Keywords interleukin-6 - oral vitamin D supplementation - oxidative stress - plasminogen activator
inhibitor-1 - type 2 diabetes mellitus - vitamin D deficiency
Introduction
Oxidative stress (OS) and inflammation are the underlying mechanisms in type 2 diabetes
mellitus (T2DM), which cause endothelial dysfunction leading to cardiovascular disease
(CVD) risk.[1 ] Elevation in plasminogen activator inhibitor-1 (PAI-1) levels has been considered
as a marker of general endothelial dysfunction with elevated levels reported in diabetic
retinopathy, diabetic nephropathy, and coronary heart disease in type 2 diabetes.[2 ] Vitamin D is now under precise investigation due to the expression of vitamin D
receptors (VDR) in body tissues such as endothelial cells, vascular smooth muscle
cells, β cells of the pancreas, T helper cells, macrophages, muscles, and adipose
tissues. The active form of vitamin D is also produced in endothelial cells through
the activity of a specific endothelial α-hydroxylase on circulating 25(OH) D.[3 ] A strong and independent association between vitamin D and cardiovascular events
such as angina, myocardial infarction, and stroke has been observed.[4 ] Low serum levels of 1, 25(OH)2 D predicted acute myocardial infarction and stroke after a 10-year follow-up in an
elderly population-based survey.[5 ] Some cross-sectional studies have shown a relationship between 25(OH)2 D and markers of inflammation. In the largest report, on more than 15,000 subjects,
it was found that 25(OH) 2 D levels less than 53 nmol/L levels were inversely associated with inflammatory biomarker
C-reactive protein (CRP).[6 ] Vitamin D was found to regulate the expression of pro-inflammatory cytokines and
adhesion molecules in the vasculature.[7 ]
Vitamin D is also found to be positively correlated with insulin sensitivity.[8 ] Indian studies found that the prevalence of vitamin D deficiency (< 20 ng/mL) in
South Indian patients with type 2 diabetes mellitus was 83%, with a higher prevalence
of vitamin D deficiency found in pre-diabetics and in diabetics with poor glycemic
control.[9 ]
[10 ]
[11 ] These low vitamin D levels were found to correlate with insulin resistance, risk
of development of both type 1 and type 2DM, hypertension, hyperlipidemia, and CVD.[12 ] An inverse and independent relationship between circulating 25(OH) D levels and
the prevalence of microvascular complications in patients with T2DM was found. Low
vitamin D status was reported to be associated with diabetic nephropathy.[13 ] Vitamin D deficiency could be an indirect risk factor, causing fatal outcomes of
the disease linked rather than being the direct cause of fatality. Hence, by itself,
vitamin D deficiency and T2DM, are associated with CVD risk independently and when
they occur together the risk is compounded.
Subsequent studies that undertook vitamin D supplementation found an improvement in
inflammatory biomarkers in patients with heart failure, attenuation of oxidative stress,
and inflammation in vitamin D-deficient T2DM patients.[14 ]
[15 ] A single dose of vitamin D was found to improve endothelial function and low-grade
inflammation in patients with T2DM.[16 ] Some studies in diabetic patients have reported improvements in clinical parameters
such as central glycemia, insulin sensitivity, and lipid profile, with some finding
improvement of endothelial function and inflammatory status.[17 ]
[18 ]
[19 ]
[20 ]
[21 ] Hence, in the light of these findings, it was postulated that vitamin D with its
pleiotropic effects, may be the elixir for the attenuation of disease processes. However,
a clinical agreement was not found with reports of no improvement in oxidative stress
and inflammation with vitamin D supplementation of 5,000 IU/day for 12 weeks in T2DM
patients.[22 ] Similarly meta-analyses have found no significant changes in inflammatory status
with vitamin D supplementation, attributed to differences in vitamin D dosage, ranging
from 400 to 200,000 IU/day, different duration of supplementation, ranging from weeks
to years, and also due to lack of information on vitamin D status at baseline in the
subjects receiving supplementation.[23 ]
Taking into consideration the variations in available study designs, we undertook
the study in subjects with T2DM having vitamin D deficiency. The aim was to observe
the effect of oral supplementation of vitamin D of dosage 2,000 IU/day for a period
of 6 months on markers of oxidative stress, inflammation, and acute phase reactants.
The biomarker levels at baseline were measured and the changes in the levels were
studied at two study points, one, at the end of the third month of supplementation
and the second, at the end of the study period that is at the end of the 6 months.
The effects of vitamin D supplementation at both the study points were compared with
baseline levels and also between the two study time points.
Materials and Methods
This interventional prospective study included 100 subjects selected from the patients
attending the outpatient Clinic of Endocrinology and Metabolism at Sri Venkateswara
Institute of Medical Sciences (SVIMS), Tirupati, Andhra Pradesh, India ([Fig. 1 ]). The subjects were diagnosed with T2DM as per the revised American Diabetic Association
(ADA) criteria[24 ] with a duration of T2DM ranging from 1 to 5 years and having vitamin D deficiency
(vitamin D < 20 ng/mL).[25 ] It was ensured that subjects receiving treatment were on oral hypoglycemic agents,
were receiving stable statin therapy for a minimum period of 3 months, with stable
treated hypertension. Patients with other forms of diabetes (type 1 DM, gestational
DM), known history of thyroid disorders, malignancy, cerebrovascular diseases, myocardial
infarction, chronic kidney disease, acute and chronic inflammatory diseases, smokers,
alcoholic, pregnant and lactating women, patients who were on insulin, corticosteroids
and vitamin D or calcium supplementation were excluded from the study. The study was
approved by the Institutional Ethics Committee (Human Studies), and was registered
in Clinical Trials Registry of India (CTRI/2017/03/008236). The study was conducted
in accordance with the principles of the Declaration of Helsinki. Informed written
consent was obtained from subjects prior to their enrollment into the study. The subjects
were given 90 vitamin D tablets (cholecalciferol) of dosage 2,000 IU and instructed
to consume one tablet daily for a period of 3 months. The patients were instructed
to hand over the empty tablet strips at the end of the third month for securing proof
of regular consumption of the vitamin D supplement. The compliance of supplementation
was verified over the telephone on alternate days. At the end of the third month,
blood was collected for biochemical analysis, and serum vitamin D levels were measured
to check for vitamin D toxicity (> 100 ng/mL). As none of the patient's vitamin D
levels reached the toxic levels at the end of the third month, they were given 90
tablets for the next 3 months of the study period with the same instructions carried
forward. The patients were instructed to follow the same routine of daily activities
and food intake over the study period and to notify any changes in treatment or routine
activities. At the end of 6 months of vitamin supplementation, blood was collected
from the subjects for biochemical analysis and measurement of vitamin D levels following
which vitamin D supplementation was stopped ([Fig. 2 ]).
Fig. 1 Selection of study subjects.
Fig. 2 Patient follow-up and dropouts at the study points.
Sample Collection
After an overnight fast of 8 to 10 hours, 8 mL of venous blood sample was collected,
of which 1 mL was transferred into sodium fluoride and potassium oxalate containing
tube, 1 mL into sodium citrate anticoagulant containing tube, 1 mL into sodium ethylene
diamine tetraacetic acid (Na-EDTA) anticoagulant containing tube, and 5 mL into an
additive-free tube. Serum and plasma samples were aliquoted and stored at –80°C in
a deep freezer (Thermo Fischer Scientific, Marietta, OHIO 45750) until biochemical
analysis.
Methods
Plasma and serum samples were analyzed immediately for plasma glucose by glucose oxidase
peroxidase (GOD-POD) method (Pathozyme Diagnostics, Kolhapur, India) on Synchron Unicel
DxC 600 auto analyzer, glycosylated hemoglobin (HbA1C) by ion-exchange high-performance
liquid chromatography (HPLC) method (Bio-Rad Laboratories India Pvt. Ltd., Gurgaon)
and vitamin D measured as 25 hydroxy cholecalciferol by chemiluminescence method (Beckman
system pack, Ireland Inc., Lismeehan, Ireland) on Access 2 auto analyzer, Beckman
Coulter, USA. Serum malondialdehyde (MDA) and ferric reducing ability of plasma (FRAP)
were measured by spectrophotometric methods[26 ]
[27 ] on a UV Spectrophotometer (Llantrisant CF728YW, United Kingdom), high sensitive
C-reactive protein (hsCRP) (Beckman System Pack, USA) on Synchron Unicel DxC 600 auto
analyzer, Beckman Coulter, USA, plasma fibrinogen by immunoturbidimetry method (Tulip-Quantia,
Goa) on Awareness Technology Chemwell Automated EIA and Chemistry analyzer, USA, serum
OxLDL, IL-6, and PAI-1 by enzyme-linked immunosorbent assay method (ELISA) (Genxbio,
Gurgaon, India) on ELISA reader (Transasia Bio-Medicals Ltd., Mumbai, India) and ELISA
Washer (ERBA Diagnostics Mannheim, Germany). Taking into consideration the analyte
stability, serum OxLDL was analyzed within 3 months of blood collection.
Statistical Analysis
Data distribution was studied using Kolmogorov–Smirnov test. Data obtained was expressed
as mean ± standard error. The data were converted to percentages with the baseline
value taken as 100%. The percentage change in the biomarker levels from the baseline
to the end of the third month and from the baseline to the end of the sixth month
was calculated taking the baseline as 100%. Repeated measures analysis of variance
(ANOVA) was used for comparison between baseline and third- and sixth-month data after
vitamin D supplementation. Linear regression was performed using generalized estimating
equations (GEE), which groups repeated measures for each subject and accounts for
correlations that may occur from multiple observations within subjects. The model
with the best goodness of fit was selected. A p -value less than 0.05 was considered as statistically significant. All statistical
analysis was performed using Statistical Package for the Social Studies (SPSS) windows
version 16.0. (SPSS Inc, Chicago, IL, USA), and MedCalc (Version 12.1, Ostend, Belgium)
and Microsoft excel spreadsheets.
Results
[Table 1 ] depicts the time-course changes in the parameters compared with the baseline that
showed a significant increase in vitamin D levels, decrease in Ox-LDL, IL-6, PAI-1,
fibrinogen, and increase in FRAP levels, observed both the end of the third month
and sixth month when compared with the baseline. A decrease in MDA and hsCRP levels
was observed at the end of the sixth month of vitamin D supplementation when compared
with baseline levels and the levels at the end of the third month. The percentage
changes from the baseline to the end of the third month and from the baseline to the
end of the sixth month were calculated taking the baseline as 100%. A greater percentage
of increase in serum levels of vitamin D at the end of the third month and FRAP levels
at the end of the sixth month was observed, and a greater percentage of decrease for
serum MDA and PAI-1 at the end of the sixth month was observed. The rate of percentage
change was double at the end of the sixth month than that observed at the end of the
third month for MDA, PAI-1, and FRAP when compared with the baseline.
Table 1
Time course changes in vitamin D levels, oxidant, antioxidant, and inflammatory biomarkers
with vitamin D supplementation
Parameter
Baseline
Mean ± SE
(n = 86)
Third month
Mean ± SE
(n = 86)
Sixth month
Mean ± SE
(n = 86)
[* ]
p -Value
Vit D (ng/mL)
15.28 ± 0.42
30.95 ± 0.60[† ]
37.90 ± 0.40[‡ ]
[§ ]
< 0.001
Percentage change compared with baseline
–
120.31 ± 10.01
173.21 ± 12.89
OxLDL (ng/L)
2105.8 ± 95.16
1217.2 ± 49.30[† ]
612.35 ± 25.72[‡ ]
[§ ]
< 0.001
Percentage change compared with baseline
–
−32.61 ± 3.84
–65.19 ± 2.28
MDA (µmol/L)
3.67 ± 0.08
3.59 ± 0.07 NS
2.48 ± 0.07[‡ ]
[§ ]
< 0.001
Percentage change compared with baseline
–
–1.08 ± 2.79
–29.15 ± 2.53
FRAP (mmol/L)
0.51 ± 0.02
0.59 ± 0.01[† ]
0.85 ± 0.01[‡ ]
[§ ]
< 0.001
Percentage change compared with baseline
–
29.03 ± 5.39
82.37 ± 8.75
HsCRP (mg/dL)
1.13 ± 0.07
0.93 ± 0.05 NS
0.69 ± 0.05[‡ ]
[§ ]
< 0.001
Percentage change compared with baseline
–
–10.03 ± 9.17
–17.80 ± 8.36
IL-6 (ng/L)
7.71 ± 0.21
6.59 ± 0.20[† ]
5.29 ± 0.17[‡ ]
[§ ]
< 0.001
Percentage change compared with baseline
–
–14.88 ± 0.80
–31.62 ± 1.06
PAI-1 (Au/mL)
4.54 ± 0.11
3.89 ± 0.09[† ]
2.56 ± 0.06[‡ ]
[§ ]
< 0.001
Percentage change compared with baseline
−
–10.98 ± 2.60
–40.94 ± 1.92
Fibrinogen (mg/dL)
145.15 ± 3.64
130.30 ± 2.95[† ]
105.66 ± 2.03[‡ ]
[§ ]
< 0.001
Percentage change compared with baseline
–
–10.44 ± 1.37
–26.74 ± 1.31
Abbreviations: Au/mL, arbitary units/microliter; FRAP, ferric reducing ability of
plasma; HsCRP, high-sensitivity C-reactive protein; IL-6, interleukin-6; MDA, malondialdehyde;
OxLDL, oxidized low density lipoprotein; PAI-1, plasminogen activator inhibitor-1;
Vit D, 25 hydroxy cholecalciferol D.
Statistical tool used: Repeated measures ANOVA test, followed by Bonferroni's multiple
comparisons. Data are expressed as mean ± standard error.
* Statistically significant.
† Statistically significant at the end of the third month compared with baseline.
‡ Statistically significant at the end of the sixth month compared with the baseline.
§ Statistically significant at the end of the sixth month compared with the third month
(p = 0.001).
NS, Not significant.
[Table 2 ] depicts the linear regression analysis performed using the GEE that showed that
the time course changes in vitamin D levels showed a significant negative association
with Ox-LDL, Hs-CRP, IL-6, PAI-1, and fibrinogen.
Table 2
Association of time-course changes in 25 hydroxyvitamin D levels with oxidant-antioxidant
and inflammatory markers
β ± SE
Wald chi-square
95% Wald confidence interval
[* ]
p -Value
Lower
Upper
Oxidant-antioxidant markers
Model 1
MDA (µmol/L)
–4.81 ± 0.45
114.14
–5.69
–3.93
< 0.001
OxLDL (ng/L)
–0.01 ± 0.00
297.98
–0.01
–0.01
< 0.001
FRAP (mmol/L)
30.39 ± 1.96
240.83
26.55
34.22
<0.001
Model 2
MDA (µmol/L)
–1.06 ± 0.56
3.58
–2.17
0.04
0.06NS
OxLDL (ng/L)
–0.01 ± 0.00
122.52
–0.007
0.005
< 0.001
FRAP (mmol/L)
16.70 ± 2.77
36.24
11.26
22.14
< 0.001
Inflammatory markers
Model 1
HsCRP (mg/dL)
–5.90 ± 1.00
34.42
–7.87
–3.93
< 0.001
IL-6 (ng/L)
–1.84 ± 0.26
49.52
–2.35
–1.33
< 0.001
PAI-1 (Au/mL)
–5.22 ± 0.45
131.46
–6.12
–4.33
< 0.001
Fibrinogen (mg/dL)
–0.15 ± 0.02
77.04
–0.18
–0.12
< 0.001
Model 2
HsCRP (mg/dL)
–2.63 ± 1.03
6.49
–4.64
–0.61
< 0.011
IL-6 (ng/L)
–0.89 ± 0.24
13.49
–1.37
–0.42
< 0.001
PAI-1 (Au/mL)
–3.74 ± 0.47
64.19
–4.65
–2.82
< 0.001
Fibrinogen (mg/dL)
–0.08 ± 0.02
18.18
–0.12
–0.04
< 0.001
Interaction of oxidative stress and Inflammation
Model 1
MDA_FRAP [x ] HsCRP
0.85 ± 0.23
13.44
0.39
1.31
< 0.001
Model 2
MDA_FRAP [x ] HsCRP
0.85 ± 0.23
13.65
0.39
1.30
< 0.001
Abbreviations: β, coefficient, SE, standard error.
Model 1 = Crude; Model 2 = Generalized estimating equations adjusted for BMI and statin
use.
x Interaction.
* Statistically significant.
Discussion
The improvement in vitamin D levels was progressive with the increase in vitamin D
levels observed at the end of the sixth month, being significant when compared with
the changes observed at the end of the third month. Normal vitamin D levels were attained
at the end of the sixth month (> 30 ng/mL) ([Table 1 ]). A decrease in the biomarkers of oxidative stress, Ox-LDL, and improvement in antioxidant
status, FRAP levels, was observed both the end of the third and sixth months when
compared with the baseline. The attenuation of inflammatory status, IL-6, PAI-1, and
fibrinogen was observed both at the end of the third and sixth months when compared
with the baseline levels. These changes were progressive as was observed by significant
attenuation at the end of the sixth month when compared with the third month ([Table 1 ]). However, no changes were observed in MDA and Hs-CRP levels at the end of the third
month with a decrease observed only at the end of the sixth month of vitamin D supplementation
compared with the end of the third month and baseline levels ([Table 1 ]). These findings indicate that the supplementation of vitamin D is effective if
given over a period of 6 months as MDA and HsCRP were found to decrease only at the
end of the sixth month.
To quantify the changes in the biomarkers at the studied time points, the percentage
changes from the baseline to the end of the third month and from the baseline to the
end of the sixth month were calculated taking the baseline as 100% ([Table 1 ]). A greater percentage of increase in serum levels of vitamin D at the end of the
third month and FRAP levels at the end of the sixth month was observed, and a greater
percentage of decrease for serum MDA and PAI-1 at the end of the sixth month was observed.
The rate of percentage change was double at the end of the sixth month than that observed
at the end of the third month for MDA, PAI-1, and FRAP when compared with the baseline,
indicating that the changes were more significant from the third month onward. Overall,
an attenuation of biomarkers of oxidative stress, inflammation, and an improvement
in the antioxidant status at the end of 6 months of vitamin D supplementation when
compared with baseline levels was observed.
Because glycemic control is known to influence oxidative stress and inflammation,
the biomarkers were corrected for HbA1c, the gold standard marker for glycemic control.
It was observed that even after correcting, a similar decrease in MDA, Ox-LDL, IL-6,
PAI-1, fibrinogen, and an increase in FRAP levels was found. This indicates that the
attenuation of oxidative stress and improvement in antioxidant status are due to vitamin
D supplementation.
To study the association of time-course changes in vitamin D levels with oxidative
stress and inflammation, linear regression analysis was performed using GEE, which
groups repeated measures for each subject and account for correlations that may occur
from multiple observations within subjects. The model with the best goodness of fit
was selected. This was performed with and without adjusting for the confounding effect
of body mass index (BMI) and the use of statin, as both these factors are known to
influence oxidative stress and inflammation. The time course changes in vitamin D
levels showed a significant negative association with Ox-LDL, Hs-CRP, IL-6, PAI-1,
and fibrinogen even after adjusting for BMI and statin use ([Table 2 ]).
The presence of both oxidative stress and inflammation in T2DM patients with vitamin
D deficiency as observed in this study are well-known CVD risk factors. Hence, the
interaction between MDA and FRAP and its association with hs-CRP was studied. Significant
association was found between oxidative stress and inflammation even after adjusting
for BMI and statin use in T2DM patients having vitamin D deficiency ([Table 2 ]). A South Indian study reported a lowering of serum MDA levels and improvement in
total antioxidant status in T2DM patients with vitamin D deficiency with oral vitamin
D supplementation of 60,000 IU/week for 8 weeks.[28 ] Meta-analyses of randomized controlled trials with regard to the effect of vitamin
D supplementation in diabetic patients have revealed that vitamin D supplementation
was found to attenuate inflammation and oxidative stress with improvement in antioxidant
status by modifying adipokine concentrations, diminishing pro-inflammatory cytokines
such as TNF-α, natriuretic peptide concentrations, and blood pressure.[29 ] The biological activity of vitamin D is found to be accomplished by its binding
to a nuclear vitamin D receptor (VDR) that mediate the regulation of gene transcription
of NADPH oxidase, which catalyzes the conversion of oxygen to superoxide, suppresses
TNF-α-induced nuclear factor kappa B (NF-κB) activation and thereby prevents further
oxidative and inflammatory modifications.[30 ]
[31 ]
[32 ]
[33 ]
The oxidative stress of T2DM enhances the pro-inflammatory cytokines such as TNF-α,
IL-6, and IL-1, which act on the liver stimulating the production of acute-phase proteins,
especially hs-CRP. Chronic inflammation promotes endothelial dysfunction, which increases
procoagulant factors, and inhibits natural anticoagulant pathways and fibrinolytic
activity, leading to a hypercoagulable state in T2DM.[34 ] Pro-inflammatory and pro-coagulant factors promote vascular smooth cell proliferation
and migration in atherosclerotic lesions and cell apoptosis, which result in diabetic
cardiovascular complications. The active form of vitamin D acts on VDR of the liver
and decreases the production of hs-CRP and prevents the progression of inflammation
in T2DM. The results from clinical trials on vitamin D supplementation and its effects
on hs-CRP levels reported that 6 out of the 10 trials found a reduced level of circulating
hs-CRP after vitamin D supplementation.[35 ] ROS can directly cause irreversible oxidative modifications of lipids, proteins,
or DNA. The ROS brings about the oxidation of LDL converting it to OxLDL, which is
subsequently taken up by macrophages contributing to foam cell formation. A further
role of ox-LDL in atherosclerosis could be to initiate and affect inflammatory mediators
such as CRP, IL-6, and TNF-α. A positive correlation between CRP and Ox-LDL in humans
has been suggested. Vitamin D can suppress foam cell formation by reducing OxLDL uptake
by macrophages. Hence, vitamin D deficiency in T2DM patients disturbs the macrophage
metabolism and increases foam cell formation, which lead to atherosclerosis and CVD
risk. The lowering of oxidative stress by vitamin D supplementation may lead to a
decrease in the formation of Ox-LDL. Lower Ox-LDL levels will lead to a decrease in
the propensity of foam cell formation and thereby lower the risk of atherosclerosis.[36 ]
The proposed mechanism of action of vitamin D may be attributed to its effect on systemic
inflammation. Improvement in vitamin D levels lowers the production of an upstream
inflammatory cytokine IL-6. Reduced IL-6 levels lead to decreased stimulation of the
production of acute-phase proteins CRP and fibrinogen from the liver. Elevated fibrinogen
levels are associated with a procoagulant environment with increased viscosity of
blood due to increased aggregation and reduced deformability of erythrocytes. By lowering
CRP levels, multiple effects of CRP are regulated, which include the induction of
PAI-I expression, oxidation, and uptake of LDL by macrophages, which are all downregulated.
IL-6 and CRP are associated with cardiovascular risk independent of traditional risk
factors.[37 ]
It can be hypothesized that the inflammation and oxidative stress present in T2DM
subjects could be one of the reasons for lowered circulating vitamin D levels due
to oxidative catabolism of vitamin D, leading to vitamin D deficiency status, compounded
by nutritional deficiencies.
Vitamin D deficiency could be an indirect risk factor, causing fatal outcomes of the
disease linked by immunosuppressive effects, oxidative stress, and inflammation, rather
than being a direct cause of fatality. Hence, using vitamin D supplementation in subjects
with T2DM having vitamin D deficiency can be considered as a supportive intervention,
which may contribute to the prevention of CVD along with specific treatment modalities
of T2DM.
