1 - ResearchGate

Insights on Vitamin D’s Role in Cardiovascular Disease:
Investigating the Association of 25-Hydroxyvitamin D with the
Dimethylated Arginines
Mohamed A. Abu el Maaty(1), Sally I. Hassanein(1), Rasha S. Hanafi(1) and Mohamed Z. Gad(1)
(1)Biochemistry
Department, Faculty of Pharmacy and Biotechnology, German University in Cairo, Egypt
(2)Pharmaceutical Chemistry Department, Faculty of Pharmacy and Biotechnology, German University in Cairo, Egypt
Introduction
Total 25(OH)D
(ng/mL)
25(OH)D3
(ng/mL)
25(OH)D2
(ng/mL)
P-value
Cohen’s d
The recent evolution of vitamin D from its long-assumed, exclusive role of calcium
homeostasis to majorly influencing living things through involvement with diseases like
cancers, cardiovascular disease (CVD) and auto-immune diseases, has raised
considerable scientific curiosity in the past years [1-2].
Control
34.5 ± 2.3
(30.1-38.9)
21.9 ± 2.2
(17.4-26.4)
12.6 ± 1.5
(9.7-15.5)
0.001
1.09
CAD subjects
24.0 ± 1.3
(21.4-26.6)
9.9 ± 0.9
(8.2-11.6)
14.1 ± 1.1
(11.9-16.3)
0.003
0.51
0.0002
<0.0001
0.497
1.00
1.38
0.19
Acute
25.0±4.5
(16.2-33.8)
13.8 ± 2.6
(8.6-19)
11.2 ± 3.7
(3.9-18.5)
0.572
0.24
Chronic
23.8±1.3
(22.5-25.1)
9.2 ± 0.9
(8.5-9.9)
14.6 ± 1.1
(13.5-15.7)
0.0002
1.62
P-value
0.739
0.054
0.258
Cohen’s d
0.12
0.67
0.45
Epidemiological data strongly supports the alleged association between vitamin D and
CVD, via demonstrating the association of suboptimal 25-Hydroxyvitamin D [25(OH)D]
levels with CVD, whereas molecular studies have illustrated the influence this vitamin has
on various crucial players of the cardiovascular system, such as the renin-angiotensinaldosterone system and the nitric oxide (NO) system [1-2].
Ever since its identification as an endogenous inhibitor of NO Synthase (NOS), the
enzyme responsible for NO synthesis, in the early 1990’s, asymmetric dimethylarginine
(ADMA) has gained unmatchable reputation in the field of cardiovascular research as a
novel cardiovascular risk factor. Its regioisomer, symmetric dimethylarginine (SDMA),
does not share the ability to directly inhibit NOS, however, has been reported to decrease
the cellular uptake of the enzyme’s substrate, L-arginine and thus decreasing NO
production [3-4].
Endothelial dysfunction (ED), assessed by different parameters, has been repeatedly
associated with low 25(OH)D levels [5], however to our knowledge, only one study
investigated the relationship between ADMA and 25(OH)D levels in an ambulant, ageing
population [6], making this the first study to investigate such connection in patients with
coronary artery disease (CAD).
Objectives
P-value
Cohen’s d
Table 2:Total vitamin D status and different 25(OH)D form comparison between the
different classes of subjects (vertical) and between the same class (horizontal).
Statistical significance is obtained from the comparison of the total 25(OH)D
concentration between controls and CAD subjects. Significance is also found in the
comparison of the different 25(OH)D forms within controls and CAD subjects exhibiting
25(OH)D3 and 25(OH)D2 dominance, respectively. 25(OH)D3 concentration in controls
is significantly higher than that of CAD subjects. Finally, 25(OH)D2 is significantly
higher than its counterpart in chronic CAD subjects. 95% CIs are indicated in
parentheses following their corresponding means.
The aim of this study was to investigate the association/correlation of 25(OH)D levels
with:
1. CAD incidence
2. Endothelial function biochemical markers, namely ADMA, SDMA, NO, high-sensitivity
C-reactive protein (hs-CRP).
Methods
Subjects: Male patients (n= 69), all between 35 and 50 years of age, with single or multivessel CAD, were recruited from in- and out-patient settings of the National Heart Institute
(NHI) in Imbaba, Cairo. CAD was verified by either a history of myocardial infarction,
percutaneous coronary intervention, or coronary catheterization. Age- and sex-matched
controls (n= 20) were also recruited, provided that they presented with no diagnostic signs
of CAD. Both groups had a controlled blood pressure of below 140/90 mmHg.
25(OH)D determination: High performance liquid chromatography with ultraviolet
detection (HPLC-UV) was employed, utilizing an in-house developed and validated
method capable of providing an individualized result for both forms of the metabolite,
25(OH)D2 and 25(OH)D3.
Biochemical investigations: NO was determined using Griess reaction whereas LArginine, ADMA and SDMA were analyzed using liquid chromatography-mass
spectrometry (LC-MS). Hs-CRP was assessed using a commercially-available ELISA kit.
Figure 1: Linear regression analyses investigating the correlation of 25(OH)D
concentrations with ADMA (A) and SDMA (B) levels. An R-square of 0.002800 was
obtained for (A) and 0.006479 for (B) demonstrating minimal correlation between the
investigated parameters. F- and P-values were 0.049 and 0.783 respectively for (A),
whereas 0.038 and 0.846 respectively for (B), thus illustrating a lack of significance in
both cases.
Conclusions
Statistical analyses: Analyses were performed using GraphPad Prism statistics software
(GraphPad Software, Inc.). Correlations between two measured parameters were made
using linear regression analysis as well as comparison of their means using the t-test.
Statistical significance was defined as obtaining a P-value of less than 0.05. All results,
unless stated otherwise, are presented as means ± standard error of the means (SEM).
Means of all investigated parameters are presented along with their corresponding 95%
confidence interval (95% CI). Cohen’s d was used as a measure of effect size for all
significant and insignificant values obtained.
Results
Normal
vitamin D
Suboptimal
vitamin D
P-value
Cohen’s d
ADMA
(µmol/L)
0.61 ± 0.05
(0.52-0.70)
0.59 ± 0.02
(0.55-0.63)
0.692
0.11
SDMA
(µmol/L)
0.53 ± 0.04
(0.46-0.61)
0.53 ± 0.04
(0.47-0.61)
0.998
0.003
l-Arginine
(µmol/L)
84.75 ± 8.62
(70.11-99.39)
94.74 ± 6.24
(83.53-105.9)
0.3952
0.27
NO
(µM)
44.53 ± 5.18
(37.69-51.37)
29.47 ± 3.43
(24.53-34.41)
0.031
0.90
hs-CRP
(mg/L)
8.27 ± 3.56
(2.64-13.91)
22.89 ± 4.86
(14.92-30.86)
0.035
0.66
Table 1: Overview of the biochemical parameters investigated in CAD subjects exhibiting
normal and suboptimal vitamin D levels. Statistical significance is found in the
comparison of NO and hs-CRP concentrations between the two groups. 95% CIs are
indicated in parentheses following their corresponding means.
In view of the results presented here, this study proposes the molecular mechanism
linking vitamin D with endothelial dysfunction to be related to inflammation and not via
modulation of the dimethylated arginines. Further investigations would unveil how
vitamin D regulates inflammatory markers and in turn, vascular function. Results of this
study also add to existing data linking vitamin D with CVD on an observational level.
Bibliography
1. Holick MF. 2007. Vitamin D deficiency. N Engl J Med 357: 266-81.
2. Abu El Maaty M.A, Gad MZ. 2013. Vitamin D deficiency and cardiovascular disease:
potential mechanisms and novel perspectives. J Nutr Sci Vitaminol (Tokyo)59: 479488.
3. Boger RH. 2004.Asymmetric dimethylarginine, an endogenous inhibitor of nitric oxide
synthase, explains the "L-arginine paradox" and acts as a novel cardiovascular risk
factor. J Nutr 134:2842S-7S; discussion 53S.
4. Gad MZ. 2010 Anti-aging effects of L-arginine. Journal of Advanced Research. 1, 169177.
5. Abu El Maaty MA, Hassanein SI, Hanafi RS and Gad MZ. 2013. Insights on vitamin
D’s role in cardiovascular disease: Investigating the association of 25-Hydroxyvitamin
D with the dimethylated arginines. J Nutr Sci Vitaminol (Tokyo) 59: 172-177
6. Ngo DT, Sverdlov AL, McNeil JJ, Horowitz JD. 2010.Does vitamin D modulate
asymmetric dimethylarginine and C-reactive protein concentrations? Am J Med
123:335-41.

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