Using HPLC, our study first showed the A-V differences in amino acid concentrations in controls and ARDS patients. In the 15 different amino acids that we detected in the arterial and venous blood, the A-V differences in only Glu concentration were significantly greater in ARDS patients than in controls. However, A-V differences in Glu concentration in ARDS patients showed no significant correlation with disease severity (grouped according to the ARDS Berlin definition). Furthermore, the result revealed that A-V differences in Glu concentration in ARDS survivors were considerably higher than in non-survivors, especially in non-survivors who died within 2 weeks of ICU admission. These results indicate that A-V differences in Glu concentration may be a biomarker for ARDS patients, especially to predict the outcome of ARDS.
Clinically, acute lung injury (ALI)/ARDS manifests as decreased lung compliance, severe hypoxemia, and bilateral pulmonary infiltrates, and is associated with a high mortality[1]. Diagnosis of ARDS are mainly based on clinical characterization. Numerous studies have focused on the identification of biomarkers, which can reflect pathophysiological mechanisms, to improve diagnosis, determining disease severity and prediction for outcome of ARDS. Researches already identified several biomarkers that are associated with the highest sensitivity and specificity for the diagnosis or outcome prediction of ARDS that have been investigated in blood, pulmonary edema fluid, and exhaled air[4]. In general, these are cell-specific for epithelial or endothelial injury or involved in the inflammatory or infectious response. But currently they are not reliable enough for clinical diagnosis of ARDS or prediction of its prognosis or have not yet been confirmed[4]. The lack of a specific biomarker to define, diagnose, monitor responsiveness to therapy or predict prognosis of ARDS has limited progress in developing novel treatments for ARDS. Thus, the discovery of a specific biological marker with early diagnostic significance is desirable.
Currently, it is an emerging area to identify metabolites including amino acid as clinically relevant biomarkers and potential therapeutic targets in several diseases, such as sepsis, diabetes, cancers and so on[14–18]. One study showed elevated levels of several metabolites, such as hippurate, L-phenylalanine, creatine, methionine, L-glutamate, and L-proline in bronchoalveolar lavage fluid (BALF) of patients with sepsis-induced ARDS compared to healthy controls[19]. Another study also showed that amino acid levels in the plasma differed in patients with acute exacerbation of COPD (AE-COPD), depending on the presence of bacterial infection. They claimed that specific amino acids (i.e., asparagine, citrulline, glutamine, histidine, serine, and threonine) have a potential utility as diagnostic markers to distinguish between bacterial and nonbacterial AE-COPD[20]. In another study, significant differences in the metabolomics and metabolic pathways between control and ARDS groups were observed. This study suggests the potential utility of metabolomics to identify biomarkers that predict early ARDS onset, progression, severity, and prognosis[21] Another study focused on the plasma amino acid levels during the acute phase of endotoxin-induced lung injury in eight sheep, they found that norepinephrine, epinephrine, and alanine levels increased whereas that of isoleucine decreased. However, they did not determine the relationship between amino acid levels and ARDS severity and outcomes[14] Till now, the dynamics and clinically significant changes in amino acid levels in patients with ARDS are largely unknown.
To the best of our knowledge, to date, A-V differences in amino acid levels have not been extensively studied. In our study, we detected the A-V differences in amino acid levels between controls and ARDS patients. We found that A-V differences in Glu concentration in ARDS patients were significantly greater than in controls; however, the differences were not significant for other amino acids we study. Importantly, survivors showed greater A-V differences in Glu concentration than non-survivors, while non-survivors that died within 2 weeks following ICU admission showed significantly smaller A-V differences in Glu concentration than survivors. These results indicate that the A-V differences in Glu concentration can be a biological marker for ARDS and for outcome prediction. We tried to understand the higher A-V differences in Glu concentration in ARDS patients than in non-survivors. A-V difference in Glu concentration represents the arteriovenous difference in the levels of Glu (arterial level of Glu-venous level of Glu). Generally, venous blood becomes arterial blood in the lungs. Thus, to some extent, the A-V differences in substances can reveal how substances are released or consumed in the lung tissue. Our previous research detected 17 kinds of amino acids (phenylalanine, alanine, methionine, glycine, glutamate, arginine, lysine, tyrosine, leucine, serine, threonine, aspartate, valine, isoleucine, and histidine) in BALF of mice. The results showed that only Glu and glycine were increased in BALF of BLM-induced lung injury mice compared to the saline control group[22]. This research indicated that endogenous Glu and glycine may be selectively released from the lungs at the beginning of BLM-induced acute lung injury. If Glu is released from the injured lungs, it will also increase the Glu level in the arteries. Thus, we hypothesized that when the lung gets injured, cells selectively release Glu from the lungs to the blood or alveolar, which is consistent with the increased level of Glu in BALF of injured lungs as well as in arteries. System x(c)- is a cysteine/Glu transporter, with two subunits-4F2hc and the function unit of x (c)- system (xCT). At physiological conditions, system x(c)- mediates the release of Glu while it uptakes cysteine, which is essential for maintaining intracellular glutathione levels and cellular defenses against oxidative stress[23]. Previous researches suggested that xCT is upregulated in injured lung tissues induced by paraquat and plays a protective role against oxidative stress and lung injury[24]. As we detected in this study, ARDS patients had higher A-V differences in Glu concentration while that in non-survivors was much lower than for the survivors. We hypothesized that System x(c)- is upregulated in injured lung tissue to promote the uptake of cysteine and the release of Glu. This is important for the synthesis of glutathione, protecting the patients from oxidative stress-induced injury. System x(c)- function in non-survivors may be damaged, resulting in decreased release of Glu from the lung tissue, leading to the lower level of Glu in the arterial blood; which is consistent with the lower A-V differences in Glu concentration in non-survivors. A-V differences in Glu concentration in non-survivors were much lower than those of survivors. This may indicate a reduction in the uptake of cysteine and glutathione synthesis, causing insufficient capacity of antioxidants in the lung cells of non-survivors.
Our study has several limitations that should be addressed. First, the results were obtained from a study population with a relatively small sample size; hence, the results will need to be confirmed using a larger sample size. Second, due to the limitation of clinical sampling, it was really difficult to obtain pulmonary arterial and venous blood. The blood from the radial artery and jugular vein that we collected could not fully represent the blood flowing in and out of the lung completely; thus, our results may not have fully determined the relationship between lung injury and A-V differences in Glu concentration. Third, we hypothesized that System x(c)- plays an essential role in the A-V differences in Glu concentration between controls and ARDS patients. However, we did not detect the level of xCT and glutathione in the patients. Further research is needed to clarify the mechanism and significance of releasing Glu from the lung. Furthermore, we did not show the relationship between long-term consequences in ARDS survivors and A-V differences in amino acid levels.