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Original Contribution
A novel role for vitamin B
12
: Cobalamins are intracellular antioxidants in vitro
Catherine S. Birch
a
, Nicola E. Brasch
b
, Andrew McCaddon
a,c
, John H.H. Williams
a,
⁎
a
Chester Centre for Stress Research, University of Chester, Parkgate Road, Chester CH1 4BJ, UK
b
Department of Chemistry and School of Biological Sciences, Kent State University, Kent, OH 44242, USA
c
Cardiff School of Medicine, Cardiff University, Gardden Road Surgery, Rhosllanerchrugog, Wrecsam, North Wales LL14 2EN, UK
abstractarticle info
Article history:
Received 22 December 2008
Revised 14 April 2009
Accepted 22 April 2009
Available online 3 May 2009
Keywords:
Vitamin B
12
Cobalamin
Thiolatocobalamin
Antioxidant
Homocysteine
Free radicals
Oxidative stress is a feature of many chronic inflammatory diseases. Such diseases are associated with up-
regulation of a vitamin B
12
(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 (N90% 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 (N2 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.
© 2009 Elsevier Inc. All rights reserved.
Chronic inflammation, often accompanied by oxidative stress, is a
component of many age-related diseases, including cancer, athero-
sclerosis, neurodegenerative disease, and arthritis. As such, it is a sig-
nificant cause of morbidity and mortality.
Recent observations suggest that cobalamins (vitamin B
12
deri-
vatives) may modulate the oxidative stress responses, including
those of the inflammatory response. Inflammatory diseases are
associated with elevated blood levels of transcobalamin (a cobalamin
transport protein) [1], and its membrane receptor is up-regulated by
TNF-α[2]. Cobalamin concentration also modulates TNF-αlevels in
cerebrospinal fluid [3]. TNF-αis important in inflammatory res-
ponses, so taken together, these observations suggest that elevation
of cobalamin could be used to supplement the cellular response to
inflammation.
In the cell, two enzymes use cobalamin as a cofactor in either the
adenosylcobalamin (AdoCbl) or the methylcobalamin (MeCbl) form.
In the mitochondrial AdoCbl-dependent L-methylmalonyl-CoA
mutase (EC 5.4.99.2) reaction, L-methylmalonyl-CoA is converted to
succinyl-CoA, which then enters the Krebs cycle. In the cytosolic
MeCbl-dependent methionine synthase (EC 2.1.1.13) reaction, tetra-
hydrofolate and methionine are generated by a methyl group transfer
from methyltetrahydrofolate to homocysteine (Hcy) [4]. Hcy is a
junction metabolite that can also be catabolized by cystathionine
β-synthase, ultimately leading to synthesis of the important
intracellular antioxidant glutathione (GSH) [4]. Elevated Hcy is
associated with endothelial cell dysfunction and promotes the
formation of reactive oxygen species, primarily by a mechanism
involving endothelial nitric oxide synthase, but also by autoxidation
[5]. Hcy also inhibits the antioxidant enzymes superoxide dismutase
and glutathione peroxidase and activates endothelial proinflamma-
tory signaling pathways [6].
Thiol derivatives of cobalamin (thiolatocobalamins) such as gluta-
thionylcobalamin (GSCbl) were first identified in the 1960s [7,8].
GSCbl can be isolated from mammalian cells and is a potential precur-
sor of the cofactor forms of cobalamin, although the exact pathways
leading to the incorporation of cobalamin into its dependent enzymes
remain unclear [9–13]. GSCbl is more active than other cobalamins in
promoting methionine synthase activity [14], suggesting that thiola-
tocobalamins might be more efficacious than other cobalamins in
treating conditions associated with hyperhomocysteinemia and
oxidative stress, such as Alzheimer disease [4,15].
In this study we explored the relative effects of thiols and
cobalamins, including the novel thiolatocobalamin N-acetyl-L-
Free Radical Biology & Medicine 47 (2009) 184–188
Abbreviations: AdoCbl, adenosylcobalamin; CNCbl, cyanocobalamin; DCFH-DA,
dichlorofluorescin diacetate; GSH, glutathione; GSCbl, glutathionylcobalamin; Hcy,
homocysteine; HOCbl, hydroxocobalamin; MeCbl, methylcobalamin; NAC, N-acetyl-L-
cysteine; NACCbl, N-acetyl-L-cysteinylcobalamin.
⁎Corresponding author. Fax: +44 1244 511346.
E-mail address: john.williams@chester.ac.uk (J.H.H. Williams).
0891-5849/$ –see front matter © 2009 Elsevier Inc. All rights reserved.
doi:10.1016/j.freeradbiomed.2009.04.023
Contents lists available at ScienceDirect
Free Radical Biology & Medicine
journal homepage: www.elsevier.com/locate/freeradbiomed
Author's personal copy
cysteinylcobalamin (NACCbl) [16], in protecting cells against Hcy- and
hydrogen peroxide (H
2
O
2
)-induced oxidative stress.
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% CO
2
. 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
H
2
O
2
as oxidants for up to 24 h. Cells were preincubated with
cobalamin, thiol (NAC or GSH), or cobalamin derivative, GSCbl or
NACCbl, for 2 h.
Cell survival was measured using reduction of MS tetrazolium
compound to formazan (490 nm) as a proxy for cell number in the
CellTiter 96 AQueous One Solution cell proliferation assay (Promega,
Madison, WI, USA) [17].
Apoptosis was measured from caspase-3 activity and necrosis from
propidium iodide (PI) uptake [17]. Caspase-3 activity was measured in
cells resuspended in 100 μl of DMEM without phenol red after the
addition of 50 μl caspase-3 substrate (EnzoLyte Rh110 caspase-3 kit;
AnaSpec, San Jose, CA, USA). Plates were incubated at 37 °C for 60 min,
and formation of free 7-amino-4-trifluoromethylcoumarin was
acquired by fluorescence measurement at λ
ex
496/λ
em
520 nm. PI
uptake was measured after the addition of 50 μlof5μgml
−1
propidium iodide solution to cells resuspended in 50 μl of phenol-red-
free medium. Plates were incubated in the dark at 37 °C for 20 min and
fluorescence was measured at λ
ex
535 nm/λ
em
617 nm.
Peroxide generation was measured by the addition of the redox-
active probe 2′,7′-dichlorofluorescin diacetate (DCFH-DA) to cells at a
concentration of 10 mM for 30 min. After treatment, samples were
solubilized in NaOH (0.1 N). Activity was measured at λ
ex
488 nm/λ
em
525 nm. Glutathione was measured using a kinetic assay measuring
the reduction of 5,5′-dithiobis-(2-nitrobenzoic) acid (DTNB) to TNB at
412 nm, after deproteinization with 5% 5-sulfosalicylic acid solution
(Sigma glutathione assay kit; CS0260).
Data are presented as the means ± SEM of six separate experi-
ments. Multiple comparisons of means were carried out by ANOVA
using the Bonferroni test post hoc.
Results
Preliminary experiments established that a Hcy concentration of
30 μM achieved N90% 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
(N90%) 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 protection against Hcy-
induced (30 μM) cell death (Supplementary Figs. 1–7). The non-
thiolatocobalamins cyanocobalamin (CNCbl), hydroxocobalamin
(HOCbl), and MeCbl were least effective at protecting against Hcy-
induced death; their optimized concentrations of 12.5–17. 5 μM
provided ∼30% protection (Pb0.001; Fig. 2). At these concentrations,
thiolatocobalamins afforded greater than 80% protection (Fig.1). Cells
pretreated with a greater concentration of thiolatocobalamin (30 μM)
were not significantly different from unexposed control cells (Fig. 2).
Although cell survival was moderately enhanced (∼55%) by pre-
incubating with NAC (45 μM) or GSH (100 μM), either alone or with
any nonthiolatocobalamin, the protection provided by the thiolato-
cobalamins was significantly superior to that of their corresponding
thiol (Pb0.001; Fig. 2). Caspase-3 is a key enzyme in apoptotic cell
death pathways and a suitable marker for quantifying apoptosis after
Fig. 1. Thiolatocobalamins protect endothelial cells from the effects of Hcy. Sk-Hep-1
cells were exposed to increasing concentrations of NACCbl (■) or GSCbl (▲) for 2 h
before exposure to 30 μM Hcy for 24 h. Cell activity was measured by MTS assay at
490 nm. Data are shown as means ±SEM.
Fig. 2. The protective effects of cobalamins and thiols against Hcy toxicity. Sk-Hep-1
cells were pretreated for 2 h with CNCbl (15.0 μM), HOCbl (17.5 μM), MeCbl (12.5 μM),
NAC (45 μM), GSH (100 μM), NACCbl (30 μM), or GSCbl (30 μM) before exposure to Hcy
(30 μM) for 24 h. Cell activity was measured by MTS assay at 490 nm. Data are shown as
means±SEM, ⁎⁎⁎Pb0.001; NS, not significant. All results are significantly different
from cells treated with Hcy only (Pb0.001); these and other obvious differences are not
annotated on the graph for simplicity.
185C.S. Birch et al. / Free Radical Biology & Medicine 47 (2009) 184–188
Author's personal copy
oxidative stress [17]. Remarkably, Hcy-induced caspase activity was
almost completely prevented in cells pretreated with either NACCbl
(30 μM) or GSCbl (30 μM) (Fig. 3a). Although cells pretreated with
optimized concentrations of nonthiolatocobalamins and thiols (singly
or combined) all showed protection varying from ∼25 to ∼60%, they
were all significantly less protected than those treated with the
thiolatocobalamins (Pb0.001; Fig. 3a). The thiolatocobalamins also
prevented Hcy-induced increases in intracellular peroxide (Fig. 3b)
and decreases in intracellular glutathione (Fig. 3c) concentrations.
Again, the optimized concentrations of nonthiolatocobalamins
and thiols were less effective—ranging from ∼20 to ∼50% reduction
in Hcy-induced peroxide production (Pb0.001; Fig. 3b).
H
2
O
2
is one of the key reactive oxygen species involved in oxidative
stress-induced necrotic cell damage, as measured by PI uptake [17].
Preliminary experiments established that 25 μMH
2
O
2
resulted
in N90% cell death of Sk-Hep-1 cells. Pretreatment with NAC and
then exposure to 25 μMH
2
O
2
reduced PI uptake by ∼35% compared
with a positive control of 25 μMH
2
O
2
(Pb0.01; Fig. 4). Similarly,
pretreatment with GSH followed by exposure to 25 μMH
2
O
2
resulted
in an ∼45% decrease in PI uptake (Pb0.01; Fig. 4). When NAC or GSH
was combined with nonthiolatocobalamins as a pretreatment, PI
uptake was reduced by ∼50% compared with positive control
(Pb0.001; Fig. 4). The thiolatocobalamins provided total protection
against necrosis induced by 25 μMH
2
O
2
(Fig. 4), demonstrating their
superior antioxidant properties. At high concentrations (N250 μM)
NACCbl or GSCbl significantly reduced cell viability, but even at
2.5 mM both compounds reduced cell viability only to 75% that of
controls (Supplementary Fig. 8).
To demonstrate that the results werenot exclusive to Sk-Hep-1 cells
we repeated some of this work with human umbilical vein endothelial
cells (HUVECs). HUVECs required higher concentrations (50 μM) of
Hcy and H
2
O
2
to achieve N90% death. However, thiolatocobalamins
were again superior to nonthiolatocobalamins in protecting against
cell death (Fig. 5a), apoptosis (Fig. 5b), and necrosis (Fig. 5c).
Discussion
In this study, preincubation of Sk-Hep-1 cells with GSH or NAC only
partially protected against Hcy- or H
2
O
2
-induced damage. Their
respective thiolatocobalamin derivatives (GSCbl and NACCbl)
Fig. 3. The effectsof cobalamins and thiols on Hcy-induced(a) caspase-3 activation, (b) peroxide production,and (c) glutathione levels. Sk-Hep-1cells were pretreated for 2 h with CNCbl
(15.0μM), HOCbl (17.5 μM),MeCbl (12.5 μM),NAC (45 μM), GSH (100 μM), NACCbl(30 μM), or GSCbl (30 μM) beforeexposure toHcy (30 μM) for 24 h. (a) Caspase 3 activitywas measured
at 520 nm. (b) DCFH-DA uptake wasmeasured at 488/525 nm. (c)Glutathione was measured from the reduction of DTNB to TNB at 412 nm. Data are shown as means±SEM. ⁎⁎⁎Pb0.001;
NS, not significant. All results are significantly different from cells treated with Hcy only (Pb0.001), these and other obvious differences are not annotated on the graph for simplicity.
Fig. 4. The effects of cobalamins and thiols on H
2
O
2
-induced necrosis. Sk-Hep-1 cells
were pretreated for 24 h with CNCbl (15.0 μM), HOCbl (17.5 μM), MeCbl (12.5 μM), NAC
(45 μM), GSH (100 μM), NACCbl (30 μM), or GSCbl (30 μM) before exposure to H
2
O
2
(25 μM) for 2 h. PI uptake was measured at 617 nm. Data are shown as means±SEM.
⁎⁎⁎Pb0.001; NS, not significant. All results are significantly different from cells treated
with Hcy only (Pb0.001); these and other obvious differences are not annotated on the
graph for simplicity.
186 C.S. Birch et al. / Free Radical Biology & Medicine 47 (2009) 184–188
Author's personal copy
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 antio-
xidant defense molecule. Reduced GSH levels correlate with increased
oxidative stress, mitochondrial damage, and apoptosis [18].A
proportion of the protection afforded by the cobalamins against
Hcy-induced cell death might be attributable to its increased
intracellular clearance due to enhanced methionine synthase activity
[19]. However, the protection shown against H
2
O
2
suggests that
cobalamins also act as antioxidants via other mechanisms.
Consistent with our results, GSH and NAC reduce apoptosis in
cultured endothelial-derived hepatocytes by down-regulating oxida-
tive stress-related mechanisms such as caspase-3 activation [20].
Caspase-3 cleavage is reduced by folic acid and CNCbl in a mouse
model of amyotrophic lateral sclerosis [21]. Similarly, we show that
thiolatocobalamins efficiently prevent peroxide-induced oxidative
stress, maintain intracellular glutathione levels, and inhibit caspase-
3-mediated cell death. However, there are no previous reports of a
direct antioxidant effect of cobalamin alone. Indirect copper-mediated
protection against LDL oxidation is reported for CNCbl and attributed
to an altered in vitro equilibrium between oxidized and reduced
cobalamin forms [22]. Indeed, cob(II)alamin—a reduced form of
cobalamin—is an efficient radical trap [23], and the superior protective
effect of thiolatocobalamins compared with nonthiolatocobalamins
might relate to their more facile reduction to cob(II)alamin or cob(I)
alamin and direct scavenging-type reactions between these reduced
forms and reactive oxygen and/or nitrogen species [24–27].
Cobalamin's protective effects may also relate to other recently
described novel non-coenzymatic functions [28–30]. Cobalamin-
deficient rats exhibit increased cerebrospinal fluid levels of some
neurotoxic molecules, including TNF-α, and decreased levels of
neurotrophic molecules, including epidermal growth factor (EGF)
[30]. Similarly, patients with severe cobalamin deficiency have high
TNF-αlevels and low EGF levels in cerebrospinal fluid and serum,
which are correctable by cobalamin replacement [3]. These observa-
tions suggest that cobalamin modulates the expression of certain
cytokines and growth factors. It is possible that this occurs as a
consequence of cobalamin modifying the activity of signaling mole-
cules such as NF-κB[30].
The antioxidant properties of cobalamin probably result from a
combination of direct and indirect effects: stimulation of methionine
synthase activity [4,23], direct reaction with reactive oxygen and
nitrogen species, a glutathione sparing effect [31], and modification of
signaling molecules [30], leading to induction of stress responses. The
remarkably superior protection of thiolatocobalamins in vitro pre-
sumably relates to their enhanced function in one or more of these
potential mechanisms, the balance of which may differ between the
two compounds. In conclusion, cobalamins, and in particular the
thiolatocobalamins, exhibit a marked antioxidant activity at pharma-
cological concentrations and afford significant cellular protection
against oxidative stress. Thiolatocobalamins might have potential in
treating a number of pathological conditions in which oxidative stress
is a clinically important component.
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 appli-
cations for the use of a synthetic vitamin B
12
derivative, glutathio-
nylcobalamin,”and U.S. Application 20080076733, “Pharmaceutical
compositions and therapeutic applications for the use of a novel
vitamin B
12
derivative, N-acetyl-L-cysteinylcobalamin”) and by A.M.
(U.S. Application 20040157783, “Method for treating or preventing a
functional vitamin B
12
deficiency in an individual and medical
compositions for use in said method”). A.M. is a Scientific Advisor
and shareholder of COBALZ Ltd—a private limited company develop-
ing novel B vitamin and antioxidant supplements.
Appendix A. Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.freeradbiomed.2009.04.023.
Fig. 5. Thiolatocobalamins protect HUVECs from the effects of Hcy. (a) HUVECs were exposed to increasing concentrations of NACCbl (■) and GSCbl (▲) for 2 h before exposure to
50 μM Hcy for 24 h. (b and c) HUVECs were pretreated for 2 h with CNCbl (15.0 μM), HOCbl (17.5 μM), MeCbl (12.5 μM), NACCbl (30 μM), or GSCbl (30 μM) before exposure to (b) Hcy
(50 μM) or (c) H
2
O
2
for 24 h and measurement of (b) caspase 3 activity or (c) propidium iodide uptake. Data are shown as means ±SEM. ⁎⁎⁎Pb0.001. All results are significantly
different from cells treated with Hcy or H
2
O
2
only (Pb0.001); these differences are not annotated on the graph for simplicity.
187C.S. Birch et al. / Free Radical Biology & Medicine 47 (2009) 184–188
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