Date: 24-05-2014
Place: Karachi, Pakistan
From,
Muhammad Imran Shaikh
Faculty of Pharmaceutical Sciences, Dow University of Health Sciences, Karachi
To
The Editor-in-Chief,
World Journal of Pharmaceutical Sciences,
Web: http://www.wjpsonline.org/
Sub: Submission of Manuscript
Article Type: Research Article
Dear Editor,
With reference to the above, please find my submission of paper entitled with “Comparative
effects of vitamin E and its combination with vitamin C on endothelial function in diabetic
hypertensive patients” for possible publications in your esteemed journal.
I hereby affirm that the content of this manuscript is original. Furthermore it has been neither
published elsewhere fully or partially or any language nor submitted for publication (fully or
partially) elsewhere simultaneously and study was carried in accordance with institutional
ethical committee.
I also affirm that all the authors have seen and agreed to the submission of paper and their
inclusion of names as co-authors.
I am looking forward to hear from you.
Thanking you
Sincerely yours,
Muhammad Imran Shaikh
COMPARATIVE EFFECTS OF VITAMIN E AND ITS COMBINATION
WITH VITAMIN C ON ENDOTHELIAL FUNCTION IN DIABETIC
HYPERTENSIVE PATIENTS
Muhammad Imran Shaikh1, 2*, Abdullah Dayo1, Noor Jahan2, Muhammad Ali Ghoto1, Omair
Anwar Mohiuddin2
1
Faculty of Pharmacy, University of Sindh, Jamshoro
2
Faculty of Pharmaceutical Sciences, Dow University of Health Sciences, Karachi
*Corresponding Author: imran.sheikh@duhs.edu.pk; +92-333-7532242
ABSTRACT:
To compare the antioxidant effect of vitamin E alone and its combination with vitamin C on
endothelial dysfunction in patients with diabetes mellitus, a prospective, randomized
controlled, parallel group, interventional study was conducted to record flow mediated
dilation (FMD) of the brachial artery in 167 diabetic patients. Out of these, 61(39%) were
smokers and 97(52%) were Hypertensive. The interventional group was treated with
Vitamins E (400mg/day) for first six weeks and with both vitamin E (400mg/day) and
vitamin C (500mg/day) for later six weeks of study. No significant variation in FMD% was
recorded in the control group while for interventional group significant increase in FMD%
was recorded. Within interventional group, addition of vitamin C in existing vitamin E
therapy significantly improved endothelial function in all diabetic patients (Individual therapy
v/s combination therapy: 7.30±1.47 v/s 8.75±1.65; p≤0.05); smokers: (6.99±1.42 v/s
8.59±1.92; p≤0.05); nonsmokers (7.48±1.48 v/s 8.85±1.48; p≤0.05); hypertensive patients
(6.94±1.41 v/s 8.49±1.53; p≤0.05) and nonhypertensives (7.82±1.40 v/s 9.15±1.75; p≤0.05).
Results obtained from the current study clearly indicate the beneficial effects of vitamin E on
FMD in patients with diabetes and were further augmented by the addition of vitamin C in
therapy. Maximum improvement in FMD was recorded in nonsmoker-nonhypertensive
diabetic group.
Key words: Antioxidant, Diabetes, Hypertension, Flow mediated dilation
INTRODUCTION:
Cardiovascular outcomes can be foretold by endothelial function. The endothelium
plays a key role in maintaining homeostasis of vasculature by mediating coordination
between wall of the vessel and size of lumen, it also under physiological conditions helps in
maintaining normal vascular tone via a range of factors, such as prostacyclin, nitric oxide
(NO) and prostacyclin. However, endothelium in pathological condition can also change its
phenotype promoting vasoconstriction[1]. Dysfunction of endothelium attributes towards
reduction in vasodilaton, proinflammatory state and prothrombic properties. Diminution in
vasodilatory response of endothelium is due to loss of nitric oxide activity in the vascular
wall [2].
Oxidative stress is broadly classified as generation of a surplus and/or decreased
depletion rate of exceedingly reactive molecules as reactive nitrogen species (RNS) and
reactive oxygen species (ROS)[3]. Oxidative stress switch antiatherosclerotic NO producing
enzyme to an enzyme that may hasten the atherosclerotic process thus prevention and
treatment of atherosclerosis is an important priority[4, 5]. ROS, RNS and oxidative stress are
thought to be the starting point for acquiring cardiovascular diseases[6]. Free radical
production is boosted up with Hyperglycemia, this result in increased production of O2 [3].
Gradual increase in inflammatory disorder of arterial wall is marked by atheroma that
stay clinically silent until turn out to be large enough to impair arterial perfusion or disruption
of the lesion producing thrombotic occlusion or ulceration or embolization of the vessel [7].
Endothelial dysfunction is considered as early stage of atherosclerosis, which could have
been caused by various mechanisms, one of those being a decreased nitric oxide (NO) or
increased reactive oxygen species (ROS) chiefly superoxides production[8, 9]. Hypertension
[10]; Diabetes mellitus [11] are the common conditions predisposing to Atherosclerosis.
Accumulated evidences propose that increased antioxidant intake lessen the risk of
coronary heart disease[12]. Vitamin E and vitamin C react in haste with organic free radicals,
and induce their biological effect [13]. Antioxidant vitamin E, principal antioxidant for the
prevention of atherosclerosis, show its efficacy in a number of oxidative stress-induced
conditions and also have cell signaling and gene regulatory functions [14]. Vitamin C
supplementation may potentially be a valuable and cost effective adjunctive therapy. Oral
ascorbic acid is known to decrease arterial blood pressure and improve arterial stiffness thus
reduces cardiovascular risk in patients with type 2 diabetes. [15].
Brachial artery flow-mediated dilation (FMD), recorded by high-resolution doppler
ultrasonography, shows endothelium-dependent vasodilator function. FMD is compromised
in patients with atherosclerosis and corrected with risk-reduction therapy thus FMD
measurement proves as a useful noninvasive prognostic technique in preventive cardiology
and is effective for long-term cardiovascular risk assessment in a lower risk population, to
estimate short-term postsurgical cardiovascular outcomes in a high-risk population, and is an
tremendous experimental method to detect changes in endothelial activity in response to new
therapeutic interventions [16].
This study aims to determine the extent of endothelial dysfunction in patients with
diabetes mellitus and to study comparative effect of individual (vitamin E) and combination
(vitamin E & C) antioxidant therapy on endothelial function in diabetic patients. In addition,
effects of antioxidants on diabetic smokers and non smokers were also determined.
MATERIAL AND METHODS:
Human subjects
A total of 167 diabetic patients were recruited from general population, identified at
outpatient hospital visits in two general hospitals, Sind, Pakistan. Further, in diabetic
category, 91 patients were placed in interventional group (treated) while 76 patients in control
group (untreated). All patients were asked to complete health questionnaires in order to
record health information including history of diabetes, hypertension and other
cardiovascular diseases, smoking, regular current exercise, and medication. Patients were
instructed not to change their diet, exercise and medication routine throughout the study.
Written and verbal informed consent was obtained from all patients.
Inclusion & exclusion criteria
Diabetic patients having fasting plasma glucose concentration of greater than 126 mg/dl were
included in the study, whereas Patients already taking antioxidants were excluded.
Ethical Approval
The study was reviewed and approved by institutional review board and Ethics Committee.
Study Design
Prospective, Randomized Controlled, Parallel group, Interventional study was designed to
investigate the effect of Vitamin E alone and in combination with Vitamin C on endothelial
dysfunction in patients with diabetes mellitus. The interventional group was treated with
Vitamins E (400mg/day) for first six weeks and vitamin E (400mg/day) and vitamin C
(500mg/day) for later six weeks of study.
Flow mediated dilation (FMD) Test
In order to determine the effect of antioxidants on endothelial dysfunction, flow mediated
dilation (FMD) was examined on the brachial artery of patients. These measurements were
made by an experienced ultrasonographer, who was blinded to treatment and to whether
participants were control or interventional. Longitudinal images of the brachial artery were
obtained with a high resolution ultrasound probe (7 MHz), while the patients lied in supine
position and the arm resting in a comfortable position before and during reactive hyperemia
that was induced by cuff inflation. Patients had a three-lead ECG attached. Brachial artery
diameter measurements were obtained in end-diastole, identified by the onset of the R-wave.
The sphygmomanometer blood pressure cuff was positioned on the forearm for 5 minutes,
and the brachial artery was imaged above the antecubital fossa. The cuff was inflated to ≥50
mmHg above systolic pressure to occlude arterial blood flow. Brachial artery diameter
measurements were taken 45–60 seconds after the release of the cuff. FMD was done before
start of therapy (T0), after 6 weeks (T1) and after 12 weeks (T2). Flow Mediated Dilation was
calculated by the formula:
Statistical Analysis
All data was analyzed by statistical software, IBM SPSS Statistics 21.0. Differences among
the FMD percent readings at different time intervals were tested for significance by repeated
measures ANOVA. The paired t-test was used to compare percent FMD readings recorded
after individual and combination therapy. The independent t-test was used to compare the
effect of vitamin E and E + C between control and interventional group of all categories.
Statistical significance was accepted at the 95% confidence level (P < 0.05). Unless otherwise
stated, all data are presented as mean ±sd.
RESULTS:
The clinical characteristics of the population under study are provided in Table I. The control
and interventional subjects were matched for age and plasma glucose level.
Mean age of sampled patients (167) was 52.75 ±9.5 years with the mean plasma glucose level
of 165.7±20.36 mg/dl. Out of these 61(39%) were smokers and 97(52%) were Hypertensive.
Endothelial function was severely impaired in patients with diabetes.
All categories of interventional group showed significant increase in FMD% (p≤0.05) as
compare to control group upon administration of antioxidants furthermore, combination
vitamin therapy produces significantly higher antioxidant effect on endothelial function as
compared to individual vitamin therapy (p≤0.05).
No significant variation in FMD% was recorded in the control group with the overall To
value 2.48 ±0.68, T1 value 2.54±0.63 and T2 value 2.59±0.66; (p≤0.05) as shown in figure 1.
As for interventional group significant increase in FMD% was recorded in comparison to
control group with the mean at To: 2.798 ±0.52, T1: 7.30±1.47 and T2: 8.78±1.65; (p≤0.05) as
shown in figure 2.
Similar trends were observed in the increase in FMD% values of Smokers and Non-Smokers,
with the T1 value 6.9±1.42 and T2 value 8.59±1.92; (p≤0.05) for the former versus T1 value
7.48±1.48 and T2 value 8.85±1.48; (p≤0.05) for the later.
Hypertensive and non-hypertensive patients responsed with analogous fashion, with the T1
value 6.93±1.41 and T2 value 8.48±1.51; (p≤0.05) versus. T1 value 7.82±1.40 and T2 value
9.15±1.75; (p≤0.05) respectively.
Within interventional group, addition of vitamin C in existing vitamin E therapy significantly
improved endothelial function in all diabetic (Individual therapy v/s combination therapy:
7.30±1.47 v/s 8.75±1.65; p≤0.05); smokers (6.99±1.42 v/s 8.59±1.92; p≤0.05); nonsmoker
(7.48±1.48 v/s 8.85±1.48; p≤0.05); hypertensive (6.94±1.41 v/s 8.49±1.53; p≤0.05) and
nonhypertensive (7.82±1.40 v/s 9.15±1.75; p≤0.05) patients as shown in figure 3, 4 & 5.
Vitamin E administration raised Flow mediated dilation percent up to 11.5% in nonsmokernonhypertensive diabetic group while vitamin E in combination with vitamin C improved
endothelial function with the maximum value 13.60% in nonhypertensive-smoker diabetic
group moreover minimum raise in FMD 3.80% and 4.60% was recorded in nonsmoker group
after administration of vitamin E alone and in combination with vitamin C respectively.
Maximum To to T1 difference of 7.6% in FMD was recorded among nonhypertensivenonsmoker diabetic group along with T1 to T2 difference of 4.9% among nonhypertensive
diabetic group.
Both vitamin E and its combination with vitamin C turned out superior results in diabetic
patients with age of ≤ 50 years as compare to patients with age more than 50years (7.92% and
9.94% versus 6.89% and 7.98%; p≤0.05).
DISCUSSION:
Vascular function in diabetic patients has been studied widely. Studies in patients with
diabetes mellitus have found impaired endothelial function when compared to nondiabetic
subjects. Further antioxidant vitamins administration significantly improved endothelial
function in forearm resistance vessels of patients with diabetes mellitus [17] and treatment
with antioxidant vitamins reduces the risk of macrovascular and microvascular
complications[18-21].
Vitamin E supplementation results in a significant diminution in the LDL oxidative
susceptibility and recovers endothelial vascular activity in patients with type I diabetes
mellitus[22]. Our study supports previous observations that impaired endothelium-dependent
vasodilation in patients with diabetes mellitus is significantly improved by administration of
vitamin E and its combination with vitamin C. The important new finding is that endothelial
function was further improved by the addition of vitamin C in existing vitamin E therapy.
Possible mechanism for significant improvement in endothelial function by addition of
vitamin C is the ability of this vitamin to scavenge excessive superoxide anions and, thereby,
decrease inactivation of nitric oxide. Ascorbic acid binds and makes stable endotheliumderived nitric oxide, increasing its availability by a mechanism not depending on free radical
scavenging. Further vitamin C restored aprox 60% of the compromised endotheliumdependent vasodilation observed in diabetic patients compared with age-matched nondiabetic
patients [17, 23]. Further ascorbic acid is considered as a part of antioxidant protection
system. Reactive alpha tocopherol radical, produced after scavenging lipid peroxy radicals is
reduced back to alpha tocopherol in the presence of ascorbic acid.[15, 24, 25].
In current study, endothelial function was found to be less compromised in nonsmoker and
nonhypertensive patients with respect to smoker and hypertensive patients and maximum
improvement in FMD was recorded in nonsmoker-nonhypertensive diabetic group with both
individual as well as combination therapy. This supports the previous studies that cigarette
smoke is one of a major cause of atherosclerosis and tissue vitamin E and C levels were much
lower and lipid peroxidation products considerably higher in smokers than in non-smokers.
Lipid peroxidation products were inversely related to vitamin E in tissue and to vitamin C in
plasma, showing the antioxidant primacy of vitamin E and vitamin C in the arterial tissue
compartments and plasma, respectively [25, 26].
CONCLUSION:
The salient features of the current study is that the impaired FMD in patients with diabetes is
improved by the oral antioxidant therapy (vitamin E and its combination with vitamin C),
whereas no significant variation in FMD was recorded in control diabetic patients.
Furthermore, this study indicates that the beneficial effect of vitamin E on FMD in patients
with diabetes was further increased by the addition of vitamin C in therapy.
ACKNOWLEDGEMENTS:
We are grateful to the Dr. Afzal Qasim and staff of radiology department for their valuable
support and thankful to all volunteers who participated in the study.
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TABLE 1: CLINICAL CHARACTERISTICS OF STUDY POPULATION
Clinical characteristics
Control
Interventional
Number
76
91
Age (years)
52.91±9.83
52.62±9.28
Gender (male/female)
40/36
63/28
Smoker %
36.84
36.26
Hypertensive %
56.57
59.34
Plasma Glucose Level (mg/dl)
166.17±22.46
165.30±18.53
Diabetic Control Patients
FMD%
6
4
To
2
T1
0
1 5 9 13
17 21 25
T2
29 33 37 41
45 49 53 57
61 65 69
73
Number of Patients
Figure 1: percentage of flow mediated dilation in diabetic control patients
Diabetic Interventional Patients
FMD%
15
10
To
5
T1
0
1 6 11 16
21 26 31
T2
36 41 46 51
56 61 66
71 76 81
86 91
Number of Patients
Figure 2: percentage of flow mediated dilation in diabetic interventional patients
Effect of individual therapy
(vitamin E)
FMD %
15
10
5
Control
0
Interventional
1 7 13 19
25 31 37
43 49 55 61
67 73 79
85 91
Number of Patients
Figure 3: effect of vitamin e therapy on flow mediated dilation
Effect of combination therapy
vitamin E & C
FMD %
15
10
5
Control
0
interventioal
1 7 13 19
25 31 37 43
49 55 61 67
73 79 85
91
Number of Patients
Figure 4: effect of vitamin e in combination with vitamin c therapy on flow mediated
dilation
Comparison of vitamin E with
vitamin E & C
10.0000
8.0000
6.0000
4.0000
2.0000
T1
0.0000
T2
Figure 5: comparative effect of vitamin e with vitamin e and c therapy on flow mediated
dilation
TABLE AND FIGURE TITLES AND LEGENDS:
TABLE 1: Clinical Characteristics Of Study Population
Figure 1: Percentage of Flow Mediated Dilation in Diabetic Control Patients
Figure 2: Percentage of Flow Mediated Dilation in Diabetic Interventional Patients
Figure 3: Effect of Vitamin E Therapy on Flow Mediated Dilation
Figure 4: Effect of Vitamin E in Combination with Vitamin C Therapy on Flow Mediated
Dilation
Figure 5: Comparative Effect of Vitamin E with Vitamin E and C Therapy on Flow Mediated
Dilation