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.