Original Communication

Oral administration of alpha-lipoic

acid did not affect lipid peroxidation
and antioxidant biomarkers in
rheumatoid arthritis patients
Sousan Kolahi1, Elham Mirtaheri2, Bahram Pourghasem Gargari3, Alireza Khabbazi1,
Mehrzad Hajalilou1, Mohammad Asghari-Jafarabadi4, and Mehran Mesgari Abbasi5
1 Connective Tissue Diseases Research Center, Tabriz University of Medical Sciences, Tabriz, Iran
2 Student Research Committee, Faculty of Nutrition and Food Sciences, Tabriz University of Medical Sciences, Tabriz, Iran
3 Nutrition Research Center, Department of Biochemistry & Diet Therapy, Faculty of Nutrition and Food Sciences,
Tabriz University of Medical Sciences, Tabriz, Iran
4 Road Traffic Injury Prevention Research Center, Faculty of Health, Tabriz University of Medical Sciences, Tabriz, Iran
5 Drug Applied Research Center, Tabriz University of Medical Sciences, Tabriz, Iran

Received: December 27, 2015; Accepted: April 8, 2016

Abstract: Rheumatoid arthritis (RA) is a chronic inflammatory disease in which oxidative stress could play a substantial pathological role.
Alpha-lipoic acid (ALA) has been known as a “universal” and “ideal” antioxidant. The purpose of this study was to investigate the effects of oral
administration of Alpha-lipoic acid (ALA) on lipid peroxidation and antioxidant biomarkers in Rheumatoid arthritis (RA) patients. The study was
a randomized, double-blinded, placebo-controlled clinical trial. 70 RA patients were randomized 1:1 to two groups using blocked
randomization method and received 1200 mg/day ALA or placebo for 8 weeks. Fasting blood samples were obtained before and after the
intervention to analyze total antioxidant capacity (TAC), antioxidant enzymes [superoxide dismutase (SOD), glutathione peroxidase (GSH-Px)
and arylesterase (ARE) activities] and malondialdehyde (MDA). We observed significant increase in serum TAC (0.11 mmol/L; p=0.033) and ARE
(13.76 U/mL; p=0.046) and significant decline in MDA (
0.36 nmol/L; p=0.002), in ALA group. However, these changes in ALA-treated group
were not statistically significant when compared with placebo-treated group (p > 0.05). Also, within- and between-group differences of whole
blood SOD and GSH-Px were not statistically significant (p > 0.05). In conclusion, unexpectedly, ALA therapy did not affect the oxidative status
of RA patients in the present clinical trial. It seems that more comprehensive clinical trials in RA patients are still warranted to clarify the
effectiveness of ALA which has been known as a potent antioxidant.

Keywords: Rheumatoid arthritis, Inflammatory disease, Oxidative stress, Antioxidant, Alpha-lipoic acid

Introduction Excessive ROS could damage lipids, proteins and nucleic

Rheumatoid arthritis (RA) is a chronic systemic inflamma-
tory disease manifested by polyarthritis, progressive
articular destruction and intermittent acute inflammatory
episodes [7]. Although, the disease etiology is unknown,
oxidative stress plays a role in pathological process of RA.
Correspondingly, studies have indicated elevation of
oxidative biomarkers in serum and synovial fluid of RA
patients [1, 2].
Synovial infiltration of immune cells develops articular
and systemic inflammation and oxidative stress, through
excess production of various types of inflammatory
mediators and reactive species (ROS and RNS) [3].
acids. Also, ROS could serve as second intracellular
messenger which activates some prominent transcription
factors such as nuclear factor kappa-B (NF-κB). NF-κB
orchestrates the expression of a wide spectrum of critical
genes involved in inflammatory response and oxidative
stress [1, 4]. Regarding the complex interactions between
oxidative stress and inflammation, antioxidant treatment
seems to be a potential adjuvant therapy for oxidative
stress-related disorders such as RA. However, study on
antioxidants in RA is still in its initial stages.
Current pharmaceutical therapies for RA cannot afford
the complete alleviation of the symptoms. Moreover, the
medications often could bring about some serious side

Ó 2019 Hogrefe Int J Vitam Nutr Res (2019), 89 (1–2), 13–21

https://doi.org/10.1024/0300-9831/a000550
14
effects [5]. On the other hand, adjuvant therapies devoid of
such disadvantages have received increasing attention
recently [6].
Alpha-lipoic acid (ALA), produced by mitochondrial
ALA synthase, is an essential cofactor for alpha-keto-
dehydrogenases in mitochondria [8]. Aside from its role
as a metabolic cofactor for energy metabolism, evidence
suggests that exogenous source of ALA may elicit several
antioxidant properties in both lipid and aqueous phases.
Some antioxidant functions of exogenously supplied ALA
are ROS quenching activity, metal chelating and regenera-
tion of other antioxidants such as glutathione, vitamin E and
vitamin C [9]. Furthermore, ALA might exert its antioxidant
and anti-inflammatory functions, at least in part, through
effects on intracellular signaling pathways and transcrip-
tion factors such as NF-κB and nuclear factor erythroid
2-related factor 2 (Nrf2). NF-κB and Nrf2 are two of key
regulators implicated in expression of many inflammatory
and antioxidant genes respectively [10, 11]. Thus, ALA as
a complimentary treatment is expected to have potential
to reduce oxidative stress in RA as well. However, to the best
of our knowledge, only a very limited number of studies,
especially in vitro and animal studies, have been carried
out to evaluate the ALA effects in RA [12–14] and no study
has investigated ALA effects on oxidative stress in RA
patients so far. Therefore, we aimed to study whether or
not oral ALA administration could alter antioxidant
biomarkers [total antioxidant capacity (TAC), superoxide
dismutase (SOD), glutathione peroxidase (GSH-Px),
arylesterase (ARE)] and malondialdehyde (MDA) in
women with RA.
Materials and methods
Study design
A randomized controlled clinical trial design was used to
conduct the study in which both the participants and the
researchers were blinded to the intervention.
The Ethics Committee of Tabriz University of Medical
Sciences approved the trial. The study was registered on the
Iranian Registry of Clinical Trials website as well (http://
www.irct.ir/, IRCT201205263140N5). The research proto-
col conformed to the ethics principles of the World Medical
AssociationDeclarationofHelsinki.
Subjects
A sample size of 30 patients in each group was calculated
which would give a power of 80% to detect target difference
at the 5% significance level. To allow an up to 15% dropout
Int J Vitam Nutr Res (2019), 89 (1–2), 13–21
S. Kolahi et al., Oral administration of alpha-lipoic acid did not affect
rate, the estimated sample size was increased to 35 patients
per group.
Patients were recruited from the rheumatology clinic of
Imam Reza Hospital, Tabriz, Iran, between February and
July 2012. We received and archived all patient informed
consent forms.
Following obtaining written informed consent, 70 female
RA patients having the inclusion criteria participated in the
trial.
Entrance criteria comprised fulfilling the American
College of Rheumatology criteria for RA [7], participants
between 20 and 50 years of age, disease activity score in
28 joints (DAS28)<5.1, not changing the treatment protocol
and not taking antioxidant/anti-inflammatory supplements
in one month prior to the enrolment.
Subjects were excluded if they had other kinds of
rheumatic diseases, severe obesity (body mass index
(BMI)>40), any type of vitamin or mineral deficiency,
uncontrolled hypertension(systolic blood pressure>140,
diastolic blood pressure>90), gastrointestinal disorders,
endocrine disorders, diabetes mellitus, thyroid disorders,
renal failure, hepatic diseases, cancer, and other inflamma-
tory and/or autoimmune diseases. Other exclusion criteria
were: pregnancy, lactation, postmenopausal, receiving
hormone replacement therapy, consuming oral contracep-
tive pill, being a cigarette smoker or a passive smoker,
changing treatment protocol or lifestyle during the inter-
vention, taking antioxidant or anti-inflammatory supple-
ments during the study, compliance with treatment<75%
and unwillingness to complete the trial.
Methods
Patients were randomly allocated to one of the two groups
to receive either ALA or placebo for 8 weeks. A block ran-
domization procedure with randomized permuted blocks
of size 4 was used in which subjects were matched to each
block based on age and disease severity.
As far we know, this study was the second one in which
ALA supplementation was used for RA patients. In the pre-
vious study performed by Bae et al. [14] dosage of 900
mg/day for 4 weeks was used and they recommended stud-
ies with higher dose and duration of supplementation to
affect the disease process. In addition, outcomes of studies
on non-RA patients testing the antioxidant effect of ALA
using dose of 600 mg/day were inconsistent. Altogether,
the dose of 1200 mg/day for 8 weeks supplementation per-
iod, which is absolutely safe, was chosen for this trial.
The ALA group received 1200 mg of ALA (2 capsules a
day, each of which containing 600 mg ALA, 30 minutes
prior to breakfast and dinner) and the control group
Ó 2019 Hogrefe
S. Kolahi et al., Oral administration of alpha-lipoic acid did not affect
received a similar amount of maltodextrin. ALA powder
(manufactured by A&A Pharmachem Inc., Ottawa, Ontar-
io, Canada) and maltodextrin were filled in identical cap-
sules and packaged in exactly the same bottles (120
capsules per bottle, by Karen Pharma & Food Supplement
Co., Tehran, Iran).
To collect information on age, disease history, medica-
tion, job, etc, a general questionnaire was used at baseline.
Volunteers were recommended not to make changes in
their medications as well as their usual life style and try to
have stable dietary intake and physical activity during the
trial period. However, before and after the intervention,
stress status, physical activity level and dietary intake were
assessed to evaluate these variables as confounding factors
[15]. For dietary assessments a three-day dietary record and
Nutritionist IV software (First Databank, San Bruno, CA,
USA) were used.
At initial and final visits, we calculated DAS28 based on
three variables: high-sensitivity C-reactive protein (hs-
CRP), the number of swollen joints and the number of tender
joints [15]. Also, body mass index (BMI) was computed
throughdividingweight(kg)bytheheightsquared(meters).
To check any probable side effects, minimize the dropout
rate and ensure the supplement consumption, patients
received a phone call every 10 days.
In the present study we evaluated oxidative stress status
by measurement of TAC, SOD, GSH-Px, ARE and MDA.
For this purpose, at baseline and final, venous blood
samples (10 mL from each patient, following an overnight
fast) were collected in two vacutainer tubes, one of which
was a heparinized tube for SOD and GSH-Px tests and the
other one was without anticoagulant for TAC, ARE and
MDA tests. Serum samples were obtained after centrifuga-
tion at 1500 g for 15 minutes. Finally, all samples (Both
serum and heparinized blood samples) were stored at

20 °C until biochemical assays.
Measurement of SOD and GSH-Px in whole blood and
TAC in serum was done using the spectrophotometric
method by commercially available kits exactly according
to the manufacturer’s protocol (TAC, SOD and GSH-Px
using RANDOX kits; RANDOX Laboratory, Crumlin,
UK), on an automatic analyzer (Abbott model Alcyon
300; Abbott Laboratories, Abbott Park, IL, USA).
ARE activity was measured using phenylacetate as a sub-
strate of ARE [16]. Therefore, in the present study 1 mmol/L
CaCl2, 750 μL of 0.1 mol/L Tris-HCl (pH 8.5), 125 μL of 12
mmol/L phenyl acetate and 125 μL of serum (1:10 diluted
with water) were used for assay of arylesterase activity.
The enzymatic activity was calculated from the absorbance
of the phenylacetate hydrolyzed which was continually
monitored at 270 nm and 37°C. The unit of ARE activity
was defined as millimoles of phenylacetate hydrolyzed
per minute and expressed as U/mL serum [16].
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15
Serum MDA level, a peroxidation product of polyunsatu-
rated fatty acids in low-density lipoprotein (LDL) consid-
ered as a marker of oxidative stress, was measured using
the reaction with thiobarbituric acid (TBA) as TBA reactive
substances (TBARS) resulting in formation of pink colored
complexes. A spectrofluorimeter (model SFM 25A;
Kontron, Milan, Italy) was used to measure the fluorescence
intensity [17].
Statistical Analysis
Data were analyzed using SPSS version 13 (SPSS Inc.,
Chicago, IL, USA). Significance level was set at 0.05. The
quantitative and qualitative data were shown as mean ±
standard deviation and median ± Interquartile Range,
respectively. Kolmogorov–Smirnov and Leven tests were
used to test the normality of variables and homogeneity of
variances respectively. The Sign test and Mann-Whitney
test were used for intra- and inter-group comparisons of
qualitative data respectively. Between-group comparison
was done using Independent t- test or Mann–Whitney test
at baseline. Within-group comparison was done using
Paired t-test or Wilcoxon test. For between-group compar-
isons at the end of the study, analysis of covariance adjusted
for baseline measurements was used.
Results
Baseline characteristics
Figure 1 depicts the trial profile. Participants did not report
any adverse effects with ALA supplementation. Table 1 pre-
sents baseline patient characteristics and their medications.
Baseline comparisons indicate that the two groups
were similar in their initial characteristics and medications
(p > 0.05) reflecting the effectiveness of randomization (p>
0.05).
No statistically significant differences in doses and types
of drugs were observed between the two groups at the
beginning of the study and drugs maintained as the same
dosage over the trial (p > 0.05). Furthermore, assessments
of stress status, physical activity level and dietary intake
showed no significant (p > 0.05) within- and between-group
changes (15).
Biochemical data
Table 2 indicates initial and final serum levels of studied
biomarkers and their comparisons within and between
ALA and placebo groups. At the beginning of the study,
Int J Vitam Nutr Res (2019), 89 (1–2), 13–21
16 S. Kolahi et al., Oral administration of alpha-lipoic acid did not affect

Women assessed for eligibility

(n=135)

Exclusion (n=65)
Not meeting the inclusion criteria (n=36)
Refusal to participate (n=29)

Randomized (n=70)

Placebo group (n= 35) Allocation Alpha-lipoic acid group (n=35)

Withdrawal (n=3); Withdrawal (n=2);

Unwillingness to swallow the
capsules (n=1)
Compliance rate<75% (n=1)
Reluctance to attend final
follow-up (n=1)
Compliance rate<75% (n=1)
Attendance at the
homeopathy treatment (n=1)

Follow-up

Women completed the trial (n=32) Women completed the trial (n=33)

Analysis

Analyzed (n=32) Analyzed (n=33)

Figure 1. Flow diagram of the study

Table 1. Baseline characteristics of study patients.

Characteristic Placebo group (n = 32) Alpha-lipoic acid group (n=33) p-value
Age (years) 38.2±8.63 36±8.77 .306*
Height (m) 1.58±.06 1.56±.07 .353*
Body weight (kg) 72.5±12.6 70.5±15.2 .576*
2
Body mass index (kg/m ) 29 ±4.71 29±6.4 .986*
DAS28 2.14±.72 2.1±.76 .850*
Disease duration (years) 6.78±4.72 7.26±4.9 .897*
Prednisolone treatment, no. (%) 28 (87.5%) 29 (87.9%) .980
Methotrexate treatment, no. (%) 29 (90.6%) 28 (84.8%) .214
Hydroxychloroquine, no. (%) 21 (65.6%) 21 (63.6) .949
Sulfasalazine, no. (%) 2 (6.3%) 1 (3%) .317
Calcium &Vitamin D supplement 27 (84.4%) 25 (75.8%) .067
Folic acid supplement 22 (68.8%) 22 (66.7%) .573
Values are presented as mean±standard deviation or the number (%) of patients.
Except where indicated otherwise, p-value was calculated using Mann-Whitney test.
*
Refers to p-values calculated using Student’s t-test.
DAS28 - Disease Activity Score in 28 joints.

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S. Kolahi et al., Oral administration of alpha-lipoic acid did not affect 17

Table 2. Supplementation effects on the antioxidant indices and MDA in studied patients.
Variables Placebo group (n= 32) Alpha-lipoic acid group (n= 33) p-valuey
TAC(mmol/L) Baseline 1.02 ± .22 .97± .24 .351
8 weeks 1± .2 1.08 ± .16 .091
Mean change
.02 .11
(95% CI) (
.12, .08) (.01, .21)
p-valueà .673 .033
SOD(U/mL) Baseline 228±90.1 225±54.8 .889
8 weeks 225±69 218±58.5 .702
Mean change
3.06
6.94
(95% CI) (
37.3, 31.1) (
22, 8.19)
p-valueà .856 .358
GSH-Px(U/L) Baseline 6202 ±1116 6356±920 .540
8 weeks 5926±688 6062±965 .618
Mean change
276
293
(95% CI) (
698, 146) (
668, 80.6)
p-valueà .192 .12
ARE(U/mL) Baseline 63.9±31 51.4±22.4 .109
8 weeks 68±29.7 65.2±27.3 .862
Mean change 4.08 13.7
(95% CI) (
7.48,15.6) (.23, 27.2)
p-valueà .476 .046
MDA§(nmol/L) Baseline 1.68 (1.42 to 2.09)y 1.9 (1.65 to 2.22)* .076
8 weeks 1.54 (1.27 to 1.84)y 1.54 (1.45 to 1.79)* .357
Median change
.18
.36
(Percentile 25,75) (
.54, .18)y (
.61,
.02)
p-valueà .102 .002
Except where indicated otherwise, values are the mean±standard deviation.
*
Refers to values which are median (percentile 25, 75).
y
p-value was calculated using Student’s t-test or Mann-Whitney test at baseline, or ANCOVA after 8 weeks.
à
p-value was calculated using paired t-test or wilcoxon test.
§
Refers to the variable with non-normal distribution which is log transformed.
TAC - total antioxidant capacity; SOD - superoxide dismutase; GSH-Px - glutathione peroxidase; ARE - arylesterase; MDA - malondialdehyde.

the two groups were not significantly different in regards to
TAC, SOD, GSH-Px, ARE and MDA levels (p> 0.05).
No significant changes in SOD and GSH-Px were
observed in either of the ALA or placebo group across the
8-week intervention and between the two groups at the
end of the study (p > 0.05). Whereas, significant increase
in TAC level (11% versus
1.9%) as well as in ARE activity
(26.74% versus 6.37%) and significant decrease in MDA
level (
18.94% versus
8.33) were revealed in ALA-
supplemented group (p < 0.05). However, these significant
within-group changes of TAC, ARE and MDA in ALA
group were not significant when compared with placebo
group (p > 0.05).
Discussion
There is a large body of evidence indicating the ALA
effectiveness as a complimentary treatment in some
inflammatory diseases with oxidative stress component [9].
Unexpectedly, in the present study on RA patients,
8-week ALA supplementation at the dose of 1200 mg/day
did not affect serum levels of antioxidant variables and
MDA compared to placebo.
Studies have indicated an elevated oxidative status in RA
patients that the level of oxidative stress is much higher in
patients with more active forms of the disease [1, 2]. The
pro-oxidant environment of the synovium with continuous
self-perpetuating cycle of oxidative stress and inflamma-
tion in RA, has prompted clinical investigations into the
use of antioxidant-based interventions for RA management
[18, 19]. However, as far we know, effects of ALA on oxida-
tive stress have not been investigated in RA patients.
In current study, a significant increase in TAC was
detected in ALA-supplemented group, but this effect was
not significant as compared with control group. In this
regard, Deng et al. [20] in a rat model of cerebral
ischemia/reperfusion (I/R) demonstrated that the brain
level of TAC following I/Rwas increased to its normal state
by pretreatment with ALA. Also, Palaniyappan et al. [21]

Ó 2019 Hogrefe Int J Vitam Nutr Res (2019), 89 (1–2), 13–21

18
showed that ALA treatment (100 mg/kg body weight/
day for 30 days) significantly inhibited the decline in plasma
TAC level occurred in aged rats compared to young
controls. Similarly, in a clinical trial by khalili et al. [22]
1200 mg of ALA supplementation for 12 weeks significantly
improved TAC in multiple sclerosis patients compared to
controls. On the other hand, in a study conducted by
Masharani et al. [23] daily intake of ALA (600 mg/day for
16 weeks) did not result in elevated TAC in non-obese and
nondiabetic polycystic ovary syndrome subjects. Apart
from direct scavenging of the hydroxyl, peroxyl and super-
oxide radicals, there is growing evidence that ALA could
maintain antioxidant status indirectly by regenerating the
non-enzymatic antioxidants such as glutathione, vitamin
C, vitamin E and coenzyme Q through reduction of their
radicals [9]. This could be an explanation for remarkably
enhanced TAC in ALA-received group in our study.
However, probably due to the relatively low disease severity
of patients at baseline, ALA supplementation did not result
in significant differences between ALA and placebo groups
at the end of the intervention. Correspondingly, baseline
level of TAC in our study was slightly under the normal
range. At these near normal TAC levels, implying the lack
of oxidative stress in patients, ALA supplementation may
have no effect.
SOD is in the first line of antioxidant defense which
transmutes superoxide anion into the hydrogen peroxide.
Thereafter, GSH-Px, another endogenous antioxidant
enzyme, acts in combination with SOD and detoxifies the
hydrogen peroxide produced by SOD [24]. In the present
study, we did not observe significant effect of ALA con-
sumption on serum SOD and GSH-Px activity. Our finding
was in agreement with result of Sharman et al. [25] study in
which ALA treatment in a randomized cross-over design
(600 mg/day ALA or placebo, on two occasions one week
apart), did not make significant changes in SOD activity in
healthy subjects. Also, Vidovic et al. [26] in their study on
schizophrenia patients did not detect any significant effect
of ALA intervention on plasma SOD activity. Moreover, in
another study [22] daily intake of 1200 mg ALA in multiple
sclerosis patients did not alter SOD and GSH-Px activities.
Our study was in contrast to Zhao et al. [27] study which
displayed that ALA therapy (daily intravenous injection of
600 mg ALA for 3 weeks) in type 2 diabetes mellitus
patients led to significant increase in plasma SOD and
GSH-Px compared to the placebo group. Furthermore, Jia
et al. [28] through an investigation on broilers reported that
orally supplied ALA (at the dose of 50 and 100 mg/kg/day)
resulted in elevated SOD and GSH-Px activities in serum
and liver of birds. ALA displays ROS scavenging functions
via metal chelating activities and regeneration of endoge-
nous antioxidants such as vitamin C and E [9]. This removal
Int J Vitam Nutr Res (2019), 89 (1–2), 13–21
S. Kolahi et al., Oral administration of alpha-lipoic acid did not affect
of superoxide ions by ALA, as a ROS scavenger, could have
resulted in no effect of ALA on SOD activity in this study.
Arivazhagan et al. [29] indicated that ALA treatment in
aged rats time-dependently decreased hydroxyl radicals
and increased glutathione levels. Moreover, in a rat model
of diabetes, ALA declined levels of GSH-Px activity
compared with control rats [30]. Taken together,
unchanged GSH-Px activity in our intervention may stem
from the ability of ALA to recycle the reduced glutathione
from its oxidized form. Therefore, no increase in GSH-Px
activity was observed.
ARE is an antioxidant enzyme which has antiatheroscle-
rotic effects as well. It has been shown that as a result of
increased oxidative stress in RA patients, a decrease in
ARE is occurred through which development of atheroscle-
rosis besides tissue injury seems inevitable in RA patients
[31]. Although in current study ARE activity substantially
enhanced in ALA group, this increase was not significant
in comparison with placebo group. Overall, limited data
are available on ARE activity in RA patients [31, 32].
As instance, a clinical trial demonstrated that fish oil supple-
mentation (1 mg/day for three months) enhanced ARE
activity in RA patients [33]. But about ALA, we have found
no research in this regard. Regarding the findings, initial
low disease activity of patients, may have been resulted in
final insignificant between-group results of this study.
In addition, the duration of our intervention may have not
been long enough to affect ARE activity significantly.
Therefore, it seems that more clinical trials are still needed
in this area.
MDA is a biomarker of lipid peroxidation. An increase in
MDA has been reported in serum and synovial fluid of RA
patients [34]. In our study MDA was significantly reduced
in ALA group which was not significant when compared
with placebo group. In a study on aged type 2 diabetes
mellitus patients with acute cerebral infarction [27].
Intravenous administration of 600 mg of ALA once a day
for 3 weeks, significantly decreased MDA compared to
the control group. Similarly, another study in children and
adolescents with a symptomatic type 1 diabetes [35] showed
that ALA (300 mg twice daily for 4 months) decreased
serum MDA significantly as compared with placebo. Also,
Noori et al. [36] reported that a 12-week period of combined
supplementation with 800 mg lipoic acid and 80 mg
pyridoxine, in comparison with placebo, resulted in signifi-
cant decrease of MDA in diabetic nephropathy patients.
However, khabbazi et al. [37] did not observe any noticeable
change in serum levels of MDA after 8-week ALA supple-
mentation (600 mg/day) in patients with end-stage renal
disease on hemodialysis. Also, in khalili et al. study
among multiple sclerosis patients, [22] ALA consumption
(1200 mg/day for 12 weeks) was not associated with
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S. Kolahi et al., Oral administration of alpha-lipoic acid did not affect
significant decrease in MDA compared to placebo group.
Increased ROS levels, could result in increased MDA levels
via oxidative damage of lipids [24, 34]. In our study, remark-
ably decreased MDA only in ALA-received group may be
attributed to the antioxidant effects of ALA, specifically its
ROS quenching activities [38]. However, baseline MDA
levels of our studied patients were slightly higher than the
healthy normal subjects (the median in ALA and placebo
group were 1.9 and 1.68 nmol/L respectively), implying a
condition of lack of (or almost lack of) oxidative stress in
which making significant changes in oxidative stress
parameters seems to be too difficult to achieve. This could
be a reasonable explanation for insignificant between-
group results in this study.
Overall, it seems that beneficial effect of ALA could be
appeared more apparently when there are enhanced levels
of oxidative stress and inflammation. Accordingly, in
clinical trials administration of ALA to patients with non-
RA inflammatory diseases (who are not under treatment
with potent anti-inflammatory drugs which might cover
the actual effect of ALA), could result in more considerable
ameliorating effects on oxidative stress. In a similar way, in
animal studies ALA could exhibit its antioxidant and anti-
inflammatory properties more clearly than human studies,
because in animal studies ALA often is used as the only
treatment (without any anti-inflammatory medication).
In addition to the absence of anti-inflammatory drugs, we
must consider the role of gut metabolism which is
eliminated from cell culture studies. Moreover, in spite of
transient cellular accumulation of ALA in the body after
an oral dose (because of rapid appearance of ALA in the
plasma and its rapid clearance), ALA remains in cell culture
medium for relatively longer times which provide ALA with
the opportunity to make more considerable effects in cell
culture studies. Furthermore, doses of ALA in cell culture
and animal investigations are often supraphysiologic [9].
This could be another explanation for observed effective-
ness of ALA in these studies.
Altogether, especially based on baseline TAC and MDA
levels, it seems that our patients had only a slightly
increased oxidative stress at the beginning of the study
which was Negligible compared with healthy people.
Hence, in this near-normal oxidative state, making signifi-
cant changes in oxidative stress parameters using antioxi-
dant supplementation is difficult and therefore ALA had
no effect in the present study.
Several limitations need to be considered when interpret-
ing our findings. First, serum ALA level was not measured.
Second, we did not measure serum levels of endogenous
non-enzymatic antioxidants such as glutathione, vitamin
C, and vitamin E which could be affected by ALA supple-
mentation and could drive the prooxidants-antioxidants
balance in favour of the latter. Also, we could not measure
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19
serum levels of other enzymatic antioxidant factors such
as iNOS.
Third, synovial biopsies was not performed in this study;
therefore, we could not measure the levels of antioxidative
and oxidative stress biomarkers in serum and synovium
simultaneously to achieve more reliable evaluations of
ALA effects in RA. Fourth, because of our limitations, we
could not perform flow cytometry, PCR in peripheral blood
cells isolated from RA patients. Finally, dose and duration of
ALA administration in our clinical trial might have not been
adequate to make significant between-group differences.
The most considerable strength of the present study was
that, to our knowledge, this was the first clinical trial which
evaluated the effects of ALA treatment on antioxidant
biomarkers in RA patients.
Conclusion
In conclusion, in the present study changes in antioxidant
biomarkers (TAC, SOD, GSH-Px, ARE) and MDA caused
by 8-week ALA supplementation (1200 mg/day) were not
significant when compared with placebo. Meanwhile,
among the studied biomarkers, within-group (but not
between-group) changes of TAC, ARE and MDA were sig-
nificant in ALA-treated group. Thus, the present study pro-
vided additional impetus for further clinical trials with more
considerations for dose and duration of ALA treatment and
more comprehensive evaluation of antioxidant and oxidant
biomarkers in serum and synovium of RA patients. Particu-
larly, studies on RA patients with more active forms of the
disease (DAS-28  3.2) are suggested.
References
1. Aletaha, D., Neogi, T., Silman, A., Funovits, J., Felson, D.,
Bingham, C., Birnbaum, N.S., Burmester, G.R., Bykerk, V.P., &
Cohen, M.D. (2010) 2010 Rheumatoid arthritis classification
criteria: an American College of Rheumatology/European
League against Rheumatism collaborative initiative. Arthritis
Rheum. 62, 2569–2581.
2. Phillips, D.C., Dias, H.K., Kitas, G.D., & Griffiths, H.R. (2010)
Aberrant reactive oxygen and nitrogen species generation in
rheumatoid arthritis (RA): causes and consequences for
immune function, cell survival, and therapeutic intervention.
Antioxid Redox Signal. 12, 743–785.
3. Veselinovic, M., Barudzic, N., Vuletic, M., Zivkovic, V., Tomic-
Lucic, A., Djuric, D., & Jakovljevic, V. (2014) Oxidative stress in
rheumatoid arthritis patients: relationship to diseases activ-
ity. Mol Cell Biochem. 391, 225–232. https://doi.org/10.1007/
s11010-014-2006-6
4. Brennan, F., & McInnes, I. (2008) Evidence that cytokines play
a role in rheumatoid arthritis. J Clin Invest. 118, 3537.
5. Filippin, L.I., Vercelino, R., Marroni, N.P., & Xavier, R.M. (2008)
Redox signalling and the inflammatory response in rheuma-
toid arthritis. Clin Exp Immunol. 152, 415–422.
Int J Vitam Nutr Res (2019), 89 (1–2), 13–21
20
6. Wood, A.J., & O’Dell, J.R. (2004) Therapeutic strategies for
rheumatoid arthritis. N Engl J Med. 350, 2591–2602.
7. Mueller, M., Hobiger, S., & Jungbauer, A. (2010) Anti-
inflammatory activity of extracts from fruits, herbs and
spices. Food Chem. 122, 987–996.
8. Reed, L.J. (1974) Multienzyme complexes. Acc Chem Res. 7,
40–46.
9. Shay, K.P., Moreau, R.F., Smith, E.J., Smith, A.R., & Hagen, T.
M. (2009) Alpha-lipoic acid as a dietary supplement: molec-
ular mechanisms and therapeutic potential. Biochim Biophys
Acta. 1790, 1149–1160.
10. Petersen-Shay, K., Moreau, R.F., Smith, E.J., & Hagen, T.M.
(2008) Is α-lipoic acid a scavenger of reactive oxygen species
in vivo? Evidence for its initiation of stress signaling pathways
that promote endogenous antioxidant capacity. IUBMB Life.
60, 362–367.
11. Lee, H., & Hughes, D. (2002) Alpha-lipoic acid modulates NF-
κB activity in human monocytic cells by direct interaction with
DNA. Exp Gerontol. 37, 401–410.
12. Lee, E., Lee, C., Lee, K., Park, J., Cho, K., Cho, Y., Lee, H.,
Moon, S., Moon, H., & Yoo, B. (2007) Alpha-lipoic acid
suppresses the development of collagen-induced arthritis
and protects against bone destruction in mice. Rheumatol Int.
27, 225–233.
13. Hah, Y., Sung, M., Lim, H., Jun, J., Jeong, Y., Kim, H., Kim, J.,
Hur, H., Davaatseren, M., & Kwon, D. (2010) Dietary alpha
lipoic acid supplementation prevents synovial inflammation
and bone destruction in collagen-induced arthritic mice.
Rheumatol Int. 31, 1583–1590.
14. Bae, S.C., Jung, W.J., Lee, E.J., Yu, R., & Sung, M.K. (2009)
Effects of antioxidant supplements intervention on the level
of plasma inflammatory molecules and disease severity of
rheumatoid arthritis patients. J Am Coll Nutr. 28, 56–62.
15. Mirtaheri, E., Pourghassem-Gargari, B., Kolahi, S., Dehghan,
P., Asghari-Jafarabadi, M., Hajalilou, M., Shakiba-Novin, Z., &
Mesgari-Abbasi, M. (2015) Effects of alpha-Lipoic acid
supplementation on inflammatory biomarkers and matrix
metalloproteinase-3 in rheumatoid arthritis patients. J Am
Coll Nutr. 34, 310–317.
16. Aldridge, W.N. (1953) Serum esterases. 1. Two types of
esterase (A and B) hydrolysing p-nitrophenyl acetate, propi-
onate and butyrate, and a method for their determination.
Biochem J. 53, 110–117.
17. Del-Rio, D., Pellegrini, N., Colombi, B., Bianchi, M., Serafini,
M., Torta, F., Tegoni, M., Musci, M., & Brighenti, F. (2003)
Rapid fluorimetric method to detect total plasma malondi-
aldehyde with mild derivatization conditions. Clin Chem. 49,
690–692.
18. Van-Vugt, R.M., Rijken, P., Rietveld, A., Van-Vugt, A., &
Dijkmans, B.C. (2008) Antioxidant intervention in rheumatoid
arthritis: results of an open pilot study. Clin Rheumatol. 27,
771–775.
19. Kolahi, S., Hejazi, J., Mohtadinia, J., Jalili, M., & Farzin, H.
(2011) The evaluation of concurrent supplementation with
vitamin E and omega-3 fatty acids on plasma lipid per
oxidation and antioxidant levels in patients with rheumatoid
arthritis. Internet J Rheumatol. 7, .
20. Deng, H., Zuo, X., Zhang, J., Liu, X., Liu, L., Xu, Q., Wu, Z., & Ji, A.
(2015) α-lipoic acid protects against cerebral ischemia/reper-
fusion-induced injury in rats. Mol Med Rep. 11, 3659–3665.
21. Palaniyappan, A., & Alphonse, R. (2011) Immunomodulatory
effect of DL-α-lipoic acid in aged rats. Exp Gerontol. 46,
709–715.
22. Khalili, M., Eghtesadi, S., Mirshafiey, A., Eskandari, G.,
Sanoobar, M., Sahraian, M., Motevalian, A., Norouzi, A.,
Int J Vitam Nutr Res (2019), 89 (1–2), 13–21
S. Kolahi et al., Oral administration of alpha-lipoic acid did not affect
Moftakhar, S., & Azimi, A. (2014) Effect of lipoic acid
consumption on oxidative stress among multiple sclerosis
patients: A randomized controlled clinical trial. Nutr Neurosci.
17, 16–20.
23. Masharani, U., Gjerde, C., Evans, J.L., Youngren, J.F., &
Goldfine, I.D. (2010) Effects of controlled-release alpha lipoic
acid in lean, nondiabetic patients with polycystic ovary
syndrome. Diabetes Sci Technol. 4, 359–364.
24. Kasama, T., Kobayashi, K., Sekine, F., Negishi, M.,
Ide, H., Takahashi, T., & Niwa, Y. (1988) Follow-up study of
lipid peroxides, superoxide dismutase and glutathione per-
oxidase in the synovial membrane, serum and liver of young
and old mice with collagen-induced arthritis. Life Sci. 43,
1887–1896.
25. Sharman, J.E., Gunaruwan, P., Knez, W.L., Schmitt, M., Marsh,
S.A., Wilson, G.R., Cockcroft, J., & Coombes, J. (2004) Alpha-
lipoic acid does not acutely affect resistance and conduit
artery function or oxidative stress in healthy men. Br J Clin
Pharmacol. 58, 243–248.
26. Vidović, B., Milovanović, S., Dorđević, B., Kotur-Stevuljević, J.,
Stefanović, A., Ivanišević, J., Miljković, M., Spasić, S.,
Stojanović, D., & Pantović, M. (2014) Effect of alpha-lipoic
acid supplementation on oxidative stress markers and
antioxidative defense in patients with schizophrenia. Psychi-
atr Danub. 26, 205–213.
27. Zhao, L., & Hu, F.X. (2014) α-Lipoic acid treatment of aged
type 2 diabetes mellitus complicated with acute cerebral
infarction. Eur Rev Med Pharmacol Sci. 18, 3715–3719.
28. Jia, R., Bao, Y.H., Zhang, Y., Ji, C., Zhao, L.H., Zhang, J.Y., Gao,
C.Q., & Ma, Q.G. (2014) Effects of dietary α-lipoic acid, acetyl-
l-carnitine, and sex on antioxidative ability, energy, and lipid
metabolism in broilers. Poult Sci. 93, 2809–2817.
29. Arivazhagan, P., Ramanathan, K., & Panneerselvam, C. (2001)
Effect of dl-α-lipoic acid on glutathione metabolic enzymes in
aged rats. Exp Gerontol. 37, 81–87.
30. Obrosova, I.G., Fathallah, L., & Greene, D.A. (2000) Early
changes in lipid peroxidation and antioxidative defense in
diabetic rat retina: effect of DL-α-lipoic acid. Eur J Pharma-
col. 398, 139–146.
31. Isik, A., Koca, S.S., Ustundag, B., Celik, H., & Yildirim, A. (2007)
Paraoxonase and arylesterase levels in rheumatoid arthritis.
Clin Rheumatol. 26, 342–348.
32. Aryaeian, N., Djalali, M., Shahram, F., Jazayeri, S., Chamari,
M., & Nazari, S.A. (2011) Beta-carotene, vitamin E, MDA,
glutathione reductase and arylesterase activity levels in
patients with active rheumatoid arthritis. Iran J Public Health.
40, 102–109.
33. Ghorbanihaghjo, A., Kolahi, S., Seifirad, S., Argani, H.,
Hajialilo, M., & Khabazi, A. (2012) Effect of fish oil supple-
ments on serum paraoxonase activity in female patients with
rheumatoid arthritis: a double-blind randomized controlled
trial. Arch Iran Med. 15, 549–552.
34. Baskol, G., Demir, H., Baskol, M., Kilic, E., Ates, F., Kocer, D., &
Muhtaroglu, S. (2005) Assessment of paraoxonase 1 activity
and malondialdehyde levels in patients with rheumatoid
arthritis. Clin Biochem. 38, 951–955.
35. Hegazy, S.K., Tolba, O.A., Mostafa, T.M., Eid, M.A., & El-Afify,
D.R. (2013) Alpha-lipoic acid improves subclinical left ven-
tricular dysfunction in asymptomatic patients with type 1
diabetes. Rev Diabet Stud. 10, 58–67.
36. Noori, N., Tabibi, H., Hosseinpanah, F., Hedayati, M., &
Nafar, M. (2013) Effects of combined lipoic acid and pyridox-
ine on albuminuria, advanced glycation end-products, and
blood pressure in diabetic nephropathy. Int J Vitam Nutr Res.
83, 77–85.
Ó 2019 Hogrefe
S. Kolahi et al., Oral administration of alpha-lipoic acid did not affect 21

37. Khabbazi, T., Mahdavi, R., Safa, J., & Pour-Abdollahi, P. (2012) Conflict of Interest
Effects of alpha-lipoic acid supplementation on inflamma- We declare that there was no conflict of interest in our study.
tion, oxidative stress, and serum lipid profile levels in patients
with end-stage renal disease on hemodialysis. J Ren Nutr. 22, Bahram Pourghasem Gargari
244–250. Nutrition Research Center
38. Goraca, A., Piechota, A., & Huk-kolega, H. (2009) Effect of Department of Biochemistry & Diet Therapy
alpha - lipoic acid on LPS-induced oxidative stress in the Faculty of Nutrition and Food Sciences,
heart. J Physiol Pharmacol. 60, 61–68. Tabriz University of Medical Sciences
51666-14711 Tabriz
Acknowledgments Iran
We are grateful to all patients for their honest cooperation. The
present clinical trial was funded by the Research Vice-Chancellor of bahrampg@yahoo.com,
Tabriz University of Medical Sciences, Tabriz, Iran. pourghassemb@tbzmed.ac.ir

Ó 2019 Hogrefe Int J Vitam Nutr Res (2019), 89 (1–2), 13–21