**5. Ascorbic acid as a co-factor of enzymes**

Ascorbic acid is an important cofactor in numerous enzymatic reactions such as in the biosynthesis of collagen, neuropeptides, and in the regulation of gene expression. The function of ascorbic acid as a cofactor is also associated with its redox potential. Ascorbic acid aids mixed-function oxidases in the synthesis of several critical biomolecules by maintaining enzyme-bound metals in their reduced forms [12]. These enzymes are either monooxygenases or dioxygenases.

One of these enzymes is dopamine β-hydroxylase (DBH). Ascorbic acid serves as a cofactor for dopamine β-hydroxylase in the conversion of dopamine to norepinephrine through hydroxylation of dopamine in different neuroendocrine tissues synthesizes [35]. Synthesis of catecholamine is ascorbic acid-dependent as ascorbic acid levels are known to be in the millimolar range in the adrenal gland. Ascorbic acid in chromaffin granule is secreted together with catecholamines from chromaffin-cultured cells and in vivo by human adrenal glands, the latter in response to adreno-corticotrophin stimulation [36]. Ascorbic acid can act as a neuromodulator. Its release into the extracellular fluid of the brain, it regulates dopaminergic and glutamatergic transmission. Ascorbic acid is released from glutamatergic neurons as a result of the high-affinity glutamate transporter-exchanging ascorbic acid for glutamate during the glutamate reuptake process [37]. This hetero-exchange process ensures a comparatively high quantity of extracellular ascorbic acid in the forebrain, which may also occur in glial cells [38]. Therefore, it protects nerve cells against glutamate excitotoxicity. As the interaction between ascorbic acid and glutamate is crucial in neuron metabolism, ascorbic acid acts as a metabolic switch that modulates neural metabolism between resting and activation periods [39]. In addition, glutamate from astrocytes in the CNS regulates ascorbic acid release [40]. Several studies have reported the neuroprotective functions of ascorbic acid. Akbari *et al*. [41] observed that after exposure of rats to radiofrequency waves produced by the BTS mobile antenna, the rats' brains were preserved from oxidative stress with the help of ascorbic acid. Naseer *et al.* [42] showed how ascorbic acid can hinder part of the negative impacts of nervous deterioration caused by PTZ in the brain of adult rats. Another related study reported how the neuroprotective activity of ascorbic acid in matured rats may be its ability to decrease the rate of lipid peroxidation and its higher enzymatic effect following seizures and epileptics state caused by pilocarpine [43]. Shokouhi *et al*. [44] indicated how ascorbic acid possesses a remarkable impact on decreasing the rate of malondialdehyde formation after nerve injury in rats and may have the potential for healing, and the degree of protection does not depend on the dose.

As a cofactor for the enzyme folate reductase, it participates in the conversion of methemoglobin back into hemoglobin and retains folic acid in its reduced form of tetrahydofolic acid, which is necessary for red blood cell maturation. Ascorbic acid stimulates the initial step in cholesterol metabolism to bile acids through the 7-alphahydroxylase enzyme. This function may have importance in the creation of gallstones and the maintenance of normal blood cholesterol levels. It is also involved in tyrosine metabolism, carbohydrate metabolism, synthesis of proteins, resistance to infections, and cellular respiration [45].

Ascorbic acid is also an important co-factor for lysyl hydroxylase and prolyl hydroxylase, enzymes essential for collagen biosynthesis, the most abundant extracellular protein. Ascorbic acid is a required element for the fusion of hydroxyproline and hydroxylysine in collagen. Hydroxyproline acts to hold the triple helix of collagen. The absence of hydroxyproline can lead to destabilization of the collagen structure that is not produced at normal rates from the cells. Hydroxylysine is necessary for the process of development of intermolecular crosslinks in collagen. Furthermore, certain carbohydrate remnants are connected to collagen glycosidically *via* hydroxylysine, an activity that could be essential in the control of crosslink production [46, 47]. Normally, ascorbic acid is considered to control collagen formation *via* its action on prolyl hydroxylation.

Ascorbic acid supplementation has been found to improve components of the human immune system such as antimicrobial and natural killer cell activities, lymphocyte proliferation, chemotaxis, and delayed-type hypersensitivity. Many cells of the immune system such as phagocytes and t-cells can accumulate ascorbic acid as they are needed to perform their task [48]. Recently, the importance of ascorbic acid in Tet-mediated DNA demethylation has been demonstrated to restrain the growth of acute myeloid leukemia [49], to promote homeostasis of hemopoietic stem cells [50], and to boost the lineage stability of regulatory T (Treg) cells [51]. Thus, ascorbic acid deficiency can result in decreased resistance against certain pathogens, while a higher level enhances several immune system parameters. Ascorbic acid concentrations in the plasma and leukocytes rapidly decline during infections and stress.
