Original Contribution
A novel role for vitamin B12: Cobalamins are intracellular antioxidants in vitro

https://doi.org/10.1016/j.freeradbiomed.2009.04.023Get rights and content

Abstract

Oxidative stress is a feature of many chronic inflammatory diseases. Such diseases are associated with up-regulation of a vitamin B12 (cobalamin) blood transport protein and its membrane receptor, suggesting a link between cobalamin and the cellular response to inflammation. The ability of cobalamin to regulate inflammatory cytokines suggests that it may have antioxidative properties. Here we show that cobalamins, including the novel thiolatocobalamins N-acetyl-l-cysteinylcobalamin and glutathionylcobalamin, are remarkably effective antioxidants in vitro. We also show that thiolatocobalamins have superior efficacy compared with other cobalamin forms, other cobalamins in combination with N-acetyl-l-cysteine (NAC) or glutathione (GSH), and NAC or GSH alone. Pretreatment of Sk-Hep-1 cells with thiolatocobalamins afforded robust protection (> 90% cell survival) against exposure to 30 μM concentrations of the pro-oxidants homocysteine and hydrogen peroxide. The compounds inhibited intracellular peroxide production, maintained intracellular glutathione levels, and prevented apoptotic and necrotic cell death. Moreover, thiolatocobalamins are remarkably nontoxic in vitro at supraphysiological concentrations (> 2 mM). Our results demonstrate that thiolatocobalamins act as powerful but benign antioxidants at pharmacological concentrations. Because inflammatory oxidative stress is a component of many conditions, including atherosclerosis, dementia, and trauma, their utility in treating such disorders merits further investigation.

Section snippets

Experimental procedures

Sk-Hep-1 (ECACC 91091816) cells were maintained in MegaCell MEME (Sigma; M-4067) with 3% serum and 200 mM l-glutamine at 37 °C in 5% CO2. The Sk-Hep-1 cell line was chosen as it is highly sensitive to oxidative stress. Cells were plated into 96-well microtiter plates and cultured for 24 h. Medium was replaced with 100 μl fresh medium containing various concentrations of dl-homocysteine or H2O2 as oxidants for up to 24 h. Cells were preincubated with cobalamin, thiol (NAC or GSH), or cobalamin

Results

Preliminary experiments established that a Hcy concentration of 30 μM achieved > 90% cell death in Sk-Hep-1 cells. Dose-dependent protection from cell death was observed when Sk-Hep-1 cells were pretreated with increasing concentrations of either GSCbl or NACCbl (2–64 μM) for 2 h before exposure to Hcy (30 μM) (Fig. 1). Cell survival (> 90%) from Hcy toxicity was achieved with GSCbl (30 μM) and NACCbl (30 μM) (Fig. 1). Cobalamin and thiol (NAC and GSH) concentrations were optimized in terms of

Discussion

In this study, preincubation of Sk-Hep-1 cells with GSH or NAC only partially protected against Hcy- or H2O2-induced damage. Their respective thiolatocobalamin derivatives (GSCbl and NACCbl) demonstrated significantly greater protective capabilities than the thiol alone or the thiol combined with standard cobalamin derivatives.

GSH and NAC, unlike the cobalamins, are well-characterized antioxidants. GSH is a thiol-containing tripeptide and a major antioxidant defense molecule. Reduced GSH levels

Acknowledgments

GSCbl and NACCbl are currently the subjects of U.S. Patent applications by N.E.B., C.S.B., and J.H.H.W. (U.S. Application 20080113900, “Pharmaceutical compositions and therapeutic applications for the use of a synthetic vitamin B12 derivative, glutathionylcobalamin,” and U.S. Application 20080076733, “Pharmaceutical compositions and therapeutic applications for the use of a novel vitamin B12 derivative, N-acetyl-l-cysteinylcobalamin”) and by A.M. (U.S. Application 20040157783, “Method for

References (31)

  • LingC.T. et al.

    Effect of vitamin B12 on the levels of soluble sulfhydryl compounds in blood

    J. Biol. Chem.

    (1953)
  • RothenbergS. et al.

    Transcobalamin II, In Chemistry and Biochemistry of B12

  • ScalabrinoG. et al.

    High tumor necrosis factor-alpha levels in cerebrospinal fluid of cobalamin-deficient patients

    Ann. Neurol.

    (2004)
  • McCaddonA. et al.

    Functional vitamin B12 deficiency and Alzheimer disease

    Neurology

    (2002)
  • WagnerF. et al.

    New aspects of the structure of corrinoid coenzymes

    Ann. N.Y. Acad. Sci.

    (1964)
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