Melatonin prevents hypochlorous acid-mediated cyanocobalamin destruction and cyanogen chloride generation

Article abstract of DOI:10.1111/jpi.12463

Hypochlorous acid (HOCl) is a potent cytotoxic oxidant generated by the enzyme myeloperoxidase (MPO) in the presence of hydrogen peroxide (H₂O₂) and chloride (Cl^?). Elevated levels of HOCl play an important role in various pathological conditions through oxidative modification of several biomolecules. Recently, we have highlighted the ability of HOCl to mediate the destruction of the metal-ion derivatives of tetrapyrrole macrocyclic rings such as hemoproteins and vitamin B₁₂ (VB₁₂) derivatives. Destruction of cyanocobalamin, a common pharmacological form of VB₁₂ mediated by HOCl, results in the generation of toxic molecular products such as chlorinated derivatives, corrin ring cleavage products, the toxic blood agents cyanide (CN^?) and cyanogen chloride (CNCl), and redox-active free cobalt. Here, we show that melatonin prevents HOCl-mediated cyanocobalamin destruction, using a combination of UV-Vis spectrophotometry, high-performance liquid chromatography analysis, and colorimetric CNCl assay. Identification of several melatonin oxidation products suggests that the protective role of melatonin against HOCl-mediated cyanocobalamin destruction and subsequent CNCl generation is at the expense of melatonin oxidation. Collectively, this work highlights that, in addition to acting as an antioxidant and as a MPO inhibitor, melatonin can also prevent VB₁₂ deficiency in inflammatory conditions such as cardiovascular and neurodegenerative diseases, among many others.

Full text of DOI:10.1111/jpi.12463

DR HUSAM M. ABU-SOUD (Orcid ID : 0000-0003-0939-4256)
Article type : Original Manuscript
Melatonin prevents hypochlorous acid mediated cyanocobalamin
destruction and cyanogen chloride generation
Roohi Jeelani*1, Dhiman Maitra*1, Charalampos Chatzicharalampous1, Syed Najeemuddin1,
Robert T. Morris2 and Husam M. Abu-Soud 1,3¶
1Department of Obstetrics and Gynecology, Wayne State University School of Medicine, C. S. Mott
Center for Human Growth and Development, 275 E. Hancock, Detroit, MI, 48201 USA
2Division of Gynecologic Oncology, Department of Oncology, Wayne State University and Karmanos
Cancer Institute, Detroit, MI, 48201 USA
3Department of Microbiology, Immunology and Biochemistry, Wayne State University School of
Medicine, Detroit, MI, 48201 USA.
Running Title- Melatonin prevents Vitamin B12 destruction
¶Address correspondence to: Husam Abu-Soud, Department of Obstetrics and Gynecology, C. S. Mott
Center for Growth and Development, Wayne State University, 275 E. Hancock Detroit, MI 48201.
Tel: 313/577-6178; Fax: 313/577-8554; email: habusoud@med.wayne.edu
*Both authors contribute equally to the manuscript
Abbreviations: MPO, myeloperoxidase; HOCl, hypochlorous acid; vitamin B12, VB12; H2O2,
hydrogen peroxide; Cl-, chloride; CN-, cyanide; CNCl, cyanogen chloride.
This article has been accepted for publication and undergone full peer review but has not
been through the copyediting, typesetting, pagination and proofreading process, which may
lead to differences between this version and the Version of Record. Please cite this article as
doi: 10.1111/jpi.12463
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Key Words: vitamin B12, mammalian peroxidases, hypochlorous acid, cyanocobalamin, melatonin,
circadian rhythm
Supported by The National Institutes of Health grant # RO1 HL066367, and the Department of
Obstetrics and Gynecology, Wayne State University, Detroit, MI, USA.
ABSTRACT
Hypochlorous acid (HOCl) is a potent cytotoxic oxidant generated by the enzyme
myeloperoxidase (MPO) in the presence of hydrogen peroxide (H2O2) and chloride (Cl-).
Elevated levels of HOCl play an important role in various pathological conditions through
oxidative modification of several biomolecules. Recently, we have highlighted the ability of
HOCl to mediate the destruction of the metal-ion derivatives of tetrapyrrole macrocyclic
rings such as hemoproteins and vitamin B12 (VB12) derivatives. Destruction of
cyanocobalamin, a common pharmacological form of VB12 mediated by HOCl, results in the
generation of toxic molecular products such as chlorinated derivatives, corrin ring cleavage
products, the toxic blood agents cyanide (CN-) and cyanogen chloride (CNCl), and redox
active free cobalt. Here, we show that melatonin prevents HOCl-mediated cyanocobalamin
destruction, using a combination of UV-Vis spectrophotometry, HPLC analysis, and
colorimetric CNCl assay. Identification of several melatonin oxidation products suggests that
the protective role of melatonin against HOCl-mediated cyanocobalamin destruction and
subsequent CNCl generation is at the expense of melatonin oxidation. Collectively, this work
highlights that, in addition to acting as an antioxidant and as a MPO inhibitor, melatonin can
also prevent VB12 deficiency in inflammatory conditions such as cardiovascular and
neurodegenerative diseases, among many others.
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INTRODUCTION
Hypochlorous acid (HOCl) is a potent microbicidal agent generated by the
myeloperoxidase (MPO)-hydrogen peroxide (H2O2)-Chloride (Cl-) system in phagocytic cells
including neutrophils and plays a key role in innate immunity1-3. Hypochlorous acid is a
strong oxidant, modifies a wide array of biomolecules ranging from proteins, nucleic acids
and lipids through its ability to react with: 1) free amine groups in amino acids, peptides and
proteins to form toxic chloramines; and 2) tyrosine to form 3-chlorotyrosine and 3, 5-dichloro
tyrosine, both of which are specific biomarkers of HOCl induced damage 4-8. Pyrimidine
nucleotides (either free or in DNA/ RNA strands) are readily chlorinated by HOCl-induced
reaction and by this pathway HOCl causes extensive denaturation of double-stranded DNA
9,10
and also acts as an effective mutagen, clastogen and induces sister chromatid exchange
.
HOCl initiates alterations of the heme prosthetic group of both ferrous-dioxy and
methemoglobin, ultimately leading to heme destruction 11. Recently, utilizing rapid kinetic
measurements, HPLC and mass spectrometric techniques we have highlighted the mechanism
through which HOCl mediates the destruction of the metal-ion derivatives of tetrapyrrole
macrocyclic rings such as hemoproteins and vitamin B12 (VB12) derivatives 12-17. We have
shown destruction of cyanocobalamin (Fig 1A), a common pharmacological form of VB12,
mediated by HOCl, resulted in the generation of toxic molecular products such as chlorinated
derivatives, different degradation products of corrin, the organic ring system of cobalamin,
release of the toxic blood agents (CN- /cyanogen chloride (CNCl)), and redox active free
cobalt (Co) through the formation of chloro-cyanocobalamin (Fig 1B) 15 18
.
Interestingly, many of the disorders known to be associated with VB12 deficiency are
also associated with increased MPO/HOCl levels19-24. For instance, deficiency of VB12 is
associated with neurodegenerative disorders including but not limited to Parkinson and
Alzheimer disease and megaloblastic anemia, elevated blood homocysteine level (a risk
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factor for cardiovascular disease and adverse pregnancy outcomes)19,20,22-25. These negative
effects are primarily because the most prominent function of cyanocobalamin is to act as
cofactor in two major biochemical pathways; 1) in the conversion of homocysteine to
methionine by methionine synthase and 2) in the conversion of methylmalonyl CoA to
succinyl CoA by methylmalonyl CoA mutase26 (Fig. 1C).
Many
experiments
have
demonstrated
that
melatonin
(N-acetyl-5-
hydroxytryptamine) can protect against HOCl mediated damage16,27-30. This ubiquitous
molecule is found in a large cross-section of organisms, ranging from unicellular organism,
fungi, plants to higher invertebrate and vertebrate animals31. In humans, it is most commonly
known to be a modulator of circadian rhythm and is produced in the pineal gland, from the
amino acid tryptophan32-34. In addition, melatonin can be found in the gastrointestinal tract,
cerebellum, retina, skin, ovary, liver, pancreas, kidneys, and the immune competent cells 33,35-
41. Additionally, melatonin is also involved in immune enhancing35, anti-inflammatory
pathway 42,43, and can inhibit cancer progression41,44,45. Several of melatonin’s functions are
mediated by the membrane bound G-protein coupled receptors, MT1 and MT246,47. In
addition to the receptor-mediated functions, melatonin performs various biochemical
functions, for example acts as an antioxidant. Previously, melatonin has also been shown to
be a major scavenger of HOCl48. In addition, melatonin has the ability to scavenge a variety
of reactive oxygen species (ROS)/free radicals, such as, H2O2, hydroxyl radical (•OH),
superoxide (O2 ), alkoxyl radical, peroxyl radical, singlet oxygen, and nitric oxide40,49-51
.
•-
Importantly, melatonin also serves as an inhibitor for enzymes that generate reactive oxygen
species, such as MPO and eosinophil peroxidase, the major sources for hypohalous acids 29,30
.
In this work, we examined the ability of melatonin to prevent HOCl-mediated
cyanocobalamin corrin destruction and subsequent release of the blood agents CN-/CNCl, and
redox active free Co release. Our results showed that melatonin prevents HOCl-mediated
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cyanocobalamin destruction and subsequent release of toxic agents. The conversion of
several melatonin oxidation products suggests that the protective role of melatonin against
HOCl-mediated cyanocobalamin destruction is at the expense of melatonin oxidation.
Collectively, melatonin displays the ability to prevent VB12 destruction in inflammatory
conditions and could benefit patients with disorders associated with deficiency of this
vitamin.
Materials and Methods
Materials
All the materials used were of highest purity grade available and used without further
purification. Melatonin, sodium hypochlorite (NaOCl), cyanocobalamin, pyridine, 1,3-
dimethyl barbituric acid, L-methionine, and dimethylformamide (DMF) HPLC-grade were
obtained from Sigma Aldrich (St Louis, MO, USA). HPLC-grade acetonitrile was obtained
from EMD Chemicals Inc. (Gibbstown, NJ, USA).
Kinetic analysis
Kinetic measurements were performed using a 96-well plate reader (Spectra Max 190;
Molecular Devices, Sunnyvale, FL, USA). Briefly, experiments were performed in 200 μL
final volume containing 200 mM phosphate buffer, pH 7.4, at 25 °C. A fixed concentration of
cyanocobalamin (20 µM) was incubated with increasing concentrations of melatonin (0 - 450
µM) prior to addition of 450 µM of HOCl. Immediately after addition of HOCl, the increase
in absorbance at 590 nm was monitored as a function of time. Similarly, for studying the rate
of HOCl-mediated cyanocobalamin destruction in presence of melatonin, cyanocobalamin
(20 µM) was incubated with increasing concentrations of melatonin (0 – 4000 µM) prior to
addition of 4000 µM of HOCl, and the decreases in absorbance at 590 nm was monitored as a
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function of time. The rate of the reaction was calculated by the slope of the tangent drawn at
the initial linear part of the curve. All experiments were performed in triplicates.
High-performance liquid chromatography (HPLC) analysis
HPLC analyses were carried out using a Shimadzu HPLC system equipped with an SCL-10A
system controller, with a binary pump solvent delivery (LC-10 AD) module and an SIL-
10 AD autoinjector connected to an SPD-M10A diode array detector and an RF-10A XL
fluorescence detector. An Alltech 5-µm particle size, 4.6 150-mm reverse-phase
octadecylsilica (C18) HPLC column was used.
Detection of Cl-cyanocobalamin
50 µL of cyanocobalamin-HOCl reaction mixture was injected to analyze HOCl-mediated
formation of Cl-cyanocobalamin and its modulation by melatonin. The photodiode array
detector was set at 360 nm to monitor the chromatogram. The column was eluted isocratically
at a flow rate of 2.0 ml/min with 20 % acetonitrile and 80 % water. Each sample was
analyzed in triplicate.
Cyanocobalamin destruction and analysis of melatonin oxidation products
50 µL of cyanocobalamin-HOCl reaction mixture was injected to analyze HOCl-mediated
destruction of cyanocobalamin and its prevention by melatonin. The column was eluted
isocratically at a flow rate of 1.0 ml/min with 40 % acetonitrile and 60 % water. Each sample
was analyzed in triplicate. To monitor cyanocobalamin, the photodiode array detector was set
at 360 nm to obtain the chromatogram. For analyzing melatonin consumption and formation
of melatonin oxidation products, the photodiode array detector was set at 260 nm. AFMK
was detected by its intrinsic fluorescence by setting the fluorescence detector at excitation
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321 nm and emission 465 nm to monitor the chromatogram. Each sample was analyzed in
triplicate.
Colorimetric detection of CNCl
CNCl was detected colorimetrically using the pyridine-1,3-dimethyl barbituric acid reagent
as described previously15,52. In this assay, CNCl reacts with pyridine to form a dialdehyde,
glutacon dialdehyde, which then reacts with 1,3-dimethyl barbituric acid and condenses to
form a violet-colored polymethine dye. The composition of the pyridine-1,3-dimethyl
barbituric acid (coloring reagent) was as follows: 1.2 g of 1,3-dimethyl barbituric acid was
dissolved in a mixture of 12.8 ml of water and 6 ml of pyridine, and then 1.2 ml of HCl was
added to the solution to bring the total volume to 20 ml. Cyanocobalamin (100 µM) was
treated with 3000 µM of HOCl in presence or absence of 3000 µM melatonin at 10 ºC for 2
hours. Following which the reaction was stopped by adding high molar excess of methionine.
Then coloring reagent (500 µL), was added to 500 µL of cyanocobalamin HOCl reaction
mixture and was incubated for 15 min at 10ºC. The absorbance of the resulting violet-
colored solution was measured at 587.5 nm against the cyanocobalamin HOCl reaction
mixture (without addition of coloring reagent, but diluted with an equal volume of water) to
subtract the absorbance due to residual cyanocobalamin. The amount of CNCl was
1
determined from the extinction coefficient of 1.03 105 M cm 1 for the violet-colored
complex53.
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Solution preparation
HOCl preparation
HOCl was prepared following a slight modification of a published method54. Briefly, a stock
solution of HOCl was prepared by adding 1 ml of sodium hypochlorite (NaOCl) solution to
40 ml of 154 mM and the pH was adjusted to around 3 by adding HCl. The concentration of
active total chlorine species in solution, determined as [HOCl]T (where
[HOCl]T = [HOCl] + [Cl2] + [Cl3 ] + [OCl−]), in 154 mM NaCl was based on converting all
−
the active chlorine species to OCl− by adding a single bolus of 40 μl of 5 M NaOH and then
measuring the concentration of OCl−. The concentration of OCl− was determined
spectrophotometrically at 292 nm (ε = 362 M− 1 cm− 1). As HOCl is unstable, the stock
solution was freshly prepared on a daily basis, stored on ice, and used within 1 h of
preparation. For further experimentations, dilutions were made from the stock solution using
200 mM phosphate buffer, pH 7.4, to give working solutions of lower HOCl concentration.
Cyanocobalamin preparation
Cyanocobalamin stock solution was prepared by dissolving cyanocobalamin in distilled
water. The concentration of the stock solution was determined spectrophotometrically at
361 nm (ε = 27.5 mM− 1 cm− 1).
Melatonin preparation
Melatonin was prepared by dissolving 92 mg of M melatonin elt in 1 mL of DMF, to give a
solution of concentration 396 mM. Melatonin solution prepared in this manner was shielded
from light and kept on ice and used within 1 hr of preparation.
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Results
We first utilized HPLC techniques to investigate the effect of melatonin in preventing
the HOCl-mediated cyanocobalamin destruction by pre-incubating cyanocobalamin with
increasing molar ratios of melatonin:HOCl (0:1 up to 2:1) prior to HOCl addition and HPLC.
Under our experimental conditions, cyanocobalamin eluted around 3 min and was identified
based on its characteristic UV-Vis spectra (360, 540 and 550 nm) obtained from the diode
array detector (Fig. 2A & 2B). Treatment of cyanocobalamin (10 µM) with 100 µM of HOCl
led to a complete disappearance of the cyanocobalamin peak and instead a new peak
appeared with an elution time of 4.25 mins. The spectral feature of this new peak was
distinctly red-shifted, from 360 to 363 nm with an additional shift in the visible absorption
region (from 550 to 590 nm), and was identified to be as Cl-cyanocobalamin, in accordance
with our earlier observation15 (Figure 2B). Adding 20 µM of melatonin to the reaction
mixture (melatonin -HOCl) decreased in Cl-cyanocobalamin peak and the restoration of the
cyanocobalamin peak. Further increasing the melatonin concentration leads to complete
restoration of the cyanocobalamin peak in a concentration dependent manner. The area under
the curve for the cyanocobalamin peak and Cl-cyanocobalamin peak was plotted as a
function of melatonin:HOCl ratio (Fig. 2C). The relative amount of Cl-cyanocobalamin
decreased linearly until melatonin:HOCl ratio 0.4:1, after which the Cl- cyanocobalamin
level gradually plateaued off, and saturating at melatonin:HOCl ratio 1:1 (Fig. 2C, red curve).
A parallel trend was observed for the amount of cyanocobalamin (Fig. 2C, black curve). Next
to study the effect of melatonin on the kinetics of the process of HOCl-mediated Cl-
cyanocobalamin formation UV-Vis spectrophotometry was used. To phase out the effect of
melatonin on the spectra of cyanocobalamin, the increase in absorbance was monitored at 590
nm, where melatonin (at the concentrations used), had no interference. Cyanocobalamin (20
µM) was preincubated with different melatonin:HOCl molar ratios (0 to 2:1) and then 450
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µM HOCl was added to the reaction mixture and increase in absorbance at 590 nm was
recorded. Figure 2D, shows the profile of the absorbance increase at 590 nm, as a function of
time for different melatonin:HOCl ratios. In absence of melatonin, HOCl caused the
absorbance to increase exponentially, and signal saturated at approximately 500s. When
cyanocobalamin, was pretreated with melatonin, we observed a decrease in the amplitude of
the absorbance change, as well as a decrease in the slope of the curve, signifying the rate.
Indeed, when the pseudo first order rate of Cl- cyanocobalamin formation was plotted as a
function of melatonin:HOCl ratio, we observed that the rate of Cl- cyanocobalamin formation
decreased linearly until 0.6:1, after which the rate plateaued off and saturated around 2:1
melatonin:HOCl ratio. Thus, we conclude that melatonin can effectively prevent HOCl-
mediated Cl- cyanocobalamin formation.
As reported earlier, the process of HOCl-mediated cyanocobalamin destruction
involves two distinct steps, first conversion of cyanocobalamin to Cl- cyanocobalamin and
next oxidative destruction of Cl- cyanocobalamin15. Since our current results indicated that
melatonin prevents HOCl-mediated generation of Cl- cyanocobalamin we hypothesized that
melatonin will prevent HOCl-mediated cyanocobalamin destruction and subsequent
generation of CNCl. To test our hypothesis, cyanocobalamin (20 µM) was incubated with
HOCl (1000 µM) in presence/absence of melatonin (1000 µM). HPLC analyses revealed that
in absence of melatonin, HOCl treatment led to complete destruction of cyanocobalamin,
while pretreatment with 1:1 melatonin:HOCl prevented cyanocobalamin destruction by 96 %
(see Fig. 3A and Fig. 3A inset). Next, to test how melatonin modulates the kinetic of the
process of HOCl-mediated cyanocobalamin destruction UV-Vis spectrophotometry was
utilized. Experiments were performed as mentioned earlier. Briefly cyanocobalamin (20 µM)
was preincubated with different melatonin:HOCl molar ratios (0 to 1:1) and then 4000 µM
HOCl was added to the reaction mixture and decrease in absorbance at 590 nm was recorded.
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Figure 3B, shows the pseudo-first order rate of HOCl-mediated cyanocobalamin corrin ring
destruction plotted as a function of melatonin:HOCl ratio. As the melatonin:HOCl ratio
increased we observed a linear decrease in the rate of cyanocobalamin destruction, which
ultimately approached ~0 at around 0.22:1 melatonin:HOCl ratio. Since our results showed
that melatonin effectively prevents HOCl-mediated cyanocobalamin corrin destruction, we
wanted to test the role of melatonin in inhibiting HOCl-mediated CNCl generation from
cyanocobalamin. As shown in Fig. 3C, treatment of cyanocobalamin with HOCl in absence
of melatonin, led to generation of 1.65 µM of CNCl while no CNCl was detected in control
cyanocobalamin without HOCl treatment. But when cyanocobalamin was preincubated with
1:1 molar ratio of melatonin:HOCl and then HOCl was added, the amount of CNCl generated
decreased by ~72 % to 0.45 µM.
To understand the mechanism of how melatonin prevents HOCl-mediated
cyanocobalamin destruction, we compared the melatonin oxidation products that were
formed when HOCl oxidized melatonin in presence and absence of cyanocobalamin. The
chromatograms (see Fig. 4A) were monitored at 260 nm to phase out the interference from
any unreacted cyanocobalamin /Cl- cyanocobalamin derivatives. Under our chromatographic
conditions, control unreacted melatonin eluted around ~ 11 min, and was identified from its
characteristic spectra (purple trace in Fig. 4A). As expected, cyanocobalamin alone and
cyanocobalamin treated with HOCl in absence of melatonin did not show any major peaks
(Fig. 4A, traces labeled as ‘cyanocobalamin’ and ‘cyanocobalamin +HOCl’). In contrast the
melatonin +HOCl and cyanocobalamin + melatonin +HOCl reaction mixtures displayed,
three major UV absorbing peaks labeled as ‘Peak I’, ‘Peak II’ and ‘Peak III’. The retention
time and spectral characteristics of the Peak I-III are listed in Table 1. The spectral features of
the compounds eluting as ‘Peak II’ and ‘Peak III’ are very similar, while ‘Peak I’ shows a
distinct red-shift (Fig. 4B). We also detected the formation of the fluorescent melatonin
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oxidation
product
N-[3-[2-(formylamino)-5-methoxyphenyl]-3-oxypropyl]-acetamide
(AFMK) (chromatogram not shown) as reported earlier by Dellegar et. al. 51 and Ximenes et
al who also described the formation of a melatonin dimer55. Although previous reports
suggested formation of 2-hydroxymelatonin as a product of HOCl-induced melatonin
oxidation51, we failed to identify 2-hydroxymelatonin, from the spectral features of the three
peaks. 2-hydroxymelatonin is relatively unstable and could easily convert to its keto
tautomer, an indolinone, a rather lipophilic molecule56. Detailed mass-spectrometric and
NMR studies are currently underway in our laboratory to characterize these novel melatonin
oxidation products. The relative amount of the melatonin oxidation product (as judged by the
area under the curve for each peak), formed when melatonin reacted with HOCl in presence
or absence of cyanocobalamin is shown in Fig. 4C. We observed similar trend for the
melatonin +HOCl and cyanocobalamin + melatonin +HOCl reaction mixtures respectively,
indicating that melatonin protects HOCl-mediated cyanocobalamin corrin ring destruction by
virtue of its ability to compete and scavenge HOCl.
Discussion
Here, we showed that melatonin is capable in preventing HOCl-mediated VB12
destruction and the generation of toxic molecular products such as chlorinated derivatives,
corrin ring cleavage products, the toxic blood agents CN- and CNCl, and redox active free
cobalt. The identification of several melatonin oxidation products clearly suggests that corrin
ring protection was at the expense of melatonin. Our work may establish a direct mechanistic
link by which melatonin exerts its antioxidant protective effect of VB12 under chronic
inflammatory conditions where MPO chlorinating activity is elevated.
In human, melatonin and VB12 complement each other as antioxidants and in the
regulation of circadian rhythm. Vitamin B12 exerts a direct influence on melatonin causing an
earlier release of melatonin at night, which resets the sleep-wake cycle, by acting directly on
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the pineal gland to provoke a faster release of melatonin 57. Vitamin B12 also causes
melatonin to drop off faster by sensitizing the body to morning light 58-60. Very serious sleep-
wake disorders have been successfully treated with vitamin B12 in the methylcobalamin
form58-61. Here we show a novel mechanistic role of melatonin, in which it can act to prevent
the corrin ring destruction under inflammatory diseases where HOCl is elevated therefore
preventing VB12 deficiency under such conditions.
Vitamin B12 is not only used for treating and preventing vitamin B12 deficiency, but
also used for treating several conditions related to memory loss, immune system, aging, heart
disease, male infertility, diabetes, sleep disorders, depression, mental disorders, and
inflammation 62,63. The safety in the 64 use of cyanocobalamin supplementation, versus other
VB12 derivatives, is a concern because: 1) biosynthesis of the B12 coenzyme requires the
release of CN- that can be used by the body’s cells manifesting acute cyanide poisoning 65,66
unwanted inflammation, reactions associated with the generation of higher levels of HOCl,
can also be a major destructive force of the vitamin corrin ring generating CNCl, free active
Co, and corrin degradation products15. Indeed, our current and previously published results
suggest that the degradation of cyanocobalamin mediated by HOCl is largely modulated by
the concentration of HOCl in the reaction milieu which could be partially or completely
prevented by the presence of melatonin. Thus, any dysregulation of neutrophils/macrophages
derived-HOCl contributes to inflammation, or reduction in HOCl consumption might get
manifested as increased cyanocobalamin destruction and CNCl generation15. Therefore, it is
of enormous therapeutic and pharmacologic importance, to prevent HOCl-mediated damage,
especially in chronic inflammation, where
a
higher rate of infiltration of
monocytes/macrophages over a longer period leads to pathologic alterations.
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Besides HOCl mediated corrin ring destruction, there are several other causes that can
lead to VB12 deficiency. For example, two widely studied causes of VB12 deficiency are:
Pernicious anemia, an autoimmune condition, and food-bound VB12 malabsorption67-70. Both
diseases have been associated with atrophic gastritis, a condition characterized by chronic
71
inflammation of the gastric mucosa . It has been generally perceived that the development
of atrophic gastritis is due to chronic Helicobacter pylori (H. pylori) infection, which leads to
extensive infiltration of the gastric mucosa by neutrophils72. MPO in
neutrophils/macrophages amplifies the oxidative potential of H2O2 that induces gastric
mucosal damage, and thus MPO is suspected to play a role in H. pylori-induced gastric
injury73. The development of atrophic gastritis subsequent to H. pylori infection is determined
by host-bacterial interaction with studies favoring host-related factors74. It has been shown
that host MPO genetic polymorphism may play an important role in the pathogenesis of
atrophic gastritis upon H. pylori infection75. In addition to lack of intrinsic factor, VB12
deficiency in pernicious anemia and atrophic gastritis, could be explained in part by
enhancement of MPO activity that leads to distortion of the corrin ring of cyanocobalamin by
HOCl 15. Destruction of cyanocobalamin tetrapyrrole macrocycle by HOCl is also believed
to be a leading event in cell injury, because it is tightly coupled with the generation of several
undesired toxic molecules such as redox-active transition metals (e.g. cobalt and iron), which,
76-81
through Fenton reaction forms highly reactive free radicals such as •OH
.
VB12
destruction will also lead to the elevation of plasma homocysteine and methylmalonyl CoA
levels by inhibiting methionine synthase and L-methyl malonyl CoA mutase 82.
A scheme of MPO catalysis that incorporates our current findings is presented in Fig.
5. In this working model, stimulation of neutrophils activates the membrane bound NADPH
oxidase, which converts molecular oxygen to O2•- 1. Superoxide either spontaneously or by an
enzyme-catalyzed process (through SOD, superoxide dismutase) is converted to H2O2.
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Concurrent to NADH oxidase activation there is also release of MPO 83. MPO utilizes H2O2
in the presence of Cl-, through the formation of a resonance-stabilized porphyrin π cation
radical (Compound I), to generate HOCl54. HOCl plays an important role in the innate
immune response, but sustained high levels of HOCl are implicated in several pathological
conditions1. Recently we have demonstrated that HOCl can oxidatively destroy tetrapyrrole
macrocycles such as free heme 13, heme moiety in hemoproteins (hemoglobin and
12,14
lactoperoxidase)
and cyanocobalamin15. Melatonin modulates HOCl-mediated
cyanocobalamin destruction in a concentration-dependent fashion by at least through two
distinct pathways (Fig. 5). Firstly by serving as an inhibitor of MPO by its ability to act as a
1e- substrate for MPO Compounds I and II (MPO-Fe(IV)=O complex) , or binding directly to
MPO above the heme iron forming melatonin -MPO-Fe(III) complex and secondly, by direct
scavenging of HOCl30,48,84. In the presence of melatonin, rapid kinetic studies suggested that
the Compound I oxidizes melatonin through two sequential 1-electron steps, forming
Compound II and MPO-Fe(III), respectively85. Through this mechanism, melatonin inhibits
MPO chlorinating activity but not the peroxidase activity85. Alternatively, melatonin binds
directly to MPO-Fe(III) presumably above the heme moiety, to readily promote reversible
inhibition of peroxidase activity, to form a dead-end melatonin -MPO-Fe(III) complex85, and
secondly, by its ability to directly scavenge HOCl may protect cyanocobalamin from HOCl-
mediated degradation 40,51. Thus, one mechanism through which melatonin might prevent
VB12 mediated HOCl-damage in vivo is by minimizing the accumulation of HOCl during
steady-state catalysis. As depicted in Fig. 4, the effect of melatonin on MPO-Fe(III)
peroxidase activity will depend on multiple factors, including the bioavailability of
melatonin versus H2O2, the affinity of MPO-Fe(III) for melatonin versus H2O2, and the rate
of melatonin -MPO-Fe(III) breakdown.
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Melatonin functions as an antioxidant has been proven not only through its capacity to
scavenge a variety of free radical and ROS, but also through its ability to chelate transition
metals (e.g. iron (III), copper and zinc), a major source of ROS86-88. Thereby resulting in the
lowering of ROS harmful effects such as lipid peroxidation, protein oxidation, and DNA
damage27,35,49,87-89. Importantly, melatonin oxidation products have no biologically harmful
consequences when it reacts with HOCl, therefore, its use as anti-oxidant is exceptional
among other HOCl scavengers such as taurine, cystine, cysteine and uric acid),29,90. For
example, 2-hydroxymelatonin is produced54 at a sufficient rate by the reaction of melatonin
with HOCl to protect catalase against HOCl-mediated inactivation48. Collectively, inhibiting
the MPO chlorinating activity or scavenging HOCl by melatonin might be a useful
therapeutic approach in reducing VB12 deficiency, and eliminating the subsequent sequelae of
the toxic cyanocobalamin degradation products generated by HOCl in a wide variety of
inflammatory conditions.
Figures Legends
Figure 1: Function of vitamin B12 (A) and structures of cyanocobalamin (B) and Cl-
cyanocobalamin (C).
Figure 2: Melatonin prevents HOCl mediated α-axial ligand replacement and
subsequent formation of Cl-cyanocobalamin. (A) HPLC analysis of cyanocobalamin-
HOCl reaction mixtures in presence different molar ratios of melatonin:HOCl (ratios shown
in inset). Chromatograms were monitored at 360 nm. The position of the cyanocobalamin and
Cl-cyanocobalamin peaks are marked by blue and black arrows, respectively. The data shown
is a representative of three independent experiments. (B) UV-Vis spectra of the
cyanocobalamin (dotted line) and Cl- cyanocobalamin (solid lines) peaks, as obtained from
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the diode array detector. The Cl-cyanocobalamin spectra was plotted as a function of
melatonin:HOCl molar ratios (ratios shown in inset). (C) Relative amount of cyanocobalamin
and Cl- cyanocobalamin as calculated from the area under the curve for each peaks, were
plotted as a function of melatonin:HOCl molar ratios. For cyanocobalamin, the area under the
curve for cyanocobalamin alone was defined as 100 %, while for Cl- cyanocobalamin, the
area under the curve at 0 melatonin was defined as 100 % and subsequent areas were
normalized to those values. The data points are the average of three independent experiments,
and the error bars represent the standard error of measurements. (D) Kinetic traces for the
cyanocobalamin-HOCl reaction monitored at 590 nm, showing the formation of Cl-
cyanocobalamin, as a function of different molar ratios of melatonin:HOCl. These data are
average of three independent experiments. (E) Pseudo-first order rate constants of formation
of Cl-cyanocobalamin, plotted as a function of melatonin:HOCl molar ratios. The data points
are the average of three independent experiments, and the error bars represent the standard
error of measurements.
Figure 3: Melatonin prevents HOCl mediated corrin destruction and CNCl generation
from cyanocobalamin. (A) HPLC analysis of cyanocobalamin-HOCl reaction mixtures in
presence/absence of 1:1 melatonin:HOCl molar ratio. Chromatograms were obtained at 360
nm. Relative amount of cyanocobalamin remaining was calculated from the area under the
curve for the cyanocobalamin peak, and is shown in inset. The data shown is a representative
of three independent experiments and the error bars are represent the standard error of
measurements. (B) Pseudo-first order rate constants of HOCl-mediated cyanocobalamin
destruction, plotted as a function of melatonin:HOCl molar ratios. The data points are the
average of three independent experiments, and the error bars represent the standard error of
measurements. (C) cyanocobalamin (100 µM) was treated with 3000 µM of HOCl in
presence/absence of melatonin (3000 µM) and CNCl generation was assayed colorimetrically
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as detailed under Materials and Methods section. The data are representative of three
independent experiments with the error bars representing the standard error measurements.
Figure 4: Analysis of melatonin oxidation products generated when melatonin was
oxidized by HOCl in presence or absence of cyanocobalamin. (A) HPLC analysis of
melatonin oxidation products generated by HOCl in presence and absence of
cyanocobalamin. Final concentration of reactants cyanocobalamin (10 µM), melatonin (1000
µM) and HOCl (1000 µM). Chromatograms were monitored at 260 nm. The peak
corresponding to unreacted melatonin is labeled as “Melatonin”. Three other, major UV
absorbing peaks are labeled as I, II and III respectively. The data shown is a representative of
three independent experiments. (B) UV-Vis spectrum of peak I, II and III as obtained from
the diode array detector. (C) Relative amount of each product generated as calculated from
the area under the curve for each peak. AFMK was detected via fluorescence detector
(chromatogram not shown) as mentioned in the Materials and Methods section. The data
shown is a representative of three independent experiments and the error bars are represent
the standard error of measurements.
Figure 5: Schematic model describing the role of melatonin in preventing HOCl-
mediated cyanocobalamin destruction.
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Author Contributions
H.M.A-S., D.M. and R.J. conceived and designed the experiments; D.M., R.J., C.C. and S.N.
conducted the experiments. D.M., R.J., C.C., S.N. and R.M. analyzed the data; and R.J.,
D.M. and H.M.A-S wrote the manuscript. All authors reviewed the manuscript.
Acknowledgement
This work was supported by a grant to H.M.A-S. from the National Institutes of Health, RO1
HL066367 and Children’s Hospital of Michigan, Detroit, MI, USA.
1.
Davies MJ, Hawkins CL, Pattison DI, Rees MD. Mammalian heme peroxidases: from
molecular mechanisms to health implications. Antioxid Redox Signal.
2008;10(7):1199-1234.
2.
3.
Klebanoff SJ. Myeloperoxidase: friend and foe. J Leukoc Biol. 2005;77(5):598-625.
Podrez EA, Abu-Soud HM, Hazen SL. Myeloperoxidase-generated oxidants and
atherosclerosis. Free Radic Biol Med. 2000;28(12):1717-1725.
4.
5.
6.
Curtis MP, Neidigh JW. Kinetics of 3-nitrotyrosine modification on exposure to
hypochlorous acid. Free Radic Res. 2014;48(11):1355-1362.
Green JN, Kettle AJ, Winterbourn CC. Protein chlorination in neutrophil phagosomes
and correlation with bacterial killing. Free Radic Biol Med. 2014;77:49-56.
Senthilmohan R, Kettle AJ. Bromination and chlorination reactions of
myeloperoxidase at physiological concentrations of bromide and chloride. Arch
Biochem Biophys. 2006;445(2):235-244.
7.
8.
9.
Cook NL, Moeke CH, Fantoni LI, Pattison DI, Davies MJ. The myeloperoxidase-
derived oxidant hypothiocyanous acid inhibits protein tyrosine phosphatases via
oxidation of key cysteine residues. Free Radic Biol Med. 2016;90:195-205.
Peskin AV, Winterbourn CC. Kinetics of the reactions of hypochlorous acid and
amino acid chloramines with thiols, methionine, and ascorbate. Free Radic Biol Med.
2001;30(5):572-579.
Prutz WA. Interactions of hypochlorous acid with pyrimidine nucleotides, and
secondary reactions of chlorinated pyrimidines with GSH, NADH, and other
substrates. Arch Biochem Biophys. 1998;349(1):183-191.
10.
11.
12.
Shishido N, Nakamura S, Nakamura M. Dissociation of DNA double strand by
hypohalous acids. Redox Rep. 2000;5(4):243-247.
Gebicka L, Banasiak E. Hypochlorous acid-induced heme damage of hemoglobin and
its inhibition by flavonoids. Toxicol In Vitro. 2012;26(6):924-929.
Souza CE, Maitra D, Saed GM, et al. Hypochlorous acid-induced heme degradation
from lactoperoxidase as a novel mechanism of free iron release and tissue injury in
inflammatory diseases. PLoS One. 2011;6(11):e27641.
This article is protected by copyright. All rights reserved.

13.
14.
Maitra D, Byun J, Andreana PR, et al. Mechanism of hypochlorous acid-mediated
heme destruction and free iron release. Free Radic Biol Med. 2011;51(2):364-373.
Maitra D, Byun J, Andreana PR, et al. Reaction of hemoglobin with HOCl:
mechanism of heme destruction and free iron release. Free Radic Biol Med.
2011;51(2):374-386.
15.
16.
17.
Abu-Soud HM, Maitra D, Byun J, et al. The reaction of HOCl and cyanocobalamin:
corrin destruction and the liberation of cyanogen chloride. Free Radic Biol Med.
2012;52(3):616-625.
Maitra D, Abdulhamid I, Diamond MP, Saed GM, Abu-Soud HM. Melatonin
attenuates hypochlorous acid-mediated heme destruction, free iron release, and
protein aggregation in hemoglobin. J Pineal Res. 2012;53(2):198-205.
Maitra D, Shaeib F, Abdulhamid I, et al. Myeloperoxidase acts as a source of free iron
during steady-state catalysis by a feedback inhibitory pathway. Free Radic Biol Med.
2013;63:90-98.
18.
19.
Maitra D, Ali I, Abdulridha RM, et al. Kinetic studies on the reaction between
dicyanocobinamide and hypochlorous acid. PLoS One. 2014;9(11):e110595.
Qureshi GA, Qureshi AA, Devrajani BR, Chippa MA, Syed SA. Is the deficiency of
vitamin B12 related to oxidative stress and neurotoxicity in Parkinson's patients? CNS
Neurol Disord Drug Targets. 2008;7(1):20-27.
20.
21.
22.
23.
24.
25.
26.
27.
Teismann P. [Myeloperoxidase in the neurodegenerative process of Parkinson's
disease]. Dtsch Med Wochenschr. 2014;139(3):99-102.
Rizzo G, Lagana AS, Rapisarda AM, et al. Vitamin B12 among Vegetarians: Status,
Assessment and Supplementation. Nutrients. 2016;8(12).
Nicholls SJ, Hazen SL. Myeloperoxidase and cardiovascular disease. Arterioscler
Thromb Vasc Biol. 2005;25(6):1102-1111.
Ohyama W, Yamaoka M, Yokoi K, et al. [Maternal Crohn's disease-related vitamin
B12 deficient megaloblastic anemia in an infant]. Rinsho Ketsueki. 2016;57(1):15-19.
Gulley ML, Bentley SA, Ross DW. Neutrophil myeloperoxidase measurement
uncovers masked megaloblastic anemia. Blood. 1990;76(5):1004-1007.
Varela-Moreiras G, Murphy MM, Scott JM. Cobalamin, folic acid, and homocysteine.
Nutr Rev. 2009;67 Suppl 1:S69-72.
Banerjee RV, Matthews RG. Cobalamin-dependent methionine synthase. FASEB J.
1990;4(5):1450-1459.
Shaeib F, Khan SN, Ali I, et al. Melatonin prevents myeloperoxidase heme
destruction and the generation of free iron mediated by self-generated hypochlorous
acid. PLoS One. 2015;10(3):e0120737.
28.
29.
Maitra D, Banerjee J, Shaeib F, Souza CE, Abu-Soud HM. Melatonin can mediate its
vascular protective effect by modulating free iron level by inhibiting hypochlorous
acid-mediated hemoprotein heme destruction. Hypertension. 2011;57(5):e22; author
reply e23.
Lu T, Galijasevic S, Abdulhamid I, Abu-Soud HM. Analysis of the mechanism by
which melatonin inhibits human eosinophil peroxidase. Br J Pharmacol.
2008;154(6):1308-1317.
30.
31.
Galijasevic S, Abdulhamid I, Abu-Soud HM. Melatonin is a potent inhibitor for
myeloperoxidase. Biochemistry. 2008;47(8):2668-2677.
Pandi-Perumal SR, Srinivasan V, Maestroni GJ, Cardinali DP, Poeggeler B,
Hardeland R. Melatonin: Nature's most versatile biological signal? FEBS J.
2006;273(13):2813-2838.
This article is protected by copyright. All rights reserved.

32.
Stehle JH, Saade A, Rawashdeh O, et al. A survey of molecular details in the human
pineal gland in the light of phylogeny, structure, function and chronobiological
diseases. J Pineal Res. 2011;51(1):17-43.
33.
34.
35.
36.
Hardeland R. Melatonin: signaling mechanisms of a pleiotropic agent. Biofactors.
2009;35(2):183-192.
Reiter RJ. Melatonin: the chemical expression of darkness. Mol Cell Endocrinol.
1991;79(1-3):C153-158.
Carrillo-Vico A, Guerrero JM, Lardone PJ, Reiter RJ. A review of the multiple
actions of melatonin on the immune system. Endocrine. 2005;27(2):189-200.
Skinner DC, Malpaux B. High melatonin concentrations in third ventricular
cerebrospinal fluid are not due to Galen vein blood recirculating through the choroid
plexus. Endocrinology. 1999;140(10):4399-4405.
37.
Yu HS, Yee RW, Howes KA, Reiter RJ. Diurnal rhythms of immunoreactive
melatonin in the aqueous humor and serum of male pigmented rabbits. Neurosci Lett.
1990;116(3):309-314.
38.
39.
Tan D, Manchester LC, Reiter RJ, Qi W, Hanes MA, Farley NJ. High physiological
levels of melatonin in the bile of mammals. Life Sci. 1999;65(23):2523-2529.
Nakamura Y, Tamura H, Takayama H, Kato H. Increased endogenous level of
melatonin in preovulatory human follicles does not directly influence progesterone
production. Fertil Steril. 2003;80(4):1012-1016.
40.
41.
Galano A, Tan DX, Reiter RJ. Melatonin as a natural ally against oxidative stress: a
physicochemical examination. J Pineal Res. 2011;51(1):1-16.
Markus RP, Ferreira ZS, Fernandes PA, Cecon E. The immune-pineal axis: a shuttle
between endocrine and paracrine melatonin sources. Neuroimmunomodulation.
2007;14(3-4):126-133.
42.
43.
Jung KH, Hong SW, Zheng HM, et al. Melatonin ameliorates cerulein-induced
pancreatitis by the modulation of nuclear erythroid 2-related factor 2 and nuclear
factor-kappaB in rats. J Pineal Res. 2010;48(3):239-250.
Chahbouni M, Escames G, Venegas C, et al. Melatonin treatment normalizes plasma
pro-inflammatory cytokines and nitrosative/oxidative stress in patients suffering from
Duchenne muscular dystrophy. J Pineal Res. 2010;48(3):282-289.
44.
45.
46.
Jung-Hynes B, Reiter RJ, Ahmad N. Sirtuins, melatonin and circadian rhythms:
building a bridge between aging and cancer. J Pineal Res. 2010;48(1):9-19.
Park SY, Jang WJ, Yi EY, et al. Melatonin suppresses tumor angiogenesis by
inhibiting HIF-1alpha stabilization under hypoxia. J Pineal Res. 2010;48(2):178-184.
Reppert SM, Weaver DR, Ebisawa T. Cloning and characterization of a mammalian
melatonin receptor that mediates reproductive and circadian responses. Neuron.
1994;13(5):1177-1185.
47.
Reppert SM, Godson C, Mahle CD, Weaver DR, Slaugenhaupt SA, Gusella JF.
Molecular characterization of a second melatonin receptor expressed in human retina
and brain: the Mel1b melatonin receptor. Proc Natl Acad Sci U S A.
1995;92(19):8734-8738.
48.
49.
Marshall KA, Reiter RJ, Poeggeler B, Aruoma OI, Halliwell B. Evaluation of the
antioxidant activity of melatonin in vitro. Free Radic Biol Med. 1996;21(3):307-315.
Banerjee J, Maitra D, Diamond MP, Abu-Soud HM. Melatonin prevents
hypochlorous acid-induced alterations in microtubule and chromosomal structure in
metaphase-II mouse oocytes. J Pineal Res. 2012;53(2):122-128.
50.
Reiter RJ, Tan DX, Osuna C, Gitto E. Actions of melatonin in the reduction of
oxidative stress. A review. J Biomed Sci. 2000;7(6):444-458.
This article is protected by copyright. All rights reserved.

51.
Dellegar SM, Murphy SA, Bourne AE, DiCesare JC, Purser GH. Identification of the
factors affecting the rate of deactivation of hypochlorous acid by melatonin. Biochem
Biophys Res Commun. 1999;257(2):431-439.
52.
53.
Lundquist P, Rosling H, Sorbo B. Determination of cyanide in whole blood,
erythrocytes, and plasma. Clin Chem. 1985;31(4):591-595.
Gumus G, Demirata B, Apak R. Simultaneous spectrophotometric determination of
cyanide and thiocyanate after separation on a melamine-formaldehyde resin. Talanta.
2000;53(2):305-315.
54.
Wang L, Bassiri M, Najafi R, et al. Hypochlorous acid as a potential wound care
agent: part I. Stabilized hypochlorous acid: a component of the inorganic
armamentarium of innate immunity. J Burns Wounds. 2007;6:e5.
Ximenes VF, Silva SO, Rodrigues MR, et al. Superoxide-dependent oxidation of
melatonin by myeloperoxidase. J Biol Chem. 2005;280(46):38160-38169.
Hardeland R. Taxon- and Site-Specific Melatonin Catabolism. Molecules.
2017;22(11).
Ikeda M, Asai M, Moriya T, Sagara M, Inoue S, Shibata S. Methylcobalamin
amplifies melatonin-induced circadian phase shifts by facilitation of melatonin
synthesis in the rat pineal gland. Brain Res. 1998;795(1-2):98-104.
Yamadera H, Takahashi K, Okawa M. A multicenter study of sleep-wake rhythm
disorders: therapeutic effects of vitamin B12, bright light therapy, chronotherapy and
hypnotics. Psychiatry Clin Neurosci. 1996;50(4):203-209.
Okawa M, Takahashi K, Egashira K, et al. Vitamin B12 treatment for delayed sleep
phase syndrome: a multi-center double-blind study. Psychiatry Clin Neurosci.
1997;51(5):275-279.
Hashimoto S, Kohsaka M, Morita N, Fukuda N, Honma S, Honma K. Vitamin B12
enhances the phase-response of circadian melatonin rhythm to a single bright light
exposure in humans. Neurosci Lett. 1996;220(2):129-132.
55.
56.
57.
58.
59.
60.
61.
62.
63.
64.
65.
Dodson ER, Zee PC. Therapeutics for Circadian Rhythm Sleep Disorders. Sleep Med
Clin. 2010;5(4):701-715.
Mikkelsen K, Stojanovska L, Apostolopoulos V. The Effects of Vitamin B in
Depression. Curr Med Chem. 2016;23(38):4317-4337.
Smith AD, Refsum H. Vitamin B-12 and cognition in the elderly. Am J Clin Nutr.
2009;89(2):707S-711S.
Bourre JM. [The role of nutritional factors on the structure and function of the brain:
an update on dietary requirements]. Rev Neurol (Paris). 2004;160(8-9):767-792.
Weissbach H, Redfield BG, Peterkofsky A. Biosynthesis of the B12 coenzyme:
requirements for release of cyanide and spectral changes. J Biol Chem.
1962;237:3217-3222.
66.
67.
68.
69.
Nelson L. Acute cyanide toxicity: mechanisms and manifestations. J Emerg Nurs.
2006;32(4 Suppl):S8-11.
Stabler SP. Clinical practice. Vitamin B12 deficiency. N Engl J Med.
2013;368(2):149-160.
Finkelstein JL, Layden AJ, Stover PJ. Vitamin B-12 and Perinatal Health. Adv Nutr.
2015;6(5):552-563.
Lanska DJ. Chapter 30: historical aspects of the major neurological vitamin
deficiency disorders: the water-soluble B vitamins. Handb Clin Neurol. 2010;95:445-
476.
70.
Toh BH, van Driel IR, Gleeson PA. Pernicious anemia. N Engl J Med.
1997;337(20):1441-1448.
This article is protected by copyright. All rights reserved.

71.
72.
Kulnigg-Dabsch S. Autoimmune gastritis. Wien Med Wochenschr. 2016;166(13-
14):424-430.
Valenzuela MA, Canales J, Corvalan AH, Quest AF. Helicobacter pylori-induced
inflammation and epigenetic changes during gastric carcinogenesis. World J
Gastroenterol. 2015;21(45):12742-12756.
73.
74.
75.
Suzuki H, Miura S, Imaeda H, et al. Enhanced levels of chemiluminescence and
platelet activating factor in urease-positive gastric ulcers. Free Radic Biol Med.
1996;20(3):449-454.
Cadamuro AC, Rossi AF, Maniezzo NM, Silva AE. Helicobacter pylori infection:
host immune response, implications on gene expression and microRNAs. World J
Gastroenterol. 2014;20(6):1424-1437.
Roe I, Nam S, Kim J, Shin J, Bang W, Yang M. Association of the myeloperoxidase -
463G-->A polymorphism with development of atrophy in Helicobacter pylori-
infected gastritis. Am J Gastroenterol. 2002;97(7):1629-1634.
76.
77.
78.
79.
Kumar S, Bandyopadhyay U. Free heme toxicity and its detoxification systems in
human. Toxicol Lett. 2005;157(3):175-188.
Clark RA. Oxidative stress and "senescent" fibroblasts in non-healing wounds as
potential therapeutic targets. J Invest Dermatol. 2008;128(10):2361-2364.
Crichton RR, Wilmet S, Legssyer R, Ward RJ. Molecular and cellular mechanisms of
iron homeostasis and toxicity in mammalian cells. J Inorg Biochem. 2002;91(1):9-18.
Ong WY, Halliwell B. Iron, atherosclerosis, and neurodegeneration: a key role for
cholesterol in promoting iron-dependent oxidative damage? Ann N Y Acad Sci.
2004;1012:51-64.
80.
81.
Trinder D, Fox C, Vautier G, Olynyk JK. Molecular pathogenesis of iron overload.
Gut. 2002;51(2):290-295.
Mates JM, Segura JA, Alonso FJ, Marquez J. Roles of dioxins and heavy metals in
cancer and neurological diseases using ROS-mediated mechanisms. Free Radic Biol
Med. 2010;49(9):1328-1341.
82.
Takahashi-Iniguez T, Garcia-Hernandez E, Arreguin-Espinosa R, Flores ME. Role of
vitamin B12 on methylmalonyl-CoA mutase activity. J Zhejiang Univ Sci B.
2012;13(6):423-437.
83.
84.
Birben E, Sahiner UM, Sackesen C, Erzurum S, Kalayci O. Oxidative stress and
antioxidant defense. World Allergy Organ J. 2012;5(1):9-19.
Ximenes VF, Paino IM, Faria-Oliveira OM, Fonseca LM, Brunetti IL. Indole ring
oxidation by activated leukocytes prevents the production of hypochlorous acid. Braz
J Med Biol Res. 2005;38(11):1575-1583.
85.
86.
Morris SM. The genetic toxicology of 5-fluoropyrimidines and 5-chlorouracil. Mutat
Res. 1993;297(1):39-51.
Tekbas OF, Ogur R, Korkmaz A, Kilic A, Reiter RJ. Melatonin as an antibiotic: new
insights into the actions of this ubiquitous molecule. J Pineal Res. 2008;44(2):222-
226.
87.
Gomez FJ, Raba J, Cerutti S, Silva MF. Monitoring melatonin and its isomer in Vitis
vinifera cv. Malbec by UHPLC-MS/MS from grape to bottle. J Pineal Res.
2012;52(3):349-355.
88.
89.
90.
Gulcin I, Buyukokuroglu ME, Kufrevioglu OI. Metal chelating and hydrogen
peroxide scavenging effects of melatonin. J Pineal Res. 2003;34(4):278-281.
Reiter RJ, Korkmaz A, Ma S, Rosales-Corral S, Tan DX. Melatonin protection from
chronic, low-level ionizing radiation. Mutat Res. 2012;751(1):7-14.
Ogino T, Than TA, Hosako M, Ozaki M, Omori M, Okada S. Taurine chloramine: a
possible oxidant reservoir. Adv Exp Med Biol. 2009;643:451-461.
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Table
Table 1: Retention time and spectral features of UV absorbing melatonin oxidation
products.
Retention
Absorbance
time (mins)
maxima (nm)
Peak I
2.06
362, 540, 576
258, 302
Peak II 3.30
Peak III 5.40
256, 290
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This article is protected by copyright. All rights reserved.

This article is protected by copyright. All rights reserved.