# L-Citrulline: A Non-Essential Amino Acid with Important Roles in Human Health
## 2 L-Citrulline Biochemistry
### L-Citrulline Metabolism
#### 2.2.2 Nitric Oxide Biosynthesis  
Nitric oxide (NO) can be synthesized endogenously in two ways: by a reduction of inorganic nitrate or through NOS enzyme action from Arg, producing CIT as a byproduct (Figure 2) [26]. For this reaction to take place, oxygen must be present as well as reduced nicotinamide adenine dinucleotide phosphate (NADPH) as cosubstrates and the collaboration of cofactors flavin adenine dinucleotide (FAD), flavin mononucleotide (FMN), tetrahydrorobiopterin and heme is required. ASS action can recycle the CIT generated as a byproduct to Arg, thereby increasing the Arg available for NO production [27]. There are three isoforms of NOS, located in different cells of the body: neuronal NOS (NOS1) which is present in neuronal cells, inducible NOS (NOS2) in macrophages and endothelial NOS (NOS3) in endothelial cells [1]. NOS2 is inducible, Ca2+ independent, and expressed by macrophages and other tissues during inflammation or when stimulated with bacterial endotoxins or inflammatory cytokines. NOS1 and NOS3 are constitutive enzymes regulated by intracellular calcium concentration and by the Ca2+/c calmodulin complex. Thus, NOS1 is involved in synaptic plasticity in the central nervous system (CNS), central regulation of blood pressure, smooth muscle relaxation, and vasodilitation via peripheral nitroergic nerves, which are involved in the relaxation of corpus cavernosum and penile section [28]. NO generated from NOS1 acts as a neurotransmitter, while NO generated by NOS3 acts as a vasodilator. NOS3 maintains blood vessel dilation, blood pressure control, as well as several other vasoprotective and anti-atherosclerotic effects. Numerous cardiovascular risk factors cause oxidative stress, NOS3 uncoupling, and endothelial dysfunction in the vasculature. NO regulates vascular tone and blood flow by activating soluble guanylate cyclase (sGC) in the vascular smooth muscle. NO relaxes vascular smooth muscle by binding to the heme moiety of cytosolic guanylate cyclase, activating guanylate cyclase and increasing intracellular levels of cyclic guanosine 3\({}^{\prime}\),5\({}^{\prime}\)-monophosphate, producing vasodilation [29].  
It also inhibits platelet aggregation and prevents the adhesion of leukocytes throughout the endothelial cell layer of blood vessels, inhibiting the proliferation of vascular smooth muscle cells by the inhibition of cytochrome oxidase, regulating the consumption of mitochondrial oxygen [30; 31]. Abnormal vascular NO production and transport cause endothelial dysfunction with several cardiovascular pathologies such as hypertension, atherosclerosis and angiogenesis disorders [19]. NO synthesis depends on Argavailability, which can be affected by: intracellular synthesis of Arg from CIT (which in turn depends on CIT being available), transport of extracellular Arg (cationic amino acid) and arginase activity (which competes with NOS for Arg). Additionally, asymmetric dimethylarginine (ADMA) is an endogenous inhibitor for all three NOS isoforms and has been reported as a risk factor for all causes of cardiovascular mortality [32].