Riboflavin as the glutathione reductase coenzyme

Glutathione reductase (GR) requires riboflavin in the FAD coenzyme form for its activity⁽ Reference Gallagher, Mahan, Escott-Stump, Raymond and Krause ^{16 )}. GR converts oxidised glutathione to the reduced form⁽ Reference Dringen, Gutterer and Hirrlinger ^{17 )} (Fig. 1). FAD transports hydrogen from NADPH to oxidised glutathione to convert it into the reduced form⁽ Reference Schulz, Schirmer and Pai ^{18 )} (Fig. 2). Reduced glutathione acts as an endogenous antioxidant in different cell types⁽ Reference Pompella, Visvikis and Paolicchi ^{19 )} and deactivates reactive oxygen species. Through its action, this peptide is deactivated as it is converted to the oxidised form⁽ Reference Dringen, Gutterer and Hirrlinger ^{17 )}. Therefore, oxidised glutathione should be reduced by GR again to recover its antioxidant properties, the process in which riboflavin has a key role. Consequently, there is a possibility that riboflavin deficiency could affect the antioxidant properties of glutathione and lead to an impaired antioxidant potential of cells. One of the most important antioxidant activities of glutathione is the deactivation of peroxides such as hydroperoxide. This activity of glutathione is mediated by the action of GPx⁽ Reference Hayes and McLellan ^{20 )}. GPx transfers a hydrogen ion from reduced glutathione to lipid peroxide and produces oxidised glutathione and alcohol⁽ Reference Mulherin, Thurnham and Situnayake ^{21 )}. According to the mentioned mechanisms, it is expected that riboflavin deficiency could increase lipid peroxidation.

Fig. 1 Conversion of oxidised glutathione (GSSG) to the reduced form (GSH) by glutathione reductase. Glutathione reductase requires riboflavin in the FAD coenzyme form for its activity. G-6P-D, glucose-6-phosphate dehydrogenase.

Fig. 2 Transportation of hydrogen from reduced NADPH to oxidised glutathione (GS–SG; GSSG) by FAD for conversion into the reduced form (GSH). Enz, enzyme. (A colour version of this figure can be found online at http://www.journals.cambridge.org/bjn).

Effect of riboflavin status on glutathione content in tissues

The effect of riboflavin status on reduced glutathione content in tissues has been investigated in a limited number of studies. Taniguchi & Hara⁽ Reference Taniguchi and Hara ^{22 )} reported that liver reduced glutathione levels are decreased in riboflavin deficiency in rats. In an experimental study, a reduction in reduced glutathione levels was observed in rats fed a riboflavin-deficient diet for over 6 weeks⁽ Reference Liang, Liu and Xu ^{23 )}. Similar results have been reported by some other studies⁽ Reference Dutta, Rivlin and Pinto ^{24 ,} Reference Horiuchi, Hirano and Ono ^{25 )}. However, to our knowledge, there are a limited number of human studies that have examined the effect of riboflavin on reduced glutathione status. The only human study published has reported no significant difference in erythrocyte glutathione concentrations between normal and riboflavin-deficient subjects⁽ Reference Powers and Thurnham ^{26 )}. Furthermore, some animal studies also did not report any change in glutathione content in different tissues (such as the liver, lens and erythrocytes) in riboflavin-deficient animals⁽ Reference Bates ^{27 –} Reference Rao and Bhat ^{29 )}. A mechanism for the unchanged tissue glutathione content in riboflavin deficiency could be the increased biosynthesis of glutathione from its precursor amino acids as a compensatory action⁽ Reference Kaplowitz, Aw and Ookhtens ^{30 )}. Another possible mechanism could be the ability of GR to maintain reduced glutathione concentrations in tissues even at low activity levels⁽ Reference Rogers and Augusteyn ^{31 )}.

Effect of riboflavin status on the activity of antioxidant enzymes

As expected, some reports have indicated that riboflavin status can affect the activity of antioxidant enzymes including GPx, superoxide dismutase (SOD) and catalase. The results of a study that has investigated the effect of riboflavin therapy on diabetic cardiomyopathy indicated that riboflavin can increase SOD activity in the heart tissue⁽ Reference Wang, Li and Lu ^{32 )}. A reduction in liver and muscle GPx activity in riboflavin-deficient pigs was reported by Brady et al. ⁽ Reference Brady, Brady and Parsons ^{33 )}. However, erythrocyte GPx activity was not found to be affected by riboflavin deficiency in this study. Another study that has investigated the effect of dietary riboflavin on the antioxidant defence mechanism of fish has reported a significant reduction in the SOD and catalase activities of riboflavin-deficient (12 weeks) fish compared with those of the control fish⁽ Reference Huang, Tian and Wu ^{28 )}. Moreover, some other animal studies have reported a similar effect of riboflavin on SOD⁽ Reference Liang, Liu and Xu ^{23 ,} Reference Hirano, Hamajima and Horiuchi ^{34 ,} Reference Tumkiratiwong, Tungtrongchitr and Migasena ^{35 )}, GPx⁽ Reference Tumkiratiwong, Tungtrongchitr and Migasena ^{35 ,} Reference Wang, Mei and Chen ^{36 )} and catalase⁽ Reference Tumkiratiwong, Tungtrongchitr and Migasena ^{35 ,} Reference Lee, Ye and Jones ^{37 )} activities. However, some animal studies did not observe any association between riboflavin status and antioxidant enzyme activity⁽ Reference Adelekan and Thurnham ^{38 ,} Reference Dutta, Seirafi and Halpin ^{39 )}. The results of the study carried out by Adelekan & Thurnham⁽ Reference Adelekan and Thurnham ^{38 )} indicated that the mean erythrocyte GPx activity in riboflavin-deficient rats was slightly, but not significantly lower than that in the control rats. In a study carried out by Dutta et al. ⁽ Reference Dutta, Seirafi and Halpin ^{39 )}, although riboflavin deficiency alone did not affect the activities of GPx and GR and increased the content of glutathione in the liver, ethanol administration (which can exacerbate oxidative stress) significantly decreased the activities of these enzymes and the content of glutathione in the liver of riboflavin-deficient rats compared with those in the liver of the riboflavin-sufficient control rats. However, there is a controversy regarding the results of different studies. In these studies, an increased GPx activity in the liver⁽ Reference Taniguchi and Hara ^{22 )}, lens⁽ Reference Rao and Bhat ^{29 )} and blood cells⁽ Reference Levin, Cogan and Levy ^{40 )} due to riboflavin deficiency has been reported. Increased lipid peroxidation has also been observed concurrent with an increased GPx activity. Therefore, it has been suggested that increased GPx activity could be a response to increased lipid peroxidation, resulting from riboflavin deficiency⁽ Reference Rao and Bhat ^{29 )}. The possible allosteric effect of reactive oxygen species on GPx enzyme can lead to an increased activity through increased lipid peroxidation⁽ Reference Levin, Cogan and Levy ^{40 )}.

Finally, results indicate that riboflavin status could affect the activity of antioxidant enzymes, but some studies do not agree with this. Furthermore, all studies in this area have been limited to animals, recommending further investigations in human populations.

Animal studies

The results of several animal studies indicate not only the adverse effects of riboflavin deficiency on lipid peroxidation but also the desirable effects of riboflavin administration on it⁽ Reference Taniguchi and Hara ^{22 ,} Reference Liang, Liu and Xu ^{23 ,} Reference Bates ^{27 –} Reference Rao and Bhat ^{29 ,} Reference Wang, Li and Lu ^{32 ,} Reference Hirano, Hamajima and Horiuchi ^{34 ,} Reference Levin, Cogan and Levy ^{40 –} Reference Yagi, Komur and Yoshino ^{42 )}. In studies in which riboflavin deficiency was induced in animals using a riboflavin-deficient diet, lipid peroxidation in different tissues was found to be significantly increased compared with that in the control groups⁽ Reference Taniguchi and Hara ^{22 ,} Reference Liang, Liu and Xu ^{23 ,} Reference Bates ^{27 –} Reference Rao and Bhat ^{29 ,} Reference Hirano, Hamajima and Horiuchi ^{34 ,} Reference Levin, Cogan and Levy ^{40 –} Reference Yagi, Komur and Yoshino ^{42 )}. In another study, it was shown that riboflavin administration could reduce the production of lipid peroxides (such as malondialdehyde (MDA)) or/and protein carbonyls in diabetic rats⁽ Reference Wang, Li and Lu ^{32 )}. However, only one study⁽ Reference Bates ^{27 )} has reported that riboflavin deficiency increased MDA levels in the lens but did not affect MDA content in the liver in rats.

Human studies

A limited number of human studies have investigated the effect of riboflavin status on lipid peroxidation and confirmed the mentioned effect of riboflavin on lipid peroxidation. In a case–control study⁽ Reference Das, Thurnham and Patnaik ^{43 )} on Indian children, it was found that plasma MDA levels of malaria patients with riboflavin deficiency were significantly higher than those of malaria-infected children with normal riboflavin status. However, in healthy children without malaria infection, no difference in MDA levels was observed between riboflavin-deficient and riboflavin-sufficient children. Furthermore, in this study, riboflavin status was found to be inversely correlated with plasma MDA levels in malaria patients but not with those in healthy subjects. A possible mechanism explained by the researchers is the increased oxidative stress in malaria-infected patients, which causes a reduction in glutathione levels⁽ Reference Das, Thurnham and Patnaik ^{43 )}. In a cross-sectional study carried out in Moscow, it was shown that there is a significant negative linear correlation between serum MDA levels and riboflavin intake⁽ Reference Kodentsova, Vrzhesinskaia and Beketova ^{44 )}. The results of the study carried out by George & Ojegbemi⁽ Reference George and Ojegbemi ^{45 )} confirm these findings.

According to the findings of animal and human studies, riboflavin status appears to have an effect on the oxidative state of the body, in particular, on lipid peroxidation. However, it seems that the mechanism through which riboflavin can exert its antioxidant effect cannot be limited to the glutathione redox cycle and its relevant antioxidant enzymes. Although in most of the reviewed studies riboflavin status was found to be inversely related to lipid peroxidation, only in some of these studies, this relationship could be attributed to the role of riboflavin in the activities of glutathione reductase and related antioxidant enzymes. In these cases, a change in lipid peroxidation was found to occur simultaneously with an inverse change in the activity of antioxidant enzymes⁽ Reference Liang, Liu and Xu ^{23 ,} Reference Wang, Li and Lu ^{32 ,} Reference Hirano, Hamajima and Horiuchi ^{34 )}. In spite of increased lipid peroxidation due to riboflavin deficiency, any change in the activity of antioxidant enzymes was not observed in the study carried out by Huang et al. ⁽ Reference Huang, Tian and Wu ^{28 )}. In this study, increased liver MDA levels were observed in riboflavin-deficient fish without any change in liver GR and GPx activities and glutathione content. However, in this study, riboflavin deficiency was found to lead to reduced SOD and catalase activities. As H₂O₂ and superoxide (O₂ ⁻) ions have been reported to have an inhibitory effect on SOD and catalase activities, respectively, the authors have suggested that riboflavin deficiency may lead to an overproduction of reactive oxygen species (obviously through a mechanism independent of the glutathione redox cycle) beyond the scavenging ability of SOD and catalase. Therefore, SOD and catalase could be inhibited by these free radicals. Bate et al. ⁽ Reference Bates ^{27 )} also reported elevated lipid peroxidation in riboflavin deficiency with no change in glutathione levels and GPx activity. Moreover, in some studies, an increased GPx activity was observed simultaneously with increased lipid peroxidation in riboflavin deficiency⁽ Reference Taniguchi and Hara ^{22 ,} Reference Rao and Bhat ^{29 ,} Reference Levin, Cogan and Levy ^{40 )}. As has been mentioned previously, this increased GPx activity, most probably, is a compensatory response to increased lipid peroxidation occurring in riboflavin deficiency.

It has been suggested that riboflavin may have antioxidant properties independent of its action in the glutathione redox cycle. Durusoy et al. ⁽ Reference Durusoy, Karagoz and Ozturk ^{46 )} reported that the antimutagenic effect of riboflavin is independent of antioxidant enzyme activity. They suggested that the antimutagenic effect of riboflavin can, at least in part, result from its direct scavenging activity on free radicals produced by mutagens. In vitro studies have also indicated that riboflavin itself has an antioxidant nature, independent of its action as the GR coenzyme⁽ Reference Toyosaki and Mineshita ^{47 )}. The suggested mechanism could be the deactivation of hydroperoxide through the reversion of riboflavin from the reduced form (dihydroriboflavin) to the oxidised form (Fig. 3) ⁽ Reference Toyosaki ^{48 )}. It is also possible that riboflavin exerts its antioxidant effect by reinforcing the effect of other antioxidants such as vitamin C⁽ Reference Rao and Bhat ^{29 )}. In this study carried out by Rao & Bhat⁽ Reference Rao and Bhat ^{29 )}, a reduced concentration of vitamin C in the lens of riboflavin-deficient rats was reported, which is concurrent with increased lipid peroxidation. However, there is no strong evidence about the effect of riboflavin on other dietary antioxidants, suggesting further investigations.

Fig. 3 Conversion of reduced riboflavin to the oxidised form – a possible mechanism for its antioxidant nature. (A colour version of this figure can be found online at http://www.journals.cambridge.org/bjn).