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Advanced Glycation End (AGE) products are formed both endogenously in humans and found exogenously in food products. AGEs have been implicated in pathophysiological conditions such as diabetes, aging, neurological diseases, kidney disease, cataracts and cardiovascular diseases. Thus, it is important to control the production of endogenous AGE. Previous studies using natural compounds such as vitamin C, vitamin E, quercetin, green tea and aminoguanidine have shown to decrease the formation of AGE in vitro studies. Thus, our study determined the inhibiting efficiency of L-ascorbic acid (vitamin C) on formation of AGE in vitro.
In the year 1912, Louis-Camille Maillard for the first time described a non-enzymatic reaction between a reducing carbohydrate and an amino acid. This reaction was called as Maillard reaction (Hellwig M & Henle T, 2014). The mixtures of amino acids and sugars on heating together become intensely brown in color during cooking, baking and roasting process (Luevano-Contreras C & Chapman-Novakofski K, 2010). Maillard reaction is observed to occur even in the organisms and has pathophysiological significance in diabetes and ageing process. Thus, after hundred years of its discovery, even today this complex reaction has retained its relevance.
The advanced glycation end products are the heterogenous, complex group of compounds. These are formed in a non-enzymatic reaction when a reducing sugar reacts with the amino acid in the proteins, DNA and lipids (Brownlee M, 1984). This reaction occurs endogenously (in humans) and exogenously (in foods). It is also called as Glycation reaction or Maillard reaction and is different from glycosylation reaction which is catalyzed enzymatically.
Formation of AGEs by Maillard reaction is a complicated multistep process which takes place in three phases. It was described in detail by John E Hodge (Hellwig M & Henle T, 2014). In the first phase the sugar (glucose) gets reacts with a free amino acid (mainly a lysine and arginine) of a protein to form a Schiff’s base. The starting of this reaction depends on glucose concentration. If the glucose concentration is high then it takes place immediately. The second phase is characterized by an unstable Schiff’s base undergoing chemical rearrangement which takes places over few days to form Amadori products. These are ketoamines and are very stable glycation products. During the third phase, the Amadori products undergo complicated chemical rearrangements and form crosslinks with the proteins. The formation of protein adducts interferes with the protein functions. This process occurs over weeks or months and is irreversible. Finally formed brownish products are called as Advanced Glycation end (AGEs) products. Some of these AGEs are fluorescent. Examples of AGEs include carboxymethyllysine (CML), pentosidine, carboxymethyl-hydroxy-lysine and pyrraline. Some of the AGEs like pentosidine are fluorescent and can be easily detected. Similarly, glycalated haemoglobin (Hb1Ac) is an AGE product observed in diabetes (Raghav A, 2016).
AGEs are known to occur in two forms as high molecular weight AGE protein aggregates of size upto 650kDa and low molecular weight AGE of size 12kDa (Grzebyk E & Piwowar A, 2016).
In addition to Maillard reaction, there are other pathways which give rise of AGEs. Oxidative stress which leads to auto-oxidation of glucose and peroxidation of lipids. In addition, in the polyol pathway the glucose is converted to sorbitol by the enzyme aldose reductase. This sorbitol is then converted by action of sorbitol dehydrogenase to fructose.
AGE exert their activity by two different mechanisms. One which is independent of the receptor and the other which involves the receptor for advanced glycation end products (RAGE) (Ahmed N, 2005). One AGE gets associated with receptors RAGE, it stimulates activation of mitogen-activated protein kinases (MAPKs) and phosphatidylinositol-3-kinase (PI3-K) pathways. Both these pathways in turn activate the transcription factor NF-κβ (nuclear factor-kappa beta). Once it is formed, it gets translocated to the nucleus. It then causes transcription of genes involved with production of cytokines, growth factors and adhesive molecules. Thus, NF-κβ increases RAGE expression which causes more production of inflammation promoters. In addition, AGE-RAGE interaction activates NAD(P)H oxidase and it increases oxidative stress (Wautier MP,2001).
The concentration of AGE in body depends on their endogenous production, exogenously intake and their excretion from the body. The enzymes glyoxalaseI, II and carbonyl reductase, receptors (AGER1) have been shown to play role in detoxification and in prevention of glycation effects. It is known that renal excretion effectively removes excess of AGEs from the body.
Exogenous AGE enter our body through the food which we eat or these can be endogenously produced at lower rates during normal metabolic processes. The rate of AGEs formation is determined by the environmental factors such as diet and smoking habit. The main factors which are important for formation of AGEs are the rate of protein turnover for glycooxidation, the degree of hyperglycemia and extent of oxidative stress in the environment. If any of these conditions is present, then it leads to glycation and oxidation of both intracellular and extracellular proteins. Once AGEs are formed they are irreversible. Presence of AGEs may lead to modification of extracellular matrix (ECM) proteins, alter the action of hormones, cytokines and free radicals via attachment to the cell surface receptors and also bring changes in the function of intracellular proteins. They alter protein half-life, immune system and enzyme functions.
The harmful effects of AGEs in different tissues are due to their chemical, pro-oxidant and inflammatory actions. AGEs are resistant to proteolysis and are more reactive, which results in synthesis of free radicals, reactive oxygen species, nitrogen and chlorine species and protein radicals. Further, these macromolecules can undergo subsequent oxidative modifications themselves which leads to disturbances and different pathological conditions such as Cardiovascular disease, renal disease, diabetes, cataracts, Parkinson’s disease, Alzheimer’s disease, arthritis, ageing and several other pathological conditions (Dasgupta S, 2015).
The complications of diabetes are the main concern. It is known that hyperglycemia increases the glycation process which produces AGEs. The haemoglobin A1C is a well-known early glycation product and is used as an indicator of regulation of blood glucose levels in case of patients with diabetes. It is reported that increase blood glucose levels leads to increased glycation in red blood cells, endothelial cells, and eye lens cells. The long term complications are result of protein alterations due to glycation and result in irreversible tissue damage (Goldin A, 2006). The glycation of proteolytic enzymes in diabetes decrease its efficiency to function properly. There is reduction in aldolase reductase activity and deactivation of superoxide dismutase (Oda A, 1994) and decreased insulin binding to the insulin receptors. All these modifications contribute to diabetic complications such as cataracts, nephropathy, vasculopathy, retinopathy, delayed wound healing and atherosclerosis (Brownlee M, 1984). One of the mechanisms implicated in progression of diabetes induced complication is due to binding of AGE with RAGE (receptors) (Ahmed N, 2005). This association initiates intracellular signalling and leads to disruption of cellular functions.
Currently, a lot of concern regarding fructose in development of metabolic diseases has been reported. The rate of intracellular glycation of fructose is rapid than that of glucose (Suárez G, 1989) and fructose is important precursor for intracellular AGE formation. Thus, fuctosamine is clinically used as an indicator for short term control of blood sugar in diabetic patients. The reduction of frcutosamine in diabetic patients is therapeutic way to delay vascular complications.
Overall, the increased amounts of AGEs during hyperglycemia increase pathogenesis and aggravate biochemical disturbance and clinical complications of diabetes.
A number of mechanisms have been proposed for AGE related cardiovascular disease. First one is due to crosslinking of collagen which leads to collagen–AGE. This will produce stiffness in blood vessels (Sims T.J, 1996). Another mechanism is glycation of the low density lipoproteins (LDL). The LDL-AGE cannot attach to the LDL receptors and reduces their uptake into the cells. The crosslinked LDL attaches to the collagen on the arterial wall and this leads to atheroma (Bucala R, 1994). The third mechanism is decreasing the nitric oxide (NO) activity which causes damage to the cardiovascular system (Xu B, 2003). Thus, the accumulation of AGEs can be probable reasons for different cardiovascular changes such as vascular vessels stiffening, diastolic dysfunction and endothelial dysfunction.
The correlation between renal disease and AGEs has been previously investigated. There is excessive accumulation of AGE and Advanced Oxidation Protein Products (AOPPs) and they have been implicated in kidney dysfunction (Bohlender J, 2005, Witko-Sarsat V, 1999). These were found in high levels in patients with uremia and end stage renal disease.
The exact causative factor of this disease is still not known. A study found that the expression of AGE and RAGE were found to be elevated in the brain samples of the patients with Alzheimer ’s disease. Further, some studies have indicated that RAGE mediates the blood brain barrier transport of amyloid peptides (Candela P, 2010). Hence AGEs and aging may have association in pathogenesis of this disease.
Oxidative stress is one of the etiological factors of Sarcopenia (loss of muscle mass and strength) and this leads to formation of AGEs. It was found by a study that pentosidine concentration was very high in a group of older population as compared to the younger population (Haus JM, 2007). Further women with higher amount of AGE show more muscle weakness (Dalal M, 2009). However, none of the studies could conclusively indicate that AGE leads to sacropenia.
Thus, the presence and accumulation of AGEs in many different cell types affect extracellular and intracellular proteins structure, functions and properties.
Aging is defined as the progressive accumulation of damage to an organism over the time which leads to disease and then death. One of the risk factors of aging is the generation and presence of high amounts of AGEs in older aged individuals (Luevano-Contreras C & Chapman-Novakofski K, 2010). Further, formation of AGEs has been implicated in loss of bone density and lessening of muscular mass.
Overheating of heating leads to protein degradation. It also leads to Maillard reaction which adds favour, color and aroma (Luevano-Contreras C & Chapman-Novakofski K, 2010). In food industry caramel production, bread baking occur by Maillard reaction. The protein containing foods when overheated lead to increased amounts of AGE which get consumed by humans (Goldberg T, 2004). Foods obtained from the animals are high in fat and proteins are usually AGE-rich and form more AGE during process of heating. Different processes such as broiling, roasting, frying and grilling increase AGE production in these foods. Thus, AGEs are exogenously introduced into the body which get transported into blood circulation. Some adhere to the tissues while only one third are removed by the kidneys. Some of the AGEs obtained due to protein and lipid glycooxidation are inert Carboxylmethyllysine (CML) and highly reactive derivatives of methyl-glyoxal (MG) (Fu MX, 1996, Abordo EA, 1999). The foods such as vegetables, fruits and milk which are carbohydrate rich contain relatively less amounts of AGEs and these remain low even after cooking and do not harm us much (Uribarri J, 2010). However, one can take precautions by restricting diet of AGEs and by physical exercises both can reduce circulating AGE in the body. Also one should try other methods of cooking such as steaming, boiling, poaching and stewing. One can also make use of acidic marinades, such as lemon juice and vinegar before cooking which limits the dietary AGE generation (Uribarri J, 2010).
There are reports of increased AGEs in smokers as compared to the non-smokers. Tobacco curing under certain environmental conditions lead to formation of glycation products (Cerami C, 1997). These are found in aqueous tobacco extract and in tobacco smoke in form that react rapidly with proteins to from AGE in vivo. These were termed as glycotoxins and exposure to these glycotoxins was found to increase the incidence of atherosclerosis and lead to high prevalence of cancers in smokers.
There are a number of studies conducted to find a compound with anti-glycation properties which can be used in treatment. Some of the therapeutic agents have been studied as blockers of AGE-crosslinking or inhibitors of AGE actions in, in-vitro, in-vivo and human models. Metformin used in treatment of diabetes patients has shown to decrease the circulating levels of AGEs (Isoda K, 2006). A study conducted showed that an angiotensin receptor blocker Candersatan which when administered for three months reduced levels of CML in patients with diabetic renal disease (Saha SA, 2010). One of the most successful agent has been aminoguanidine which limits process of early glycation and that of AGE formation (J.A. Vinson & T.B. Howard III, 1996). Aspirin (Urios P, 2007), acetaminophen, ibuprofen have shown to decrease the glycation of lens proteins and prevent diabetic cataracts in rats.
Further some studies have shown anti-glycation activity of quercetin, Vitamin E, Carnosine and polyherbal formulations(Grzebyk E & Piwowar A, 2014, Grzebyk E & Piwowar A, 2016) and green tea (Lavelli V, 2011 ). In addition, a study used red grape skin extract (RGSE) and showed it to significantly inhibit the formation of AGEs (Jariyapamornkoon N, 2013). It exhibited its anti-oxidant properties against fructose mediated protein oxidation.
An in-vitro study conducted by Vinson and Howard determined the action of L-ascorbic acid on protein-glucose and protein-fructose as AGE products as relative to the control. They reported that L-ascorbic was a very potent inhibitor of glycation and decreased AGE production within time span of 3 days to 30 days. Further they also reported that combination of vitamin C and Vitamin E were more potent inhibitors of glycation as compared to a single vitamin (J.A. Vinson & T.B. Howard III, 1996). By using vitamin C, Tarwadi and Agte demonstrated a 20% inhibitory effect on glycation. They suggested that the effect of vitamin C lead to reduction of oxidative stress, which decreased further oxidation and glycation (Tarwadi KV & Agte VV, 2011). Similarly, Lavelli et al also reported that vitamin C inhibited glycation and indicated further studies for confirmation (Lavelli V, 2011). While, Vinson and Howard demonstrated that vitamin C at a concentration of 20 mM inhibited the process of glycation and AGE formation by about 73% (J.A. Vinson & T.B. Howard III, 1996). While E.Grzelyk & A.Piwowar confirmed a strong inhibitory effect of vitamin C at a concentration of 10 mM on both oxidation and glycation process (Grzebyk E & Piwowar A, 2016).
The exact mechanism of inhibition is not known. Vitamin C (L-ascorbic acid) is known as an antioxidant. Ascorbate and its free radical semihydroascorbate can form ionic bonds with proteins and prevent glucose from binding to the protein (Bensch KG, 1981). This further would inhibit AGE formation. The antioxidant function would decrease the concentration of free radicals and this will not produce highly reactive oxidants from glycated proteins.
Ribonuclease (RNase) is a type of nuclease that catalyzes the degradation of RNA into smaller components. There are different types of RNases as RNaseA, RNase L and RNase H. RNase L enzyme is part of the RNA degradation pathway. RNase L and interferons protects cells from viral infection by degrading the double stranded RNA (Silverman RH, 1996). RNaseL has been implicated for its role in immune responses and has a role as a tumor suppressor gene in cancer (Zhou A, 1998). RNase L also regulates the expression of certain proinflammatory genes in the pancreas under the special condition. The absence or inactivation of RNase enzyme has been implicated in pathogenesis of type 1 diabetes mellitus. It has been observed that high levels of both ribose and glucose lead to AGE formation and inactivation of RNase enzyme. Therefore Rnase enzyme is one of the potential targets in therapy of type 1 diabetes. The Inhibition of glycation RNase A enzyme by Vitamin B has been studied. However, the inhibitory action of Vitamin C (L-ascorbic) on glycation of RNase enzyme has not been studied. Hence this study was undertaken.
Overall the production of AGE is associated with declining organ functioning. However the field being new, the deleterious effects of AGE on health still needs to be explored thoroughly. Previous studies have determined the anti-AGE effects by using different types of natural or herbal compounds. The results of some studies especially with respective to L-ascorbic acid needs to be validated to determine if it promotes or inhibits AGE activity.
The aims and objectives of this study are to investigate the ability of L-ascorbic acid (Vitamin C) to inhibit the formation of advanced glycation end product in RNase A enzyme in glucose solution.
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