Clinical characteristics of the subjects enrolled
This is a single-center nested-cohort study enrolled a total of 162 patients diagnosed of asthma admitted to the First Affiliated Hospital of Xi’an Jiaotong University from January 2018 to December 2019. Among them, we included 30 patients with chronic persistent asthma between 20 to 35 years old; 30 health control with evenly distributed age and sex were then selected. 15 asthma patients had blood eosinophilia count within normal range and 15 with higher levels of eosinophilia count (Figure 1A). We evaluated serum metabolic profile of 30 healthy and 30 chronic persistent asthma adults aging from 20 to 35 years old. Baseline information of participants enrolled was shown in Table 1. No significant difference in age, female and body mass index (BMI) were seen between chronic persistent asthma patients and healthy control. Of note, eosinophil count was increased in the patients with chronic persistent asthma compared to control, and asthma control test (ACT) was 22.5±2.1 points in the patients with chronic persistent asthma.
Comprehensive metabolomics profile and composition in asthma patients and control.
To further investigate metabolic profile alteration in the chronic persistent asthma, the untargeted metabolomics profiling was performed and a total of 280 metabolites were detected, including amino acids, organic acids, carbohydrates, nucleotides, and so on (Figure 2A). The compositions of each metabolite kinds indicated by respective percentage were generally the same between control, asthma, asthma with high eosinophilia and asthma with normal eosinophilia groups (Figure 2B). Variable importance in projection (VIP) and correlation coefficients assessment of significant metabolites and ratio between control and asthma was calculated using an OPLS-DA model (Figure 2C, Supplementary Table 1). The most significantly altered metabolites and its ratio and classes were ratio of ornithine/L-arginine, ratio of citrulline/ornithine, aminoadipic acid, L-arginine, citrulline, ratio of pyruvic acid/L-alanine, and alpha-ketoisovaleric acid belonging to amino acids, malic acid, fumaric acid and pyrophosphate belonging to organic acids, L-arabitol, sorbitol, L-arabinose, D-threitol, ribonolactone belong to carbohydrates, and tetracosanoic acid and palmitoleic acid belonging to fatty acids.
Significantly altered pathways during chronic persistent asthma
To further investigate disturbed metabolic pathways in chronic persistent asthma patients, we performed the pathway analysis to identify significantly altered pathways based on metabolites involvement in the same biological pathways. In line with metabolites identification, the arginine biosynthesis pathway was found mostly differentially altered (Figure 3A) with down regulation of L-arginine and citrulline, and up regulation of Citrulline (Figure 3B).
Significantly dysregulated amino acids and their ratios in asthma and control cohort
Since the enrichment analysis points to the mostly differentially regulated arginine metabolism pathway. We carried on to evaluate the differential associated metabolites. The volcano-plot combining the strength of both contribution (VIP) and variable reliability (correlation coefficients, Corr.) has been proposed for reliable metabolite marker selection. Of note, both the metabolites L-arginine and citrulline and the metabolites ratio of Ornithine/L-Arginine and citrulline/Ornithine exhibited VIP score greater than 1 with significance corresponding Corr. Values, indicating these metabolites and ratios were reliable metabolite markers for asthma (Figure 4A). We next compared the relative levels of L-arginine, citrulline and ornithine among asthma, asthma with normal eosinophilia, asthma with high eosinophilia and control participants. L-arginine and citrulline, but not ornithine were significantly lower among asthma patients (Figure 4B). Interestingly, the ratio of ornithine/L-arginine is significantly lower, and ratio of citrulline/ornithine significantly higher in the control as compared to asthma with normal or high eosinophilia count (Figure 4C).
Coefficients between significantly altered metabolites and serum eosinophilia counts.
From the metabolites investigation, the altered arginine biosynthesis pathway along with down regulation of L-arginine and citrulline were identified in young chronic persistent asthma patients. We therefore asked the question whether those metabolites were correlated to disease activity during asthma. To this end, we performed correlation analysis between all the significantly altered metabolites and blood eosinophilia counts (Table 2). The ratio of citrulline/ornithine was found significantly negatively correlated to eosinophilia counts, indicating potential interrelation between citrulline metabolism and asthma severity.
Addition of L-citrulline prevented asymmetric dimethylarginine (ADMA)-mediated reduction of NO metabolites in HBECs.
The cohort metabolomics data showed the reduced systemic levels of L-arginine and L-citrulline in young patients with chronic persistent asthma, but how this imbalance leads to reduced nitric oxide (NO) formation in asthma and greater oxidative stress is unknown. We then cultured human bronchial epithelial cells (HBECs) line BEAS-2B and found that the addition of 100 μM of ADMA significantly decreased NOx concentration in HBECs, but L-citrulline could reverse ADMA-mediated reduction in NOx at lower (25 μM) L-arginine concentration in the media (Figure 5A), correlating to the previous study[15]. However, L-citrulline was ineffective at higher (100 μM) L-arginine concentration in the media (Figure 5B) indicating that HBECs could synthesize L-arginine from L-citrulline under specified conditions.