Vitamin-C as anti-Helicobacter pylori agent: More prophylactic than curative- Critical review.

Potential of nonantibiotic therapies for treatment of Helicobacter pylori-related acid peptic disease remains underexplored. Several clinical studies have shown that higher prevalence of H. pylori infection is associated with low Vitamin C (Vit C) level in serum and gastric juice. However, there is no consensus regarding the usefulness of Vit C supplementation in the management of H. pylori infection. Surveying the existing literature we conclude that high concentration of Vit C in gastric juice might inactivate H. pylori urease, the key enzyme for the pathogen's survival and colonization into acidic stomach. Once infection established, urease is not very important for its survival. The role of Vit-C as anti-H. pylori agent in peptic ulcer diseases appears to be preventive rather than curative. Rather than supplementing high dose of Vit C along with conventional triple therapy, it is preferable to complete the conventional therapy and thereafter start Vit C supplementation for extended period which would prevent reinfection in susceptible individuals, provided the patients are not achlorhydric. Further studies are required to prove the role of Vit C in susceptible population.

Introduction

In spite of initial success of eradication treatment for Helicobacter pylori in peptic ulcer diseases using combination of a proton pump inhibitor and antibiotics, emergence of antibiotics resistance in recent years, leads to frequent treatment failure in at least 10-20% of the patients.[1] Nonantibiotic therapies, including phytomedicines, probiotics and antioxidants have been increasingly investigated as potential adjuvants for the treatment of H. pylori.[2] Several clinical studies have shown that high H. pylori infection rate is related to low ascorbic acid (AA)/Vitamin C (Vit C) level in serum as well as in gastric juice.[3-5] On the other hand high dose of Vit C have been shown to inhibit H. pylori growth, colonization or even eradication of H. pylori infection in few studies while others gave nonconclusive results.[26-11] But still it is not clear how Vit C level can affect the course of H. pylori infection in stomach and how the infection affects the level of Vit C in serum and gastric juice. Moreover doses of Vit C used in those experiments were extremely higher compared to the physiological concentration of Vit C in the stomach. These high concentrations cannot be achieved in physiological condition due to its pro-oxidant activity at high concentration (500 mg/day or more) particularly in presence of high body iron stores leading to several adverse effects including DNA damage and an increase risk of cancer.[1213] So the actual role of Vit C at physiological or therapeutic level, in the course of H. pylori infection in the stomach is still inconclusive. One study shows that AA does not inhibit growth of H. pylori at a concentration (400 Mm) within the physiological range.[14] This experiment was carried out in buffered condition at pH 7.4, but in vivo H. pylori have to counter unbuffered acidic pH (pH 3) to colonize successfully into the stomach. At low pH the effect of AA on survival of H. pylori might be different. So these ex vivo studies also do not rule out the role of AA as anti-H. pylori agent.

At this juncture a new query emerges; whether antioxidant effect of Vit C has any effect on the survival of H. pylori at low pH? Till date very limited studies have been carried out in this direction. A systematic analysis of the available information would lead to some interesting logical derivations which might explain the existing confusion associated with the role of Vit C in H. pylori infection. This would help us to design more clinical studies in a logical way and to formulate appropriate antipylori regimen.

Mechanism of H. pylori Survival At Low Gastric pH

H. pylori synthesizes large amount of urease, which is found in its cytosol. The cytosolic urease is released into the gastric juice upon spontaneous autolysis of a subpopulation of H. pylori and subsequently it is adsorbed onto the surface of intact bacteria. The urease catalyses the hydrolysis of urea present in the gastric juice, to yield carbonic acid and ammonia. Thus H. pylori makes a cloud of ammonia on its surface to neutralize the gastric acid which enables it to colonize the gastric epithelium.[1516] Once successfully colonized, H. pylori resides below the gastric mucus layer which has a higher pH than gastric lumen.[1718] So in chronic infection, the role played by urease, in survival of the bacteria seems less important. However, besides protecting from acid, urease also aids in colonization by providing ammonia for bacterial protein synthesis.

Structural Features of H. pylori Urease

In the active site of urease there are two Ni (Ni++) centers held by co-ordination bonding. Ni(1) is coordinated by two imidazole ligands from two histidine residues and a water molecule. Ni(2) is coordinated by two histidine residues as well as with an aspartate and a water molecule.[19] It has been found that purified urease is inactivated at pH<5 (in buffered solution). So it had long been unanswered that how extracellular urease on H. pylori cell surface remains active in low gastric pH. Moreover cytosolic pH (pH 6) of H. pylori is also suboptimal for maximal urease activity. Ha et al; 2001 have convincingly shown that supramolecular assembly of urease create a pore within the complex which serves as a pathway for diffusion of urea toward the 12 clustered active sites which protect each other from acid inactivation by producing localized cloud of ammonia from breakdown of urea.[20]

Vit C as Biological Antioxidant

Vit C is an acidic molecule with strong reducing activity and is an essential component of most living tissues. It has two major redox forms: AA and DHA, the reduced and oxidized form, respectively, and they are all interconvertible. Within the cell DHA is rapidly converted to AA by the specific enzyme systems like DHA reductase, glutaredoxins and protein disulfide isomerase in presence of glutathione or other thiols as electron donors[2122] Unlike AA, DHA is relatively unstable and undergo rapid spontaneous irreversible hydrolysis particularly at a pH > 4.[23]

Vit C as a Reducing Agent for Transition Metals

Few transition metals like Fe(III), Cu(II), Hg(II), Cr(VI) are able to accept electron from AA, resulting in their reduction and simultaneous oxidation of AA to DHA. In presence of strong biological oxidants like oxygen, hydrogen peroxide the oxidation of AA is accelerated, where the metal ions act predominantly as catalyst. In aqueous solutions at neutral pH, metal ions, having relatively low redox potential like Zn(II), Ni(II), Co(II), Pb(II) unable to oxidize AA by themselves nor serve as a catalyst for oxidation by molecular oxygen. However, in biological solutions (e.g.: tissue fluid, blood, serum) redox chemistry of these metal ions is different due to presence of different natural metal complexing agents like amino acids, peptides, proteins, nucleotides, etc. These natural ligands are also able to form ternary complexes with the metal ions and Vit C. Among all biological ligands, histidine or histidine containing peptides are shown to be more efficient in binding transition metals like Ni(II) and form stable ternary complexes with AA. The assembly favors oxidation of AA by the metal ion and the process is relatively faster in presence of oxygen.[24-26]

Vit-C as an Anti-H. pylori Agent

Normally AA is actively secreted from plasma to the gastric juice.[2728] High concentration of AA in gastric juice favor reduction of Ni++ centers, coordinated to the histidine residues of the urease, secreted from H. pylori, leading to inactivation of the enzyme followed by acid denaturation [Figure 1]. This reduction of the transition metal ion by AA, also accelerated at low pH of gastric juice as in case of reduction of ferric iron by AA.[1011] Recently Krajewska et al. showed that in a buffered system neither AA nor DHA themselves are inhibitors of urease. The inhibitory effect of AA and DHA was seen in the presence of Fe(3+) ions and, unlike reported in the literature, they found that it was mediated by H2O2. Interestingly the resulting inhibition by DHA-Fe(3+) consisted of enzyme thiol oxidation and its effectiveness grew with increasing pH.[29] This could be another interesting mechanism by which AA can act against H. pylori especially in the initial stages of infection.

Figure 1

Inactivation of H. pylori urease by Vit.C in H. pylori-infected gastric mucosa. Abbreviation: Ni – nickel (reduced form), AA- ascorbic acid/Vit. C, DHA- dehydroascorbic acid, HP- H. pylori

In low gastric pH, once urease is inactive it becomes difficult for H. pylori to survive and colonize in stomach. But once it successfully colonizes into stomach wall, H. pylori stays within the gastric mucosa, where the pH is suitable for the survival of the bacteria, due to the bicarbonate buffer from gastric epithelium secreted into the luminal surface.[1718] Moreover after chronic infection, a proportion of patients develop relative achlorhydria leading to higher pH of gastric juice.[30] So at this stage of infection, role of urease for its survival is relatively less. This might explain the fact that even a high dose AA supplementation is not able to eradicate H. pylori infection in a significant percentage of cases[11] though AA have been shown to inhibit urease in vitro.[2931] In chronically infected stomach, the bacteria continue to produce urease, which capture the free Ni++ from the gastric juice. As discussed above, this bound Ni++ is more preferred substrate for reduction by AA than free Ni++. As a consequence of reduction of the Ni++ centers of urease, AA itself oxidized to DHA and rapidly exhausted by spontaneous hydrolysis particularly when passed through alkaline medium of intestine. This might be one of the explanations of low serum and gastric juice AA level in chronically H. pylori-infected patients. Though local ROS, generated by chronic gastritis may also be responsible partially for conversion of AA to DHA. At the site of inflammation extra cellular DHA is taken up by leucocytes and rapidly converted to AA and thus recycled.[3233] So the local gastric inflammation may not have significant effect on Vit C deficiency in H. pylori induced gastritis. There is also evidence that, in Ni++ toxicity, there is depletion of serum AA and the toxic effect is reversed by addition of AA.[26] This seems to be due to reduction of Ni++ by AA in the biological solution in vivo. On the other hand, patients with already low serum AA likely to be more prone to get infection by H. pylori, because low AA in gastric juice might favor colonization by the bacteria as explained above. Moreover low antioxidant level in chronically infected gastric mucosa, causes elevation of ROS contributing perpetuation of inflammation and infection cycle.[3435] Thus it is possible that AA can have an anti-H. pylori effect during initiation, spreading and perpetuation of the infection although major effect could be during initiation of colonization.

Few clinical studies have been carried out where different conventional anti-H. pylori regimens supplemented with Vit C were used. The results of these studies[36-41] range from no benefit on Vit C supplementation with conventional anti-H. pylori regimens to studies which reported that Vit C decreased effectiveness of conventional anti-H. pylori particularly where a triple therapy containing metronidazol was used.[3637] In all those anti-H. pylori regimens proton pump inhibitor was one of the critical components which increased the gastric pH. Ex vivo studies using physiological concentration of AA and pH (7.4) in H. pylori culture media also support these views. Moreover it have been reported that PPI reduce bioavailability and stability of Vit C in gastric juice.[4243] Secondly in the anerobic micro-organisms, metmronidazol enter into the bacterial cell through passive diffusion and is reduced to generate active free radicals. Thus a flow of metronidazol is maintained into the cells along a concentration gradient.[94445] In presence of high dose of AA extracelluar reduction of metronidazole might take place, hampering effective accumulation of the drug into the bacterial cells. More over active free radical forms of metronidazol might be stabilized by high concentration of Vit C which leads to reduced H. pylori eradication rates when this combined regimen is used. Vit C supplementation at a dose of 500 mg b.d. following triple therapy seems to be quite effective as shown from a preliminary study by Sezikli et al.[38] More clinical trials are required in this direction to determine the optimum dosage and regimen keeping in mind the increasing pro-oxidative role of Vit C at higher dose (500 mg/day) as observed by Podmore et al.[13] It is important to particularly consider long-term Vit C supplementation therapy. However, a few recent studies report an increased eradication of H. pylori when triple therapy was supplemented with Vit C.[38-41] Yet there is no convincing evidence if supplementation of Vit C along with triple therapy could be beneficial in eradicating resistant H. pylori infection.[41] Interestingly, in one study Vit C was supplemented two more weeks following completion of triple therapy showing better eradication rate than achievable by triple therapy alone.[38]

Conclusions

The role of AA as anti-H. pylori agent in peptic ulcer diseases is most likely to be preventive rather than curative. Rather than supplementing high doses of AA along with conventional antipylori regimen it is preferable to complete the standard course of antipylori regimen, which might then be followed by Vit C supplementation therapy for extended period which would prevent from reinfection in susceptible subjects and also might eradicate the residual H. pylori infection, provided the patients are not achlorhydric or under prolonged acid suppressive therapy. It is also essential to maintain a constant protective level of Vit C in gastric juice or in serum to inhibit colonization or reinfection by H. pylori in gastric epithelium. Considering the short biological half life of this water soluble vitamin and the chances of toxicity from single daily bolus dose, split multiple doses/sustained release formulations would be more effective to maintain constant protective level of Vit C in plasma and gastric juice. More clinical trials are required in this direction to determine the actual dosage and regimen. It would be interesting to study the incidence of H. pylori infection among the large cohorts of population having varied concentrations of plasma Vit C. Future studies should be directed toward comparison of the reinfection rates in cohorts undergone the standard course of anti-H. ylori pylori regimen versus those who are maintained on Vit C supplementation after the completion of the standard therapy. It is clear from this review that well-controlled, well-designed studies are required in this direction as H. pylori infection and consequent gastric cancer is a major public health problem.