Archives of Biochemistry and Biophysics 627 (2017) 21e29

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Archives of Biochemistry and Biophysics

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Inhibitory effect of vitamin B3 against glycation and reactive oxygen

species production in HSA: An in vitro approach
K.M. Abdullah a, Faizan Abul Qais b, Iqbal Ahmad b, Imrana Naseem a, *
a
Department of Biochemistry, Faculty of Life Sciences, Aligarh Muslim University, Aligarh, 202002, India
b
Department of Agricultural Microbiology, Aligarh Muslim University, Aligarh, 202002, India

a r t i c l e i n f o a b s t r a c t

Article history: Hyperglycaemia is a key factor for the formation of advanced glycated endproducts (AGEs). Inhibition of
Received 23 March 2017 glycation may play key role in minimizing the diabetes related complications. We have tried to explore
Received in revised form the glucose and methyl glyoxal mediated glycation and antiglycation activity of niacin using human
26 May 2017
serum albumin as model protein. Protein was incubated with glucose for 28 days at physiological
Accepted 12 June 2017
Available online 15 June 2017
temperature to achieve glycation. Antiglycation activity was evaluated by assessing free lysine, carbonyl
content, AGE specific fluorescence. Molecular docking and isothermal titration calorimetry was deployed
to study the interaction of niacin with HSA and get a detailed insight of binding site and thermodynamics
Keywords:
Niacin
of interaction. Niacin reduced the glycation significantly which was evident from the estimation of free
Antiglycation lysine and carbonyl content. Niacin binds with HSA in a spontaneous manner with the binding constant
AGEs in the range of 104 M
1. Niacin also prevented the loss in secondary structure induced by glycation.
Molecular docking Reactive oxygen species were also effectively quenched by niacin leading to protection from DNA
ROS damage. Niacin was found to be located at Sudlow's site I with binding energy of 5.3 kcal/mol. These
Human serum albumin results clearly highlight the antiglycation activity of niacin and its potential in preventing disease pro-
gression in diabetes.
© 2017 Elsevier Inc. All rights reserved.

1. Introduction amplified up to three-folds under hyperglycaemia in diabetes [7].

Glycation is a process in which free reducing sugars react non-
enzymatically with free amino group of proteins DNA and lipids.
The early unstable product is characterized as Schiff's base that
later results in formation a stable Amadori product [1]. This reac-
tion proceeds by oxidative modifications and rearrangements
triggering the proteins to cross-link and resulting in formation of
advanced glycation endproducts (AGEs) [2]. Hyperglycaemia asso-
ciated with type 2 diabetes favours glycation and accumulations of
AGEs in vivo. AGEs alter different signalling pathways leading to
generation of oxidative stress as well as other severe pathological
conditions including neuropathy, nephropathy, retinopathy and
micro-vascular complications [3,4]. Once glycation starts, forma-
tion of AGEs continues for whole life span that cannot be stopped
even after normalization of blood glucose level [5,6]. At normal
blood glucose level, modification of 6e10% of lysine chains of hu-
man serum albumin (HSA) occurs non-enzymatically which is
* Corresponding author.
E-mail address: imrananaseem2009@gmail.com (I. Naseem).
The major in vivo AGEs characterized till date are product of
highly reactive carbonyl intermediates which include glyoxal,
methyl glyoxal, 3-deoxyglucosone pentosidine N-ε-carbox-
ymethyllysine (CML) etc. collectively called as dicarbonyls or
oxoaldehydes [8,9]. AGEs are highly reactive heterogenous class of
compounds which are difficult to be characterized by single tech-
nique. Some specific properties of AGEs are exploited for their
characterization such as fluorescence, specific antibodies, mobility
in polyacrylamide gel depending on their cross-linking [10e12].
Type 2 diabetes is a major global health problem and 90% of its
cases are related to defects in glucose metabolism [13]. The
mechanisms involved in diabetes are required to be scrutinized at
molecular, genetics and physiological levels. Irrespective of the
advancements in diabetes treatment, the rate of mortality and
morbidity related to the disease is still quite high. One of the known
mechanisms responsible for tissue damage in diabetes is non-
enzymatic glycosylation [14]. The AGEs formed gets accumulated
in vascular tissues where they bind to AGE specific receptors called
RAGE. Hyperglycaemia accelerates binding of AGEs to RAGE
altering enzyme activity, immunogenicity and modify the half-life

http://dx.doi.org/10.1016/j.abb.2017.06.009
0003-9861/© 2017 Elsevier Inc. All rights reserved.
22 K.M. Abdullah et al. / Archives of Biochemistry and Biophysics 627 (2017) 21e29
of protein [15]. Although a large number of drugs and interventions
are known to manage diabetes, but they are either expensive or
cause adverse effects on normal tissues. This had led the re-
searchers to focus on natural products with combined antioxidant
and antiglycation properties that may reduce the tissue damage. It
is now well known that dietary components inhibit formation of
AGEs both in vitro and in vivo [16]. Previously, a brief comparative
study on antiglycation effect of various micronutrients including
niacin has been reported [17]. The present study is focussed on
much detailed investigation of antiglycation potential of niacin
using various biophysical and computational techniques. Vitamins
are one such essential component. Niacin which is also known as
vitamin B3 and nicotinic acid is used for treatment of hypercho-
lesterolemia pellagra. Niacin reduces the triglyceride level that
have been found to be useful in diabetic dyslipidemia [18]. In this
article, we have explored the antiglycation activity of niacin and its
possible mode of interference.
2. Materials and methods
2.1. Materials
Fatty acid and globulin free human serum albumin (HSA), DTNB
and glucose were purchased from SRL chemicals (India). TNBSA
(2,4,6-trinitrobenzene sulfonic acid) solution was obtained from
Sigma-Aldrich (USA). Nicotinic acid (Niacin or vitamin B3) and
DNPH (2,4-Dinitrophenylhydrazine) was purchased from HiMedia
Laboratories (India). Rest of the reagents and chemicals were of
analytical grade and therefore, used without further purification.
2.2. Human serum albumin (HSA) in vitro glycation assay
The in vitro glycation assay of test compound was adopted from
Qais et al. (2016) with minor modifications [19]. Briefly, 300 mM
HSA was incubated with 165 mM glucose in 10 mM PBS (pH 7.4) in
absence and presence of test compound with 0.02% NaN3 to avoid
microbial contamination. Aminoguanidine (10 mM) was taken as
positive control and a varying concentrations of niacin (50, 100, 200
and 500 mM) were taken for test group. All samples were incubated
for 28 days at 37  C. After incubation period, all samples were
dialyzed against same buffer for 24 h to remove unbound and
excess amount of glucose. Samples were stored at
20  C for
further study and diluted in same buffer wherever stated.
2.3. UV-visible absorption study
The UV-visible absorption spectra were monitored on Shimadzu
1800 UV-vis spectrophotometer, Shimadzu, Japan. The protein
samples were diluted to 5 mM in 10 mM PBS (pH 7.4) and absorption
spectra were recorded in range of 200e700 nm.
2.4. Analysis of fluorescent AGEs
The fluorescence emission profile of all samples was recorded
on RF-5301 spectrofluorometer, Shimadzu, Japan. Each sample was
diluted to 3 mM in 10 mM PBS for detection of fluorescent AGEs. All
the samples were excited at 370 nm and the fluorescent emission
signal was recorded in the range of 375e600 nm with slit width of
5 nm each for excitation and emission. The percent inhibition of
fluorescent AGEs was determined from the equation-1:
 
FIg
FIt
% Inhibition ¼  100
FIg
FIn
where, FIg, FIt and FIn are fluorescent intensities of glycated sample,
treated sample and native HSA sample respectively.
2.5. Secondary structure analysis by circular dichroism (CD)
To evaluate the effect of niacin on secondary structure of HSA,
Circular dichroism spectra of samples were monitored using JASCO
spectropolarimeter (J-815). The spectral measurement was carried
out at 25  C with the help of thermostatically controlled cell holder
attached to Neslab's RTE 110 water bath having a temperature ac-
curacy of ±0.1  C. Each protein sample was diluted to 5 mM in
10 mM PBS (pH 7.4) for far-UV CD analysis and spectra was recorded
with 1 mm path length quartz cuvettes.
2.6. MethylglyoxaleHSA reactivity
MethylglyoxaleHSA reactivity analysis method was adopted
from Alam et al. (2015) with slight modifications [20]. Briefly, HSA
(600 mM) was incubated with 40 mM methyl-glyoxal (MG) in
absence and presence of niacin (50, 100, 200 and 500 mM) and
10 mM aminoguanidine (AG) was taken as positive control. All the
preparations were made in 10 mM PBS and samples were incubated
at 37  C for 14 days. The percent inhibited of MG mediated glycation
was estimated from equation-2:
 
FIt
% AGEs Inhibition ¼ 1
 100 (2)
FIc
where FIt and FIc are florescence emission signals of test samples
and control sample respectively.
2.7. Estimation of carbonyl content
The carbonyl content in native, glycated and treated HSA sam-
ples were estimated to find out the degree of protein oxidation. The
Levine method was adopted for the same with few modifications
[21]. Briefly, 1.0 ml of diluted sample was mixed with 400 ml of 40%
ice cold trichloro acetic acid (TCA) followed by centrifugation at
10,000 rpm for 1 min. Pellet obtained was dissolved in 200 ml PBS,
200 ml distilled water and 400 ml of 20 mM 2,4 dinitrophenylhy-
drazine (DNPH). The reaction mixture was allowed to incubate for
90 min at 37  C. On completion of incubation, 350 ml TCA was added
and centrifuged at 10,000 rpm for 2 min. The pellet was washed
thrice with 1 ml of ethanol and ethyl acetate (1:1 v/v) to remove
excess of DTNB. The remaining pellet was dissolved in 2 ml
guanidine-HCl (6M) and absorbance was read at 360 nm. The final
amount of carbonyl content in each was determined by taking
22,000 M
1 cm
1 as molar extinction coefficient and results are
presented in nmole per mg protein.
2.8. Estimation of free lysine
Free ε-amino groups of lysine amino acids in native and treated
samples were examined by 2,4,6-trinitrobenezene sulphonic acid
(TNBS) method [22]. Each protein sample was diluted to 0.2 mg/ml
in 100 mM sodium bicarbonate buffer of pH 8.5 followed by addi-
tion of 0.25 ml of 0.01% (w/v) TNBSA solution and the reaction
mixture was incubated 37  C for 2 h. After incubation, the sample
was solubilized in 0.25 ml of 10% SDS, and 0.1 ml of 1N HCl was
added. For blank, double distilled water was used in place of protein
sample and the absorbance was recorded at 335 nm against the
same blank.
(1)
2.9. Reactive oxygen species production
For the detection of reactive oxygen species (ROS) in different
 K.M. Abdullah et al. / Archives of Biochemistry and Biophysics 627 (2017) 21e29
test samples, Nitro blue tetrazolium (NBT) assay was adopted with
few modifications [23]. Briefly, 100 ml sample, 300 ml NBT (1 mM),
300 ml Triton-X-100 (0.06%), 300 ml EDTA (1 mM) were mixed in
300 ml sodium phosphate buffer (100 mM). The final volume of each
sample was made to 3.0 m by double distilled water. The samples
were incubated at 37  C and absorbance was recorded at 560 nm
after 4 h. Double distilled water was used for setting the reference.
2.10. Sodium dodecyl sulphate polyacrylamide gel electrophoresis
(SDS-PAGE)
To evaluate the changes in mobility caused by glycation, samples
were subjected to 10% SDS-PAGE as described earlier [24]. Twelve
microgram of control and treated samples were loaded into wells of
polyacrylamide gel. Electrophoresis was carried out 100 V at room
temperature for 3 h. Staining was performed using Coomassie
Brilliant Blue (CBBR-250) for 30 min and gel was destained over-
night in 20% glacial acetic acid.
2.11. Transmission electron microscopy (TEM)
Transmission electron micrograph of native, glycated and
treated HSA was obtained using transmission electron microscope
(JOEL-2100, Tokyo, Japan). Slides were made by placing 10 ml of
sample to TEM grid. Excess of protein was removed and samples
were allowed to dry overnight at room temperature. Images were
recorded at 200 KV at magnification range of 20,000X-100,000X.
2.12. Lymphocyte isolation
Approximately 4 ml heparinised blood was obtained from
healthy donor by venepuncture followed by dilution in PBS (Ca2þ
and Mg2þ free). Isolation of lymphocyte was performed with His-
topaque1077 by standard protocol. Isolated lymphocytes were
cultured in RPMI 1640 media for the treatment [20].
2.13. Estimation of lipid peroxidation
The major stable product of lipid peroxidation is melandi-
aldehyde (MDA) which was estimated in lymphocytes by the
method developed by Beuge and Aust [25]. Briefly, 0.4 ml of iso-
lated lymphocytes were treated with 100 ml of native, glycated and
niacin treated HSA samples and incubate for 2 h. The reaction
mixture containing 0.5 ml treated lymphocytes, 0.5 ml TCA (30%)
and 0.5 ml TBA (0.67%) was incubated for 20 min in boiling water
bath. Tubes containing reaction mixture were centrifuged at
4000 rpm for 15 min. The pink supernatant obtained was gently
taken out and the absorbance was read at 530 nm. The amount of
MDA level in each sample is expressed in nmole/mg protein that
was calculated by using molar extinction coefficient of
1.56  10
5 M
1 cm
1.
2.14. Comet assay (single cell alkaline gel electrophoresis)
Comet assay was performed as per the method developed by
Singh et al. (1988) under alkaline conditions with minor changes
[26]. Fully frosted microscopic slides were coated on one side with
1.0% normal melting agarose (NMA) and left overnight at room
temperature to dry. 100 ml of isolated lymphocytes were treated
with the 100 ml of each protein sample i.e. native HSA, glycated HSA
and niacin treated HSA and were incubated for 3 h in an incubator
at 37  C. 100 ml of each treated suspension was overlaid on the base
layer of slide containing 1% NMA and coverslips were placed. For
control set, untreated lymphocytes were taken. Slide were solidi-
fied by placing on ice-pack and the coverslips were removed. A final
23
layer of 0.5% low melting point agarose (LMPA) was placed and
allowed to solidify by placing on ice-packs. The coverslips were
removed and slide were immersed in electrophoretic tank con-
taining freshly prepared cold lysis solution of pH 10.0 for 1 h at 4  C
to lyse the cells. The slides were washed twice with normal saline.
DNA of lymphocytes was allowed to unwind in electrophoretic
buffer (pH 13) for next 30 min. The electrophoresis was performed
for 30 min at 300 mA current and field strength of 0.74 V/cm. After
electrophoresis, each slide was washed in saline before placing in
neutralizing buffer (pH 7.5). The slides were finally stained with
80 ml ethidium bromide (20 mg/ml) for 5 min and washed to remove
excess amount of ethidium bromide. Coverslips were placed and
kept in humidified slide box at 4  C. Scoring was performed with
Komet 5.5 software attached to Olympus fluorescent microscope
(Olympus Co., Japan) with integrated camera (COHU, San Diego,
USA). Fifty cells from each slide were scored and DNA damage in
lymphocytes was expressed as the average tail length.
2.15. Isothermal titration calorimetry
ITC experiment was done using MicroCal VP-ITC (Northampton,
MA, USA). HSA solution, niacin solution and reference buffer were
first degassed in thermovac for 20 min to remove air bubbles at
310 K temperature. Niacin (1 mM) was loaded into rotatory syringe
and then injected to isothermal chamber. The reference cell was
filled with 10 mM PBS and sample chamber was loaded with
1.4235 ml of 20 mM HSA solution. 29 consecutive injections (10 ml
each) of niacin was titrated to isothermal sample chamber con-
taining HSA. The spacing time between two injection was 180 s
with initial delay of 60 s and time for each injection was set to 20 s.
The stirring speed of syringe was at 307 rpm and reference power
was 16 mcal s
1. Area associated with each curve was calculated
with Origin 7.0 software that yielded heat associated with in-
jections. The heat of injections were plotted as function of molar
ratio of niacin to HSA ([niacin]/[HSA]) and the curve was fitted for
one set of binding sites with Origin 7 software. After curve fitting, it
yielded binding affinity (Kb), binding stoichiometry (N), entropy
change (DS ) and enthalpy change (DH ). Gibb's free energy change
(DG ) was calculated using thermodynamic equation-3:
DG ¼
RTlnðKb Þ (3)
2.16. Molecular docking
Molecular docking of niacin with HSA was performed by Auto-
Dock vina software to search for best binding sites in HSA [27]. This
molecular docking program has been documented as more accu-
rate molecular docking system than AutoDock 4 and is able to
perform faster using multiple processors [28]. The structure of
ligand i.e. niacin [CID: 938] was downloaded from https://
pubchem.ncbi.nlm.nih.gov in three-dimensional SDF format. The
pdf file of ligand molecule was converted using Chimera 1.10.2. The
ligand molecule was made flexible to attain most fit conformation
and was saved into pdbqt format. Three-dimensional crystal
structure of HSA was obtained from Protein Data Bank [PDB: 1AO6].
All water molecules surrounding the receptor were deleted to avoid
hindrance while docking. Non-polar hydrogen atoms were merged
and Kollman charges were added [29,30]. The energy minimization
of niacin and HSA was done using Avogadro 1.1 and SPDBV-Swiss-
pdb Viewer respectively. The coordinate file of HSA was then saved
into pdbqt format. The grid was kept at maximum spacing of 1 Å
and size was set to 82  108  86 Å to cover all the active sites. The
centre of the grid was set to x ¼ 25.797, y ¼ 8.363 and z ¼ 22.537.
24 K.M. Abdullah et al. / Archives of Biochemistry and Biophysics 627 (2017) 21e29
Rest of docking parameters were set to default and analysis of
ligand receptor complex was performed by PyMol and Accelrys
Discovery Studio 2016 Client.
3. Results and discussion
3.1. UV-visible spectral analysis
The changes induced by glycation was preliminarily investi-
gated by recording the UV-visible absorption spectra of all samples
after 28 days of incubation. It is clear from Supplementary Fig. S1
that native HSA exhibited a characteristic peak at 280 nm. Glyca-
tion of HSA resulted in increased absorption around this region
with approximately two-fold hyperchromicity. This increase in
absorption is considered to arise due to structural changes in HSA
leading to partial unfolding. The perturbation in native conforma-
tion alters the normal carrying capacity of HSA. Treatment of 50,
100, 200 and 500 mM niacin resulted in 11.55%, 16.75%, 26.78% and
34.29% decrease in absorbance at 280 nm signifying the protection
against glycation. Absorbance at 280 nm in protein is due to aro-
matic amino acids which was found in all samples. Only the extent
of absorption was changed that is attributed to exposure of these
aromatic amino acids that may either be caused by fragmentation
or unfolding of protein resulting from glycation [31]. Thus, it can be
elucidated from absorption studies that niacin inhibited unfolding
of HSA induced by glycation.
3.2. Analysis of fluorescent AGEs
AGEs are group of heterogenous compounds, many of them
exhibit strong fluorescence at 440 nm when excited at 370 nm.
Fig. 1 (inset) shows the fluorescence emission spectra of native,
glycated and niacin treated HSA. It is evident from figure that HSA
exhibited a weak fluorescent signal while its was found to be
increased substantially in case of glycated sample with a prominent
peak around 440 nm. The presence of fluorogenic AGEs in glycated
sample was confirmed by monitoring AGEs specific fluorescence
[32]. Niacin was found to exhibit dose dependent effect on glucose
mediated glycation. There was 3.06%, 19.18%, 41.49% and 64.36%
inhibition in post-Amadori glycation with the treatment of 50, 100,
200 and 500 mM niacin. The percent inhibition was plotted as
function of concentration of niacin to obtain the IC50 value which
Fig. 1. Fluorescence emission spectra of native HSA, glycated HSA, and HSA with
different concentrations of niacin (A). Inhibitory effect of niacin in formation of fluo-
rescent AGEs (B). Protein concentration in each sample was 3 mM. The slit width for
excitation and emission was 5 nm each.
was found to be 288 mM (Fig. 1). HSA contains only one tryptophan
(Trp-214) residue which is located in subdomain II of HSA. Glyca-
tion mainly occurs at ε amino group of lysine that is reported to
alter the micro environment of Trp-214 of HSA [24].
3.3. Effect of niacin on secondary structure of HSA
Circular dichroism (CD) is one of the most sensitive technique
which is used to study the changes in secondary structure of pro-
tein. The far-UV CD spectra of all samples is presented in Fig. 2 that
clearly shows the presence of two negative bands at 208 nm and
222 nm. The two negative bands in the CD spectra are associated
with a-helical content of protein [33]. The mean residue ellipticity
(MRE) value in deg cm2 dmol
1 was calculated using following
equation-4 [34]:
observed CD ðmdegÞ
MRE ¼ (4)
Cp nl  10
where, n is the number of amino acid residues (585 for HSA), Cp is
the molar concentration of the protein and l is the path length of
cuvette (0.1 cm). The percentage of a-helix was calculated form
mean residue ellipticity values at 208 nm using equation-5 [34]:
ð
MRE208
4000Þ
% a
helix ¼ X 100 (5)
33000
4000
where, MRE208 is mean residue ellipticity (MRE) at 208 nm, 4000 is
the MRE of the random coil conformation and b-form at 208 nm
and 33,000 is the MRE value of a pure a-helix at 208 nm. The
amount of a-helix and MRE at 208 nm in all samples calculated
using above equation-5 is given in Table 1. It is evident from the
data that native HSA had 60.83% a-helix which is consistent with
earlier reports [35]. The incubation of HSA with glucose for 30 days
has drastic decrease in a-helix that decreased to 20.40% showing
approximately three-fold loss. With increasing concentration of
niacin, there is restoration in the a-helical content to 29.43%,
42.61% and 46.08% at 100 mM, 200 mM and 500 mM niacin con-
centration respectively. This demonstrates the dose dependent
protective effect of niacin to secondary structure of HSA caused by
glycation.
Fig. 2. Far UV-CD spectra of native, glycated and niacin treated HSA. The protein
concentration used was 0.3 mg/ml. Each spectrum represents the average of three
scans.
 K.M. Abdullah et al. / Archives of Biochemistry and Biophysics 627 (2017) 21e29
Table 1
a-helical content in native HSA, glycated HSA and niacin treated HSA. 5 mM HSA in
each case was used to obtain CD spectra.
Sample MRE208 nm % a-helix
Native
21642.02 60.83
Glycated
9916.63 20.40
Aminoguanidine
19615.85 53.84
Niacin (100 mM)
12537.38 29.43
Niacin (200 mM)
16357.69 42.61
Niacin (500 mM)
17365.96 46.08
3.4. MethylglyoxaleHSA reactivity assay
In hyperglycaemia, the enhanced efflux of glucose causes the
formation and accumulation of AGEs [36]. One of the most reactive
a-ketoaldehydes produced through glycolytic pathway is MG
which is formed in vivo that leads to modification in lysine residues
of serum proteins. This again causes formation of fluorescent AGEs
mainly argpyrimidine [37]. MG-HSA reactivity assay was performed
for quantitative evaluation of MG-mediated glycation of HSA. The
result demonstrated dose dependent inhibition of methyl glyoxal
mediated AGEs formation by niacin (Supplementary Fig. S2). The
percent inhibition was found to be 7.01%, 9.61, %25.12%, and 31.50%
in presence of 50, 100, 200 and 500 mM niacin. Aminoguanidine (a
known glycation inhibitor) inhibited MG mediated glycation by
48.55%. The reduction in both glucose mediated as well as MG
mediated glycation could be due to antioxidant ability of niacin.
3.5. Carbonyl content estimation
Early reaction between glucose and protein leads to formation
of unstable product i.e. Schiff's base that further converted to stable
ketoamines called Amadori products. The Amadori products
further undergoes through enediol reaction to produce protein
carbonyl compounds [38]. The final product of these reactions are
superoxide radicals which are converted to highly reactive hy-
droxyl radical through Fenton reaction and ultimately generating
oxidative stress and cellular damage [39]. As shown in Fig. 3, the
level of carbonyl content in native HSA was found to be 3.86 nmol/
mg protein that was increased to more than three-folds
(11.90 nmol/mg protein) in glycated protein. Such high level of
Fig. 3. The carbonyl content present in native, glycated and niacin treated HSA.
(Aminoguanidine is positive control). All the data have been expressed in mean ± SEM
of three independent experiments. * indicates significantly different from native at
p  0.05. # indicates significantly different from glycated HSA at p  0.05.
25
carbonyl content in glycated sample suggests the formation large
amount of reactive carbonyl species. Treatment of varying con-
centration of niacin (50, 100, 200 and 500 mM) decreased the level
of carbonyl content by 8.71%, 19.48%, 42.05% and 48.71% respec-
tively, as compared to glycated HSA. This demonstrates the ability
of niacin to quench free carbonyl group which is apparent from
reduced carbonyl content as detected in niacin treated HSA
samples.
3.6. TNBSA assay to determine lysine modification
Free amino group containing amino acids such as lysine and
arginine have been documented as major site for glycation in many
proteins including haemoglobin, albumin and lens crystallin and
lysozyme. Out of 59 lysine present in albumin protein, 34 have been
reported to be involved in glycation in various studies [40]. Glyca-
tion at these sites leads to the formation of heterogenous group of
intermediates and end products such as vesperlysine (VESP), car-
boxyethyllysine (CEL) and carboxymethyllysine (CML) [41]. One of
the mechanisms of glycation inhibition is the masking of free amino
groups of lysine and arginine residues. TNBSA (2,4-trinitro benzene
sulfonic acid) assay was performed for the determination of free
lysine present in glycated and niacin treated HSA and the result
obtained is shown in Fig. 4. Percent lysine modification in glycated
HSA was found to be 68.39% that reduced in concentration
dependent manner with addition of niacin. At 50, 100, 200 and
500 mM niacin, the free lysine modification was found to be 62.42%,
58.26%, 47.79% and 35.41%. The masking of free lysine group by
niacin indicated the protective effect of niacin in the formation of
AGEs. Similar trend in free lysine modification was shown by
vanillin and sinirgin [12,41].
3.7. Reactive oxygen species (ROS) quenching by niacin
NBT assay was performed for detection of ROS in different
samples. Glycation leads to formation of diverse class of reactive
oxygen species both intracellular and extracellular [42]. Level of
ROS found in different samples are shown in Supplementary Fig. S3
in which glycated sample showed maximum ROS production. The
ROS production by glycated HSA was found to be more than three-
Fig. 4. Effect of niacin on free ε-NH2 group of lysine determined by TNBSA assay in
native HSA, glycated HSA and niacin treated HSA. (Aminoguanidine is positive control).
All the data have been expressed in mean ± SEM of three independent experiments. *
indicates significantly different from control at p  0.05. # indicates significantly
different from group B at p  0.05.
26 K.M. Abdullah et al. / Archives of Biochemistry and Biophysics 627 (2017) 21e29

folds higher compared to native HSA. The concentration dependent
decrease in ROS was observed with varying concentration of niacin.
At 500 mM niacin concentration, there was 52.16% decrease in ROS
level compared to glycated HSA sample. Niacin is known antioxi-
dant that may be the reason for quenching of ROS [43]. These re-
sults explain the role niacin in inhibiting the ROS production under
hyperglycaemia. We have previously reported similar activity of
quercetin on lymphocytes in which DNA damage was protected
[20].
3.8. SDS-PAGE
living system. The effect of niacin on MDA level in lymphocytes is
shown in Fig. 6. It's evident that niacin had a dose dependent
inhibitory effect on lipid peroxidation. The MDA level in lympho-
cytes treated with native HSA was found to be 2.41 nmol/mg pro-
tein that increased to approximately four-folds in glycated HSA
treated cells. It can be seen in Fig. 6 that on treatment with 50, 100,
200 and 500 mM niacin, there was 4.31%, 11.46%, 38.44% and 46.72%
decrease in the MDA level compared to glycated HSA. Amino-
guanidine (10 mM) inhibited the same by 59.9%. These results are in
agreement with the earlier report where quercetin treated human
serum albumin inhibited lipid peroxidation in lymphocytes [20].

The electrophoretic mobility of native HSA, glycated HSA and
niacin treated HSA was analysed on 10% SDS-PAGE as shown in
Supplementary Fig. S4. It is evident from the image that native HSA
exhibited a single band while upon glycation protein is fragmented
giving three bands that might be due to production of ROS by AGEs
as a result of glycation. The parental band of HSA in glycated sample
showed reduced mobility due to attachment of glucose molecules
that increased the overall molecular weight of protein. Similar
trend has been reported by Perera et al. (2014) where glycation of
BSA with glucose reduced the electrophoretic mobility [44].
Treatment with niacin clearly inhibited the protein fragmentation
as evident from single band on polyacryl amide gel. The rate of
migration was also restored to a significant extent upon treatment
with niacin.
3.11. Comet assay
Super oxide radicals produced by glycated HSA generate ROS
causing oxidative stress and cellular damage. The DNA damage
caused by ROS generated by glycated HSA was studied and esti-
mated by measuring the tail length of lymphocytes by single cell
alkaline gel electrophoresis. The average tail length of lymphocytes
treated with glycated sample (23.12 mm) was significantly greater
than lymphocytes treated with native HSA (4.47 mm) which is
shown in Fig. 7. This signifies the DNA damaging tendency of su-
peroxide radicals induced by glycation. It was found that DNA
damage was inhibited by 25.09% and 36.82% at 200 mM and 500 mM
niacin respectively in lymphocytes treated with niacin

3.9. Transmission electron microscopy

Transmission electron micrograph of native, glycated and niacin

treated HSA is shown in Fig. 5 (A, B and C). In native HSA that there
is no aggregate. On contrary, TEM image of glycated protein
exhibited dark patches which are aggregates caused by glycation.
Treatment with niacin reduced the formation of aggregates. TEM
images of native and niacin treated proteins are comparable to say
that niacin has partially restored the native structure. Protein gly-
cation is known to cause Amyloid Cross-b Structure in albumin [45].
Although, the underlying mechanism through which glycation in-
duces aggregation is still not properly known. A study has found
that there is a series of steps involved including chemical modifi-
cation in lysine side chains changing the overall charge on protein
and increasing the hydrophobic surface. These changes in protein
leads to formation of small oligomers that gradually evolve to
bigger insoluble particles [46].

Fig. 6. Level of MDA in lymphocytes treated with native HSA, glycated HSA and niacin
3.10. Estimation of lipid peroxidation treated HSA. All the data have been expressed in mean ± SEM of three independent
experiments. * indicates significantly different from native HSA at p  0.05. # indicates
MDA is the final quantifiable product of lipid peroxidation in significantly different from glycated HSA at p  0.05.

Fig. 5. Transmission electron micrographs of native HSA (A), glycated HSA (B) and 500 mM niacin treated HSA (C) after 28 days of in 10 mM phosphate buffer at pH 7.4. Ten
microliter of each sample was kept on TEM grid and images were recorded at 200 KV.
 K.M. Abdullah et al. / Archives of Biochemistry and Biophysics 627 (2017) 21e29
Fig. 7. The average tail length of lymphocytes treated with native HSA, glycated HSA
and niacin treated HSA obtained by single cell gel electrophoresis.
supplemented HSA samples. This can be concluded that niacin has
protective role in DNA damage against reactive oxygen species
generated by advanced glycation endproducts.
3.12. Isothermal titration calorimetry
ITC is an effective tool to decipher the thermodynamics of drug-
protein interaction. It is a sensitive technique as it measures the
overall energy change involved in the reaction. ITC delivers com-
plete thermodynamic profile as well as binding stoichiometry and
binding affinity. ITC titration of niacin to HSA is shown in Fig. 8. The
upper panel if this figure denotes the raw data obtained by
isothermal calorimetric titration of niacin to HSA which was per-
formed at 37  C. Individual burst curves shown in this panel de-
notes binding isotherm of each injection of niacin. The area under
each curve was calculated by Origin 7 that produced heats associ-
ated with every injection. The resultant heat thus obtained was
plotted as function of molar ratio of niacin to HSA as depicted in
lower panel. These data points are the experimental heats of in-
jection and the solid curve is best fit of these data points for one set
of binding sites. The binding stoichiometry (n), binding constant
(Ka), entropy change (DS ) and enthalpy change (DH ) were directly
obtained from the fitted curve with lowest c2 value. The value of
Gibb's free energy (DG ) was obtained from standard thermody-
namic equation and the result obtained is mentioned in Table 2. The
negative DG (
5.55 kcal/mol) value confirms that reaction is
thermodynamically favourable and spontaneous. The positive
values of both enthalpy and entropy change signifies that interac-
tion of niacin with HSA is entropically and thermodynamically
favoured [47,48]. Positive delta H value also explains the endo-
thermic nature of this reaction. After fitting ITC data, value of
binding constant (Ka) for one set of binding sites was fund to be
2.32  104 M
1.
3.13. Molecular docking
To get a closer insight into binding of niacin to HSA, molecular
modelling was deployed that provides the details of various bonds
along with their bond lengths. HSA contains 585 amino acids with
three domains naming domain I, domain II and domain III whose
amino acids ranges 1e195, 196e383 and 384e585 respectively.
Each domain is further divided in to two sub-domains i.e. A and B.
Crystallographic data have revealed that the most common binding
27
Fig. 8. Isothermal titration calorimetry profile of HSA and niacin interaction performed
at 25  C using MicroCal VP-ITC (Northampton, MA, USA). The concentration of niacin
and HSA was 10 m and 20 mM respectively.
Table 2
Isothermal titration calorimetry profile of interaction of niacin with
HSA at pH 7.4.
Parameters Values
pH 7.4
Temp. (K) 310
Kb (  104 M
1) 2.32 ± 0.306
n 0.9258 ± 0.317
DG (kcal mol
1)
5.55
DH (kcal mol
1) 38.47 ± 1.011
DS (cal mol
1 K
1) 142
site in HSA for drugs are located in sub-domain IIA sub-domain IIIA
[49]. The ligand molecule (niacin) was made flexible to get ther-
modynamically most fit conformation and to predict more accurate
binding mode using detailed molecular mechanics. AutoDock vina
resulted in 9 conformations ranked with increasing binding en-
ergies and the best conformation is shown in Fig. 9 [27]. Macular
docking results showed that niacin directly formed hydrogen bond
with Arg257 at distance of 4.12 Å. There were two hydrophobic
bonds between niacin and Leu238 & AlaA291 of HSA [PDB ID:
1AO6]. Four amino acids (Tyr150, HisS242, Arg222 and Ser287)
surrounding niacin were attached by van der Waal's interactions.
Although, no lysine residue was found interacting directly with
niacin but LysS199, Lys212 and Lys225 were in close proximity. The
binding of niacin molecule to arginine occupying the site sur-
rounding lysine might have reduced the extent of glycation. Bind-
ing energy of niacin-HSA interaction was found to be
5.3 kcal/mol.
28 K.M. Abdullah et al. / Archives of Biochemistry and Biophysics 627 (2017) 21e29
Fig. 9. Molecular models of HSA complexed with niacin. (A) Detailed view of the docking poses of the HSAeniacin complex, selected protein side-chains are shown as ribbons. (B)
niaicn is shown in the binding pocket of HSA with interacting amino acids. (C) niacin in hydrophobic pocket of HSA surrounded by hydrophobic amino acids. (D) 2-dimensional view
by Discovery Studio 2016.
It is interesting to note that the binding energy of niacin with HSA
was found to be similar as obtained by ITC.
4. Conclusion
Niacin, an essential dietary component, was found to inhibit
glycation. Preliminary investigation showed that level of AGEs was
reduced by addition of niacin to HSA containing glucose. Increased
absorption at 280 nm showed the exposure of aromatic amino acids
which was decreased in niacin treated samples. Free lysine content
was restored and secondary structure of HSA was retained in
presence of niacin. Niacin was found to be effective quencher of ROS
produced as result of glycation and inhibited DNA damage in
lymphocytes and may be beneficial in diabetes related complica-
tions. The strong affinity of niacin towards HSA was thermody-
namically favourable and spontaneous one. Niacin was bound in
subdomain IIA of HSA which is one of the two major binding sites.
Glycation of HSA resulted in ROS production that led to protein
damage. Far-UV CD data indicated that HSA resisted change in its
native conformation caused by glycation in presence of varying
concentration of niacin. After analysing these data, we can conclude
that niacin successfully inhibited AGEs formation and may be
useful in hyperglycaemia. To further validate its efficacy, study on
rats are already underway.
Conflict of interest
The authors declare that there is no conflict of interest in this
work.
Acknowledgements
KMA and FAQ are thankful to University Grants Commission,
New Delhi, for the award of UGC-Non-NET Fellowship. The authors
also thanks to the Department of Biochemistry, Aligarh Muslim
University, Aligarh, and Department of Agricultural Microbiology,
Aligarh Muslim University, Aligarh for providing the necessary
facilities.
Appendix A. Supplementary data
Supplementary data related to this article can be found at http://
dx.doi.org/10.1016/j.abb.2017.06.009.
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