, however, no significant decrease was seen in CAD without MI patients. Since 4-Hydroxyproline is related to collagen stability, this suggests that decreased serum 4-Hydroxyproline levels are associated with ventricular remodeling . Our results show that there is a significant negative correlation between 4-Hydroxyproline and cardiac troponin I levels, suggesting a potential relationship between 4-Hydroxyproline and myocardial injury. We expect 4-Hydroxyproline will become a novel biomarker for predicting fibrosis and ventricular remodeling after MI.
Our study provides a profile of the serum amino acid changes in EOCAD patients, however, it has some limitations. Firstly, due to funding reasons, we have not measured all the identified amino acids individually, and the relationship between other amino acids and EOCAD still needs to be verified by large sample size studies. Secondly, because amino acid determination requires fresh samples and female patients with EOCAD are rare, this study did not include female patients. Finally, coronary angiography was not performed in healthy controls as they were age- and sex-matched individuals with no signs or symptoms of CAD.
In conclusion, we initially quantified serum levels of 25 amino acids levels in EOCAD patients and identified eight amino acids associated with disease status. Compared to the control group, serum levels of L-Arginine, L-Methionine, L-Tyrosine, L-Serine, L-Aspartic acid, L-Phenylalanine, and L-Glutamic acid were higher in the EOCAD group, and 4-Hydroxyproline levels were lower. 4-Hydroxyproline appears to be a promising amino acid for further biomarker studies of predicting fibrosis and ventricular remodeling after MI.