Lavoro comune di Federica Collino, Silvia De Francia, Antonina Germano, Simona Perga, Monica Pradotto.
Recent years have witnessed the discovery that amino acids (AA) are not only cell signaling molecules but are also regulators of gene expression and the protein phosphorylation cascade. Among more than 300 AA in nature, only 20 of them (α-AA) serve as building blocks of protein. However, non-protein α-AA (e.g., ornithine, citrulline, and homocysteine) and non-α AA (e.g., taurine and β-alanine) also play important roles in cell metabolism (Amino acids: metabolism, functions, and nutrition, 2009).
The organic compound citrulline is an α-amino acid. It is a key intermediate in the urea cycle, the pathway by which mammals excrete ammonia.
Citrulline is generated from ornithine and carbamoyl phosphate in one of the central reactions in the urea cycle. (Figure 1).
Figure 1. The urea cycle
Citrullina may also be produced from arginine as a product of the reaction catalyzed by NOS family (Figure 2). Arginine is first oxidized into N-hydroxyl-arginine, which is then further oxidized to citrulline concomitant with release of nitric oxide.
Figure 2. NO synthesis
The urea cycle is the metabolic pathway by which most vertebrates excrete nitrogen derived from amino acid catabolism. In the urea cycle, amino groups of urea are donated by carbamoyl phosphate and aspartate, while the carbon atom of urea is supplied by bicarbonate. The last reaction in the urea synthesis is catalyzed by arginase, using arginine and water as substrates. Urea is secreted into the bloodstream, from which it is ultimately eliminated by the kidneys for excretion. The ornithine produced in this last step is shuttled into the mitochondrial matrix, completing the cycle (Figure 1). Otherwise citrulline may also generated as intermediate of NO synthesis mediated by NO syntheses (NOS) (Figure 2).
Several proteins are known to contain citrulline as a result of a posttranslational modification. These citrulline residues are generated by a family of enzymes called peptidylarginine deiminases (PADs), which convert arginine into citrulline in a process called citrullination or deimination. Proteins that normally contain citrulline residues include myelin basic protein (MBP), filaggrin, and several histone proteins, whereas other proteins, such as fibrin and vimentin are susceptible to citrullination during cell death and tissue inflammation.
Figure 3. The citrullination process involves enzymatic conversion of arginine to citrulline.
The enzyme catalyzing this reaction is peptidylarginine deiminase (PAD). During the reaction, the arginine is attacked by the Cys residue of the enzyme establishing a tetrahedral adduct while ammonia is released. The adduct is then cleaved by the nucleophilic attack of a water molecule that regenerates the Cys residue and forms the keto-group.
Detectable antibodies against proteins containing citrulline were found in patients with rheumatoid arthritis. Although the origin of this immune response is not known, detection of antibodies against citrulline (anti-citrullinated protein antibodies) containing proteins is becoming an important aspect in the diagnosis of rheumatoid arthritis.
Rheumatoid arthritis (RA) is the most common autoimmune rheumatic disease, but specific and practicable tests for its diagnosis are lacking. The detection of anti-cyclic citrullinated peptide (anti-CCP) antibodies is becoming an important diagnostic tool for the identification of RA.
Thyroid and Rheumatological Disorders
A close link between systemic arthritis and autoimmune thyroid disease (ATD) exists. Because symptoms of arthritis are common in autoimmune hypothyroidism, joint pain has long been listed as a symptom of hypothyroidism. Some researchers are questioning whether hypothyroidism is responsible for arthritic symptoms and suspect that these symptoms may be caused by other co-existing autoimmune rheumatological conditions. Another theory is that thyroid autoantibodies are responsible for rheumatologic symptoms. This is supported by the fact that antibodies to thyroid hormone are commonly seen in autoimmune rheumatological disorders (Thyroid hormone autoantibodies in primary Sjögren syndrome and rheumatoid arthritis are more prevalent than in autoimmune thyroid disease, becoming progressively more frequent in these diseases,2002).
Overlap Syndrome hypothesis
Just as there are overlapping autoimmune liver diseases, such as autoimmune hepatitis and primary biliary cirrhosis, and an overlap between systemic lupus and undifferentiated connective tissue disorders, some researchers suggest that there are overlapping thyroid and rheumatological disorders that may elude a definitive diagnosis and are characterized by a systemic inflammatory reaction associated with thyroiditis. In fact, it is assumed that patients with joint pain that occurs when thyroid hormone levels are low may be expressing latent conditions of rheumatoid arthritis or undifferentiated connective tissue disease. This theory would explain why not all patients with hypothyroidism exhibit joint pain (Chronic autoimmune thyroiditis and rheumatic manifestations, 2004).
As described before the presence of anti-citrulline antibody in the serum of patient with rheumatoid arthritis (RA), is becoming an import index to define this pathology. Otherwise no real correlation between the anti-CCP antibody and ATD has been demonstrated. The only correlation is based on the concomitance of hypothyroidism and latent conditions of rheumatoid arthritis.
Autoimmune thyroid diseases (ATD) are organ-specific autoimmune disorders characterized by the presence of antibodies against the thyroglobulin, thyroid peroxidase, or thyrotropin receptor auto-antigens ( Autoimmune thyroid disease: further developments in our understanding, 1994). The association between rheumatologic and thyroid disorders has long been known, the most common being the association of rheumatoid arthritis, Sjogren’s syndrome (SS) and ATD (Autoimmune thyroid disease in systemic lupus erythematosus, 2002, Autoimmune thyroid disease in primary Sjogren’s syndrome, 1995). However, the occurrence of rheumatic diseases among patients with ATD remains unclear.
Recently Soy et al., investigated the frequency of rheumatic diseases in patients suffering of autoimmune thyroid diseases (ATD) (Frequency of rheumatic diseases in patients with autoimmune thyroid disease, 2007). They found a high frequency of rheumatic diseases in patients with ATD. The most frequently associated disease was fibromyalgia, which was detected in 31% of patients with ATD. They also found a good correlation between ATD and other autoimmune diseases, other than Sjögren’s syndrome (Frequency of rheumatic diseases in patients with autoimmune thyroid disease, 2007) supporting the importance of a regular checking for rheumatic diseases in patients developing ATD.
Thyroid and NOS function
Thyroid disease has profound effects on cardiovascular function. Hypo- and hyperthyroidism, for example, are associated with reduced and increased maximal endothelium-dependent vasodilation respectively.
Thyroid disorders are accompanied by important changes in haemodynamic and cardiac functions and renal sodium handling. Nitric oxide (NO) plays a crucial role in regulating vascular tone and renal sodium excretion. Therefore understanding whether changes in the activity of NO synthase (NOS) may participate in the cardiovascular and renal manifestations of thyroid disorders is an important issue. In 2002, Nitric oxide synthase activity in hyperthyroid and hypothyroid rats, 2002 observed an up-regulation of NOS activity in tissues primarily related to blood pressure control (like heart, vessels and kidney) in hyperthyroid rats. Previously, Fernandez et al. demonstrated that hyperthyroidism leads to a significant and reversible enhancement in rat liver NOS activity (Influence of hyperthyroidism on the activity of liver nitric oxide, 1997). These data support the idea that increased NO production may participate in cardiovascular manifestations of hyperthyroidism. On the other hand, the group of Quesada demonstrated an heterogeneous tissue response to NOS activity in hypothyroid rats (Nitric oxide synthase activity in hyperthyroid and hypothyroid rats, 2002). More recently, McAllister et al. showed that the hyperthyroid state was associated with increased capacity for NO formation by vascular endothelium, but reduced capacity for responding to NO, compared with the hypothyroid state (Thyroid status and nitric oxide in rat arterial vessels, 2005).
The level of citrulline, the catabolic product of NOS activity, has been demonstrated to be a good parameter in studying NOS activity in pathophysiological conditions, like thyroid disorders (Citrulline immunohistochemistry for demonstration of NOS activity in vivo and in vitro, 2000).
Interestingly, McAllister et al., observed also a lower citrate synthase activity in the soleus muscle of hypothyroid rats and a higher citrate synthase activity in hyperthyroid rats in respect to control animals (Thyroid status and nitric oxide in rat arterial vessels, 2005). The mechanism of citrate synthesis from acetyl-CoA and oxaloacetate should be favored in the hyperthyroid state as a consequence of the elevation in mitochondrial oxaloacetate concentration and the decline in the level of long chain acyl-CoA, an inhibitor of the enzyme (Starvation-induced changes of hepatic glucose metabolism in hypo- and hyperthyroid rats in vivo, 1981). Increasing level of intra-mitochondrial concentration of citrate, has been correlated with an inhibition of citrulline synthesis (Relationship between intramitochondrial citrate and the activity of carbamoyl-phosphate synthase (ammonia), 1977). This inhibition was related to a reduced activity of carbamoyl-phosphate synthase, important enzyme of the urea cycle, involved in carbamoyl phosphate generation, used as substrate for the citrulline production (Relationship between intramitochondrial citrate and the activity of carbamoyl-phosphate synthase (ammonia), 1977).
Indeed Marti et al., found that the capacity of the liver to synthesize urea was increased in hypothyroid rats, as well the activities of the urea cycle enzymes (Effect of thyroid hormones on urea biosynthesis and related processes in rat liver, 1988). Isolated hepatocytes from these rats showed an increased capacity for urea synthesis. In hyperthyroid rats the picture was described as more complicated, since there was no change in the urea-synthesizing capacity of the liver, although there were changes in some enzymes and metabolites.
In different metabolic pathways, citrulline deregulation may be responsible (directly or indirectly) or be used as marker of different pathological states observed in hypothyroid or hyperthyroid patients, such as the concomitance of autoimmune rheumatological conditions, cardiovascular and renal diseases.