2. Ind J Clin Biochem
1 3
movements of joints which develops pain and fatigue [1].
There are different types of arthritis [2]. The most common
type of joint disorders are osteoarthritis (OA) or degenera-
tive joint, rheumatoid arthritis (RA), gouty arthritis (GA),
calcium pyrophosphate deposition disease (CPPD), psoriatic
arthritis (PA), infectious arthritis (IA) and spondyloarthritis
(SA) [2, 3].
Causes of arthritis may vary according to the type of
arthritis. In osteoarthritis, obesity, advancing age and dis-
comfort in joints show a contributory role. Some genetic
modification such as mutations in genes encoding types
II, IV, V, and VI collagens have also been found to cause
osteoarthritis. On the other hand, RA is an autoimmune
disorder. Some studies showed that exposure of air pollu-
tion may increase the risk of RA [4]. Some gases such as
SO2, NO2, carbon monoxide (CO), and O3 are the main
pollutants that induce occurrence of RA. Further, the results
from the epidemiological studies have indicated that expo-
sure of an individual to many other environmental factors
including dioxin, cigarette smoke, noise, and air pollutants
[5]. Some other factors such as viral diseases (CoVID-19)
and prolonged medication may also cause development of
arthritis [6, 7]. In another study carried out by Gong et al.
[3], it was found that plant based lectins may be involved
in the pathogenesis of some inflammatory disorders in
arthritis via activation of NLRP3 inflammasome [8]. Tsuda
et al. [9] has reported that cross reaction of serum ACPA
of RA patients’ citrullinated proteins derived from viruses
(EBNA-1), bacteria (Micrococcus luteus, Mycobacterium
gastri, Nocardia brasiliensis), fungi (Aspergillus fumiga-
tus, Candida albicans, Cryptococcus neoformans) and
plants (Oryza sativa, Solanum lycopersicum, Glycine max,
with tissue antigens may induce RA through cross-reactivity
[10]. In addition, the excess consumption of high-fructose
corn syrup and fruit drinks may cause three-fold higher risk
of development arthritis [11].
The corticosteroids, the steroidal hormones, possess anti-
inflammatory, vasoconstrictive and immunosuppressive
properties [12]. They are prescribed to treat chronic arthritis
patients to combat emergence of any serious clinical compli-
cations [13–16]. The influence of steroids on inflammatory
processes has long been established since the interaction
of glucocorticoids and their receptors were found to inhibit
several inflammatory pathways. Inflammatory processes are
known to be accompanied by an increased oxidative burden
followed by a depletion of antioxidants [17]. The oxidative
stress is found to be associated with major health complica-
tions [18–20]. Some workers have previously shown that
RA induces oxidative stress. However, it is not well known
that the intake of steroids by arthritis patients may further
augment this process [21]. Chronic usage of corticosteroids
has been linked to the negative side effects such as weight
gain, osteoporosis, hypertension, diabetes mellitus, cataract,
dyspepsia, and psychiatric issues [12, 22].
The usage of corticosteroids is considered to be safe, in
terms of hepatotoxicity, however, the clinical impact of this
not well known. Some workers have shown liver injury in
those related to high-dose methylprednisolone [23]. How-
ever, the implication of steroidal drugs in generating oxida-
tive stress and inducing hepatotoxicity has not been system-
atically investigated in human system.
In the present study, an attempt has been made to evaluate
the effect of steroidal drugs (deflazacort and methylpredniso-
lone) on the levels of antioxidant defense system and hepatic
markers in the blood plasma of the arthritis patients using
these drugs for a long duration. The results from present
investigation suggested that the long term treatment with
steroidal drugs may induce imbalance in the endogenous
antioxidant system and develop hepatotoxicity.
Materials and Methods
Chemicals
Ethylene diaminetetraacetic acid (EDTA), potassium dichro-
mate, pyrogallol, sodium bicarbonate
(NaHCO3), sodium
dihydrogen phosphate
(NaH2PO4), disodium hydrogen
phosphate (Na2HPO4), sodium acetate, sodium carbonate
(Na2CO3), sodium hydroxide, sulfuric acid, Merk Limited
(Mumbai, India),1-Chloro 2, 4 dinitrobenzene (CDNB), Fol-
lin’s reagent, glutathione (GSH),5, 5′-Dithiobis (2-nitroben-
zoic acid) (DTNB), glycerol, NADH, sodium pyruvate,
sodium chloride, and Tris-base were from SRL. Pvt. Ltd.
(India). Bovine serum albumin (BSA) was from LobaChe-
mie (LobaChemie Pvt. Ltd., Mumbai, India). All other
chemicals used were analytical grade.
Subjects
This study included blood samples from 30 patients suf-
fering from arthritis, reactive arthritis, and joint pain and
receiving steroidal therapies using deflazacort and methyl-
prednisolone for different durations such as 7, 14, 28, 32, 84
and 168 days. The blood samples of these subjects were col-
lected from different hospitals/ clinics. These patients were
prescribed to take short-term and long-term therapies with
corticosteroids i.e., deflazacort and methylprednisolone for
the improvement of their clinical conditions. The deflazacort
is prescribed in 12–18 mg divided dose in a day whereas
methylprednisolone is prescribed as 16-24 mg in a day. The
blood is collected into the heparinized sterile vials and kept
inside the ice box.
3. Ind J Clin Biochem
1 3
Inclusion and Exclusion Criteria
The inclusion criteria followed by the patients included in
this study contained age 30–55 years, suffering from arthritis
for at least one month or more, taking steroids (methylpred-
nisolone or deflazacort) for therapy, inhabiting in any social
structure, and agreeing to extend informed consent for the
said purpose. The exclusion criteria for the recruitment of
patients in this study were subjects intolerant or allergic to
any therapeutic agents, diagnosed positive for viral, bacte-
rial, fungal infections or parasite infestation, uncontrolled
diabetes mellitus (DM), gastrointestinal problems or gastro-
intestinal bleeding (GIB) history, heart failure (HF), active
malignancies and received any immune-suppressor agents.
The study also excluded pregnant or lactating women.
Sample Preparation
The blood samples collected in the sterile heparinized tubes,
centrifuged at 10,000 rpm for 15 min using refrigerated cen-
trifugation machine (Sigma 2-16KL). After centrifugation,
the supernatant (plasma) was collected and stored at 4 °C
for future uses.
Biochemical Assays
Determination of Protein Content
The total protein content in different samples was deter-
mined by the method of Lowry et al. [24] using BSA as a
standard.
Estimation of Malondialdehyde (MDA) Level
Lipid peroxidation (LPO) in terms of level of malondialde-
hyde (MDA) was measured in the blood plasma samples fol-
lowing the method of Niehaus and Samuelsson (1968) and
the results were expressed as n mol MDA/mg protein using
1.56× 105
M−1
cm−1
as an extinction coefficient.
Determination of GSH Content
The Glutathione (GSH) content in blood plasma was esti-
mated according to the method describe Ellman et al. (1959)
modified by Sedlak and Lindsay (1968). The optical density
of the yellow color complex developed in the reaction mix-
ture was measured at 412 nm. The reaction mixture con-
taining 50 mM Tris-HCl buffer pH 8.2, 0.06 mM DTNB,
absolute methanol, and suitable amount of plasma was incu-
bated with intermittent shaking and centrifuged at 800×g for
15 min. The results were expressed as microgram (µg) of
GSH/mg protein. The GSH was used as a standard.
LDH Activity Assay
The activity of LDH in blood plasma was measured by the
method of Horecker and Kornberg (1948). Briefly, The total
reaction mixture volume (3 ml) contained 50 mM Tris-HCl
buffer, pH 7.4, 3 mM KCl (0.15 ml), 1 mM sodium pyruvate,
0.16 mM NADH and suitable amount of enzyme protein.
The enzyme activity was monitored at 340 nm for 3 min as
a decrease in its absorbance at the regular interval of 30 s.
The absorbance of the reaction mixture recorded in absence
of enzyme protein was considered as a control. The spe-
cific activity of the enzyme was calculated using the extinc-
tion coefficient of NADH (6.22×
103
μmol−1
L−1
min) and
denoted as IU/ mg protein. The assays were performed on
UV visible double beam spectrophotometer (Spectroscan
UV 2700).
SOD Activity Assay
The activity of superoxide dismutase (SOD: E.C. 1.15.1.1)
in plasma was measured by the method of Marklund and
Marklund (1974). The method allows measuring the optical
density of colored complex involving the autooxidation of
pyrogallol at 412 nm for 3 min at the 30 s interval on spec-
trophotometer. The unit for enzyme activity was expressed
as 50% inhibition of pyrogallol autooxidation per min.
CAT Activity Assay
The activity of catalase (CAT: E.C.1.11.1.6) in blood plasma
was measured according to Beers and Sizer (1952) method.
The decrease in absorbance for
H2O2 consumption was mon-
itored at 240 nm for 3 min with an interval of 30 s. The unit
for CAT activity was expressed in terms of μmol of
H2O2
hydrolyzed per min. The enzyme activity was calculated by
using molar extinction coefficient (43.6
M−1
cm−1
).
Assay for Liver Function Enzymes
The activities of transaminases such as aspartate transami-
nase (AST: EC 2.6.1.1) and alanine transaminase (ALT: EC
2.6.1.2), also known as SGOT and SGPT, respectively, were
monitored in the blood samples using the method of Reitman
and Frankel (1957). A suitable amount of blood plasma was
incubated in 0.25 ml of substrate (200 mM aspartate with
2 mM α-ketoglutarate) for AST and (200 mM Malanine with
2.0 mM α-ketoglutarate) for ALT in 0.1 M phosphate buffer,
pH 7.0, for 30 min. An aliquot of 0.25 ml of DNPH (1 mM)
was used to stop the reaction and allowed incubation for
20 min at room temperature (about 26 °C). The reaction
4. Ind J Clin Biochem
1 3
mixture was added with 2.5 ml of 0.4 N NaOH followed by
gentle vortex. The optical density of the colored complex
was monitored at 510 nm. The total reaction mixture volume
(TRMV; 3 ml) in the absence of enzyme protein was used as
a control in each case.
Statistical Analysis
The Graph Pad Prism version 5.01 (San Diego, CA) was
used for analyzing the data using one way analysis of vari-
ance (ANOVA) and calculating the values as mean±standard
deviation.
Results
Level of Blood Plasma Protein in the Arthritis Patients
The results obtained after analysis of protein concentration
in the blood plasma from the patients suffering from arthritis
indicated almost no difference from the normal subjects.
Also, the level of blood plasma protein from the arthritis
patients taking steroids (methylprednisolone and deflaza-
cort) did not reflect any change in the values of protein (data
not shown).
Effect of Deflazacort and Methylprednisolone
on the Level of MDA and GSH in the Plasma of Human
Blood Samples
The effect of steroidal drugs on the levels of MDA and GSH
were determined and the results are shown in Table 1. It
was found that there is a significant increase (about two-
fold) in the level of MDA in the patients suffering from
arthritis in comparison to the normal subjects. The level of
MDA was also higher in the steroid treated arthritis patients
as compared to controls but these values were lower than
those from patients not using steroids. The level of GSH
was found to be significantly decreased in the patients tak-
ing steroidal drugs. However, there was no change in the
level of GSH in the arthritis patients as compared to the
normal control.
Effect of Deflazacort and Methylprednisolone
on the Level of LDH Activity on the plasma of Human
Blood Samples
The effect of deflazacort and methylprednisolone was evalu-
ated on the activity of LDH in the blood plasma of arthritis
patients. The results shown in Fig. 1 indicated that there was
a slight increase in the LDH activity both in the patients suf-
fering from arthritis as well as those using (A) deflazacort
or (B) methylprednisolone as therapeutic agents for longer
duration.
Effect of Deflazacort and Methylprednisolone
on the Activity of SOD on the Plasma of Human Blood
Samples
The effect of deflazacort and methylprednisolone was
evaluated on the activity of SOD in the blood plasma of
arthritis patients. The results shown in Fig. 2 indicated
that there was a decrease in SOD activity by 22% in arthri-
tis patients in comparison to the control. After treatment
with methylprednisolone and deflazacort, there was no
significant alteration was seen with respect to the arthritis
patients. However, the slight increment in the activity of
enzyme was seen in case of deflazacort treatment.
Effect of Deflazacort and Methylprednisolone
on the Activity of Catalase on the Plasma of Human
Blood Samples
The effect of deflazacort and methylprednisolone was
evaluated on the activity of catalase in the blood plasma
of arthritis patients. The results shown in Fig. 3a indicated
that there was only mild decrease (22%) in CAT activity in
the arthritis patients. When the arthritis patients received
deflazacort for 7 days, the activity of catalase was found
to be near normal subjects. There was almost no further
change in the activity of catalase when the patients received
deflazacort up to 168 days. In contrast, the treatment of
Table 1 Effect of steroids on the levels of MDA and GSH in the
human blood plasma
The data presented in the table reflect the values as mean ±SEM of
three independent experiments
Treatment Time duration Plasma MDA
level
Plasma GSH
level
Control – 3.19±0.16 1.01±0.012
Arthritis patients – 6.14±0.20 1.09±0.13
Arthritis patients
using deflaza-
cort
7 Days 5.78±0.18 1±0.97
14 Days 5.71±.155 0.80±0.065
28 Days 5.09±0.53 0.78±0.031
84 Days 5.0±0.39 0.77±0.05
168 Days 5.79±0.049 0.72±0.019
Arthritis patients
using methyl-
prednisolone
7 Days 3.24±0.198 0.77±0.037
14 Days 4.59±0.27 0.76±0.036
28 Days 5.8±0.31 0.72±0.041
84 Days 6.33±0.36 0.70±0.026
168 Days 6.49±0.38 0.70±0.037
5. Ind J Clin Biochem
1 3
Fig. 1 Effect of deflazacort (a) and methylprednisolone (b) on the
activity of lactate dehydrogenase (LDH) in human blood plasma. The
effect was observed for different treatment durations (7–168 days) as
mentioned in the Materials and Methods section. The IU represents
µmoles of substrate converted into product by the enzyme in one
min. The data has been displayed as mean±SD of three independ-
ent experiments. C=normal blood plasma, C1=blood plasma from
arthritis patients. Treatment durations in days reflect the intake of
steroids by arthritis patients (C1) for varying periods
Fig. 2 Effect of deflazacort (a) and methylprednisolone (b) was
evaluated on the activity of superoxide dismutase (SOD) in human
blood plasma. The effect was observed for different treatment dura-
tions (7–168 days) as mentioned in the Materials and Methods sec-
tion. The IU represents µmoles of substrate converted into product by
the enzyme in one min. The data has been displayed as mean±SD of
two independent experiments. C=normal blood plasma, C1=blood
plasma from arthritis patients. Treatment durations in days reflect the
intake of steroids by arthritis patients (C1)
Fig. 3 Effect of deflazacort (a) and methylprednisolone (b) on the
activity of catalase (CAT) in human blood plasma. The effect was
observed for different treatment durations (7–168 days) as mentioned
in the Materials and Methods section. The IU represents µmoles of
substrate converted into product by the enzyme in one min. The data
has been displayed as mean±SD of three independent experiments.
C=normal blood plasma, C1=blood plasma from arthritis patients.
Treatment durations in days reflect the intake of steroids by arthritis
patients (C1)
6. Ind J Clin Biochem
1 3
patients with methylprednisolone from 7 to 168 days dem-
onstrated a continuous decrease in the catalytic activity
of catalase ranging from 30 to 45% in comparison to the
control (Fig. 3b)
Effect of Deflazacort and Methylprednisolone
on the Activity of Alanine Transaminase (ALT)
and Aspartate Aminotransferse (AST) on the Plasma
of Human Blood Samples
The effect of deflazacort and methylprednisolone was eval-
uated on the level of ALT and AST in the blood plasma of
arthritis patients. The results shown in Table 2 indicated
that there was no significant change in the level of ALT in
arthritis patients in comparison to the control. The level
of ALT recorded on day zero remained unchanged over
the period up to 168 days in comparison to the control.
However, after treatment with steroids (methylpredniso-
lone and deflazacort), the level of ALT was found to be
increased by 30–35% up to 168 days of treatment of arthri-
tis patients. Similar to ALT, the level of AST was also
found to be unchanged in arthritis patients from day 0 to
168 days. After treatment with the steroids (methylpred-
nisolone and deflazacort), the level of AST increased by
1.5-fold up to 0–168 days of treatment period in compari-
son to the control. There was significant increase in the
ratio of AST: ALT as observed from day 28 to 168 after
the treatment with deflazacort. This ratio increased from
the day 84 to 168 after the treatment with methylpredni-
solone (Table 2).
Discussion
Arthritis, a very common chronic autoimmune disease,
needs long term treatment with the therapeutics involving
steroids, especially the methylprednisolone and deflazacort.
The treatment of arthritis patients with higher doses of drugs
combined with steroids for the extended period of time has
been shown to develop certain adverse effects [25–27].
In some previous studies, the applications of steroids have
been demonstrated to induce oxidative stress and to develop
alterations in clinical parameters including hepatotoxicity
[22, 28]. In the present investigation, a biochemical analy-
sis of different clinical parameters including the markers of
oxidative stress and liver function tests has been carried out
in the blood plasma of arthritis patients. The results from the
present study indicated significant perturbations in the levels
of oxidative indices, protein and the activities of antioxidant
enzymes, transaminases as well as lactate dehydrogenase,
a key enzyme of energy metabolism, in the blood plasma
of arthritis patients using steroids (methylprednisolone and
deflazacort) with other therapeutics up to 168 days.
The significant decrease in the level of GSH and increase
in the level of MDA as observed in the arthritis patients
indicated the development of oxidative stress due to chronic
treatment of patients with steroidal drugs up to 168 days.
Similar observation has been recorded by other workers who
have studied on the arthritis patients and found perturba-
tions in the levels of GSH and MDA [29]. The Hassan et al.,
(2011) have observed increased level of oxidative species
and drastic reduction in the level of anti-oxidative factors in
Table 2 Effect of deflazacort
(D) and methylprednisolone
(M) on the level of Alanine
transaminase (ALT) and
Aspartate transaminase (AST)
[U/mg of protein] in human
blood plasma
The effect was observed for different treatment durations (0–168 days) as mentioned in the Materials and
Methods section. The data has been displayed as mean±SEM of three independent experiments. C=nor-
mal blood plasma, C1=blood plasma from arthritis patients. Treatment durations in days reflect the period
of intake of steroids by arthritis patients (C1)
Groups Treatment duration (days)
0 7 14 28 84 168
AST (C) 32.71±0.10 33.50±0.09 36.09±0.10 34.86±0.15 33.98±0.09 35.70±0.18
ALT (C) 45.08±0.50 47.01±0.01 49.90±0.10 47.48±0.08 45.09±0.13 45.34±0.18
AST/ALT (C) 0.71±0.30 0.712±0.15 0.723±0.09 0.734±0.12 0.753±0.13 0.787±0.09
AST (C1) 34.56±0.22 36.08±0.19 35.78±0.25 34.45±0.21 35.68±0.24 36.90±0.30
ALT (C1) 42.09±0.48 45.60±0.20 43.09±0.30 43.75±0.15 43.98±0.19 46.01±0.20
AST/ALT (C1) 0.81±0.35 0.80±0.16 0.79±0.30 0.78±0.27 0.81±0.31 0.80±0.19
AST (C1+D) 33.61±0.30 34.75±0.25 52.25±0.15 59.95±0.22 61.65±0.17 63.6±0.19
ALT (C1+D) 45.05±0.23 47.31±0.30 53.5±0.05 54.8±0.50 55.7±0.50 61.05±0.51
AST/ALT (C1+D) 0.75±0.15 0.73±0.026 0.976±0.010 1.09±0.36* 1.11±0.03* 1.04±0.35*
AST (C1+M) 34.45±0.05 49.95±0.02 51.75±0.04 54.05±0.02 77.1±0.05 80.75±0.20
ALT (C1+M) 42.28±0.07 55.05±0.30 54.0±0.09 59.1±0.03 66.3±0.02 66.95±0.07
AST/ALT (C1+M) 0.81±0.02 0.90±0.16 0.95±0.020 0.91±0.01 1.16±0.04* 1.20±0.01*
7. Ind J Clin Biochem
1 3
the patients suffering from rheumatoid arthritis and systemic
lupus erythematosus and taking treatment with methoxarate
[29]. Some authors have studied the effect of methylpredni-
solone in the experimental animal models and reported sig-
nificant perturbations in the levels of oxidative indices [7].
The patients under this treatment conditions indicated
differential response of deflazacort and methylprednisolone
on the activities of antioxidant enzymes i.e. SOD and CAT.
These drugs caused significant decrease in the activities of
SOD and CAT in blood plasma samples; the effect with the
administration of deflazacort being less than that of meth-
ylprednisolone. In a study conducted by Sunitiparpluacha
(2018) et al., the development of oxidative stress in the oste-
oarthritis patients using glucocorticoid has been reported
[30]. Some workers have reported the excess production
of ROS and reduction in the activities of ROS scavenging
enzymes in the patients suffering from eye diseases and tak-
ing corticosteroids as a therapeutic agent [31, 32].
Tansaminases (AST and ALT) are key marker enzymes of
liver involved in biotransformation of xenobiotics and detox-
ification process [3]. The increased level of these enzymes
has been shown to be associated with the liver toxicity [3].
The significant increase in the level of transaminases (AST/
ALT) observed in the arthritis patients taking steroids (meth-
ylprednisolone and deflazacort) in the present study suggests
the adverse impact of these drugs on the liver function of
patients. Corticosteroids also have major effects on the liver,
particularly when given for long term and in higher than
physiologic doses [23, 26]. High doses and long-term use
has been associated with the development or exacerbation of
nonalcoholic steatohepatitis with elevations in serum ami-
notransferase levels and liver histology resembling alcoholic
hepatitis with steatosis, chronic inflammation, centrolobular
ballooning degeneration [23, 26]. In one of such study con-
ducted by Gutkowski et al. (2011) the high dose of meth-
ylprednisolone has been reported the cause hepatotoxicity
[28].
Conclusion
The results from the present study reflected that the long-
term treatment of arthritis involving steroids elicit marked
perturbations in some key biochemical parameters in the
patients. The significant increase in the level of MDA and
decrease in the anti-oxidative indices such as GSH and
activities of SOD and CAT indicated the development of
oxidative stress. The significant alterations in the levels AST
and ALT as observed in the blood plasma indicated hepatic
damage in the patients taking steroids with the anti-arthritis
therapeutics. The mild change in the activity of LDH in the
blood plasma of such patients is an indicative of impact of
these therapeutic agents on the energy metabolism. These
results suggest that the application of steroids either should
be avoided or minimized during treatment of arthritis. The
use of antioxidants as supplements to the anti-arthritis agents
could play a role in suppressing the oxidative stress medi-
ated adverse effects. However extensive research is required
to explore for safer medication devoid of steroids to cure
arthritis.
The detail illustration of present work is presented in
graphical abstract.
Acknowledgements PT and NS are grateful to the University Grant
Commission, New Delhi, for providing financial assistance in the form
of a Research Fellowship. Authors acknowledge UGC-SAP and DST-
FIST for support to Department of Biochemistry, University of Alla-
habad, Prayagraj India.
Funding No funding was received for conducting this study.
Declarations
Conflict of interest Corresponding author on behalf of all authors
declare no conflict of interest.
Ethical Approval Vide office no. IERB/2016/03 by institute of eth-
ics committee of population resource and research centre Allahabad.
Consent to Participate Consent was taken from the patients.
Consent for Publication Consent was obtained from the patients for
publication of this case.
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