Keywords:
Migraine Disorders - Cystatin C - Headache
Palavras-chave:
Transtornos de Enxaqueca - Cistatina C - Cefaleia
Migraine is a primary headache accompanied by neurological, gastrointestinal and autonomic
disorders that usually appear as severe, throbbing and unilateral. Migraine is characterized
by recurrent headache attacks and affects about 10–20% of the world population[1]. It is well known that migraine attacks have a negative impact on social relationship
and productivity of the patient, both within the family and in the workplace. Depending
on the costs of treatment and loss of labor, migraine brings economic burden to people
and society[2].
As a clinical condition, migraine is diagnosed by the International Headache Society
(IHS) diagnostic criteria. Common symptoms of migraine without aura are as follows:
pain on one side of the head, throbbing pain, sensitivity to light, photophobia, sensitivity
to sound, phonophobia, pain or discomfort that is made worse by physical activities[3]. One study concluded that about 64% of migraines are classified as migraine without
aura type[4]. Vascular, neurogenic and biochemical theories have been proposed in the pathogenesis
of migraine attacks[5]. However, the underlying causes of the pathophysiology of migraine remain unclear.
Sensitive and predictive biomarkers in migraine are important for early diagnosis,
prognosis and treatment.
New investigations suggest that some peptides and cytokines may play a role in the
pathogenesis of migraine[6]. A study performed by Fan et al. revealed that calcitonin gene-related peptide (CGRP)
level can differentiate migraine from non-migraine headache[7]. Another study concluded that pentraxin 3 (PTX 3) levels were significantly higher
in patients with migraine[8]. As seen from the literature, recent studies have focused on the relation between
migraine and new peptides.
Cystatin C (formerly named gamma trace, post gamma-globulin) is a peptide, member
of the cystatin superfamily from proteinase inhibitors, with a molecular weight of
about 13 kDa and composed of 122 amino acids. It is produced by nearly all human cells
and excreted into the bloodstream[9]. As a potent inhibitor of lysosomal proteinases and cysteine proteases, cystatin
C (Cys C) modulates inflammatory response, extracellular matrix degradation and phagocytic
functions. Also, Cys C is a sensitive indicator for various chronic inflammatory diseases
linked to oxidative stress and apoptosis[10]. It has been previously shown that inflammation and oxidative stress play a role
in the pathophysiology of migraine[11].
To the best of our knowledge, the present study is the first to investigate the relation
between the serum Cys C level and migraine. This study was carried out to measure
serum Cys C levels in patients migraine without aura and to determine prognostic value
of Cys C in migraine.
METHODS
Eighty participants were included into the study as two groups: 40 patients with migraine
without aura and 40 healthy controls, who were admitted to the Neurology department
in the period April-November, 2018.
Migraine patients without aura were diagnosed by an experienced neurologist according
to the International Headache Society (IHS) diagnostic criteria (3rd edition, beta version). The diagnostic criteria for migraine without aura were as
follows: 1. At least five headache attacks lasting 4–72 hours under/without treatment;
2. Headache episodes displaying at least two of the following characteristics: a.
Pulsating quality, b. Unilateral location, c. Pain to moderate or severe intensity
to prevent daily living activities, d. Increased pain with physical activity; 3. At
least one of the following symptoms during pain episodes: a. Phonophobia and photophobia,
b. Nausea and/or vomiting (3).
The group of healthy controls was composed by healthy volunteers without any headache
complaint or history of migraine. All voluntary participants were aged 20 to 59 years;
ages were categorized in 10-year intervals. The exclusion criteria of both groups
were hypertension, diabetes, psychiatric diseases, endocrinopathies, anti-inflammatory
and steroid drugs or analgesic medication for at least two months before the study.
The patients and healthy controls had no metabolic diseases before the study. Informations
about participants were obtained from self-reported questionnaires. The visual analogue
scale (VAS) was applied to migraine patients without aura; the rating pain intensity
ranged from 0 (painless) to 10 (the worst painful)[12]. Body mass index (BMI) was calculated as weight (kg) divided by the square of the
height (m), according to the World Health Organization (WHO)[13].
Blood samples (10 mL) taken from each patient were transferred to biochemistry tubes
to determine Cys C levels. The samples were separated after coagulation. Within 20
minutes samples were centrifuged at 3000 rpm for 10 minutes and stored in deep freeze
at -80°C until assay. After that, the serum levels of Cys C were measured by enzyme
linked immunosorbent assay (ELISA). The present study was approved by the Ethics Committee
of Necmettin Erbakan University Medicine Faculty, Turkey and conducted based on the
principles of the Declaration of Helsinki. A written informed consent was obtained
from each participant prior to the study.
Cystatin C measurement
Concentration of serum Cys C levels was measured by using an ELISA kit (Sunred Biological
Technology, China, Human (Cys-C) ELISA Kit:). The minimum detectable concentration
(sensitivity) was 0.427 ng/mL; assay range was 0.6–100 ng/mL, and intra-assay variation
was <8%. We followed the manufacturer’s instructions for all the analyses. The absorbances
of the samples were measured at 450 nm and recorded by Absorbance Microtiter Plate
Reader (ELx800TM, BİO-TEK instruments, USA).
Statistical analysis
The statistical analyses were performed by Statistical Package for the Social Sciences
(SPSS) software package Version 22.0 (SPSS Inc, Chicago, IL, USA). Differences between
migraine patients without aura and healthy control group were evaluated with the Mann-Whitney
U test. Data were expressed as mean ± standard deviation (X±SD). One-way analysis
of variance and the Kruskal-Wallis test were used for age categories, VAS score and
disease duration time. Results were considered to be statitically significant at p<0.05.
RESULTS
In the study, the mean ages of the 40 patients with migraine without aura and of the
40 healthy controls were 37.47 and 38.93 years, respectively. As seen in [Table 1], serum levels of Cys C were 73.88 ng/mL in the migraine without aura group and 24.92
ng/mL in the healthy control group (p=0.000). However, BMI and frequency of migraine
attacks were not significantly different between the two groups (p=0.136 and p=0.198,
respectively). Cys C levels were significantly different among age subgroups within
the migraine group (p=0.049), with no significant difference among healthy controls
(p=0.728) ([Table 2]). As shown in [Table 3], no significant differences were found in Cys C levels at VAS score and disease
duration (p=0.707 and p=0.725, respectively).
Table 1
Comparison of sociodemographics, Cys C levels and some clinical findings between migraine
patients without aura and healthy controls.
|
Parameter
|
MWA
|
Healthy control group
|
p-value
|
|
Age (years), mean
|
37.47
|
38.93
|
-
|
|
Sex (n)
|
Male
|
10 (25%)
|
10 (25%)
|
-
|
|
Female
|
30 (75%)
|
30 (75%)
|
-
|
|
Serum Cys C (ng/mL)
|
Mean
|
73.88
|
24.92
|
0.000*
|
|
n
|
40
|
40
|
|
St. Dev
|
44.01
|
29.66
|
|
BMI (kg/m2)
|
21.64±0.74
|
22.08±0.82
|
0.136
|
|
Frequency (per month)
|
6.025
|
-
|
0.198
|
MWA: migraine patients without aura; *p<0.05.
Table 2
Comparison of the levels of Cys C according to age categories in migraine patients
without aura and healthy controls.
|
Age categories
|
MWA (Cys C)
|
Healthy group (Cys C)
|
|
20–29
|
Mean
|
91.87
|
3.41
|
|
n
|
9
|
8
|
|
St. Dev
|
35.46
|
4.43
|
|
30–39
|
Mean
|
47.86
|
5.82
|
|
n
|
15
|
12
|
|
St. Dev
|
44.13
|
5.86
|
|
40–49
|
Mean
|
87.22
|
3.10
|
|
n
|
11
|
13
|
|
St. Dev
|
36.61
|
4.31
|
|
50+
|
Mean
|
90.22
|
3.97
|
|
n
|
5
|
7
|
|
St. Dev
|
45.99
|
7.67
|
|
p-value
|
|
0.049*
|
0.728
|
MWA: migraine patients without aura; *p<0.05.
Table 3
Comparison of the levels of Cys C according to VAS score and duration of disease in
migraine patients without aura.
|
value
|
df
|
p-value
|
|
VAS score
|
Cys C
|
133.333
|
144
|
0.707
|
|
Duration (MWA)
|
Cys C
|
378.778
|
396
|
0.725
|
|
N
|
40
|
|
|
DISCUSSION
Migraine is a highly prevalent neurological disorder and affects approximately 10–20%
of the world’s population[14]. While migraine reduces the quality of life of individuals, it leads to economic
burden on people and society due to treatment costs. Although the etiology of migraine
has yet to be fully understood, the pathogenesis of the disorder is considered to
be multifactorial. In this sense, vascular, neurogenic and biochemical events play
a role in the pathogenesis of migraine[5]. Among the several hypotheses related to migraine, the leading ones are connected
to the activation of the trigeminovascular system (TS), the cortical hyperexcitability
and the neuronal and glial interactions[15].
Recent studies show that some peptides and cytokines may play a role in the pathogenesis
of migraine and neurogenic inflammation, whereas proinflammatory cytokines are associated
to pain[15],[16]. Neurogenic inflammation (NI) is a locally induced inflammatory response and characterized
by vasodilation, increased vascular permeability, mast cell degranulation, and release
of neuropeptides, including SP and calcitonin gene-associated peptide (CGRP). NI also
plays an important role in the pathogenesis of many diseases, such as migraine, fibromyalgia,
dystonia and multiple chemical susceptibility[17].
Cys C is a nonglycosylated cysteine proteinase inhibitor and consists of 122 amino
acids with a single polypeptide chain[18]. It is encoded as the “housekeeping type” Cys 3 (CST3) gene, and produced by all
nucleated cells at a constant rate. This protein is freely filtered in the renal glomeruli
and reabsorbed and catabolised in the proximal tubules[19]. Besides that, Cys C is expressed in virtually all organs of the body, such as adrenal
medulla, pancreas, thyroid gland, adenohypophysis and brain cortical neurons[20]. Investigations imply that Cys C is a typical secretory protein and can be found
in high concentrations in cerebrospinal fluid, blood, saliva, and semen. Although
the primary structure, physicochemical and immunological properties of Cys C have
been determined, their biological roles have not yet been fully understood. However,
members of the cysteine proteinase family are involved in many cellular events, such
as; catabolism of intracellular proteins, proteolytic degradation of prohormones,
collagen metabolism and penetration of malignant cells into tissues[21].
Several studies concluded that Cys C has a potent regulator role in the inflammatory
process, defense against viral and bacterial infections[22]. In a previous study, it has been suggested that Cys C modulates leukocyte chemotaxis
and phagocytosis and, therefore, plays a regulatory role in the inflammatory process[23]. Moreover, Bäcklund et al. proposed that Cys C have a protective role in chronic
inflammatory disorders, such as rheumatoid arthritis and atherosclerosis[24]. Although Cys C antagonizes TGF-beta signal transformation, it is not an acute phase
protein and is affected by cellular damage and immune response[25].
Newly investigations emphasize that the levels of Cys C are increased or decreased
in many diseases. Xiao et al. reported that higher serum Cys C levels were observed
in cerebral stroke patients compared to healthy controls[26]. Interestingly, Cys C levels are increased in the cerebrospinal fluid of patients
with Alzheimer's disease and Creutzfeldt-Jakob disease[27],[28].
Our study revealed a remarkable increase in serum CysC levels in migraine patients
without aura compared to healthy controls (p=0.000). Zao et al. evaluated the relation
between Cys C and cerebral infarction and determined that Cys C level was significantly
lower in the cerebral infarction group than in the control one (p<0.01)[29]. In a recent study, lower serum levels of Cys C were observed in patients with acromegaly
in comparison to controls[30]. One previous study showed no association between BMI and Cys C levels[31]. In our study no significant difference was observed between migraine patients and
controls in relation to BMI.
Many studies have been conducted on the inflammatory aspects of migraine and associations
between various inflammatory biomarkers and migraine have been noted. The level of
C-reactive protein (CRP), an inflammatory biomarker, has been associated to coronary
heart disease and stroke previously[32], which was also found to be elevated in migraine patients[33]. In addition, a study emphasized that CRP levels are increased in migraine patients
without aura[34]. Interestingly, Shlipak et al. concluded that Cys C levels has a significant linear
correlation with CRP[35]. This may be an explanation for the increased Cys C levels in our study. Association
between inflammation and migraine has been previously observed and serum levels of
fibrinogen and D-dimer have been linked to migraine[36].
Recent investigations suggest that Cys C levels also have a diagnostic value in different
diseases and metabolic disorders, such as chronic kidney disease[37], breast cancer[38], obesity[39] and multiple sclerosis[40].
Predicting circulating biomarkers may facilitate the diagnosis of migraine and evaluation
of prognosis. To the best of our knowledge, we report the first study to determine
the serum levels of Cys C in patients with migraine without aura. However, we acknowledge
that our study has some limitations, the main one being the small sample size.
Future studies should address whether Cys C will have a diagnostic value in migraine.
Serum levels of Cys C may differ during migraine attacks. In this sense, we suggest
that serum Cys C levels during migraine attacks shall be investigated.
In summary, high serum levels of Cys C were found in patients with migraine without
aura. Cys C levels could be an useful marker to screen patients with the diagnosis
of migraine. The determination of Cys C in migraine patients with aura may provide
a diagnostic value.
