Discussion
BD is a chronic, systemic inflammatory vasculitis presented with oral and genital ulcers, uveitis and arthritis (10). VBD is known to be seen in nearly 10-40% of BD patients and is a major cause of morbidity and mortality in BD (11). The diagnosis of BD disease is challenging because of the lack of specific or serological biomarkers. Especially, the detection of vascular involvement in BD is presented by clinical vascular incidences like deep vein thrombosis, pulmonary embolism or arterial aneurysms. Unfortunately, there is no predictive or identifying biomarker for early detection and prevention of disease (12,13). Metabolomics is a new and promising method for the detection of disease-specific biomarkers and the metabolomic approach has come to the front for identifying biomarkers in various rheumatic diseases such as lupus, rheumatoid arthritis (RA), ankylosing spondylitis and also BD ( 14-17).
In this study, we examined the metabolomic profile changes in BD patients who did or did not have vascular involvement. A total of 168 metabolites in the plasma samples of three study groups (BD, VBD and Healthy controls) were identified. A total of 29 amino acids and biogenic amines were detected among participants.
This study revealed significant amino acid metabolic disturbances among BD patients and healthy controls. When we compared the overall metabolomic profiles of BD patients with or without vascular involvement with healthy controls in terms of amino acid biosynthesis and utilization pathways; we observed that the most common changes have occurred in glutamate, glutamine, alanine, α-ketoglutarate, cysteine, oxaloacetate and homoserine levels (Figure 1). The levels of all these amino acids and amino acid derivatives were higher in the control group and decreased in BD patients. The decrease in levels of these amino acids and their derivatives in BD patients may suggest the utilization of these amino acids in this patient group. The common amino acids affected in BD patients with and without vascular involvement are glutamate, α-ketoglutarate and homoserine. Besides being a major amino acid in protein synthesis, glutamate is also involved in various metabolic processes in a variety of organisms. Glutamate is produced by the conversion of α-ketoglutarate by glutamate dehydrogenase (GHD) in the citric acid cycle which is the key element of oxidative phosphorylation in the human body. This can be a sign of decreased aerobic respiration in BD patients, which may be caused by tissue hypoxia (18). Another key function of glutamate is being a building block of glutathione which is a tripeptide composed of cysteine, glycine and γ- glutamate. Glutathione functions in redox homeostasis of the cell and protects the cells against oxidative stress (19). In previous studies, it has been shown that serum antioxidant status decreases in patients with BD (20). The findings of our study are in concordance with these results. The fold changes in both α-ketoglutarate and glutamate were similar in both BD patients with and without vascular involvement when compared to healthy controls. L-cysteine levels were also found to be lower in BD patients without vascular involvement when compared to healthy controls. As cysteine is another amino acid found in the structure of glutathione, this also supports the idea that antioxidant mechanisms are diminished in BD patients. Homoserine was another commonly decreased metabolite in both BD and VBD patients compared to healthy controls. Homoserine is the first step precursor of methionine synthesis. Succinylation of homoserine serves to activate the molecule to condense with cysteine to form cystathionine (21). Decrease in levels both cysteine and homoserine in BD patients can be accepted as a sign of interrupted amino acid synthesis in these patients. Another commonly decreased substance in all BD patients was 5-Oxoproline (L-pyroglutamic acid). This natural amino acid derivative is in fact a metabolite in the glutathione cycle that is converted to glutamate by 5-oxoprolinase (22). The first two steps in the glutathione cycle, are for glutathione biosynthesis. The oxidized glutathione (together with it’s conjugates) can be pushed out of the cytoplasm by specific transporters for oxidized glutathione or it’s conjugates under oxidative or xenobiotic stress. This mechanism is also helpful in the regulation of cellular redox potential (23, 24). The lower levels of oxoproline together with cysteine and glutamate in BD patients once more point out altered glutathione metabolism, reduced redox potential and increased oxidative stress in this patient group.
Methyl myristate levels were decreased in all BD patients regardless of vascular involvement. Myristic acid (tetradecanoic acid) is a commonly known saturated fatty acid that is known to serve as a lipid anchor in biological membranes. In the body myristic acid may have positive effects on high-density lipoprotein (HDL) cholesterol and so improves HDL/total cholesterol ratio (25). It is known that BD is a disease that affects vascular structures and in one of our previous studies, we also focused on increased atherogenic risk in patients with vascular BD (26). HDL is known as the cardioprotective lipid in circulation. Decreased levels of HDL increase the incidence of cardiovascular events. The findings of the present study about decreased tetradocanoic acid in BD patients are in concordance with previous studies that it will cause a lowering in circulating HDL levels and increases atherogenic risk.
The glutamine level decreased in BD patients while the alanine level decreased in VBD. Both glutamine and alanine are synthesized in the skeletal muscle from the essential branched-chain amino acids (BCAAs). These BCAAs (valine, leucine and isoleucine)are known to have key functions in severe illness. Glutamine is the most abundantly found amino acid in free form within human body, and it comprises nearly 20% of the free amino acid pool in circulation. Glutamine is also important for fast dividing cells like red blood cells and cells of the immune system and it also functions in nucleotide synthesis. Alanine is also important in gluconeogenesis (27, 28). In the disease state, protein turnover is increased and BCAAs are degraded which causes an increase in glutamine and alanine levels. But the demand for alanine and glutamine in catabolic illness increases so their use exceeds their production and there occurs starvation in alanine and glutamine in plasma and tissues (29). A number of studies have revealed the protective effects of alanine-glutamine in inhibiting inflammatory response and preventing tissue injuries. Also significantly, several reports have shown that alanine-glutamine have protective roles towards ischemia–reperfusion injury. They do this by improving microcirculation, reducing inflammation, and elevating GSH levels in the liver (30-32). So, these findings are in concordance with the decreased GSH levels and increased oxidative stress in BD patients we previously mentioned above.
We observed a decrease in malic acid and citramalic acid levels in BD and VBD groups respectively. Citramalic acid is a malic acid analogue with an extra methyl group. These organic acids are basically found in fruits and vegetables but in recent years, they were found to have pharmacological effects such as anti-inflammatory, anti-oxidant responses anti-platelet aggregation, and direct cardioprotective effects (33-35). We can conclude that these organic acids may function in protection during ischemia/reperfusion injury. The decrease in levels of malic and citramalic acid in BD patients also supports our hypothesis that these patients are more prone to inflammation-based atherosclerotic and cardiovascular diseases.
Shikimic acid levels were lower in VBD patients when compared to the control group. Shikimic acid pathway is the common pathway for biosynthesis of aromatic amino acids and metabolites like folic acid cofactors, coenzyme q10, together with vitamins E and K. This biosynthetic pathway is found in bacteria, fungi, and higher plants but it does not present in mammals. However, the human gut flora contains more than 100 trillion bacterial colonies (36). The disruption of gut microflora may result in decreased antioxidant activity in VBD patients in our study.
2-hydroxybutyric acid, an intermediate produced during threonine metabolism and glutathione synthesis, was higher than control subjects in both patient groups. It is known to be elevated in patients with energy metabolism deficits or in lactic acidosis. Its increase may be attributed to increased oxidative stress in BD patients (37).
Trehalose levels were also found to be elevated in both patient groups. Trehalose is a non-reducing disaccharide of glucose. In recent years, it has been found to be a protective agent against reactive oxygen species (ROS), and chornic inflammation. As BD is a chronic, auto-inflammatory disease and increased oxidative stress seems to be a key feature of this disease; an increase in trehalose levels can be explained as a compensatory response to increased oxidative stress and inflammation (38, 39).
The citramalic acid, oxaloacetic acid, homoserine, and shikimic acid levels were lower in VBD group compared to BD. As discussed above, the changing amino acid profile can be attributed to inflammatory mechanisms and lower antioxidant capacity. The comparison between the two patient groups also supports our previous findings. As we highlighted, the levels of these amino acids were lower than that of the control group in BD patients. Even lower levels are observed in VBD patients. Especially low levels of oxaloacetate and homoserine point to a deeper impairment in amino acid synthesis in these patients. As BD is also a chronic, inflammatory disease and vascular involvement alleviates the inflammatory processes, our findings are in concordance with previous studies.
The further decreased level of citramalic acid and schimic acid in VBD patients indicates that VBD patients are more prone to atherosclerosis as oxidation and inflammation trigger endothelial damage (26,40). Since both organic acids have anti-inflammatory and cardioprotective effects.
The most promising metabolites revealed in our study can be listed in glutathione synthesis, which is the major thiol pool of antioxidant mechanisms in the human body. Homoserine, oxaloacetate, citramalic acid and shikimic acid can be listed as the most prominent metabolites as they can be discriminative not only between BD patients and healthy controls but also between BD and VBD patients.
Our study does pose some limitations. One of which is a single-center study. Also, the patient number is relatively limited. But to our knowledge, there are no published reports on metabolomic profiles in BD patients with and without vascular involvement yet. And our findings propose some potential biomarkers especially related to oxidative stress pathways in BD patients.