Figure 1. Serum and urine proteomic biomarkers of the medication-overuse headache.
L-PGDS is expressed in various tissues, such as the brain, retina, cochlea, and male reproductive organs, and it is found in different biological fluids, such as cerebrospinal fluid (CSF), ascites, seminal plasma, serum, urine, and amniotic fluid. Alongside hematopoietic-type PGDS (H-PGDS), L-PGDS is related to a group of prostaglandin D synthases (PGDS) responsible for converting PGH2 to PGD2 
, which is involved in a variety of central nervous system (CNS) functions, such as sedation, nonrapid eye movement (NREM) sleep and PGD2-allodynia. In addition to synthesizing PGD2, a potent endogenous nociceptive modulator 
within the cells, in the extracellular space and body fluids L-PGDS binds various small nonsubstrate lipophilic molecules such as retinal, retinoic acid 
, bilirubin, biliverdin 
, gangliosides 
, amyloid β peptides 
. Therefore, L-PGDS can potentially be a promising proteomic biomarker as the entire prostaglandin system plays a huge role in pain and central sensitization development.
Vitamin D-binding protein (VDBP) is a monomeric glycoprotein synthesized and secreted predominantly by the liver. VDBP can be found in various body fluids, such as plasma, ascitic fluid, CSF. It was originally known as a group-specific component (GC) because of its worldwide polymorphisms 
. It is also known as macrophage-activating factor (GcMAF/DBP-MAF) because it initiates macrophage activity, a key part of the host defense system 
. VDBP has multiple functions, such as actin binding and neutrophil chemotaxis 
. However, the main function of VDBP is to bind vitamin D and its plasma metabolites and transport them to target tissues. There are several studies showing the relation between Vitamin D deficiency or insufficiency and chronic headache development 
. Possible mechanisms include the anti-inflammatory role of vitamin D, specifically, decreased production of inflammatory substances which activate trigeminal nerve, the main structure involved in migraine development 
. Moreover, by inhibiting nitric oxide (NO) synthase expression, vitamin D reduces the production of NO, a key endogenous mediator in headaches, such as migraine 
and tension-type headaches 
, deficient levels of VDBP in MOH patients can be associated with a decreased level of vitamin D, which can be one of the mechanisms of chronic headache development.
Apolipoprotein E (APOE) is a multifunctional protein which participates in lipid metabolism and carries lipids in different tissues of the body, including both the peripheral and the central nervous system 
. APOE mediates the binding of APOE-containing lipoproteins and lipid complexes to specific cell-surface receptors, such as the low-density lipoprotein (LDL) receptors, the LDL receptor-related protein (LRP), the very low-density lipoproteins (VLDL) receptor, the APOE receptor-2, gp330. Moreover, APOE demonstrates genetic polymorphisms containing three common alleles, ε2, ε3, ε4 that encode three isoforms (APOE2, APOE3, APOE4) 
. According to the available literature, NO synthesis is dependent on APOE polymorphisms. Thus, the APOE4 gene is involved in the production of NO through increased uptake of arginine in the microglia compared to APOE3 gene 
. APOE polymorphisms also influence the expression of cytokines which play a huge role in inflammation, pain modulation and central sensitization 
Alpha-1 antitrypsin (A1AT), also known as alpha-1 proteinase inhibitor, is an acute phase reactant and serine protease inhibitor (serpin) whose targets are elastase, plasmin, thrombin, trypsin, chymotrypsin, plasminogen activator, and is mostly produced in the liver and expressed by hepatocytes 
. It is important to note that A1AT inhibits NO production 
, a key molecule in the pathophysiology of primary headaches 
Hemopexin (HEMO) is an acute-phase plasma glycoprotein with the highest affinity to heme among all known proteins and is responsible for transporting heme from the plasma to the liver for breakdown and iron recovery. Moreover, it has intracellular antioxidant activities and, therefore, is involved in protecting cells from scavenging and oxidative stress. HEMO is expressed in various tissues such as the nervous system, skeletal muscle, retina, kidney, but mainly in the liver 
. HEMO was found at significantly different levels in the CSF of patients with leptomeningeal metastases of breast cancer with neurological complications 
and with other neuropathologically confirmed diseases 
Haptoglobin (HPT) is an acute-phase protein whose main function is to bind hemoglobin (Hb) during hemolysis, forming the Hb-Hp complex, which is crucial for the elimination of free Hb by the macrophage CD163 scavenger receptor expressed on the liver Kupfer cells surface, therefore preventing kidney damage. HPT also acts as an antioxidant and plays a huge role in the neutralization of oxidative stress.
Retinol binding protein (RETBP) is specific carrier protein whose only known function is to transport retinol (vitamin A) from hepatic stores to target tissues 
Transthyretin (TTHY) is a homotetrameric protein mostly produced in the liver and choroid plexus of the brain. The main function of TTHY, the transport of thyroxine and RETBP, is well-known. However, other functions of this protein, namely in the nervous system, have emerged.
Urinary biomarkers are increasingly used in the diagnosis, classification and prognosis of kidney diseases. Uromodulin (UROM), also known as Tamm-Horsfall protein, is the most abundant protein in urine. It can also be found in serum in lower amounts. UROM is exclusively produced by renal epithelial cells in the kidney. Amongst all the functions of UROM, the most important ones are the regulation of ion transport in the thick ascending limb, immunomodulation and protection against urinary tract infections and kidney stones 
. In clinical practice, UROM is used as a valuable biomarker of tubular damage and kidney diseases, including drug-induced nephrotoxicity caused by medication overuse.
Alpha-1-microglobulin (AMBP) is one of the urinary microproteins that are becoming more and more important in clinical diagnostics and practice. AMBP is a tubular glycoprotein, mostly expressed in liver, blood and kidney, which is used for detecting acute lesions of proximal tubules. It was first discovered 40 years ago in human urine 
. Functions of AMBP are still unknown. However, some reports have suggested that AMBP may be involved in oxidant-scavenging and have enzymatic reductase properties as an antioxidant 
. Altered plasma and urine levels of AMBP are usually markers of impaired liver or kidney functions. Therefore, nowadays, urinary AMBP is considered as a promising inexpensive alternative biomarker for the early detection and diagnosis of urinary tract disorders 
, including kidney damage, caused by acute headache medication overuse.
Zinc-alpha-2-glycoprotein (ZAZG) is a single-chain polypeptide secreted in various body fluids, such as serum and urine. Despite the fact that functions of ZAZG still remain unknown, some reports suggest that ZAZG has a lot of important functions in the human body, including fertilization and lipid mobilization, therefore it is considered as a multidisciplinary protein 
. As its structural organization and folding characteristics are similar to the MHC class I antigen-presenting molecule, it may have a biological role in the immune response. ZAZG is used as a tumor biomarker for various carcinomas 
. However, some immunohistochemical analyses have shown predominant expression in the kidney tubules of the human 
. Therefore, urinary ZAZG may be a potential biomarker of renal damage, including drug-induced nephrotoxicity.
Inter-alpha-trypsin heavy chain H4 (ITIH4) is a liver-produced glycoprotein belonging to the liver-restricted serine protease inhibitor family. Its biological role is still unknown, as ITIH4 is cleaved in a number of different pathologies. However, it plays important role in various biological processes, such as inflammatory responses to trauma, liver formation or regeneration 
Immunoglobulin kappa constant (IGKC) is a constant region of immunoglobulin light chains, also known as antibodies, membrane-bound or secreted glycoproteins produced by B lymphocytes. The main function of IGKC is to serve as receptors which, upon the binding of a specific antigen, trigger the clonal expansion and differentiation of B lymphocytes into immunoglobulin-secreting plasma cells. Secreted immunoglobulins play a significant role in the mediation of the effector phase of humoral immunity, which results in the elimination of bound antigens 
Nonsecretory ribonuclease (RNAS2) is a pyrimidine specific nuclease with a slight preference for cytotoxin and helminthotoxin. RNAS2 is selectively chemotactic for dendritic cells and possesses a wide variety of biological activities.
Cystatin C (CYTC) is a nonglycosylated basic protein encoded by the CST3 gene found in all nucleated cells. CYTC was first discovered in 1961 and formally named in 1984. CYTC is a potent inhibitor of lysosomal proteinases and extracellular inhibitors of cysteine proteases that play a huge role in human vascular pathophysiology 
. Nowadays, CYCT is used as a more accurate alternative to serum creatinine for measuring glomerular filtration rate (GFR), one of the main parameters in the estimation of kidney function 
. CYCT is increasingly used as an earlier biomarker for acute kidney injury, a superior marker of kidney transplant function, cardiovascular disease risk and transplant failure. Therefore, CYTC may be a promising proteomic biomarker for MOH and drug-induced nephrotoxicity, caused by acute headache medication overuse.