Mol Neurobiol
DOI 10.1007/s12035-016-9736-2
Oxidative Stress Markers in Vitamin B12 Deficiency
Usha Kant Misra 1 & Jayantee Kalita 1 & Sandeep Kumar Singh 1 &
Sushil Kumar Rahi 1
Received: 30 November 2015 / Accepted: 20 January 2016
# Springer Science+Business Media New York 2016
Abstract In this study, we report the status of oxidative stress
markers in vitamin B12 deficiency and their relation to clini-
cal, laboratory, and neurophysiological findings. Fifty-one
subjects with serum vitamin B12 deficiency (<211 pg/ml)
were included. Plasma glutathione (GSH), malondialdehyde
(MDA) and serum total antioxidant capacity (TAC) were mea-
sured in the patients and 53 controls. These markers were also
compared between subacute combined degeneration (SACD)
and non-SACD vitamin B12 deficiency patients groups as
well as with normal controls. In the patients, GSH, MDA
and TAC were correlated with demographic, clinical, hemato-
logical, biochemical, nerve conduction study (NCS), visual
evoked potential (VEP) and somatosensory-evoked potential
(SEP) findings. In the study group, 20 (39.2 %) patients had
SACD manifesting with myeloneuropathy, cognitive or
behavioral abnormalities, and 31(60.8 %) patients had non-
SACD neurological manifestations. The GSH (2.46 ± 0.32 vs.
2.70 ± 0.36 mg/dl; P = 0.002) and TAC (2.13 ± 0.38 vs. 2.33
± 0.24 nmol Trolox eq/l, P = 0.005) levels were lower, and
MDA levels (4.01 ± 0.69 vs. 3.00 ± 0.45 nmol/ml, P < 0.001)
were higher in B12 deficiency group compared with controls.
Similar trend was found in SACD and non-SACD vitamin
B12 deficiency groups. GSH levels correlated with abnormal
VEP (r = 0.54; P < 0.01), TAC with female gender (r = 0.43;
P = 0.002) and joint position impairment (r = −0.34; P = 0.01),
and MDA with LDH (r = 0.41; P = 0.01). Vitamin B12 defi-
ciency was associated with reduction in GSH and TAC and
* Usha Kant Misra
drukmisra@rediffmail.com
1
Department of Neurology, Sanjay Gandhi Post Graduate Institute of
Medical Sciences, Raebareily Road, Lucknow 226014, India
increase in MDA levels which were more marked in SACD
compared to non-SACD group.
Keywords Vitamin B12 . Oxidative stress . Subacute
combined degeneration . Glutathione . Malondialdehyde .
Total antioxidant capacity . Nerve conduction study . Visual
evoked potential
Introduction
Cobalamin deficiency occurs in pernicious anemia, veganism,
malabsorption syndrome, gastrointestinal surgery, chronic re-
nal failure, multiple myeloma, and acquired immune deficien-
cy syndrome [1, 2]. The prototype neurological manifestation
of cobalamin deficiency is subacute combined degeneration
(SACD) which is characterized by the involvement of pyra-
midal and posterior columns in the spinal cord, peripheral
nerve, and rarely in the brain [3]. Various neurobehavioral
and cognitive abnormality, evoked potentials, nerve conduc-
tion, and MRI changes have been reported [1, 4, 5].
Hematological manifestations of cobalamin deficiency occur
more frequently than neurological manifestations and include
m e ga l o b l a s t i c a ne m i a a n d h e m o l y s i s l ea d i n g t o
hyperbilirubinemia and elevated serum lactate dehydrogenase
(LDH). The clinical, evoked potential, and radiological chang-
es are the result of a series of biochemical and molecular
changes. In the mammalian cells, cobalamin (Cbl) is essential
for two functions: (1) Adenosylcobalamin (Ado Cbl) is a co-
factor for conversion of methyl melonyl-CoA to succinyl-
CoA by the enzyme methyl melonyl-CoA mutase; (2) methyl
cobalamin also is a cofactor for conversion of homocysteine to
methionine and methyl tetrahydrofolate to tetrahydrofolate by
enzyme methionine synthase [6]. In Cbl deficiency, therefore,
the activity of methyl melonyl-CoA (MM-Co) mutase and

methionine synthase (MeS) is reduced leading to accumula-
tion of methyl malonic acid (MMA) and homocysteine
(HCY). Increased HCY results in overstimulation of N-meth-
yl-D-aspartate (NMDA) receptors resulting in excitotoxicity.
There have been reports of imbalance of cytokine and epider-
mal growth factor in Cbl deficiency [6–8]. Cytokine release is
dependent on imbalance between oxidative stress markers and
antioxidants. Glutathione (GSH) is a major water soluble in-
tracellular antioxidant which directly reduces most of the re-
active oxygen species. Total antioxidant capacity (TAC) has
been recently used to assess the antioxidant status in different
diseases [9–11]. Malondialdehyde (MDA) is the end product
of lipid peroxidation and has been used as a marker of oxida-
tive stress.
In SACD, there are demyelinating changes in subcortical
white matter and spinal cord [12]. The prototype of demyelin-
ating disease is multiple sclerosis in which the role of oxida-
tive stress in blood and multiple sclerosis plaque has been
reported [13]. There is no report evaluating the oxidative
stress markers in cobalamin deficiency patients. In the present
communication, we report the status of oxidative stress and
antioxidant levels and their relation to clinical, laboratory, and
neurophysiological findings in the patients with vitamin B12
deficiency syndromes.
Subjects and Methods
Inclusion Criteria
Fifty-one patients with low serum vitamin B12 levels
(<211 pg/ml) attending the neurology service of Sanjay
Gandhi Post Graduate Institute, India, were included. Fifty-
three healthy volunteers with normal neurological examina-
tion and normal serum B12 levels (>211 pg/ml) were included
as control.
Exclusion Criteria
Patients with pregnancy, vasculitis, malignancy, previous neu-
rological diseases, on chemotherapy, radiotherapy and immu-
nosuppression, and those not giving consent were excluded.
Clinical Evaluation
Detailed medical history including dietary habit, malabsorp-
tion, gastrointestinal surgery, alcohol consumption, and liver
and kidney failure was noted. Examination findings such as
pallor, edema, hepatosplenomegaly, ascites, lymphadenopa-
thy, and icterus were recorded. The cognitive functions were
evaluated using Mini Metal State Examination (MMSE) scale
and were considered abnormal on the basis of cutoff points
based on age and education [14]. Cranial nerve examination
Mol Neurobiol
including fundus findings was noted. Muscle power was
assessed by the Medical Research Council (MRC) scale.
Muscle tone and tendon reflexes were grouped as normal,
increased, or decreased. Sensations of joint position, touch,
and vibration were noted. HCY level is measured by chemi-
luminescence methods (normal range 5–15 μmol/l).
Investigations
Hemoglobin, blood counts, red blood cell indices, fasting and
postprandial blood sugar, blood urea nitrogen, serum creati-
nine, transaminases, lactate dehydrogenase (LDH), protein,
albumin, bilirubin, and electrolytes were measured. Thyroid
function test (thyroid stimulating hormone, T3 and T4) and
human immunodeficiency virus serology were done. Fasting
serum vitamin B12 and folate levels were measured using
vitamin B12 assay kit (Siemens Healthcare Diagnostics
Technical Services). Antiparietal cell antibody was assessed
by enzyme-linked immunosorbent assay. Spinal magnetic res-
onance imaging (MRI) was done on a 3T MRI scanner (Sigma
GE Medical System, Wisconsin USA). Pattern reversal visual
evoked potential (VEP) and somatosensory evoked potential
(SEP) were done using standard techniques and compared
with our laboratory normative data [15, 16]. Sural and pero-
neal nerve conductions were done using standard techniques
and compared with our laboratory normative data [1, 16].
Oxidative Stress Markers
Glutathione Assay
Plasma GSH was measured by spectrophotometer at 412 nm
[17]. Plasma was added to 10 % trichloroacetic acid (TCA)
and was allowed to stand at 4 °C for 2 h. This mixture was
centrifuged at 2000×g for 15 min, and the supernatant was
added to Tris-buffer (0.4 M, pH 8.9) containing ethylenedi-
aminetetraacetic acid (EDTA) (0.02 M). Finally 5,5-dithiobis
was added to the mixture which was reduced to the yellow
product 5-thio-2-nitrobenzoic acid (TNB). This was measured
by spectrophotometer at 412 nm. A standard curve of reduced
GSH was plotted to determine the amount of GSH in the
plasma sample.
Total Antioxidant Capacity Assay
Serum TAC was measured by the method described by
Koracevic et al. [18]. In this method, the hydroxyl radical is
produced by the Fenton reaction and reacts with benzoate
resulting in release of thiobarbituric acid reactive substances,
which is yellowish-brown in color. On addition of a serum
sample, the oxidative reactions initiated by the hydroxyl rad-
ical present in the reaction mixture are suppressed by the
Mol Neurobiol

antioxidant components of serum, which prevents the color Karl-Pearson correlation test. The variable having a two tailed
change and thereby providing an effective measure of the P value of <0.05 was considered significant. The statistical
TAC. This was measured by spectrophotometer at 532 nm analysis was done using SPSS version 16 software and
and the inhibition of color development defined as the TAC. GraphPad Prism 5.

Plasma Lipid Peroxidation Assay

LPO was measured by assessing MDA level, which is the end
product of LPO [19]. Plasma was mixed with EDTA, ascor-
bate (10 mM), and FeSO4 (16.7 mM) and incubated at 37 °C
for 60 min. The reaction was stopped by adding ice-cold 10 %
trichloroacetic acid (TCA). The mixture was centrifuged at
2000×g for 10 min, and supernatant was aspirated. The super-
natant was mixed with equal volume of 0.67 % thiobarbituric
acid (TBA) and was kept in a boiling water bath for 15 to
20 min. MDA level was measured with the absorption coeffi-
cient of MDA-TBA complex at 532 nm using spectrophotom-
eter. GSH, TAC, and MDA levels were also measured in 36
age (44.28 ± 2.20 vs 45.78 ± 2.19 years; P = 0.36) and gender
(male/female 28:8 vs 36:15; P = 1.00)-matched healthy con-
trols whose mean (±SD) serum vitamin B12 levels were 412
± 289.48 pg/ml.
The patients were categorized as SACD who had charac-
teristic neurological symptoms and sign (myelopathy with or
without neuropathy and cognitive abnormality), and non-
SACD vitamin B12 deficiency who manifested with vague
symptoms such as anxiety, anorexia, and insomnia.
Statistical Analysis
GSH, MDA, and TAC levels were compared between patients
and controls using independent t test. These variables were
also compared between SACD and non-SACD patients using
independent t test. GSH, MDA, and TAC levels in the patients
were correlated with age, duration of illness, mean corpuscu-
lar volume, hemoglobin, bilirubin, LDH, transaminase, VEP,
SEP, and nerve conduction parameters using Spearman and
Results
Clinical Findings
There were 51 subjects with vitamin B12 deficiency whose
mean age (±SD) was 45.76 years (±15.70) and 15 (29.4 %)
were females. Thirty-seven (72.5 %) patients were vegetarian,
and 11 (24.4 %) patients drank alcohol and smoked cigarettes.
In the study group, 31 (60.7 %) patients were in non-SACD
group who presented with vague symptoms. Twenty (39.2 %)
patients had SACD with typical symptoms and signs consis-
tent with vitamin B12 deficiency; 14 had myelocognitive and
6 had neuropsychiatric abnormality.
Biochemical Findings
Twenty-one (41.2 %) patients were anemic (Hb < 12 g/dl) and
14 (27.4 %) had macrocytosis. Serum bilirubin was elevated
in 2 (3.9 %), transaminase in 5 (9.8 %), and LDH in 11
(21.6 %) patients. Serum creatinine was normal in all (median
0.83 mg/dl; range 0.10–1.40 mg/dl). The median serum ho-
mocysteine (HCY) level was 19.9 pg/ml (8.01–122.58), and
19/30 (63.3 %) patients had raised HCY (>15 μmol/l) level.
The comparison of demographic, hematological, and bio-
chemical parameters in SACD and non-SACD patients was
not significantly different (Table 1).
Oxidative Stress Parameters
Plasma GSH (2.46 ± 0.32 vs 2.70 ± 0.36 mg/dl, P = 0.002) and
TAC (2.13 ± 0.38 vs 2.33 ± 0.24 nmol Trolox eq/l, P = 0.005)

Table 1 Comparison of
demographic, hematological, and Variable SACD (N = 20) Non-SACD (N = 31) P value
biochemical parameters in SACD
and non-SACD vitamin B12 Age (year) 48.49 ± 17.71 44.06 ± 14.30 0.36
deficiency groups MCV (fl) 91.17 ± 12.83 91.31 ± 11.25 0.97
Hemoglobin (g/dl) 12.32 ± 2.05 12.32 ± 1.14 0.99
Platelet count (thousand/mm3) 209.33 ± 70.33 235.43 ± 55.79 0.54
Reticulocyte (%) 1.56 ± 0.82 1.19 ± 0.31 0.11
Serum Bilirubin (mg/dl) 0.78 ± 0.72 0.76 ± 0.32 0.90
SGPT (U/l) 45.86 ± 32.54 27.75 ± 14.44 0.20
LDH (U/l) 432.00 ± 64.74 425.05 ± 67.10 0.75
Serum B12 (pg/ml) 173.30 ± 21.75 171.74 ± 25.44 0.81
Serum homocysteine (μmol/l) 33.23 ± 33.31 22.27 ± 12.76 0.75

LDH lactate dehydrogenase, MCV mean corpuscular volume, SGPT serum glutamate pyruvate transaminase,
SACD subacute combined degeneration, Non-SACD non-subacute combined degeneration

Fig. 1 Plasma glutathione (GSH) and total antioxidant capacity (TAC) in
the patients were significantly reduced (P < 0.001), and malondialdehyde
(MDA) was significantly increased (P < 0.001) compared to the controls
levels in the patients were reduced, and MDA (4.10 ± 0.69 vs
3.00 ± 0.45 nmol/ml, P < 0.001) levels were increased com-
pared to the controls (Fig. 1). In the SACD group, GSH and
TAC levels were lower and MDA level was higher compared
to non-SACD vitamin B12 deficiency group (Table 2).
Table 2 Comparison of
oxidative stress markers in SACD Variable
and non-SACD vitamin B12
deficiency groups TAC (nmol Trolox eq/l)
GSH (mg/dl)
MDA (nmol/ml)
GSH glutathione, MDA malondialdehyde, TAC total antioxidant capacity, SACD subacute combined degenera-
tion, Non-SACD non-subacute combined degeneration
Mol Neurobiol
Correlation
Plasma GSH level correlated with VEP (r = 0.54; P < 001),
TAC with gender (r = 0.43; P = 0.002) and joint position sen-
sation (r = −0.34; P = 0.01), and MDA with LDH (r = 0.41,
P = 0.01). The duration of illness, MMSE, clinical evidence
of myeloneuropathy, mean corpuscular volume of red blood
cell, and HCY were not correlated with GSH, TAC, or MDA
levels. The details are summarized in Table 3, and correlations
of significant variables are shown in Fig. 2.
Discussion
In the present study, the patients with vitamin B12 deficiency
had reduction in GSH and TAC, and increase in MDA levels
compared to the controls. SACD patients had significantly
more oxidative stress and reduced antioxidants compared to
the non-SACD vitamin B12 deficiency group. GSH level cor-
related with VEP, TAC with gender and joint position, and
MDA with LDH level. This is the first study evaluating oxi-
dative stress markers in the patients with SACD and non-
SACD vitamin B12 deficiency patients. The role of vitamin
B12 deficiency in producing oxidative stress was evaluated in
elderly individuals. In this study, out of 473 subjects, 37 %
patients had vitamin B12 level above 400 pg/ml. The clinical
evidence of oxidative stress was diabetes mellitus, smoking,
alcohol consumption, neurodegenerative disorders, malignan-
cy, chronic infection, heart failure, hematological disorders,
pregnancy, asthma, hyperthyroidism, cirrhosis, and high
ESR. When no oxidative stress risk factor was present, indi-
vidual ≥70 years had higher MMA (methyl malonic acid)
compared to the younger ones. MMA levels were significant-
ly higher in the elderly with one oxidative risk, and in younger
subjects, MMA was higher when two or more oxidative risk
factors were present. HCY values were also higher in the sub-
jects with two or more oxidative risks. Cobalamin therapy
irrespective of vitamin B12 level decreased MMA and HCY
levels in more than three-fourths of the subjects. The subjects
with two or more oxidative risk factors had lesser suppression
of MMA and HCY levels. This study emphasized the func-
tional cobalamin deficiency in elderly subjects especially in
SACD (N = 20) Non-SACD (N = 31) P value
1.90 ± 0.31 2.27 ± 0.35 <0.001
2.35 ± 0.27 2.54 ± 0.33 <0.04
4.33 ± 0.77 3.82 ± 0.57 0.01
Mol Neurobiol

Table 3 Correlation between

oxidative stress markers with GSH (mg/dl) R value TAC (nmol Trolox eq/l) MDA (nmol/ml)
various clinical and laboratory
parameters Age (year) 0.14 0.19 0.17
Gender 0.07 0.43** −0.15
Duration of illness (year) −.0.25 −.0.14 0.25
MMSE −0.04 −0.06 −.0.13
Dietary habit 0.12 −0.09 −0.17
Joint position −0.01 −.0.34** 0.02
MCV (fl) −0.04 −.0.08 −0.05
LDH (U/l) −.0.15 −0.16 0.41**
Serum B12 (pg/ml) −0.10 0.04 −0.10
Serum folic acid mg/dl 0.22 0.04 0.06
Homocysteine (μmo/l) −0.23 −0.15 −0.24
VEP −0.54** −0.10 0.30
SEP 0.06 −0.13 0.02

LDH lactate dehydrogenase, MCV mean corpuscular volume, MMSE Mini Mental State Examination, SEP
somatosensory evoked potential, VEP visual evoked potential
**P < 0.01; *P < 0.05

the presence of oxidative stress [20]. This study however did
not evaluate the biological markers of oxidative stress.
HCY and MMA are surrogate markers of vitamin B12
deficiency. Vascular injuries such as coronary artery disease,
stroke, and multi-infarct dementia have been reported in asso-
ciation with hyperhomocysteinemia [21]. Homocysteine
thioacetone could induce endothelial dysfunction mediated
by endoplasmic reticulum (ER) stress. ER stress results in
downstream enhancement of oxidative stress and inflam-
mation which produces endothelial dysfunction. Two ER
stress inhibitors—4-phenylbutyric acid (4-PBA) and Tudka—
significantly reduce reactive oxygen species (ROS),
nicotinamide adenine dinucleotide phosphate (NADPH) ac-
tivity, and MDA levels [22]. In another study on diabetic
obese and non-obese females, plasma oxidative stress and
nitric oxide levels have been reported and correlated with
HCY. This study highlights the permissive role of HCY in
endothelial damage through free radical generation [23].
In an experimental study producing cobalamin deficiency
by nitrous oxide exposure (1.5 l/d for 1 month), there was
hyperhomocysteinemia, focal myelin loss, and vacuolar
changes in the white matter of brain and spinal cord which
were associated with reduced GSH and TAC. There was a
relationship between oxidative stress markers and HCY

Fig. 2 Error bar diagram and

regression curve show significant
relationship of total antioxidant
capacity (TAC) with gender and
joint position sensation,
glutathione (GSH) with visual
evoked potential (VEP) and
malondialdehyde (MDA) with
lactate dehydrogenase (LDH)

level suggesting the role of oxidative stress in the patho-
genesis of cobalamin deficiency-related biochemical and
histophatological changes [24]. White matter changes in
the spinal cord and subcortical white matter are found char-
acteristically in multiple sclerosis. In the autopsy study of
multiple sclerosis, the role of oxidative stress parameters in
demyelinating plaques resulting in inflammation and apopto-
sis has been reported [13]. Low serum TAC in multiple scle-
rosis has also reported highlighting the important role of oxi-
dative stress in producing demyelinating changes [10]. Total
antioxidant capacity is a measure of combined antioxidant
effect of nonenzymatic defense in the biological fluid and
can be used for assessing the redox status [25].
Methylcobalamin acts as a cofactor with methionine syn-
thase and helps in converting HCY to methionine by donating
the methyl group. Increased HCY in the presence of pyridox-
ine deficiency or cystathione β synthase mutase deficiency
may not be able to convert cysteine, leading to accumulation
of HCY [26]. The neurological manifestations of vitamin B12
deficiency are due to myelin dysfunction in the central and
peripheral nervous system. Due to reduced methionine, there
is less S-adenosyl methionine (SAM) which is the sole methyl
donor for numerous reactions in the brain involving nucleo-
protein, protein, membrane phospholipids, and neurotransmit-
ters. Spinal cord dysfunction, peripheral neuropathy, and cog-
nitive abnormalities in vitamin B12 deficiency may be due to
the above mentioned mechanisms which may be contributed
by the oxidative stress [24]. In our study, GSH correlated with
VEP and TAC with joint position impairment. Prolongation of
P100 latency in SACD has been reported even without visual
abnormality which suggests myelination dysfunction of optic
nerve [15]. Abnormal proprioception suggests myelin dys-
function of posterior column of the spinal cord. Sensory ataxia
is a dominant feature of SACD [27]. These phenomena high-
light the role of oxidative stress in myelin dysfunction. Higher
MDA level with increasing age has also been reported [28].
In our study, HCY level did not correlate with GSH, TAC,
MDA, and serum vitamin B12 level. The level of HCY was
however above the reference value in most of the patients.
This may be due to inclusion of cases of low serum B12 level
(<211 pg/ml) rather than patients with clinical syndrome of
vitamin B12 deficiency.
Limitations
The present study is limited by a small sample size and lack of
MMA and S-adenosyl methionine assay. The estimation of S-
adenosyl methionine and methyl malonic acid might have
provided additional supportive evidence for the neurological
complications of vitamin B12 deficiency. We have not esti-
mated other vitamins like A, C, and E, which are known to
have antioxidant properties, and their estimation could have
provided additional information. Further studies are needed to
Mol Neurobiol
learn about the mechanism of oxidative stress in vitamin B12
deficiency and effect of vitamin B12 supplementation on ox-
idative stress markers.
Acknowledgments We acknowledge the Indian Council of Medical
Research, Government of India, for supporting senior research fellowship
to Mr. Sandeep Kumar Singh.
Compliance with Ethical Standards
Ethics Approval The study was approved by the Institution Ethics
Committee, Sanjay Gandhi Post Graduate Institute of Medical Sciences,
Lucknow, India (IEC code 2014-49-IP-75).
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